Typical information processing is thought to depend on the integrity of neurobiological oscillations that may underlie coordination and timing of cells and assemblies within and between structures. The 3–7 Hz bandwidth of hippocampal theta rhythm is associated with cognitive processes essential to learning and depends on the integrity of cholinergic, GABAergic, and glutamatergic forebrain systems. Since several significant psychiatric disorders appear to result from dysfunction of medial temporal lobe (MTL) neurochemical systems, preclinical studies on animal models may be an important step in defining and treating such syndromes. Many studies have shown that the amount of hippocampal theta in the rabbit strongly predicts the acquisition rate of classical eyeblink conditioning and that impairment of this system substantially slows the rate of learning and attainment of asymptotic performance. Our lab has developed a brain–computer interface that makes eyeblink training trials contingent upon the explicit presence or absence of hippocampal theta. The behavioral benefit of theta-contingent training has been demonstrated in both delay and trace forms of the paradigm with a two- to fourfold increase in learning speed over non-theta states. The non-theta behavioral impairment is accompanied by disruption of the amplitude and synchrony of hippocampal local field potentials, multiple-unit excitation, and single-unit response patterns dependent on theta state. Our findings indicate a significant electrophysiological and behavioral impact of the pretrial state of the hippocampus that suggests an important role for this MTL system in associative learning and a significant deleterious impact in the absence of theta. Here, we focus on the impairments in the non-theta state, integrate them into current models of psychiatric disorders, and suggest how improvement in our understanding of neurobiological oscillations is critical for theories and treatment of psychiatric pathology. Recent findings suggest that an estimated 18.1–36.1% of the global population will suffer from a mental disorder, as classified by the Diagnostic and Statistical Manual of Mental Disorders, during their lifetime (1). Onset of these conditions can begin as early as childhood or not appear until late adulthood. One of the primary areas affected by mental illness is cognitive functioning, including attention and memory. Cognitive disruption is seen in a wide range of psychiatric disorders, including, but not limited to, major depressive disorder (MDD) (2), schizophrenia (3), and Alzheimer’s disease (AD) (4). Due to its efficacy in both humans and animal models, eyeblink conditioning (EBC) has proven valuable as a behavioral marker of cognitive impairment in mental illness. Through studies of human patients and animal models, researchers have identified disruptions in electrophysiological activity in each of these disorders (5–8). This review summarizes a series of findings on the relationship between theta oscillations in the hippocampus and EBC in the rabbit. We propose that EBC, which is remarkably similar behaviorally and neurobiologically in humans, can be a productive model system that can serve as a marker for psychiatric disorders and allow invasive local field potential (LFP) and single-unit analyses to investigate their neural substrates. We have developed a brain–computer interface that allows us to give training trials in the explicit presence (T+) or absence (T−) of theta in the CA1 region of dorsal hippocampus. A major feature of this interface is that, unlike drug, lesion, or genetic manipulations, our method allows the phasic increases and decreases of theta that characterize intact hippocampal function and may be a critical aspect of theta’s influence on cognitive processes. We will show that EBC training in the explicit absence of theta reproduces several important behavioral and electrophysiological dysfunctions similar to what is observed in major psychiatric disorders. We argue that the electrophysiological markers at the cellular level during disordered behavioral performance will aid in our understanding of these pathologies and set the course for manipulations or treatments that can restore function or prevent the progression of disease. A major theme will be that neurobiological oscillations, especially theta, serve as important coordinators and facilitators of distributed cognitive brain systems and that the disintegration of these areas is responsible for cognitive impairment and, in extreme cases, psychiatric disorders. We conclude with recommendations for the directions such research may take. Rabbit classical EBC is a widely used model of associative learning. It has been used in research involving humans (9) and non-human animals to investigate the neural substrates of associative learning (10). The EBC paradigm typically involves the presentation of a relatively neutral conditioned stimulus (CS), such as a tone, paired with, but preceding, the presentation of behaviorally relevant unconditioned stimulus (US), such as a corneal airpuff. After sufficient pairings, the subject learns to perform an adaptive eyeblink conditioned response (CR) to the CS, prior to the arrival of the airpuff US. EBC is most commonly presented in one of two general paradigms, delay or trace conditioning. In delay EBC, the CS and US overlap and coterminate. The essential neural circuitry for delay EBC is well established and is contained within the cerebellum [for review: (11)]. The primary site of plasticity has been localized in the interpositus nucleus (IPN). Lesions of the IPN completely prevent acquisition of CRs and eliminate responding in previously trained animals without preventing eyeblinks to the UR (12). In addition to the IPN, the cerebellar cortex has also been shown to be necessary for delay EBC (13), playing a role in the precise timing and amplitude of the CR. Information about the US projects from the inferior olive (IO) to Purkinje cells in the cerebellar cortex and granule cells of the IPN via climbing fibers. CS-related information projects from the lateral pontine nuclei (LPN) to the cerebellar cortex and IPN through the mossy fiber pathway. This cerebellar pathway is essential for delay EBC acquisition and performance, but there are also structures that seem to play a modulatory role. The hippocampus, a structure strongly implicated in learning and memory, is unnecessary for learning the delay paradigm (14), though electrophysiological studies have shown conditioning-dependent changes in cellular response profiles over training (15, 16). Additionally, lesions of the amygdala have been shown to disrupt reflex facilitation in rabbits (17). Lee and Kim (18) provide evidence that the amygdala and hippocampus modulate the emotional and muscular components of EBC, respectively, interacting to allow for the overall learned behavior. The trace form of EBC alters the paradigm by introducing a stimulus-free period between CS offset and US onset. This form of EBC still requires the cerebellar pathway discussed above (19), but lesion and inactivation studies have shown that it is influenced by the amygdala (20, 21), and requires the medial prefrontal cortex (22) and hippocampus (23). Pharmacological inactivation of the hippocampus with scopolamine, a muscarinic acetylcholine (ACh) receptor antagonist, prevented learning; however, a day of training with saline infusions resulted in a gradual acquisition of the paradigm as if training had just begun (24). Disruption of hippocampal functioning via lesions or pharmacological inactivation of major inputs has also been shown to cause behavioral deficits (25–27). Additionally, electrophysiological studies have identified conditioning-related changes in hippocampal cellular responding during the trace paradigm. Multiple-unit recordings have demonstrated gradual increases in response magnitude during the late half of the trace period as training progresses (28). McEchron and Disterhoft (29, 30) have identified several unique response profiles for hippocampal pyramidal cells at the single-unit level. The response profiles most associated with CR learning show increases in pyramidal cell firing to both the CS and US early in training; however, as the animal approaches behavioral asymptote, the response to the US, but not to the CS, begins to decrease. Additionally, recent work has shown that conditioning-related increases in single-unit firing continue through retrieval of the consolidated memory (31). Eyeblink conditioning does not serve solely as an animal model, having been used in human subjects for over a century (32). As in rabbits, patients with cerebellar damage are impaired in learning the delay and trace forms of EBC (33–35). Those suffering hippocampal damage fail to acquire trace, but are able to learn delay EBC (9, 36–38). Additionally, neuroimaging work has implicated a role for the prefrontal cortex in trace EBC (39, 40). Due to the well-defined circuitry necessary for successful EBC performance, this paradigm is able to provide critical input into the neural regions affected in several psychiatric disorders. Early research in patients with MDD implicated cerebellar dysfunction primarily through neuroimaging studies (41–43). The behavioral effects identified with EBC serve to corroborate regional dysfunction observed in neuroimaging studies. Training patients on both delay and trace EBC, Greer et al. (44) provided behavioral evidence indicating abnormalities in cerebellar processing. They found a significant decrease in the number of CRs in MDD patients compared to controls across both forms. While these results do not allow for differentiation of cerebellar and hippocampal dysfunction, comparison of the delay and trace paradigms has been used in other disorders to differentiate functional regions. Grillon et al. (45) compared performance on both the delay and trace EBC paradigms in patients suffering from panic disorder. There was no difference in performance between patients and control subjects on the delay task; however, patients performed significantly worse on the trace paradigm, showing a delayed acquisition rate. This pattern of results indicates hippocampal dysfunction and potential deficits in declarative memory in panic disorder patients. As panic disorder requires unexpected panic attacks, the authors posit that these deficits may underlie a patient’s inability to identify predictive cues. Results have been less clear in studies of schizophrenia. Early work indicated a possible enhancement of delay EBC, with patients demonstrating faster acquisition rates than controls (46, 47). More recently, several studies have found impaired delay EBC performance through decreased acquisition rates (48–53), decreased CR amplitude (54), and less adaptively timed CRs (50) compared to controls, as well as linking those deficits to decreased cerebellar volume (49) and blood flow (52). Additionally, Marenco et al. (55) demonstrated an increase in short latency (non-adaptively timed) CRs during trace EBC in schizophrenic patients. Eyeblink conditioning has been especially prominent in the study of AD, being used both in animal models and in human patients. Studies have shown deficits in acquisition rate for both the delay (33, 56–58) and trace paradigms (59–61), with a larger effect in the delay paradigm (59). Papka and Woodruff-Pak (62) identified the number of trials necessary to accurately assess delay EBC in AD patients, providing a more efficient test of cognitive performance that may serve as a diagnostic tool in differentiating normal aging from dementia (63). While delay EBC can be acquired normally after hippocampal removal, pharmacological disruption of the septo-hippocampal cholinergic system leads to deficits in performance (26, 64). As cholinergic disruption is a key component of AD pathology (65–67), parallel findings between rabbits with cholinergic dysfunction and AD patients provide validation of the animal model. Furthermore, galantamine, a cholinesterase inhibitor, facilitates EBC performance in aged, but not young, animals, suggesting that it counteracts the decrease in cholinergic activity associated with aging (68). Cholinergic systems have long been associated with cognitive functions, such as attention and memory, that are often affected in psychiatric disorders (69). The basal forebrain cholinergic system is deserving of particular attention due to the target structures of its separate cholinergic neuron populations. The first originates in the horizontal limb of the diagonal band of Broca (DBB) and nucleus basalis and projects to areas of the cortex, such as the mPFC (70), an area involved in sustained attention (71). A separate population of cholinergic projections originates in the medial septum and vertical DBB targeting the dorsal hippocampus, an essential region for encoding of declarative memory. Numerous lines of research have converged to show deficits in cholinergic functions underlying the cognitive deficits of several psychiatric disorders. In AD patients, postmortem studies have indicated a loss of cholinergic neurons in the nucleus basalis (72), a finding supported recently using MRI (73). Additionally, the primary treatments for AD involve acetylcholinesterase inhibitors as a means of increasing cholinergic activity (74–76). Other disorders linked to cholinergic dysfunction include schizophrenia and MDD. In humans, muscarinic antagonists have been shown to increase the severity and duration of both positive and cognitive symptoms in schizophrenic patients (77, 78). Furthermore, anti-muscarinics can lead to a temporary psychosis resembling schizophrenia in healthy subjects (79). Postmortem studies have shown a decrease in muscarinic ACh receptors in schizophrenia patients (80, 81). Additionally, acetylcholinesterase inhibitors have been useful in treating hallucinations (82). These findings have been corroborated in animal models where muscarinic antagonists have led to cognitive impairments and psychosis indicating behaviors in rodent models (78). Though less research has been conducted in MDD patients, recent studies have shown antidepressant effects of scopolamine, a muscarinic receptor antagonist (83), and decreased levels of muscarinic receptors in MDD. As hippocampal theta power is positively correlated with ACh activity (84, 85), it may be possible to use our model system, in which the non-theta group likely shows diminished cholinergic activity immediately preceding conditioning trials, to explore electrophysiological and behavioral bases of these disorders. Neurobiological oscillations have been associated with memory processes, feature binding, and consciousness through their ability to synchronize across and within brain regions, though a definitive function has not been established (86–89). Synchronization of cellular activity within a region can be clearly seen in the strong relationship of single-units and neurobiological oscillations with many cells having preferred phases of the oscillation to increase their firing rates (90–93). Oscillatory potentials can be divided into a several frequency bands based on functional behaviors with which they are associated, as well as cellular and pharmacological mechanisms underlying their generation (88). It is important to note that these different oscillations do not operate in isolation, with multiple theories proposing an interaction between two frequency bands being essential for cognitive processes (89, 94, 95). As normal functioning requires the complex interplay of oscillatory activity across brain regions, lack of synchrony or perturbations of these endogenous signals can lead to detrimental effects associated with several psychiatric disorders. In recent years, research into causes and potential treatments for schizophrenia has increasingly emphasized a basic understanding the neural circuits and processes leading to the myriad of symptoms. Due to the large-scale network believed to be involved in the disorder, abnormalities in oscillatory dynamics seem poised to play a major role in explaining the cognitive deficits (5). At a relatively broad level, schizophrenia has been associated with alterations in the relative power of several oscillatory frequencies associated with cognitive processes, including theta (4–7 Hz), alpha (8–12 Hz), beta (15–30 Hz), and gamma (40–100 Hz) (5–8, 96, 97). Some research has also indicated the importance of understanding different frequency oscillations in the context of their cross-frequency modulation, particularly in regard to gamma and theta (97). Researchers have also attempted to examine disruptions in neural dynamics and relate them to specific disruptions of behavioral tasks (6). A common finding in electrophysiological research is phase locking of oscillatory activity following stimulus presentation, a phenomenon typically allowing for coordination of neuronal firing across a distributed system. However, schizophrenic patients have shown delays in phase locking following auditory (98) and visual stimulation (99), with the degree of phase locking correlated with the extent of visual hallucinations and thought disorders (100). Additionally, while increases in frontal midline theta are typically seen following initiation of working memory tasks (101), schizophrenic patients show no increase, and at times a decrease, of evoked theta at various degrees of working memory load (102). These disturbances have been linked to a lack of theta coherence between left frontal and temporal EEG recordings in schizophrenics compared to controls (103). At the cellular level, a loss of synchrony may affect the optimal balance between excitation and inhibition, particularly in regard to activity of GABAergic interneurons (96). Similarly, MDD has been characterized by alterations in oscillatory activity across theta, alpha, and beta bandwidths, but has also shown decreases in delta (0.5–3 Hz) activity (104, 105). These patterns result in changes of the relative ratio of each frequency, creating a highly heterogeneous state (104). MDD patients show a convoluted pattern of effects in terms of oscillatory synchronization. While MDD is characterized by increased synchronization of alpha and beta, as well as frontal theta (105, 106), several studies have also demonstrated a decrease in frontal theta power relative to controls (107–109). Furthermore, increases in theta power following deep brain stimulation have been shown to predict long-term clinical efficacy of treatment (110). Extending beyond frontal theta, animal models of MDD have revealed the effects of theta in the medial temporal lobe (MTL). Zheng and Zhang (111) found a decrease in theta phase coupling between the ventral hippocampus and medial prefrontal cortex that was associated with decrease in synaptic plasticity of the pathway. Furthermore, Sauer et al. (112) have shown reduced synchrony of theta and gamma oscillations in the prelimbic cortex attributed in part to a decrease of output from prelimbic GABAergic interneurons. Finally, it is important to consider neurobiological oscillations in AD, a disorder most commonly noted for the presence of amyloid beta (Aβ) plaques. Recent work has shown the potential of oscillatory activity as a means of early AD diagnosis. Compared to controls, AD patients have shown lower theta phase locking to stimuli (8), as well as decreased functional connectivity as measured by phase synchronization (113–115). Utilizing Granger causality and stochastic event synchrony, Dauwels et al. (116) demonstrated that loss of EEG synchrony can accurately predict occurrence of AD based on pre-dementia data. Using EEG synchrony as a screening tool can potentially be improved upon by applying principal component analysis before estimating synchrony (117). Animal models of AD are also being used to characterize the cellular basis of maladaptive alterations in oscillatory and cellular activity. Increasing disruption of hippocampal theta oscillations has been shown in Aβ overproducing transgenic mice as a function of age (118). Guitérrez-Lerma et al. (119) found that the two different types of hippocampal theta are affected differentially by a variety of Aβ peptides. Hippocampal pyramidal cells are disrupted in normal aging, showing a decrease in excitability over time (120, 121), as well as in AD models in which desynchronization of action potential generation leads to a shift in the excitatory/inhibitory equilibrium (122). Hippocampal Aβ also impacts functioning in target structures. For example, investigating a decrease in hippocampal theta power, Villette et al. (123) showed a reduction of firing activity in GABAergic neurons in the medial septum. Importantly, this reduction in firing was not caused by a loss of neurons, but rather an alteration in their normal firing pattern. Our model system permits analysis of specific electrophysiological responses to the conditioning stimuli in terms of LFP synchrony and cellular reactivity with precise control of hippocampal theta state. Though psychiatric disorders are accompanied by disruptions in several frequency bands, work in our lab has focused on the hippocampal theta rhythm (3–12 Hz). Across a range of species and tasks, hippocampal theta has been implicated in spatial (90, 91, 124–126), declarative (127–129), and working (101, 130, 131) memory processes. Within the theta band, Kramis et al. (132) identified two types of theta that are pharmacologically and behaviorally different, cholinergic (3–7 Hz) and non-cholinergic (8–12 Hz) theta. Cholinergic theta is present during alert immobility and is eliminated by the muscarinic ACh receptor antagonist, atropine. Non-cholinergic theta appears during voluntary movements and is unaffected by atropine. Both types of theta have been shown in the rabbit depending on the task (132, 133), with cholinergic theta being the dominant frequency during EBC. In 1978, Berry and Thompson (134) identified a cognitive benefit of hippocampal theta that would serve as the foundation of the future development of our brain–computer interface (BCI). They found a strong positive correlation between pre-training hippocampal theta and learning rate, a finding that was recently replicated in rabbits (135) and extended into human spatial learning (136, 137). Several studies have shown that lesions to the MS reduce hippocampal theta power and significantly slow learning of an EBC task (25, 26, 64, 138). Additionally, eliciting theta through MS stimulation or water deprivation has led to increases in learning rate (139, 140). It is important to note, however, that all of these studies utilized non-physiological alterations to the LFP, disrupting the natural ebb and flow that some believe to underlie the role of theta in cognitive processes (88, 141, 142). Also, it has been shown that artificial stimulation of the MS distorts the normal physiological response patterns of theta-related cells in the hippocampus (143). Thus, allowing the normal fluctuations of theta and non-theta states, as our interface does, may be a key to understanding the natural role of oscillations in behavioral learning and cellular response profiles. To address that important issue, Seager et al. (144) developed a BCI capable of making training trials contingent on fluctuations in the naturally occurring oscillations. For a comprehensive overview of the BCI design and methodology, see Hoffmann et al. (145). Briefly, the BCI uses real-time spectral analysis to restrict EBCC trials to the explicit presence (T+) or absence (T−) of hippocampal theta (Figure 1). To accomplish this, either chronic monopolar electrodes or independently moveable tetrodes are implanted in area CA1 of the hippocampus. During training, a custom LabView program calculates a ratio of power at bandwidths specified by the experimenter. For our work that involves calculating the ratio of theta (3.5–8.5 Hz) to non-theta (0.5–3.5 Hz and 8.5–22 Hz) in real time. The ratio is calculated for 640-ms running time intervals, offset by 160 ms to allow for partially overlapping samples. In the T+ condition, a trial is triggered if the ratio of theta to non-theta exceeds 1.0 for three consecutive intervals. A trial is triggered in the T− condition if the ratio falls below 0.3 for three consecutive intervals. This methodology allows for the different training groups to receive trials under opposite theta conditions while still allowing for the natural fluctuation between trials. Figure 1. Surface plots of power spectral density (PSD) of the pre-CS period triggering a trial for 1 day of training for an animal in the T+ (A) and T− condition (B). Trials in the T+ condition were consistently triggered under conditions of high theta and low delta and alpha. The T− condition was triggered by periods of low theta and high delta or alpha. Note that the T− condition is more heterogeneous than T+, with trials being triggered under both high delta and high alpha conditions. Figures created from data published in Cicchese et al. (146). The initial BCI study examined the effects of theta-contingent training during a delay EBC paradigm (144). Subjects were divided into four groups: (1) trials triggered in the explicit presence of theta (T+); (2) trials in the explicit absence of theta (T−); (3) T+ yoked controls, inter-trial intervals matched to the T+ subjects regardless of theta state; and (4) T− yoked controls. Animals trained under T− conditions learned significantly slower than those in the T+ condition (Figure 2A), requiring more trials to reach asymptotic performance (eight CRs out of nine consecutive trials; 8/9 CRs) and showing a lower percentage of CRs across training. Additionally, T− subjects required significantly more trials to the 8/9 criterion than their yoked controls (Figure 2B), highlighting the detrimental effects of T− training. This is important to note when considering non-theta-contingent training as a natural model of a dysfunctional hippocampus, as these results coincide with the previous findings that pharmacologically disrupting hippocampal functioning is more detrimental to delay EBC than having no hippocampus (64). These findings have been extended to trace EBC in several studies. Utilizing the same four groups (T+, T−, T+ yoked, and T− yoked), Griffin et al. (28) showed that T− animals required significantly more trials to reach early (fifth CR) and late (8/9 CRs) learning criteria, demonstrated a lower percentage of CRs on the first 4 days of training, and required more trials to reach fifth CR than their yoked control counterparts. These results have been replicated by our lab with T− animals reaching the fifth CR criterion later than T+ animals (146, 147) and T− animals showing a lower percentage of CRs across the first 4 days of training (148). Taken together, the deficits seen in both delay and trace EBC mirror the patterns seen in patients and animal models of several psychiatric disorders. This is particularly relevant for disorders in which the cholinergic system is affected, such as AD, as the T− condition reflects a period where the cholinergic system is not engaged. Figure 2. (A) Average number of trials required to reach behavioral criteria in delay (8/9 CRs) and trace (fifth CR and 8/9 CRs) forms of EBC. Animals trained under T− conditions required significantly more trials than T+ animals to reach asymptotic performance (8/9 CRs) in delay conditioning, as well as more trials to reach early (fifth CR) and asymptotic (8/9 CRs) behavioral markers. (B) Average difference in the number of trials to reach behavioral criteria from yoked controls. T− animals needed more trials than controls to reach asymptotic performance of delay conditioning and more trials to reach the early learning criteria of trace conditioning. The differences from yoked controls provide evidence of detrimental performance in the T− condition, showing that T−/T+ differences are not simply an effect of improved performance in the T+ condition. *p< 0.05. Delay figures adjusted from Seager et al. (144), trace figures adjusted from Griffin et al. (28). Furthermore, our BCI findings point to a potential treatment for cognitive deficits seen in aging and AD. Asaka et al. (149) examined the effects of theta-contingent training on aged animals, those that typically show learning deficits (150, 151). Four groups of animals were trained, young T+, young yoked controls, aged T+, and aged yoked controls. As expected, aged yoked controls performed significantly worse than young yoked controls, taking longer to reach several late learning behavioral criteria (including 8/9 CRs and 80% CRs in a session). However, aged T+ animals learned significantly faster than aged yoked controls, and showed no difference in learning rate from young yoked controls (Figure 3). Importantly, the benefit of T+ training persisted past behavioral indicators of asymptotic performance in aged animals, suggesting that sustained accurate performance, a cerebellar-dependent function, is also affected by oscillatory state. While aging is accompanied by a decrease in cholinergic activity, the presence of 3–7 theta in the hippocampus demonstrates that periods of relatively normal cholinergic activity persist that can be engaged as a non-pharmacological intervention for cognitive deficits. Figure 3. Average number of trials to reach the late learning criteria (8/9 CRs and 80% CRs) for young yoked controls, aged T+ triggered, and aged yoked control animals. Aged yoked controls required more trials to reach both criteria than young yoked controls, indicating disrupted performance in aged animals. Aged animals trained under T+ conditions performed better than their yoked control counterparts and showed no difference from the young yoked controls. Thus, theta-contingent training alleviated the cognitive deficits seen in aged controls. *p< 0.05. Figure adapted from data published in Asaka et al. (149). These behavioral results are consistent with recent studies in human subjects. Using magnetoencephalographic (MEG) recordings, Guderian et al. (152) found a positive correlation between pretrial theta amplitude in the MTL and recall rate in an episodic learning task. Following this demonstration, Fell et al. (153) recorded bilaterally along the longitudinal axis of the MTL with intracranial EEG. Enhancement of hippocampal theta predicted successful encoding of a word recognition task. Similarly, Lega et al. (154) recording from the hippocampus of neurosurgical patients showed higher theta power during encoding. Interestingly, the researchers identified a slow and fast center in the theta rhythm, and only the slow theta (~3 Hz) showed this pattern. In addition to deleterious behavioral effects, training in the explicit absence of theta has been shown to have negative effects on hippocampal electrophysiology at the LFP, multiple-unit, and single-unit levels. Previous work in rats has demonstrated a phase reset of the local theta rhythm following stimulus presentation (155, 156). Using the trace EBC paradigm, our lab has replicated this phase reset and shown coherent rhythmicity at theta frequencies in T+ animals following both CS and US presentation (147, 148); however, animals trained under T− conditions display a delayed onset of phase reset, as well as decreased rhythmicity in theta frequency compared to T+ animals. These results in the T− condition are important to consider as McCartney et al. (156) have shown that the phase reset produced by relevant stimuli provides ideal conditions for LTP to occur, suggesting a decrease in neural plasticity when trained in the absence of theta. Additionally, this delayed phase reset is comparable to that seen in schizophrenic patients in response to both auditory (98) and visual (99) stimuli. Coinciding with the effects on LFPs, T− training impairs both the magnitude and rhythmicity of hippocampal multiple-units. During trace EBC, multiple-units in T− animals inhibited below baseline firing during presentation of the tone and through the 500-ms trace interval, while those in T+ animals showed excitation (28). Note that this indicates an active suppression or inhibition of unit firing under T− conditions rather than simply the absence of an excitatory response. While this effect was seen on the second and third days of training, Darling et al. (147) linked this decrease in activity of T− units to behavioral criterion, showing significant inhibition at the early (fifth CR) and late (8/9 CRs) learning markers. Furthermore, similar to what has been seen in LFPs, T− multiple-units lack rhythmicity in firing during the trace interval, whereas T+ units fired coherently at 6.25 Hz (147). Early work in rabbit EBC showed that conditioning-dependent changes in multiple-unit activity were the result of changes in pyramidal cell activity (16, 157). To replicate this, our theta-triggered work was continued with single-unit recordings of hippocampal pyramidal cells. To determine whether changes in multiple-unit activity were caused by large firing rate changes in a few critical cells or by a change in the overall number of cells responding in a particular way (firing rate increasing or decreasing), Cicchese et al. (146) analyzed pyramidal cell responses by their qualitative (rate increasing or decreasing) and quantitative (response magnitude) properties. Early in learning, putative pyramidal cells were more likely to decrease their firing rate during the tone period in T− than in T+ animals and more likely to increase their firing rate during both the tone and trace periods in T+ compared to T− (Figure 4). Importantly, there were no theta-contingent differences in the magnitude of either firing rate increases or decreases. These findings suggest that the role of theta in cellular firing is related to the recruitment of additional units firing a particular pattern, rather than a drastic change in rate of relatively few cells. This implies that an optimal hippocampal ensemble response for EBC consists of more widespread excitation of pyramidal cells rather than a sparse code of heightened responses by a few cells. Thus, theta may serve to optimize the ratio of cells showing excitation or inhibition, leading to a dysfunctional balance in the absence of theta. This conclusion would agree with findings from models of schizophrenia (96) and AD (122), implicating a shift in the excitatory/inhibitory equilibrium as a potential cellular mechanism. Additionally, Rutishauser et al. (158) found a positive correlation between performance of a memory task and coordination of hippocampal spike timing to the local theta rhythm. This is consistent with our results showing a learning deficit in T− subjects accompanied with less coherence of pyramidal cell response direction. Figure 4. Surface plots showing the standard scores (10-ms bins) of all rate decreasing (A) and rate increasing (B) cells averaged across the entire training session (truncated to 4 for illustration purposes). Note that rate decreases and increases during tone presentation and are sustained past airpuff presentation. (C) A greater percentage of cells in the T− condition were rate decreasing during the tone than in the T+ condition. (D) Cells in the T+ condition were more likely to increase their firing during the tone and trace periods than those in T−. *p< 0.025. Figure adapted from data published in Cicchese et al. (146). Due to the distributed memory system involved in trace EBC, it is important to consider how non-theta-contingent training may negatively affect processing in other necessary regions. LFP recordings taken from hippocampal CA1, and cerebellar IPN and HVI, have revealed striking theta-contingent differences in both rhythmicity and synchronization between areas that may underlie dysfunctional processing during training (148). Coinciding with improved behavioral performance, T+ animals showed theta rhythmicity time-locked to conditioning stimuli in the cerebellum and precise theta antiphase (180^o) synchronization between CA1 and IPN/HVI LFPs. By contrast, T− performance deficits were accompanied by an absence of theta oscillations in IPN and HVI, as well as a lack of synchronization with CA1. These results are consistent with human studies showing an increase in theta synchrony across distributed regions following induction of MTL theta oscillations (159), as well as with fear conditioning studies in rats showing a synchronized theta activity between the lateral amygdala and hippocampus following training (160). The lack of synchronization across areas is of particular interest in light of psychiatric research. Animal models of MDD have implicated the absence of ventral hippocampus–mPFC theta phase coupling with decreased synaptic plasticity (111), while a loss of cortical EEG synchrony is a fundamental feature in AD (113–116). These oscillatory disruptions likely cause a decline in functional connectivity, failing to coordinate activity across regions necessary for cognitive processes. The hippocampus does not directly project to the cerebellum, but may have an indirect influence through its effects on the mPFC. The mPFC is necessary for trace EBC (161, 162) and projects to the lateral pontine nucleus, which conveys important CS-related mossy fiber input to the cerebellum (163). Previous work has identified a mPFC cellular response profile characterized by inhibition followed by a period of persistent excitation in response to tone presentation (164). This pattern is thought to increase the salience of the tone by increasing the signal-to-noise ratio. Darling et al. (147) capitalized on our theta-triggered paradigm by recording simultaneously from area CA1 and the mPFC (caudal anterior cingulate region) under T+ and T− conditions. Interestingly, though the inhibitory/excitatory pattern was replicated in T+ animals, it was absent in those trained under T− conditions. This finding implies that mPFC processing is highly related to hippocampal theta state and that our T− animals may fail to apply proper motivational salience to the conditioning stimuli. Importantly, the increased theta synchrony between hippocampus and amygdala during Pavlovian conditioning (160) raises the possibility that motivational and emotional input from the basolateral amygdala normally converges on the mPFC in synchrony with hippocampal input to modulate salience; thus, in the absence of hippocampal theta, a lack of converging input disrupts processing of the stimuli. A similar effect is seen in schizophrenia where patients show maladaptive motivational salience when rating reinforcements (165) and when learning to discriminate between a predictive CS+ and neutral CS− (166, 167). Additionally, compared to controls, schizophrenia patients show increased neural activity to the CS− in regions associated with learning (166, 167). Thus, our T− condition appears to replicate some important findings from the human literature and relate them to neuronal response patterns in important structures. As the study of cognitive processes has moved away from discrete functional regions to distributed neural networks (168), it is essential to understand the oscillatory activity capable of synchronizing these anatomically disparate regions (88, 141, 142). Similarly, a focus on electrophysiological disruption in psychiatric disorders is proving invaluable as loss of synchronization across regions is a common feature underlying their pathology (8, 112–114). Using our BCI, we have shown that training in the explicit absence of hippocampal theta produces deficits in EBC expected of a number of psychiatric conditions. Furthermore, these behavioral deficits are accompanied by electrophysiological disruptions at the LFP (147, 148), multiple- (28, 147), and single-unit (146) levels that are characteristic of conditions as disparate as schizophrenia, MDD, and AD. Of particular interest are the patterns seen across the regions necessary for EBC, with a lack of synchrony between hippocampus and cerebellum (148) and the absence of relevant response patterns in mPFC units (147). Though our non-theta-triggering has proven effective at modeling the electrophysiological correlates of a disrupted system, it is important to note that it still has room to grow. The BCI allows for trials to be delivered in the presence of a specific brain state, but does not give control of that activity. Thus, fluctuations in pretrial activity that may typically be abnormal in disorders cannot be controlled for. However, the ability of our non-theta-triggering to model interruption of distributed neural networks without lesions or pharmacological intervention provides a tool for studying psychiatric disorders in a more natural way, allowing for decreased levels of the given frequency, as is typical in illness, rather than complete abolition. An important challenge to our findings has recently been published in the form of a failure to replicate the benefits of theta-contingent EBC (169). The authors found that animals trained under T− conditions were more likely to acquire the paradigm than yoked controls or those trained in the presence of theta; however, it should be noted that T− animals required more sessions to reach behavioral criterion than their yoked controls, consistent with our findings. These findings seem to contradict numerous studies in animals (28, 135, 144, 146–148) and humans (152–154), showing beneficial learning effects of increased hippocampal/MTL pretrial theta. Due to a fundamental methodological difference, it is possible that the study by Nokia and Wikgren (169) does not directly apply to our work. Specifically, in their study, all subjects were presented with a full session of unpaired conditioning before training began. This introduces latent inhibition as a major confound to later learning effects. While T+ and T− animals each received the unpaired session, work has not been completed to investigate how effects of latent inhibition may interact with theta-contingent learning conducted after unpaired presentations. For example, unpaired presentations of CS and US have been shown to cause a baseline EEG shift from pre- to post-exposure (170), and latent inhibition produces significantly reduced hippocampal unit responsiveness to a tone CS (171). An effect of the unpaired session is suggested by the unusually low percentage of animals that successfully acquired the CS–US association. Additionally, T+ animals that reached criterion took an average of ~5 fewer sessions than their yoked control counterparts; however, that difference was not significant, likely due to insufficient power (T+: n = 4, yoked control: n = 2; 0.05 < p< 0.10). While these results highlight the complex relationship between oscillatory potentials and different learning paradigms, potential differences in hippocampal functioning caused by latent inhibition, as well as low statistical power, prevent a direct comparison to our theta and non-theta-contingent findings. Knowing the established effect of theta on cognitive processes, it will be critical to further study its role. In particular, further exploration of mPFC theta activity could serve to bridge the gap between animal and human recording studies. Much of the theta work in human subjects has centered on frontal midline theta, but it is still unclear what the neural correlates underlying these oscillations are (101). By understanding the relationship between oscillations in subcortical structures and those recorded by scalp EEG, it would be possible to utilize neurofeedback training as a possible treatment for psychiatric conditions, similar to what has been done in patients with ADHD (172, 173). Though our BCI does not allow for direct manipulations of theta, new research methods, such as optogenetics, may make this possible. Using optogenetic stimulation of the medial septum could provide precise temporal control of theta rhythm induction. During this stimulation, simultaneous recordings from relevant areas (hippocampus, mPFC, and cerebellum) could provide further insight into the electrophysiological relationship of the distributed network. Specifically, this methodology would allow for precise control over theta phase during stimulation presentation. Considering the prominent model of separate encoding and retrieval phases of theta (128), our T+ group could be further studied by looking at trials triggered consistently on either the peak or through of theta. It is possible that triggering during the retrieval phase of the theta rhythm could be equally detrimental to training in the absence of theta, an idea recently supported using theta-contingent training in conjunction with threshold values to target specific phases (174). Furthermore, optogenetic manipulation of theta state could be used in conjunction with conditional genetic knockout animal models to identify potential benefits of inducing synchronous neural activity in animals that are typically lacking. Initial studies into this possibility could utilize classical conditioning to allow for discrete learning points. By doing so, optogentic stimulation of the medial septum at theta frequency could be initiated prior to CS delivery, ensuring synchronous and homogeneous neural activity when learning is expected to occur. Dependent on the results, additional work should be completed to examine the amount of time asynchronous activity must be disrupted for alleviation of behavioral deficits. While research has shown physiological difference in cellular responding to naturally occurring and artificially stimulated theta (143), it is likely that optogentically induced theta would still provide benefits in animals with genetically disrupted theta oscillations. Several studies using the Morris water maze support this notion. Deficits in performance caused by disruption of hippocampal theta via pharmacological inactivation of the medial septum (175–177) or fimbria-fornix lesions (178) were overcome by artificial stimulation at theta frequency. Conversely, recent contextual fear conditioning work found a decrease in performance as a result of artificial theta stimulation (179). The authors propose, however, that the continuous stimulation provided at a fixed frequency may have interrupted the normal oscillatory processes of the rat; specifically, the constant theta likely interfered with the natural theta entrainment experienced during walking and sniffing as the rat explores its environment. Furthermore, they suggest that stimulation coinciding with an external cue, such as a tone CS, may show enhancement in performance similar to the aforementioned studies. Although our work has focused on the theta to non-theta [3.5–8 Hz/(0.5–3.5 Hz + 8.5–22 Hz)] ratio, the LabView program can be set with any frequency range in the numerator and denominator. With this flexibility, future studies could utilize the BCI for training contingent on different frequency bands and exploration of different definitions of non-theta. Our non-theta state is heterogeneous, with major contributions of delta (0.5–2 Hz) and alpha (8–12 Hz) compared to the homogeneous theta band. This heterogeneity may underlie the detrimental effects seen in our non-theta conditioning. It will be important for future studies to alter the frequencies defined as non-theta, including using individual frequency bands in the denominator, to determine whether the decrease in theta or the heterogeneity of oscillatory bands is responsible for adverse learning. In work by others, triggering trials based on sharp-wave ripple oscillations (150–250 Hz) has been shown to increase EBC learning rate and increase the phase locking of theta oscillations to conditioning stimuli (180), suggesting that the heterogeneity of our non-theta state plays an important role. Therefore, it will be important to continue research into the effects of ripple-contingent training and their relation to theta. 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Stephen D. Berry, [email protected]

DOCSPER [1,3-Dioleoyloxy-2-(N⁵-carbamoyl-spermine)-propane] is a cationic amphiphile consisting of a hydrophobic 1,3 dioleylglycerol moiety and threefold positively charged spermine head group ( ). We optimised the 5-step-synthesis of the lipospermine and after up-scaling we have obtained sufficient amounts to initiate preclinical investigations. DOCSPER was tested for its ability to transfect eukaryotic cells in vitro. It has proven to possess high transfection efficiency in comparison to commercially available liposomal transfection agents. Furthermore, DOCSPER was extensively tested in several in vivo studies ( ). These studies revealed a high transfection efficiency, whereas very low toxicity levels were detected. Thus, the results clearly indicate that the cationic lipid DOCSPER is a reliable, low-risk system for broad applications in gene therapy. Cancer chemotherapy targeted on angiogenic vessels is expected to cause indirect tumor regression through the damage of the neovasculature without the induction of drug resistance. To develop a new tool for neovasculature-specific drug delivery, we isolated novel peptides homing to angiogenic vessels from a phage-displayed peptide library. After the determination of the epitope sequences in isolated peptides, liposomes composed of distearoylphosphatidylcholine and cholesterol were modified with penta-peptides. When ¹⁴C-labeled liposomes were injected into tumor-bearing mice, liposomes modified with Ala-Pro-Arg-Pro-Gly (APRPG) showed the highest accumulation in murine tumor xenografts. Similar results were also demonstrated by using positron emission tomography. APRPG-modified liposomes (PRP-Lip) encapsulating adriamycin (ADM) effectively suppressed experimental tumor growth with reducing side effects compared with control liposomes encapsulating ADM and with free ADM. Furthermore, these liposomes markedly damaged angiogenic vessels in dorsal air sac model. Next, we investigated whether the peptides selected in murine angiogenic model have affinity for angiogenic endothelium in human cancer. Confocal observation demonstrated that APRPG-modified liposome specifically bound to human umbilical vein endothelial cells (HUVECs) only when HUVECs were activated by vascular endothelial growth factor (VEGF). Furthermore, histochemical analysis demonstrated that biotinylated PRP-containing peptide specifically bound to angiogenic endothelium in human insulinoma and glioma specimens. These data indicate that PRP-containing peptides may be useful for human cancer treatment. The present study indicates the usefulness of APRPG-modified liposomes as a tool for anti-neovascular therapy, a novel modality of cancer treatment. The aim of this study was to check the adjuvanticity of squalene in a model consisting of liposomal vaccine, containing an antigenic peptide from the V3 loop of gp120 of HIV. The liposomes were composed of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, with or without cholesterol, squalene, or lipid A. We characterized the interaction between the squalene and the liposomes and measured the effect of squalene on liposome formation and stability. The CGP18 was found at least partially on the surface of the liposomes as shown by flow-cytometricanalysis using monoclonal anti-P18 antibodies. Balb/c mice were injected i.p. with 10 µg of CGP18 with 7 mole%, 43 mole% or 71 mole% squalene, with or without lipid A. Mice injected with liposomal CGP18 containing either lipid A or squalene, developed a moderate anti-CGP18 antibody response at a similar level. Liposomes containing CGP18 without lipid A or squalene failed to develop an antibody response. Splenocytes from mice immunized with liposomes containing CGP18 were tested for INF-γ secretion in an ELISPOT assay. Lipid A enhanced the IFN-γ production 15–50 fold depending on the formulation used, indicating an induction of Th1-response. No increased IL-4 secretion was detected by ELISPOT in any of the groups. In conclusion, liposomes containing squalene show moderate adjuvanticity for induction of a humoral response against CGP18. Addition of lipid A to the liposomes is necessary for IFN-γ secretion by the splenocytes. We conclude further, that squalene itself has a distinctive adjuvant activity upon incorporation into liposomes. Antibody-targeted liposomes (immunoliposomes/IL) have been regarded as very promising targeting vehicles for systemic drug delivery. A liposomal drug therapy necessarily implies a repeated application of liposomes within a certain periode. Despite a clear indication for immunological drawbacks of coupled antibodies, strongly affecting the pharmacokinetic profile, the behavior of IL upon repeated injection has attracted nearly no attention up to now. The present study investigates the pharmacokinetics as well as organ distribution of repeatedly injected IL of different design as indicator for immunogenicity in rats. To correlate IL structure with immunogenicity, the amount of conjugated antibodies was comparable in each case at about 30 µg per μmol total lipid. When injected into naive rats, an increase in elimination can clearly be attributed to the presence of antibodies, since plain pegylated liposomes display the longest circulation half-life (12.8 h). A second injection of identical liposomes into the rats after 14 days has a strong influence on the pharmacokinetic parameters. The circulation time of plain pegylated liposomes drastically droped (t_1/2 5.5 h), which indicates that a sterical barrier of the liposomes seems to be less efficient to reduce opsonization and immune reactions. An active role of PEG-PE in provoking liposomal immunogenicity could not be found. Surprisingly, the circulation time of the IL was only sligthly reduced or, in case of terminally coupled IL nearly unaffected. The differences among the various IL can neither be correlated to the accessibility of the coupled antibodies nor to the intensity of the sterical barrier. However, this study confirms that the immunogenicity of IL cannot be generalized to be very high, but depend on several structural parameters such as antibody coupling technique, liposomal size and composition. Although this study for the first time directly compares different types of IL on repeated injections, the immunological parameters can not totally be cleared at this point. These findings might have important implication for the repeated application of IL as drug carriers. Endothelial cells play an active role in various diseases such as inflammation or cancer metastasis. They undergo phenotypic modulations to an activated state which is especially marked by expression of several cell surface adhesion molecules. These adhesion receptors are attractive targets for cell selective pharmacological intervention employing drug targeting strategies since they are easily accessible due to the direct contact with the blood. Recently, we could demonstrate that immunoliposomes (IL) bearing anti-E-Selectin antibodies specifically accumulate at activated endothelial cells (HUVEC) in vitro. In order to derive therapeutical strategies from liposomal targeting, we analyzed route and degree of cellular uptake of the targeted liposomes. Results from several spectroscopical and microscopical techniques display that about 25% of targeted IL were internalized by active as well as passive mechanisms. The internalization was correlated with several liposomal parameters such as type of antibody, coupling strategy or sterical stabilization. Following the intracellular trafficking of liposomes, we modified the liposomal lipid composition in order to avoid lysosomal degradation. Therefore, we established pH-sensitive, sterically stabilized immunoliposomes that deliver their content into the cytoplasm due to liposome destabilization in the late endosomes followed by liposome-endosome fusion. Therefore, these drug carriers offer a great potential for therapeutical approaches in the treatment of inflammation, such as liposomal gene therapy. First results will be introduced. Parkinson's disease is characterized by selective loss of neurons in the substantia nigra pars compacta and significant reduction of neostriatal content of dopamine (DA) and its major acidic metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA); parkinsonian symptoms are relieved by administration of l-dihydroxyphenylalanine (l-DOPA), which is converted by neuronal aromatic l-amino acid decarboxylase (AADC) into DA, hence restoring DA levels in surviving neurons. In order to improve the bioavailability and to minimize unfavourable side effects we studied new dimeric l-Dopa derivatives, as potential prodrugs encapsulated in unilamellar liposomes (PC-CHOL). For better evaluate the influence of geometric conformation of prodrugs on entrapment efficiency and “in vivo” activity, we synthesized new maleic and fumaric amides of l-Dopa. The new compounds and drug loaded vesicular systems were characterized evaluating chemical and physical stability, entrapment efficiency and pharmacokinetic parameters. The striatal l-Dopa and DA concentrations, after i.p. administration, of new delivery systems was performed by means of a new HPLC-EC method². The use of polyethylene glycol (PEG)-modified lipids is well established for liposome encapsulated drugs and their ability to enhance the delivery of anti-cancer drugs to tumor sites has been demonstrated. We examined the effect PEG-lipid incorporation has on the fundamental in vitro and in vivo characteristics of cationic lipid based Lipid-Protamine-DNA (LPD) formulations. Pegylated lipids allowed for a higher final DNA concentration (280 µg/mL vs. 150 µg/mL for non-PEG formulations), prevented serum-induced aggregation or size increases, and effectively shielded DNA from serum nucleases. The in vivo manifestation of these properties were addressed in experiments directed at examining transfection characteristics, lipid deposition, and acute toxicity responses after systemic administration in murine models. Incorporation of pegylated lipid into LPD resulted in a 3-log reduction of luciferase expression in the lung as compared to non-PEG controls. This reduction was reflective of a trend in all organs examined. Biodistribution studies using ³H-cholesteryl-hexadecyl ether as a lipid tracer indicated that luciferase expression correlated with lipid deposition. Further, an enhanced circulatory half-life was observed for the PEG bearing formulations. Peak serum levels of TNF-α induced by PEG-LPD were up to158 fold lower than non-PEG controls in C57BL6 mice. In three murine xenograft models tumor expression generated by systemic LPD or pegylated-LPD administration was examined. Luciferase expression was evaluated at 6 or 16 h post-administration with one set of experimental groups receiving ultrasound treatment at the tumor site immediately following formulations administration. Non-PEG formulations had a peak expression 6 h post administration unaffected by ultrasound treatment. PEG-bearing LPD formulations did not generate a measurable signal at the 6 hr. time point. At 16 h post administration, expression was minimal for each formulation type, in non-ultrasound treated tumors, but an 8–150 fold enhancement was observed for PEG bearing over non-PEG formulations in ultrasound treated tumors. This data suggested greater passive accumulation of transfection competent PEG bearing formulations at the tumor site with ultrasound treatment, enhancing transit through, and into, cells resulting in enhanced expression levels. This data suggests that DSPE-PEG_5k addition to LPD formulations modulates LPD behavior in vivo generating a formulation profile with reduced non-specific deposition and transfection characteristics, coupled with reduced inflammatory effects. PEG-LPD may have clear utility for systemic treatment of cancer. This work was supported by Emerald Gene Systems. Magnetoliposomes were prepared by coating magnetite nanoparticles with a phospholipid bilayer, made of zwitterionic dimyristoylphosphatidylcholine and anionic dimyristoylphosphatidylglycerol. Since their discovery in the late 1960s, these magnetic fluids have continued to attract attention. They can be used to deliver drugs to a target organ in the body whether or not combined with electromagnetically induced hyperthermal treatment of this organ. Because these so-called magnetoliposomes can be effectively captured by magnetic forces, these colloids are very suitable material to study the sorption behaviour of e.g., medical compounds into or onto liposomal bilayers. Unlike other separation techniques, such as gel permeation chromatography, equilibrium conditions are obtained during the complete separation of bound molecules from unbound molecules. The sorption behaviour of propranolol, a nonselective beta-adrenergic receptor blocking agent, in magnetoliposomes was assessed. A clear negative influence of electrolyte addition on the sorption behaviour of this molecule in the magnetoliposomes at pH 7 was found, which indicates that the electrostatic attraction between the negatively charged phospholipid bilayer and the positively charged propranolol is at least partly governing this sorption phenomenon. The knowledge that infections caused by Mycobacterium avium Complex (MAC) affect up to 50% of AIDS patients has led to the development of efficient prophylactic strategies. Rifabutin (RFB) was approved by FDA, as a single agent, for prophylaxis of MAC. With the aim to enhance its therapeutic index, the delivery of the drug to the more vulnerable organs namely liver Kupffer cells and spleen macrophages, was tested using liposomes as a carrier system. RFB was incorporated in conventional liposomes made from phosphatidylcholine/phosphatidilglycerol (PC/PG) or phosphatidylcholine/phosphatidylserine (PC/PS) and in stealth liposomes. The prophylactic effect of liposomal formulations was tested in a Mycobacterium avium infection model. Doses of 5 and 10 mg/kg of the formulations were daily injected, starting one day before the infection induction. The reduction of the infection degree of RFB incorporated in stealth liposomes was not significantly different from the non-incorporated drug. On the contrary, conventional liposomes incorporating RFB were able to reduce to a higher extent the bacterial levels in comparison to the free drug, in a dose dependent manner. Stronger reduction on bacterial load was observed for the lipid composition PC: PG compared to PC: PS either in liver or in spleen, for the 10 mg/kg administered dose. These results indicate a different intrahepatic distribution of PG and PS liposomes and a higher affinity of PG versus PS liposomes for the spleen. Conventional liposomes, including PG in their composition, seem to be promising delivery system for RFB as a prophylactic M. avium agent. These systems will be tried in future for prophylaxis of M. tuberculosis. Small arginine- and tryptophan-rich motifs have been identified in antimicrobial peptides with different secondary structure. To analyze the role of the specific side chains and of conformational constraints we synthesized linear and cyclic hexa-peptides with substitutions of arginine (R) by lysine (K) and of tryptophan (W) by tyrosine (Y) and naphthylalanine (Nal). Measurements of the growth inhibiting activity against Gram positive and Gram negative bacteria revealed a complex relationship between peptide structure and biological activity. No hemolytic and antimicrobial activity was observed for K- and Y-containing linear and cyclic peptides. Cyclization of the R- and W-bearing hexapeptide induced pronounced antimicrobial activity while conserving low activity against red blood cells. Substitution of W by the bulky Nal conferred pronounced activity on the linear as well as cyclic peptides towards bacteria and induced moderate activity towards erythrocytes. Conformational studies of SDS- and lipid-bound peptides and investigation of the lipid vesicle permeabilizing activity served to clarify the biophysical principles involved in the modulation of the biological effects. Comparable activity pattern of the all-l peptides and their corresponding d-enantiomers point to a membrane disturbing mode of action. We previously described ion gradient loading of idarubicin driven by creation of a transmembrane pH gradient in distearoyl phophatidylcholine:distearoyl phosphatidylethanolamine conjugated polyethylene glycol (95:5 molar ratio) and demonstrated that idarubicin was retained better in cholesterol-free liposomes as compared to identical liposomes with cholesterol. When investigating the application of these cholesterol-free liposomes as carriers for other drugs, we observed that some drugs, like doxorubicin, could not be loaded below the phase transition temperature of the bulk phospholipid. We proposed to utilize ethanol incorporation in the lipid bilayer to increase the rates of drug loading in liposomes. Doxorubicin loading rates in cholesterol-free liposomes was 6.6 fold higher in the presence of ethanol. We demonstrated that >90% doxorubicin was encapsulated at 37°C within 2 h, 2.4 fold higher than in the absence of ethanol. Optimal ethanol concentration was 10–15% (vol/vol), a concentration that did not affect either liposome size, trapped volume or, most importantly, the stability of the pH gradient. At 30% (vol/vol), we observed interdigitation characterized by gelling of the sample. Ethanol-induced increases in drug loading was temperature, lipid composition and lipid concentration dependent. Collectively, our results suggest that ethanol addition during drug loading may be an improved method to incorporate high levels of entrapped drug in the absence of high temperatures. A novel approach to prophylaxis against poisoning by organophosphates (OP), the toxic components of nerve gases, is offered by combination of new OP-scavenging formulations positioned at major gates through which the OP gain entry into living systems. The new formulations consist of OP-scavenging enzymes entrapped in bioadhesive liposomes. The liposomes binding with high affinity to recognition sites at the locations desired for scavenger positioning, and remaining bound there for prolonged periods in active (enzyme) form. To provide efficient protection against a wide repertoire of OP, we propose a multi-enzyme approach, operating in more than one mechanism, in which OP is an irreversible inhibitor (Cholinesterases) or a substrate (Organophosphorous Acid Anhydrolase (OPAA), Paraoxonase (PON) and others). Feasibility of our conceptual approach was previously shown with one formulation: burytylcholinesterase (BChE) entrapped in bioadhesive liposomes that had collagen as the bioadhesive ligand. In this communication we report on: (1) optimization of BChE-encapsulating systems, through testing the relationships between the magnitude of the bioadhesive coat (9, 12 and 26 mg collagen/mmol lipid) and system properties and (2) first steps in obtaining OPAA-encapsulating liposomal systems. Our major findings with liposomal BChE are: (i) All three formulations were similar in retention of enzymatic activity, with K_m = 0.48 ± 0.18 mM and comparable V_max values. (ii) Liposomal diameter slightly decreased with the increase in coat size (1.0 to 0.6 cm) (iii) All three formulations were similar in protection of liposome-associated BChE from hostile proteolytic environment. (iv) All formulations showed good binding to recognition sites but with differences in capacity and affinity. (v) Most important all tested formulations performed well as stoichiometric OP scavengers. The cumulative data favor the liposomes with the intermediate-sized bioadhesive coat. Our preliminary findings with OPAA show that the enzyme can be entrapped in liposomes with retention of activity and that, using paraoxon as the test OP/substrate, Intact OPAA-encapsulating liposomes perform well as catalytic OP scavengers. In conclusion, the data reported here is further support for the feasibility of our prophylaxis approach, confirming the multi-enzyme approach and showing that the magnitude of the bioadhesive coat is a tool for system optimization. Liposomes were formed of highly purified membrane-spanning archaeal phospho tetraether lipid by (a) French pressure cell extrusion, (b) extrusion through polycarbonate filters, or (c) detergent dialysis resulting in unilamellar vesicles of 100–200 nm (a,b) or 300–400 nm (c) in diameter. Shelf life stability was tested at 4°C over two years: during this time, these liposomes are stable as determined by electron microscopy and size distribution measurements. Leakiness of the liposomal membrane for electrolytes, alcohols etc. is much lower than that of lecithin bilayer liposomes; permeability of protons is even about two magnitudes lower. Release of insulin and carboxyfluorescein depends on pH, from absolutely no release in acidic milieu liberation increases with rising pH. Rapid intermembrane exchange of lipophilic bromobimane and Nile red from the liposomal membrane to erythrocyte ghosts was observed. The affinity of the liposomes towards T84 colon carcinoma cells in culture was much higher than that of lecithin liposomes as measured by carboxyfluorescein fluorescence. This phenomenon may be ascribed to the sugar component, the biologically rare gulose. No cytotoxicity, no mutagenicity of the liposomes and no toxicity in mice could be determined. The calculated LD₅₀ is above 561 mg/kg BW and immunosuppressed mice lived even longer than controls if liposomes (or the lipid) were admixed to the diet. Spin labelled retinoic acid was incorporated into the liposomes and penetration into the skin of hairless mice measured by electron paramagnetic tomography (MOSS) in comparison to a hydrogel formulation. The extraordinary characteristics of these liposomes can be ascribed to the unique membrane-spanning bipolar main phospholipid from Thermoplasma acidophilum. On the one hand its conformation is much more dynamic than formerly assumed and on the other hand it forms the most stable and impermeable membranes from biological material investigated so far. The most important function of the skin is the formation and maintenance of a barrier that protects the organism against environmental damage and from desiccation. This barrier is located in the stratum corneum (SC). The SC consists of at least two phases: the proteinaceous, highly cross-linked cornified cell envelopes, (corneocytes) and the extracellular, lamellar lipid phase. The corneocytes - imbedded in the lipid phase - have a size of 20–30 micron (pig) and are mainly oriented parallel to the surface of the skin. The lipid matrix is composed of cholesterol, free fatty acids and ceramides. A number of biophysical techniques are used to study the components of the SC ( ). Most studies focuses on the molecular organization of the lipid matrix and the interactions of the different ceramides with cholesterol, fatty acids or water molecules. However, less is known of the molecular organization and distribution of the protein and lipid fraction in the SC tissue. Traditional Fourier transform infrared (FTIR) spectroscopy provides information concerning lipid structure, the formation of micro-domains and thermotropic alterations in macroscopic samples. We have used a new FTIR technology, which is based on a focal plane array detector (FPAD). This technique combines FTIR spectroscopic chemical identification and quantification with microscopic imaging technology ( ). FPAD technology performs much like a camera. It consists of an array of detectors, which are working in parallel to produce spectra from different portions of the tissue sample, and thus collecting spectra simultaneously from all the detector pixels. Any spectral parameters obtained by FTIR microscopy may be monitored, scaled, and mapped across the array, thus providing a true IR vibrational spectroscopic imaging of the analysed tissue area at a spatial resolution approaching the IR diffraction limit (˜5 micron). The images obtained from pig skin SC clearly demonstrate that the SC is a rather inhomogeneous tissue consisting of protein domains embedded in a lamellar lipid matrix. Isothermal titration calorimetry (ITC) is used to determine the critical micelle concentration (cmc) and the demicellization enthalpy (ΔH_demic) of a variety of surfactants as a function of temperature. ITC has the advantage that the cmc and the thermodynamic parameter ΔH_demic can be directly determined from one and the same experiment. In addition, the experimentally observed quantity, the reaction heat, is already a differential quantity, because changes in enthalpy due to changes in concentration are measured. This improves the precision of the cmc determination as long as ΔH_demic is sufficiently large. ΔH_demic, the cmc, and the thermodynamic parameters ΔG_demic (change in Gibbs energy) and ΔS_demic (change in entropy) are usually calculated using the pseudo-phase separation model ( ). This facilitates the comparison between different detergents and with literature data. The experimental data show that ΔH_demic is clearly temperature dependent. The heat capacity change Δcp_demic, obtained from the temperature dependence of ΔH_demic, contains information on changes in exposed hydrophobic surface area when the aggregates dissociate. This finding can be used for making suggestions about the structure of the micelles in terms of water-exposed hydrophobic surface area in the aggregates. This is a critical information for the formulation of drugs in micellar systems. For the low aggregation numbers found for different surfactants, the pseudo-phase-separation model is a crude approximation. Therefore, the aggregation process can better be described using a mass action model ( ). To derive the thermodynamic parameters for the aggregation process, the ITC titration curves have to be simulated with the aggregation number n as one of the variable parameters. We will show that with this model additional information on the aggregation number n as a function of salt concentration and temperature can be obtained. Gene transfer methodologies are emerging strategies for the treatment of both genetic and acquired diseases. The advances in understanding the mechanisms involved in the disease development have stimulated gene therapy research. CNS diseases, as many others, are also a potential target for gene therapy. Work in this area has mainly used viral vectors, mainly due to their enhanced efficiency over non-viral vectors. However, viruses can produce inflammatory and immune responses. Non-viral strategies have become increasingly important and cationic liposomes have shown to be particularly promising. In this work we conducted experiments to evaluate the potential application of cationic liposomes for gene transfection of brain cells. For this purpose, we performed in vitro studies using rat embryo hippocampal and cortical neurons as experimental models, and luciferase and GFP as reporter genes. Several parameters affecting transfection activity were examined, including liposome composition, cationic lipid/DNA charge ratio, association of the ligand transferrin, and degree of cell differentiation. The lipoplexes internalization pathway was also investigated. Our results showed that, independently of the experimental model used, complexes prepared with DOTAP: Chol liposomes were the most efficient to transfect the cells. Moreover, the presence of transferrin proved to be crucial to enhance the transfection activity mediated by these systems. The efficient transfer of genes into specific cell type remains an unresolved problem for gene therapy. In the past, several gene transport systems were tested to deliver DNA pieces into specific target cell types. Toward this goal, we have generated a new liposomal vector which is based on the composition of anionic retroviral envelopes. The packaging of plasmid DNA into these Artificial Viral Envelopes (AVE) in the presence of a suitable condensing cationic polymer yields serum-resistant liposomes of high stability. These liposomes were endowed with a specificity for a_vβ₃-integrin expressing cells (such as melanoma and tumor ECs) by attaching a cyclic RGD-containing peptide to the liposomal surface. These targeted particles are non-toxic, exhibit a very high transduction efficiency for primary ECs of >80%, and are target cell-selective. The in vivo performance of these liposomes as well as their applicability for the targeted delivery of small molecule drugs is currently under investigation. Lipid-Protamine-DNA (LPD) lipopolyplexes have successfully been used for systemic gene transfer. The use of polyethylene glycol (PEG)-modified lipids is well established for liposome encapsulated drugs and their ability to enhance the delivery of anti-cancer drugs to tumor sites has been proven (Lasic, (1998) Trends in Biotechnology 16; 307–332). Pegylated lipids are also known to control surface properties of cationic lipid-based gene transfer systems. However, pegylated lipid incorporation into lipid-DNA complexes causes a concentration dependent reduction of in vitro transfection activity, a result that can be partially attributed to a reduction in particle binding to cells (Harvie et al. J. Phar Sci. (2000) 89;5 652–663). In order to restore particle binding and target LPDs to tumor cells, we incorporated a folate-lipid-conjugate, DSPE-PEG_5k-Folate, into the formulations and compared such formulations with those containing DSPE-PEG_5k. As expected, addition of pegylated lipid without targeting functions altered the LPD transfection characteristics resulting in a concentration dependent reduction of in vitro transfection activity in KB cells. In contrast, particle binding and transfection were enhanced by the incorporation of the DSPE-PEG_5k-Folate conjugate. Addition of such lipid-conjugated-ligands was found to be compatible with LPD formation and results in a dose-dependent enhancement of luciferase expression following in vitro transfection of KB cells. A maximum of 20–50 fold enhancement of luciferase expression over the corresponding DSPE-PEG_5k base formulation was observed when 2 mol percent lipid-conjugated-ligand was incorporated into the LPD formulation. FACS analysis also demonstrated DSPE-PEG_5k-Folate increased LPD-cell binding by 2 fold over the base formulations. Moreover, transfection activity of LPD bearing DSPE-PEG_5k-Folate can be partially abolished in a competition assay using 5000 fold excess of free folate. More interestingly, folate mediated transfection enhancement can be totally inhibited using DOPC:CHOL:DSPE-PEG_5k-Folate liposomes as competition agent, while DOPC:CHOL:DSPE-PEG_5k control liposomes formulation did not block the transfection enhancement. These data suggest that this new systemic gene delivery formulation will lead to more effective in vivo cell specific targeting. This work was supported by Emerald Gene Systems. Systemic chemotherapy does not result in adequate intratumoral drug levels in the treatment of many solid tumors. High interstitial fluid pressure (IFP) and other pathophysiologic conditions at the tumor site are responsible for this. Some of these barriers may be overcome (in part) by using long-circulating liposomal formulations. We have demonstrated that systemic administration of low dose Tumor Necrosis Factor-α (TNFα) plus doxorubicin liposomes (DOXIL) results in an increased intratumoral uptake of liposomes and a significantly enhanced antitumor efficacy. We also have shown that in an isolated limb perfusion (ILP) the addition of TNFα to the perfusate resulted in significant enhanced accumulation of doxorubicin in rat sarcomas. In vitro studies demonstrated that addition of TNFα had no additive effect when tumor cells were exposed to cytostatic agents. When human umbilical vein endothelial cells (HUVEC) are exposed to TNFα a change in morphology and some gaps between the cells were observed. Apparently TNFα has an indirect effect on the tumor vasculature resulting in an increased permeability and a higher uptake of cytostatic agents. Studies are performed examining the role of TNFα on the antitumor activity of stealth DOXIL in other animal studies. In addition the effect of TNFα on Stealth liposomal Cisplatin (CDDP-SL) uptake in tumor tissue is investigated. Phospholipase A2 (PLA₂) hydrolyses phospholipids, rendering fatty acids and lysophospholipids. Secretory PLA₂ is found in inflammatory tissue and venom from snakes. PLA₂ from snake venom readily catalyses the hydrolysis of both zwitterionic and anionic lipids. However, it has previously been shown, that the activity of secretory PLA₂ type IIA towards anionic phospholipids is significantly higher than the activity towards zwitterionic phospholipids. In the present study human tear fluid has been employed to study the activity of PLA₂ type IIA towards zwitterionic phospholipids (dipalmitoyl-phosphatidylcholine and dipalmitoyl-phosphatidylethanolamine) and anionic phospholipids (dipalmitoyl-phosphatidylglycerol and dipalmitoyl-phosphatidylserine). The degree of hydrolysis of each phospholipid component in multi-component large unilamellar liposomes was determined. In agreement with previous work (Yang et al., Anal Biochem 1990; 269:278–288) the overall activity of PLA₂ was high towards liposomes with a negatively charged surface as compared to neutral liposomes. Interestingly, the activity of PLA₂ towards a specific lipid component in multi-component liposomes exhibits a non-trivial dependence on surface charge. The results presented in this study have applications in drug-delivery research in relation to the site-specific degradation of liposomes at desired pathological sites where the concentration of PLA₂ is significantly increased. Our laboratory has developed a method to isolate and identify both transcytosing and non-transcytosing, luminal exposed endothelial integral membrane proteins. These proteins can be used to derive novel and tissue-specific targeting ligands for use in drug-delivery. One of the antibodies (LVET) derived by this technique was shown following iv injection and three-color histology of rat lung tissue, to recognize a lung-specific, transcytosing vascular endothelial target. We have developed a LVET-targeted, liposomal formulation of gentamicin which may have clinical utility. Aminoglycoside antibiotics are limited in their clinical use due to their severe renal- and oto-toxicities. However, they remain an important alternative for treating nosocomial pneumonia and chronic lung infection in cystic fibrosis patients. The use of targeted-liposomal formulations of gentamicin may ameliorate some toxic side effects by rapidly distributing the drug to the lung, keeping the serum concentration low and allowing dose reduction. In our laboratory, the amount of gentamicin delivered to healthy rat lung by LVET-targeted, gentamicin-loaded liposomes prepared with egg phosphatidyl choline reached approximately 11 µg/g tissue at 30 min, exceeding the minimum inhibitory concentration for Pseudomonas aeruginosa. Importantly, the gentamicin dose was only 0.75 mg/kg, which is 26 times lower than the standard dose in rats. In contrast, the lung uptake was approximately 0 and 1 µg/g tissue for free and non-targeted liposomal gentamicin, respectively. Liposomes, sterically stabilized by surface grafted polyethylene glycol (sterically stabilized liposomes (SL)) have been shown to be attractive carriers for anti-cancer agents. The accelerated clearance on multiple injection of SL, have been recently reported ( ), could be one of problems to further develop drug-containing SL for clinical applications. Although understanding the mechanism is very important, limited data are available on the effect. In this study, therefore, we investigated in more detail biodistribution of SL in rats and mice after repeated injection. SL indicated long circulation time in both animals in the absence of first-injection. The accelerated clearance occurred in rats and most highly at 5 days after first injection, whereas it occurred in mice at 10 days after first injection. Highly increased hepatic accumulation of SL and hepatic clearance was observed in rats at 5 days post-injection and in mice at 10 days post-injection. At 14 days (2 weeks) after pre-injection, the biodistribution of SL had almost returned normal. It appears that a species difference on the innate immunity for SL clearance exist, although the underlying mechanism is uncertain in this stage. The results obtained in this study may have important implications for the design of liposomal anticancer formulations for clinical application based on animal scale up. The capacity of four different cytostatic drugs to generate free radicals was investigated and correlated to their capacity to cause PPE (palmar-plantar erythrodysestesia) or Hand-Foot Syndrome. This particular dermatologic disorder occurs during chemotherapy with liposome-encapsulated or free doxorubicin and 5-FU, less severe forms are observed with daunorubicin and no PPE occurs with mitoxantrone ( ). The production of reactive oxygen species (ROS) and organic radicals are strongly thought to be involved in the occurrence of the PPE ( ). In the present study the hydroxyl radical production as well as the lipid peroxidation were investigated in the presence of cytostatics. The production of hydroxyl radicals (·OH) was analysed by ESR spectroscopy, using a spin-trap method: OH radicals produced by Fenton reaction or by an azo-compound AAPH were trapped with DMPO in the presence of cytostatics and the ESR signal intensity was analysed. Lipid peroxidation was initiated by the AAPH system in spin labeled liposomes and red blood cells from rats. The signal intensity decay of the 5-, and 16-DXSA spin labels was analysed and the lineshapes of the obtained ESR spectra were simulated. In the presence of doxorubicin the highest amounts of ·OH radicals were produced. Lipid peroxidation was significantly increased in the presence of doxorubicin, daunorubicin, and 5-FU. The addition of glutathione inhibits lipid peroxidation in red blood cells, indicating the possibility to attenuate the PPE by supplementing the chemotherapy with liposome-encapsulated antioxidants. Entrapment of anticancer drugs in liposomes has been shown to decrease their side-effects while increasing or preserving therapeutic activity by drug targeting and elongation of the blood circulation time. Vesicular phospholipid gels (VPG)—highly concentrated, semisolid matrices of densely packed liposomes—are suitable to entrap substances with particular high encapsulation efficiencies up to 70%. In order to increase the tolerability and the clinical efficacy we are developing a VPG formulation of the anticancer drug 5-FU (FU-VPG). We have studied different formulations of VPG and analysed their main in vitro characteristics—encapsulation efficiency and 5-FU-release—with regard to the desired application that can be intratumoral as semisolid VPG or intravenous as vesicular redispersion of VPG. Encapsulation efficiency was found to depend on both the lipid amount and the lipid composition. Using mixtures of soy phosphatidylcholine and cholesterol, an increasing amount up to the upper limit of 45 mol% of cholesterol decreased the encapsulation efficiency. Also changing the pH of the FU-stock-solution influenced the encapsulation efficiency, whereby an increase of the pH led to increased encapsulation efficiency. We found that 5-FU release from redispersed FU-VPG occurs rapidly with a half-life time of approximately one hour. So the formulation would have to be injected quickly after redispersion to ensure high encapsulation efficiency. The half life time of the release of 5-FU from non-redispersed FU-VPG was found to be in the order of a few hours. In vitro data of these different 5-FU loaded VPG suggest an applicability of them as implants with controlled release properties or, after redispersion, as intravenously injected liposomal formulations. This study discloses a novel formulation of paclitaxel-liposome. A liposome made of two major phospholipids with distinct transition temperatures was prepared and characterized. The liposome showed a remarkable capacity for increasing drug loading, especially for hydrophobic drugs which are incorporated in the phospholipid bilayer. The formulated liposome can incorporate as much as 20% paclitaxel to lipid molar ratio, which has been below 3% in most of the previous literature. Shelf stability was found to depend on paclitaxel and/or lipid concentration. Liposome remained stable in liquid form at 4°C for at least 12 months when the drug/lipid molar ratio below 15%. Cell culture test displayed a similar growth inhibition efficacy as the cremophorEL/alcohol formulation (Taxol®). The AUC in rats at a dose of 5 mg/kg was comparable as well. Animal tests indicated that liposomal paclitaxel exhibited considerably lower acute toxicity than did Taxol®. Taken together, the novel liposome formulation developed in this study can incorporate a high content of hydrophobic paclitaxel. These results demonstrate that the liposomal paclitaxel is highly promising for clinical use. By the “oxidative burst” phagocytes are able to kill ingested bacteria and fungi after phagocytosis. Phagocytes of patients suffering from chronic granulomatous disease (CGD) have lost this ability due to a defective enzyme, a NADPH-oxidase. Aim of our project is to reconstitute this ability by glucose-oxidase (GOD) encapsulated in liposomes. The liposomes should be taken up by granulocytes and H₂O₂, produced from GOD, can be used for the oxidative burst. In order to follow up the uptake and the fate of liposomes inside of granulocytes, we applied different microscopic methods. Confocal laser microscopy was used to determine the intracellular location of liposomes and the potential co-localization of ingested bacteria together with liposomes in the same phagosome. For electron microscopy, ferritin was encapsulated into liposomes to increase their electron density. Spectral imaging, using different liposomal and cellular fluorescence labels, was additionally used to follow the fate of the liposomes in granulocytes. With all three methods the intracellular uptake of the liposomes and their co-localisation with ingested bacteria (S. aureus) was shown and no evidence of liposomes remaining on the cell surface was found. This work was supported by the Reinhold-Beitlich-Stiftung and the fortüne-Programm, Tübingen, Germany. Many papers have shown that immunoliposomes can deliver a large amount of encapsulated drugs into tumor cells when the liposomes were targeted to internalizable receptors. But it is unclear which liposome property affects the cytotoxicity of anti-cancer agent containing immunoliposomes. We investigated effect of membrane fluidity of immunoliposomes on cytotoxicity and their interaction of the liposomes with tumor cells. Transferrin-grafted immunoliposomes were composed by egg phosphatidylcholine (high fluidity) or hydrogenated soy phosphatidylcholine (low fluidity), cholesterol and N-[3-(2-pyridyldithio)propionyl] phosphtidylethanolamine (2:1:0.02, mol/mol). Doxorubicin was loaded into the liposomes by using ammonium-sulfate gradient method. There was significant difference between high and low fluid immunoliposomes on the cytotoxicity of liposomal doxorubicin. High fluid immunoliposomes showed much higher cytotoxicity against both cell lines. Then we examined amount of liposomes associated with the cells as well as intracellular doxorubicin level. High fluid immunoliposomes delivered a large amount of doxorubicin into not only drug sensitive but also drug resistant cell lines, while low fluid immunoliposomes did the same amount of doxorubicin as non-targetted liposomes. Our results showed that immunoliposomes having high membrane fluidity could deliver a large amount of liposomal doxorubicin into both cell lines, resulting in increased cytotoxicity. The presence of large hydrophilic polymers such as polyethyleneglycol on the liposomal surface retards elimination of liposomes from the circulation and increases accumulation in the tumor tissue. We prepared pegylated liposomes containing novel cationic lipid TRX-20, which bind preferentially to chondroitin sulfates (CS). The liposomal composition was optimized to 8 mol% of TRX-20 and 0.75 mol% of PEG-PE to achieve long retention in the circulation and enhanced binding to metastatic tumor cells such as LM8G5 and ACHN that express high levels of CS on the cell surface. In vitro, TRX-20 liposome-entrapped cisplatin showed anti-tumor cell cytotoxicity equivalent to free cisplatin. In vivo, they suppressed the subcutaneous growth of LM8G5 tumor cells more effectively than free-cisplatin or cisplatin encapsulated in pegylated liposomes without TRX-20. Furthermore, cisplatin-loaded TRX-20 liposomes markedly suppressed metastatic spreading of the LM8G5 tumor cells to the liver, and thus significantly prolonged survival of the tumor bearing mice. TRX-20 liposomes loaded with chemotherapeutic drugs may be a useful tool to prevent the growth and metastatic spreading of tumor cells that have enhanced expression of CS. Matrix metalloproteinases (MMPs) are involved in angiogenesis. Several MMPs are known to abundantly present on tumor-induced neovasculature. Especially, membrane-type (MT) MMPs may provide potential targeting guide for selective delivery of agents such as cytotoxic agents and angiogenesis inhibitor. In this study, we attempted to develop novel liposomes targeted on MT1-MMP. The peptide-ligands specific for MT1-MMP was recently obtained by use of phage displayed peptide library, and the epitope sequences were determined as a targeting probe to neovasculature. In here, we prepared liposomes modified with three kinds of candidate 6 mer peptides. In vivo behaviors of these liposomes were determined by a non-invasive method using positron emission tomography (PET). We also performed usual biodistribution study of them by labeling liposomes with [³H] cholesteryl ether. As a result, all peptide-modified liposomes accumulated in tumor compared with control liposomes after intravenous administration. These liposomes had tendency to avoid the reticuloendothelial system (RES)-trapping. We are now investigating the inhibitory effect of them on activation of MMP-2 and invasion of tumor cells. Novel liposomal formulations modified with MT1-MMP-targeted peptides would be useful for anti-neovascular therapy. Vascular endothelial cells (VEC) play a key role in various inflammatory processes. VEC are therefore an important target cell population for active drug delivery strategies. We target adhesion molecules (AM) expressed on VEC involved in the two major processes occurring in (chronic) inflammation; leukocyte recruitment and angiogenesis. Recruitment of leukocytes into inflamed areas is one of the key issues in maintaining an inflammation. Angiogenesis is known to enhance an inflammation by providing means to transport of leukocytes, oxygen and nutrients into the inflamed area. Active drug targeting strategies, specifically blocking or modulating these AM may provide a major therapeutic approach in anti-inflammatory therapy. Therefore we are developing liposomes targeted to AM on VEC for the delivery of anti-inflammatory drugs and DNA. Liposomes are targeted by attaching antibodies specific for E-selectin or ICAM-1, AM involved in leukocyte recruitment, or by coupling an RGD-peptide specific for αvβ3-integrin, an angiogenesis-associated integrin. In vitro studies demonstrated the possibility of using these liposomes with either one of the three types of ligands for intracellular drug delivery to VEC. Transfection of VEC using stabilized plasmid lipid particles (SPLP) ( ) was enhanced up to ten-fold after attachment of RGD peptides. This targeted system provides a means for active gene delivery to VEC at pathological sites. In vivo studies using fluorescently labeled liposomes and intravital microscopy demonstrated extensive binding of E-selectin targeted liposomes to the blood vessel wall in an LPS-induced inflammation in mice. In a rat model for adjuvant arthritis as well as in control animals the pharmacokinetics and biodistribution of RGD-liposomes were assessed. Decreased circulation time, increased uptake in liver and spleen and, surprisingly, equal target to blood ratios in inflamed paws of arthritic rats were observed for targeted compared to non-targeted liposomes. RGD-liposomes encapsulating an anti-inflammatory drug showed enhanced efficacy compared to free drug or non-targeted liposomal drug in a rat adjuvant arthritis model. In conclusion, VEC at inflamed sites provide a target cell population with high accessibility that can be reached by AM-targeted liposomes. Delivery of an anti-inflammatory drug by such liposomes strongly inhibited ongoing arthritis and suggests an important contribution of endothelial cell modulation on the development of this inflammatory disease. Targeting drugs or DNA to VEC at inflamed sites opens new possibilities for therapeutic intervention in (chronic) inflammatory disorders. Many studies showed significantly higher absorption rates as well as a greater pharmacological effect for drugs applied to the skin entrapped in liposomes as compared to conventional topical formulations, which depend on liposome bilayer properties and their size. Information about the role of size and composition are based primarily on measurements of drugs in different skin layers in vitro. In this work electron paramagnetic resonance (EPR) oximetry in vivo is used to measure directly the vasodilatory response produced by local application of benzylnicotinate in liposomal formulations to the skin. It increases the blood flow in skin and consequently the oxygen concentration increases. Partial oxygen pressure pO₂ in skin is measured with time after topical application of benzylnicotinate to the skin of mice. The liposomes with the entrapped benzylnicotinate (1.25 w/w%) composed of hydrogenated (HSL) or nonhydrogenated soy lecithin (NSL) and 30 w/w% cholesterol (25 mg/mL lipids) were prepared by thin film method and dispersed in hydroxyethylcellulose hydrogel. Measurements were performed on MLV with mean diameter 350 nm for NSL and 770 nm for HSL and on extruded liposomes with mean diameter 250 nm and 289 nm, respectively. It was found that the efficacy of the drug measured as the area under the curve (AUC) is about the same for all the preparations, but the time, when the oxygen concentration increases as well as the time of action depends strongly on liposome composition and their size. It is well established that over the course of HIV infection, viral particles accumulate and replicate actively in lymphoid organs. The high viral load observed in lymphoid tissues was reported to be associated with trapped HIV-1 particles on the follicular dendritic cells (FDC) located in the germinal centers. FDC, antigen presenting cells such as macrophages and activated CD4+ T-cells are abundant in lymphoid tissues and all express substantial levels of the HLA-DR determinant of MHC-II. In addition, the HLA-DR determinant is physically present on the virion surface and constitutes one of the most abundant host-derived molecules carried by HIV-1. Liposomes bearing surface-attached anti-HLA-DR antibodies thus constitute an interesting approach to target specifically HIV-1 cellular reservoirs as well as free HIV-1 particles. Our previous studies have demonstrated that sterically stabilized anti-HLA-DR immunoliposomes were efficient in delivering high concentrations of drugs to lymphoid tissues leading to a 126-fold increased drug accumulation in lymph nodes. On the other hand, we showed that incubation of HIV-1 particles with anti-HLA-DR immunoliposomes containing amphotericin B (2.5 µg/mL) completely inhibited viral replication. In this study, we have evaluated the efficacy of amphotericin B incorporated in anti-HLA-DR immunoliposomes to inhibit HIV-1 replication in an ex vivo human tonsil model. Preliminary results showed that lymphoid tissue cultures constitute an appropriate model to evaluate the efficacy of immunoliposomal drugs in a human microenvironment. Fluorescence resonance energy transfer (FRET) is a potential method for the characterisation of DNA-cationic lipid complexes. Most of the studies involving fluorescence probes are essentially qualitative. In this work we are using the methodology of resonance energy transfer within the context of parameterised models previously derived in studies of membrane biophysics. The application of these models to DNA-cationic lipid complexes allows the measurement of the distances between the fluorescent intercalator on the DNA and the membrane dye on the lipid, or to evaluate encapsulation efficiencies of this liposomal vehicle. The experiments were carried out both in steady and transient state, with DOTAP/pUC19 complexes with charge ratios (+/−) of 0.5, 2 and 4, in 30 mM Tris/HCl. We used DPH and BODIPY-PC as membranes dyes and etidium bromide and a cyanine dye as DNA intercalators. In cationic complexes (Charge ratios (+/−) ≥ 2), we verified that the cyanine dye remains bounded to DNA and energy transfer occurs to the membrane dye. This was also confirmed by anisotropy and lifetime measurements. In complexes with all the DNA bounded to the lipid, by the application of the referred biophysical models, we determined the distance between both dyes. In complexes with DNA unbound (Charge ratio (+/−)<2)) to the lipids we evaluated its fraction, and calculated encapsulation efficiencies of these complexes. In recent years, the therapeutic applications of proteins, including enzymes, have received increasing attention. However, the clinical effectiveness of these macromolecules is limited by their low stability in circulation and by unwanted reactions in the body after non-targeted administration. Biological therapeutics therefore would benefit from controlled release and/or targeted administration assuring drug concentrations within therapeutic limits. Non-invasive techniques for transdermal delivery of macromolecules are an attractive option and have the potential to compete with techniques such as continuous infusion or multiple injections. Transfersomes®, a trademark of IDEA-AG, are sufficiently deformable to cross the skin barrier and to deliver drugs, including macromolecules, into the body. We previously reported the results of work with Cu, Zn superoxide dismutase (SOD) in Transfersomes®. Here we present the data obtained with Transfersomes® associated with another enzyme, catalase, which shares antioxidant and anti-inflammatory properties with SOD, but has different structural characteristics and size. The process developed for incorporating catalase into Transfersomes® is a modification of the method used for preparing SOD in ultradeformable carriers. Such catalase-loaded carriers were characterised with regard to their loading parameters as well as the retention of catalytic enzyme activity. The free protein and protein-loaded carriers were separated using gel-permeation chromatography. The catalytic activity of the carrier-associated catalase was determined by a modified method of the standard procedure. The ability of catalase-loaded Transfersomes® to cross an artificial membrane with pores much narrower than the average carrier diameter was evaluated as well. The results confirmed functionality of protein and carriers. This work was partially financially supported by the research project POCTI/1999/FCB 35787. Tumor accumulation and therapeutic activity of Stealth® liposomes loaded with doxorubicin (DXR) were examined in Balb/c nude mice xenografts inoculated subcutaneously with the human small cell lung cancer (SCLC) cell line, H69. Mice were treated with non-targeted liposomes (SL) or liposomes targeted with the hexapeptide, H-Arg-D-Trp-N^mePhe-D-Trp-Leu-Met-NH₂, known as antagonist G, coupled to the liposome surface (SLG). SLG showed 30–44 fold higher binding to H69 cells harvested from H69 xenografts than SL. At 48 and 72 h post-injection, tumor accumulation of [¹²⁵I]-tyraminylinulin-containing liposomes was shown to be independent of the presence of the targeting ligand. Maximum tumor uptake of either SLG or SL ranged from 2–4% of injected dose/g of tissue. In therapeutic studies, mice received 3 weekly injections of 3 or 6 mg free DXR/kg, or 3 or 10 mg liposomal DXR/kg, at initial tumor volumes of either 7 or 33 mm³. The therapeutic efficacy of DXR-containing SL or SLG was significantly improved over free DXR, but SLG did not improve anti-tumor efficacy relative to SL. Stealth liposomes containing DXR have potential as a therapy against human SCLC tumors. In the context of a research project for gene therapy of glioma, novel cationic liposomes have been designed and prepared for an efficient transport of genetic material into cells. These new liposomes have been prepared from novel lipids, whose polar head group is constituted by morpholinic ring (MM43; MM45; MM73), piperazinic ring (MM46; MM69; MM76) and dimethilamine group, respectively. The non-polar moiety is constituted by different fat acid residues or triterpenic units. Liposomes (cationic lipid:DOPE = 1:1 mixtures) have been prepared by sonication and by injection methods. The diameter of vesicles, measured by dinamic light scattering (Brookhaven Spectrometer), is in the range between 100 and 200 nm. The toxicity of cationic liposomes to C6 rat glioma cells has been determined by XTT cell proliferation test. Lipoplexes were formed by adding cationic liposomes to pGL3 Luciferase Reporter Vector. Size analysis of the resulting complexes has been performed by the deconvolution of the light intensity correlation function, using CONTIN program. The formation of complexes has been demonstrated by agarose gel electrophoresis. The transfection efficiency of these new cationic liposomes in C6 rat glioma cells has been measured by Luciferase Reporter Gene Assay, using pGL3 Luciferase Reporter Vector. The transfection efficiency has been measured as function of different cationic liposome to plasmid concentration ratios and compared with that of Fugene. The cationic liposomes MM54 have a good transfection efficiency, which increases with increasing liposome: plasmid concentration ratio. We here for the first time describe the encapsulation of single liposomes within individual networks made from polymers or proteins. The resulting structures resemble biological cells, with a watery interior being surrounded by an almost impermeable lipid bilayer, that is in turn packed into a meshlike and self stable network. We here show construction and characteristics of the novel material. In particular we will emphasize the ability of the shell to protect the inner liposome against lytic components. Liposomal nanocapsules are expected being useful in the development of novel drug delivery systems that are in particular well suited for the transport of biological macromolecules such as DNA, proteins or peptides. Of particular advantage for biomedical applications is the fact, that the entire structure can be constructed from biological materials. Melanoma is a highly malignant and increasingly common tumor. Since metastatic melanoma remains incurable, new treatment approaches are needed. We previously reported that coated cationic liposomes targeted with a monoclonal antibody (mAb) against GD₂ and containing c-myc as ODN (anti-GD₂-CCL [c-myc]) resulted in a selective inhibition of the proliferation of melanoma cell lines. Further, treatments with anti-GD₂-CCL[c-myc] also reduced levels of c-myc protein in melanoma cells. In this study we show the pharmacokinetic and biodistribution results obtained after intravenous injection of ³H-c-myc-asODN, free or encapsulated in non-targeted or anti-GD₂-targeted CCLs. Free c-myc-asODN was rapidly cleared, with less than 10% of the injected dose remaining in blood at 30 m post-injection. C-myc-asODN encapsulated within either CCL or anti-GD₂-targeted CCL demonstrated a more favorable profile in blood, with around 45% of the dose of either preparations remaining in vivo at 24 h post-injection. The free and the encapsulated c-myc-asODN had different biodistribution profiles in vivo. While free c-myc-asODN was widely distributed (mainly liver, kidney and spleen) as soon as 30 m post-injection, c-myc-asODN entrapped in CCL or anti-GD₂-CCL only accumulated in the spleen to a low degree even after 24 h. These results indicate that anti-GD₂-CCL[c-myc] are long-circulating. Preliminary studies in an in vivo melanoma metastatic model show that anti-GD₂-CCL[c-myc] inhibited the development of microscopic metastasis in the lung compared to animals treated with free c-myc asODN or free anti-GD₂. Further experiments, using CCL[c-myc] and sense c-myc, either free or in a CCL-encapsulated formulation as controls, will define the efficiency of this liposomal formulation as a new therapeutic strategy for treatment of melanoma. Supported by Inex Pharmaceuticals, Burnaby, BC, Canada and Costa Crociere. Design of novel vaginal delivery systems for local drug delivery and also, systemic administration of peptides and proteins has been extensively investigated over the last decade ( ). Due to increasing incidence of sexually-transmitted diseases, especially genital HSV infections ( ), appropriate local therapy of antiviral agents has been recently given more attention. Here we report development of a liposomal delivery system for vaginal administration of acyclovir, able to provide sustained release and improved bioavailability of drug. Acyclovir was entrapped in liposomes prepared by polyol dilution method ( ), whereby different phospholipid compositions were used (EPC, EPC/SA = 9/3 and EPC/EPG = 9/1, molar ratio). All liposomal preparations were characterised for particle size, entrapment efficiency and tested for in vitro stability in buffer pH 4.5 (corresponding to normal human vaginal pH), as well as in vaginal fluid simulant (medium developed to simulate the fluid produced in the human vagina) ( ). To be closer to in vivo application of liposomes and to achieve further improvement of their stability, liposomes were incorporated in vehicles suitable for vaginal self-administration. Bioadhesive hydrogel made of Carbopol 974P NF resin with adequate pH value and desirable viscosity was chosen as vehicle for liposomes containing acyclovir. In vitro release studies of liposomes incorporated in gel confirmed their applicability as a novel vaginal delivery system in localised and sustained release of entrapped acyclovir. Even after 24 h of incubation in vaginal fluid simulant more than 35% of originally entrapped drug was retained in liposomal gel. Cationic liposomes are among the most efficient nonviral vectors used for gene delivery purposes. In our laboratory, we have explored different strategies to improve the biological activity of cationic liposome/DNA complexes (lipoplexes), namely through their association to transferrin (Tf) (ternary complexes). In this work a comparison between plain lipoplexes and ternary complexes prepared under different experimental conditions was performed. The effect of the ionic strength of the medium where the complexes are prepared and its relation to their final size and transfection activity was evaluated. In addition, the ability of the obtained complexes to condense and protect DNA was investigated. Transfection activity was drastically reduced when plain lipoplexes were produced in dextrose solution as compared to that in saline buffer (HBS), whereas no significant effect was observed for the ternary complexes. On the other hand, ternary complexes were shown to be more susceptible to DNase degradation than lipoplexes, independently of the lipid/DNA charge ratio tested, suggesting that association of Tf weakens the interaction between DNA and cationic liposomes. However, ternary complexes produced in dextrose appear to protect DNA more efficiently than those produced in HBS. A correlation between the size of the complexes and their ability to mediate transfection will also be presented. Several preparations of different carnitine liposome derivatives were evaluated both in in vitro and in in vivo models for their ability to gene trasfection. Using these approaches we identified ST983 (also named PUCE) as a liposome with optimal characteristics for gene delivery both in in vitro and in vivo assay. In addition to gene delivery, PUCE liposome was tested for its ability to deliver lipophilic antitumoral drug. To this end, using the lipid film method, we encapsulated with high efficiency a new sigma-tau camptothecin derivative, named Gimatecan (ratio 1:23 drug to PUCE respectively) in PUCE liposome. The intravenous administration of Gimatecan/PUCE liposome reduces the tumor growth of 91% respect to the control group in murine carcinoma 3LL Lewis Lung tumor-bearing mice. In addition, Gymatecan/PUCE liposome induced a good antitumoral responses in Human ovaric carcinoma IGROV-1 and human non small cell lung carcinoma H-460 xenografted in nude mice.Taken together, our results suggest that cationic PUCE liposome could be a new delivery system useful for clinical application. In order to develop a new cationic liposome for drug delivery, we synthesized a series of acyl-derivatives of l-carnitine (general formula (CH₃)₃-N⁺-CH₂-CH(OCOR₁)-CH₂COOR₂ (Patent US 5498633). In particular, we selected ST983 (where R₁ = (CH₂)₁₄CH₃, R₂ = (CH₂)₁₀CH₃) for its intrinsic ability to generate cationic liposomes in aqueous medium. In this work we present our results on ST983 (also named PUCE) and its ability to entrap antitumoral substances like Taxol and a new proprietary derivative of camptothecin named Gimatecan. Among different feasible methods to entrap drugs into liposomes, for our liposomal preparation, we selected the lipidic film method which gives an high entrapment rate (drug/lipid = 1/23 mol/mol). Moreover, we analyzed the preparation of PUCE by physical-chemical methods and as a result we obtained a homogeneous dispersion of SUV liposome with a size of 100 nm. Finally, we tested cryoprotecting agents, such as lactose, sucrose and trehalose, to support the step of lyophilization. Interestingly, after liposome reconstitution in physiological solution, all tested disaccharides induced a preservation of liposomes size analyzed by QLS method. Taken together our experiments led us to set up a new injectable formulation ready to use for clinical applications. Targeted drug delivery through the blood brain barrier could distinctly improve the therapy of many diseases and reduce peripheral side effects. The recognition and cellular uptake of apolipoprotein E bearing lipoproteins is governed by LDL receptor mediated transcytosis. Here we report the investigation of a potential drug delivery system designed as an artificial lipoprotein with the function to activate an transcytotic process in brain capillary endothelia cells. The model carrier system consists of liposomes and α-helical amphipathic vector peptides containing the LDL receptor binding sequence from human apo E (residues 141–150) and different lipid associating domains, such as (I) transmembrane helices, (II) amphipathic helices or (III) long chain carboxylic acids. Structural studies done by circular dichroism spectroscopy revealed the α-helicity of these peptides and confirmed required binding to POPC SUVs. To assess the binding efficiency and to obtain binding constants isothermal titration calorimetry (ITC) and surface plasmon resonance (BIACORE®) experiments were performed. Membrane lytic properties of the peptides were estimated in release experiments using liposomes with encapsulated fluorescent dye. The well characterised peptide-loaded liposomes provide a promising tool for further receptor binding and translocation studies. Plasmid DNA—actually in gene therapy and DNA vaccination ( ) a pharmaceutical substance by itself ( )—has a topological structure that can now be characterised by capillary gel electrophoresis (CGE) for quality control and stability assessments in storage or in application. Any process development for the manufacturing of plasmid DNA has to be accompanied by a powerful in-process-control (IPC) system to generate data on the characteristics of the plasmid molecules. In particular, methods are required for obtaining supercoiled covalently closed circular (ccc) plasmid DNA in pure form. Commonly, other plasmid topologies appear as well, which have to be separated from the desired product ( ). The innovative technology of CGE (see www.CGEservice.com for further information) is a necessary tool to be added to the actually short list of applicable quality control assays for clinical grade plasmid DNA because it is superior to simple agarose gel electrophoresis (AGE) assays ( ). Here we will focus on an aspect that turned out to be of significant importance for the formulation of plasmid based therapeutics. It allows the establishment of quality assurance standards for plasmid DNA form distribution. In comparison to AGE, CGE offers high resolution and high sensitivity. The data on plasmid stability in storage assessments or those, generated by the analysis of shear effects influencing the plasmid quality in DNA drug delivery formulation and application (e.g. gene gun or jet injection), will heavily influence the QA performed for clinical studies. The aspect for manufacturers of DNA pharmaceutical is, that now the in-line monitoring for the plasmid production ( ) staring from fermentation control until the formulated and filled product (see www.PlasmidFactory.com) will be performed the first time in a way conforming to GMP. Our attempts to design interface targeting inhibitors for HIV-1 Protease (PR) are reviewed: (a) Modified peptides: Short lipopeptides, structurally derived from the terminal segments of PR show good inhibitory activities against PR and act as interface targeting “dimerization inhibitors” (1). They have low metabolic stability and bio-availability. Therefore, the structures had to be modified either by backbone modifications or by side-chain modifications (e.g. as pro-drugs). Modified peptides were synthesised and tested (König, S., et al., accepted, Dumond, J. et al. in preparation). (d). Examples: Esterification of inhibitory peptides may even improve the activity of the parent peptide while neither peptoids nor retro-inverso compounds show strong improvement. In the case of the ß-sheet targeting “dimerization inhibitors” of PR, it may be difficult to obtain good inhibitory constants with backbone modified compounds, since the hydrogen binding of the interface ß-sheet is disturbed. (b) Receptor targeting? The use of thyronine (1), thyroxine and amino-palmitic acid as amino acids in the 3-mer lipopetide structure Pam-YEX has revealed further improvement in binding. Interface targeting anti-HIV agents should be active also against therapy mutants since the mutated active site is not involved in binding. Natural interface mutations are very rare. (c) Computer modelling suggests for ß-sheet structures targeting other modifications, e.g., D-amino acids, substituted aminoglycins, aza-glycins, depending on the position. (d) Other composite enzymes: The principle of creating potent allosteric inhibitors is applicable to many enzymes. The steps are: identification of weakly binding peptides, transformation into ‘modified peptides’ or peptidomimetics with improved activity and bio-availability, attachment of lipids as auxiliary binding groups. (e) Some PR dimerization inhibitors also interfere with other ß-sheet interactions, e.g., ß-secretase, amyloid formation (Aß(1-42) aggregation, Alzheimer). On the other hand, some serpine (ß-sheet) insertion peptides inhibit PR. The questions of specificity and toxicity arise. The results may be helpful for formulation of a ß-sheet rule for inhibitor design, e.g., adding “10” H-bonds to “rule of 5”. We present a novel colorimetric biosensor for studying a broad spectrum of biochemical processes that lead to chemical or physical disruption of membranes, such as peptide-membrane association; changes in pH, temperature, ionic strength and their effects on natural lipid bilayers. The assay is based upon generation of rapid visible color changes in liposomes composed of lipids and polymerized polydiacetylene (PDA). We have applied the assay for studying antimicrobial peptides of the α-defensin family [cryptdins] and analogs with altered antibacterial activity. We demonstrate the existence of direct relationships between the chromatic transitions and the mechanism of peptide-membrane association. A correlation is observed between the colorimetric response of the assay and the extent of membrane penetration by the peptides. The applicability of the assay for determination of membrane properties is shown by analysis of PDA vesicles containing natural lipid components, extracted from diverse sources, such as erythrocytes, yeast and E.Coli mutants with various lipid compositions. The aim of our present study is to develop an enhanced liposomal formulation containing linoleic acid in order to prevent skin hyperpigmentation disorder. For these issues, an inclusive complex of liposomes composed of soya lecithin and linoleic acid has been investigated. We have previously shown that linoleic acid decreases melanin synthesis. Further, clinical trial for melasma showed that the topical application of liposomes is the most suitable formulation for applying linoleic acid to skin. In order to clarify the mechanism of effect of liposomal formulation, the evaluation of both penetration of linoleic acid and long-time existence of the linoleic acid around melanocytes are required. The cutaneous absorption of ¹⁴C-labeled linoleic acid with liposomal formula and conventional formula has been evaluated by using brownish guinea pig. In the case of using once frozen skin, no difference in the cutaneous absorption was observed between both formulae. On the other hand, in the case of using freshly extracted skin, the topical application of liposomal formula was more remarkable for imparting the long-lasting effect of linoleic acid in the epidermis than conventional one. The influence of ultraviolet (UV) light irradiation against the permeation of linoleic acid with several type of liposomes was evaluated by 3D reconstructed skin model. Further, the distribution of linoleic acid in the epidermis was determined by the micro-autoradiography method. It was found that ¹⁴C-linoleic acid was retained in the epidermis even after 24 h by the topical application of liposomal formula. Back ground: Cancer chemotherapy, in general, accompanies side effects. To reduce them and to enhance the therapeutic efficacy, many applications have been made by using DDS technology. In this study, we challenged to encapsulate TOP-53, a novel and effective topoisomerase inhibitor, into liposomes and investigated its properties in vitro and anti-tumor efficacy in vivo. Methods: TOP-53 was encapsulated into liposomes by remote-loading method and the properties of liposomal TOP-53, such as the stability at 4 and the agglutinability in 50% serum, were examined. Anti-tumor activity of liposomal TOP-53 against solid tumor was examined in vivo by using colon 26 NL-17 carcinoma model mice. Results and Discussion: TOP-53 was effectively entrapped into liposomes, stable at 4, and acceptable aggregation in the presence of serum. Liposomal TOP-53 showed potent cytotoxic activity against tumor cell in vitro, and significant anti-tumor activity against solid tumor as well as the increase in life span of tumor-bearing mice. Improved reduction of side effects will also be discussed. Mechanisms of cationic lipid-based nucleic acid delivery are receiving increasing attention, but despite this the factors that determine high or low activity of lipoplexes are poorly understood. This study is focused on the fine structure of cationic lipid–DNA complexes (lipoplexes) and its relevance to transfection efficiency. Monocationic (DOTAP, DMRIE) and polycationic (DOSPA) lipid-based assemblies, with or without neutral lipid (DOPE, DOPC, cholesterol) were used to prepare lipoplexes of different L⁺/DNA⁻ charge ratios. Circular dichroism,cryogenic-transmission electron microscopy and static light scattering were used for lipoplex characterization, while expression of human growth hormone or green fluorescent protein was used to quantify transfection efficiency. All monocationic lipids in the presence of inverted hexagonal phase-promoting helper lipids (DOPE, cholesterol) induced appearance of Ψ-DNA, a chiral tertiary DNA structure. The formation of Ψ-DNA was also dependent on cationic lipid–DNA charge ratio. On the other hand, monocationic lipids either alone or with DOPC as helper lipid, or polycationic DOSPA-based assemblies, neither of which promotes a lipid–DNA hexagonal phase, did not induce the formation of Ψ-DNA. Parallel transfection studies reveal that the size and phase instability of the lipoplexes, and not the formation of Ψ-DNA structure, correlate with optimal transfection. This study was supported in part by two grants from the “Center of Excellence” of Israel Science Foundation of the Israel Academy of Sciences and Humanities (to Y.T. and Y.B.), and the Canada-Israel Industrial Research and Development Foundation (CIIRDF) grant. Back ground: A novel quinoline derivative, TAS-103, is known to show high anti-tumor activity through the inhibition of topoisomerases. To enhance the bioavairability of the agent, liposomalization was attempted. Methods: TAS-103 was encapsulated by remote-loading method into liposomes composed of DPPC and cholesterol. Liposomal TAS-103 was evaluated for the stability, agglutinability in the presence of 50% serum, and cytotoxic activity against Lewis lung carcinoma in vitro. Then, the anti-tumor activity against Lewis lung carcinoma-bearing mice was examined in vivo. Result and Discussion: TAS-103 was entrapped into liposomes effectively. Liposomal TAS-103 was stable for storage and in the presence of serum, and showed potent anti-tumor activity like as free TAS-103. In In vivo study, liposomal TAS-103 showed a significant anti-tumor activity and increased in the life span of Lewis lung carcinoma-bearing mice after three dose-administration. These result indicate that liposomal TAS-103 is useful for cancer therapy. Recently, it is clear that anti-angiogenic photodynamic therapy (PDT) is induced intensive suppression of tumor growth in comparison to the general tumor cell-targeted phototherapy. In this study, we developed benzoporphyrin derivative monoacid ring A (BPD-MA)-entrapped polycation liposome (PCL) as a novel angiogenic vessel-targeted phosensitizer. PCL is constructed that cationic polymer, cetylated polyethylenimine (cetyl-PEI), is located on the surface of liposome. We report here that BPD-MA-entrapped PCL induces remarkable phototoxicity for vascular endothelial cells, specific damage to angiogenic vessels for angiogenesis-model mice induced by dorsal air sac technique, and intensive suppression of tumor growth for Meth-A sarcoma-bearing mice at low BPD-MA dose (only 0.25 mg/kg in BPD-MA) following PDT treatment. The photodynamic efficacy of BPD-MA-entrapped PCL is elucidated by facilitation of photosensitizer uptake efficiency due to the strong electrostatic interaction between PCL and plasma membrane and subsequent rapid intracellular transport of liposomal photosensitizers via endocytosis. Furthermore, some degrees of photosensitizers mediated with PCL were accumulated in nuclei, so that the intranuclear photosensitization is one of the factors for induction of enhanced phototoxicity. A-fect™ is a novel liposomal drug delivery system with unique properties. Special constituents of A-fect are chemically modified membrane spanning tetraether lipids (TEL) derived from archaea. Due to their bipolar structure the membrane spanning TEL exhibit a clearly higher stability than conventional bilayer forming lipids. In addition, TEL considerably enhance the stability of conventional liposomes. These unique features allow a continuous adjustment of the release characteristics of A-fect™ liposomes in biological fluids. Release experiments using carboxyfluorescein were performed to study the stability of A-fect™ liposomes in serum. Compared to conventional liposomes A-fect™ liposomes exhibited a high stability with a slow and nearly linear release over several days at 37°C. The release rate could be adjusted continously by the amount of TEL in the A-fect™ liposomes. In vitro and in vivo studies were carried out to evaluate the potential of A-fect™ as an oral drug delivery system. Compared with conventional liposomes consisting of phosphatidylcholine (PC) A-fect™ liposomes demonstrated a significant higher stability under conditions mimicking the gastrointestinal environment. Additionally, multilamellar A-fect™ liposomes are substantially more stable towards bile salts and in low pH media than unilamellar A-fects™. Somatostatin and vasopressin were used as model compounds for oral uptake studies in rats. Peptide formulations were administered by oral gavage, subsequently the plasma levels of peptides were determined at different time points. The A-fect™ formulations led to a significant improvement of the oral resorption of both peptides in comparison to conventional liposomes or to free peptide administration. Both the in vitro data as well as the in vivo results clearly indicate that A-fect™ formulations could be developed into powerful tools for parenteral controlled release systems and for the oral delivery of compounds. Folate-diplasmenylcholine (1,2-di-O-(Z-1′-hexadecenyl)-sn-glycero-3-phosphocholine; DPPlsC) liposomes have been shown to greatly enhance the potency of water-soluble antitumor agents via a selective folate-mediated uptake and acid-catalyzed endosomal escape mechanism. We report an adaptation of this strategy for the delivery of chloroaluminum phthalocyanine tetrasulfonate ( ), a water-soluble sensitizer used in photodynamic therapy, in a binary role as tumor imaging beacon and phototoxic agent. /DPPlsC: folate liposomes (9.8 µM bulk concentration, 2.5 mM intraliposomal concentration) were substantially more phototoxic to folate-deficient KB cells than 12.5 µM free after a 30 m irradiation (630–910 nm). Experiments with /DPPC:folate and folate-free /DPPlsC liposomes (acid-insensitive and non-targeted controls, respectively) showed significantly reduced phototoxicities under the same illumination conditions. Confocal microscopy experiments indicate that these differences are due to 1) increased concentrations of water-soluble sensitizers that can be delivered to target cells using the folate receptor-mediated pathway and 2) changes in the biodistribution and intracellular localization of the beacon when acid-labile DPPlsC liposomes are used as the delivery vehicle. Preliminary results in a subcutaneous murine tumor model suggest that this is a promising in vivo detection and ablation tool for folate-positive tumor tissues. Potential advantages of this approach include the use of lower bulk concentrations, rapid plasma clearance of free , and better phototoxic responses—due to higher intracellular concentrations combined with reduced collateral photodamage arising from misguided sensitizer accumulation—thereby enhancing the selective phototoxicity of PDT treatments. The predictable pharmacokinetics and flexible formulation properties of this targeted liposomal beacon offer significant advantages over related polymer-based conjugates. Self-assembling peptides present attractive platforms for engineering stimulus-responsive materials with controlled nano- and microstructures. Here, we demonstrate the stimuli-triggered self-assembly of fluid peptide and protein precursors into highly crosslinked peptide hydrogels. Our system takes advantage of thermal or light triggered release of encapsulated salts from liposomes to trigger self-assembly of either a 16-amino acid peptide or an enzyme-crosslinkable protein substrate. The peptide consists of alternating hydrophobic and hydrophilic residues and is known to self-assemble into β-sheet structures in the presence of salts. We show that temperature and light-triggered release of calcium salts from liposomes results in rapid gelation of peptide/liposome suspensions into highly crosslinked, fibrillar, β-sheet hydrogels. The enzymatically-crosslinked system forms hydrogels upon near-infrared irradiation of calcium-containing liposomes in the presence of transglutaminase and fibrinogen. These solutions also form patterned hydrogels upon photolithographic processing. This approach may lead to useful nanomaterials that are able to undergo drastic changes in physical properties upon activation by mild triggering stimuli. The efficacy of polyethyleneglycol (PEG)-modified liposomal doxorubicin (PEG-LDOX) against tissue distribution and antitumor activity has been demonstrated. Our results on the fixed aqueous layer thickness (FALT) have suggested that sing le PEG-modification by the PEG-lipid, which had a large molecular weight, couldn't display sufficient ability, whereas the mixture of PEGs, which had a long and short polyoxyethylene chain, exhibited a stronger effect on PEG-modification. We recognized the most suitable mixed rate (PEG2000:500 = 2:1), showed the maximum FALT. In this study, we examined the effects of this mixed PEG-modification on tissue distribution and antitumor activity. With increase of FALT, the DOX concentration increased in the plasma and decreased in the livers. This result suggested that the ability to avoid trapping by RES was caused by increase of FALT. Furthermore, The administration of PEG(2:1)-LDOX caused a reduction of tumor weight as compared with PEG2000-LDOX and PEG500- LDOX. Moreover, the DOX concentration in the tumor showed PEG(2:1)-LDOX > PEG2000-LDOX > PEG500-LDOX, this order agreeing with that of FALT. Namely, the connection between increase of FALT and the enhancement of antitumor activity of these liposomes was suggested. In conclusion, it was confirmed that this mix ed PEG- modification of liposome was superior. The overproduction of biochemical mediators, and the activation of leucocytes and endothelial cells, generated in thermally injured tissue, gives rise to both local and distant effects. Highly reactive metabolites, such as oxygen derived free radicals are involved in the postburn phase of thermally damaged skin, leading to enhanced tissue loss an delayed wound healing. To capture these radicals, enzymes such as superoxide dismutase (SOD) are of great therapeutical interest. But the nature of the SOD, with a high molecular weight, the hydrophilic characteristic and the extremely short biological half life diminish its therapeutical application. The aim of our studies was to evaluate the effects of topically applied recombinant human SOD in different formulations, whereas special attention was payed on the liposomal encapsulated SOD. To evaluate the effects edema formation in the inflammatory stage and the wound healing properties were examined by histological and macroscopical observations. In all tested parameters the liposomal formulation showed statistically relevant data compared to all other groups. Therefore, we believe that the liposomal application of SOD may compensate its unfavourable characteristics. In the case of topical application, an alternative non invasive strategy, liposomes may act as drug carrier to localize the drug in the affected tissue and furthermore, the sustained release of the protein enhances its potential in radical scavenging. Liposomes represent one of the major tools for modern drug delivery strategies. We aimed to establish a novel liposome production procedure, where several features, such as simplicity, robustness, easy handling in sterilization procedures and especially scalability were of major concern. Based on the principles of the ethanol injection technique (Batzri and Korn, 1973) we developed a new technique, whereby a substantial progress was achieved, leading from the conventional batch process to a novel continuous procedure. Herein, the principal item is the crossflow injection module, especially designed for this purpose. This specially conceived unit has the benefit of liposome manufacture independent of production scale, as scale is determined only by the free disposable vessel volumes. All devices such as vessels, valves, pumps, manometers and tubes, were designed to be suitable for sterile operations. Manufacture of liposomes using the newly invented crossflow module in the multiple injection technique resulted in reproducibility regarding vesicle size and size distribution as well as high protein entrapment. Variation of lipid concentration and injection pressures resulted in narrow distributed vesicles at any desired vesicle. Subsequent continuous crossflow filtration within the same closed containment separates unentrapped material from the liposome suspension. Stability testing was performed until now for more than one year and up to date the liposomes remained stable. In addition no loss of protein activity could be detected, due to the mild and optimized preparation and filtration conditions. In previous work we reported that liposomes surface-modified with AcoHSA are massively targeted to the hepatic endothelial cells ( ). In order to modulate the function of sinusoidal endothelial cells in pathological conditions we modified stabilized lipid plasmid particles (SPLPs) developed by Cullis et al. ( ) with AcoHSA for the selective targeting of genes to these cells. SPLPs were prepared by solving PEG-lipids, cationic lipids, neutral lipids and DNA in high detergent solution. Lipid particles were formed during detergent dialysis. The DNA encoded for the reporter genes Enhanced Green Fluorescent Protein or luciferase. SPLPs of varying composition were prepared and characterized. AcoHSA was covalently coupled directly to the SPLP surface or to the distal end of a PEG-chain. The size of the SPLPs ranged from 80–120 nm and up to 60% of the DNA was encapsulated. The amount of AcoHSA coupled to the surface of the SPLPs was approximately 10 µg/µmol total lipid, but could be increased to 22 µg/µmol total lipid by coupling the AcoHSA to the distal end of the PEG-chain. Transfection efficiency in vitro with COS-7 cells (as control cells) is low but is increased in the presence of CaCl₂. In conclusion, we are able to make stable SPLPs containing high amounts of DNA that can be targeted to the hepatic endothelial cells by coupling of AcoHSA. In vivo transfection experiments are currently under investigation. consisting of phosphatidylcholine (PC), cholesterol, and (MPB-PE) in the ratio of 6:4:0.4:0.32. AcoHSA was covalently coupled to the MPB-PE present in the lipid coating. After extrusion the coated cationic lipoplexes (CCL's) were 144 ± 20 nm. CCL's containing plasmid DNA could be separated from empty vesicles by using a sucrose gradient. The CCL's were further characterised by measuring the amount of AcoHSA coupled to the CCL's, the amount of incorporated DNA, and transfection efficiency. The amount of DNA associated with the CCL's varied from 0.35 to 0.81 µg/µmol total lipid. Transfection of COS cells with the coated lipoplexes was observed after a 96 h incubation with these particles (96 h). The number of transfected cells is low. In conclusion, we are able to make CCL's containing DNA that are stable for at least several weeks, and have a low transfection efficiency in vitro and which can be used for in vivo studies. The resulting SPLPs consisted of 1,2-diacyl-sn-glycero-3-phosphoethanol-amine (DOPE), cholesterol, poly(ethylene-glycol)₂₀₀₀C14-ceramide (PEG-Cer14) or 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene-glycol)₂₀₀₀] (DSPE-PEG₂₀₀₀), 1,2-diacyl-3-trimethylammonium-propane (DOTAP), and 1,2-dioleoyl-sn-glycero-3-phophoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (MPB-PE) in the molar ratio of 37.5:40:10:12:2.5. The amounts of DOPE and cationic lipid varied. To improve liposomal gene transfer we investigated the influence of alkyl phospholipids (APLs) on gene transfer efficiency in human tumour cell lines. To approach this, liposomes were employed which consist of APLs of varying chain lengths, saturation of the chain and head groups combined with dioleylphosphoethanolamine and dioctadecyldimethylamine bromide. The membrane-interacting activity of APLs in these liposomal preparations was utilised to enhance DNA transfer into targeted cells. The transfer of the β-galactosidase (LacZ) expressing plasmid pCMV by these APL-liposomes containing the short chain tetradecylcholine led to an up to 3-fold increase in transfer efficiency in HCT15 and HCT116 human colon carcinoma cells compared to lipofectin-mediated transfection. We further evaluated the transfection potency in vivo using the LacZ reporter gene and a plasmid encoding the cytosine deaminase (CD)- gene. It was found that the LacZ activity was comparable after transfection with the TPC-lipoplex and with lipofectin. By contrast, a significantly improved therapeutic effect was observed in mice treated with the TPC-lipoplexes for intratoumoral CD-gene transfer compared to animals, which received lipofectin-lioplexes. It was found that the best in vivo transfection efficiency correlates with a higher brutto charge as it was found in vitro. The studies demonstrated that utilisation of short chain APL for the preparation of lipoplexes may be of significant benefit to generate more efficient lipid-DNA complexes for gene transfer in vitro and in vivo. The analysis of high- and low-density lipoproteins merits huge interest in the diagnosis and understanding of atherosclerosis. Significant changes are detectable towards protein as well as lipid composition under pathological conditions. Since damages of lipids lead to the formation of well-defined products like lysophospholipids or chlorohydrins, analytical methods which allow the fast and reliable determination of lipid composition of lipoproteins are strongly needed. MALDI-TOF mass spectrometry was applied to the analysis of the lipid composition of human lipoproteins. Differences between LDL and HDL in sphingomyeline and phosphatidylcholine content could be easily monitored by mass spectrometry and differences towards the extraction efficiency of different solvent systems could also be found by MALDI-TOF MS. Additionally, treatment of LDL with hypochlorite and phospholipase A₂ resulted in marked changes (formation of chlorohydrines and lysolipids) which were detectable to a different extent by mass spectrometry. Using MALDI-TOF MS the chlorohydrine of cholesterol was detectable. We conclude that MALDI-TOF mass spectrometry is able to provide fastly a reliable lipid profile of human lipoproteins, and may be useful in clinical investigations of LDL and HDL. The transfection efficiency of DOTAP and DOTAP-Cholesterol complexed with DNA encoding the luciferase reporter gene was optimized in cell culture in the presence or absence of protamine sulfate. From 164 different complexes four were selected for further analyses. Although the four selected complexes had a similar high transfection efficiency in cell culture 22-fold differences in transduction efficiencies of the complexes were observed in mice. In vivo transduction efficiency seemed to correlate with resistance to inhibition of transfection by high serum concentrations. Physical properties, such as zeta-potential, turbidity, degree of resonance energy transfer between lipids and accessability of DNA were determined to better understand the underlying cause for serum resistance. Control complexes, that contained the same compounds at the same final concentration as the selected complexes but that differed in transfection efficiency due to a change in the order of addition in compounds were also used to detect structural differences in complexes correlating with transfection efficiency. The Zeta potential and changes in turbidity or resonance energy transfer after addition of serum did not seem to correlate with transfection efficiency or resistance to serum. The percent inhibition of transfection by serum was dependent on the lipid used and the lipid concentration but not on the order of addition of components. Protamine sulfate when added to the DNA before addition to lipid did only decrease the degree of lipid membrane pertubation but also increased the capture efficiency of the complexes there by reducing the accessability of their DNA to PicoGreen and DNAse.

The therapeutic effect of Uighur prescription on abnormal Savda in asthma patients was evaluated using plasma proteomics in order to elucidate the biological mechanism and identify potential therapeutic targets of abnormal Savda. In the present study, 40 asthma patients with abnormal Savda including abnormal Savda Munziq and Savda Mushil were enrolled and treated with Uighur prescription. The effect of Uighur prescription on protein expression and potential targets was investigated by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics and bioinformatics analysis. Expression of candidate proteins was verified by an enzyme‑linked immunosorbent assay. Following treatment with the Uighur prescription, 22 proteins were differentially expressed in the plasma of patients with asthma and abnormal Savda. The majority of these proteins were localized in intermediate filaments and the cytoskeleton and acted as antioxidant enzymes and binding proteins. Furthermore, they participated in the defense and inflammatory response, and the response to oxidative stress and wound healing. Peroxiredoxin 2 and carboxypeptidase B2 expression was significantly upregulated, whereas S100A7 was considerably downregulated in the whole plasma of patients (all P Introduction Asthma is a chronic disease of airway inflammation that presents with varying and recurrent symptoms, including cough, chest tightness and shortness of breath (1). Progress has been made in understanding the pathogenesis, diagnosis and treatment of asthma (2), and novel drugs, formulations and dosing instruments have been applied to treat asthma (3,4). However, asthma still presents a considerable threat to health, due to its high morbidity and mortality rates (5). Therefore, the development of novel drugs and complementary therapies, possibly in the form of traditional medicine, such as traditional Uighur medicine, is urgently required. Uighur medicine is a well-established branch of medicine comprised of unique types of medicines, including munziq and mushily of abnormal savda. It is currently practiced by physicians and clinicians from the Xinjiang Uighur autonomous region in China (6,7). Uighur medicine shares an origin with Greco-Arab medicine and describes the incidence of illnesses associated with abnormal Hilits (representing syndromes), which are caused by an imbalance amongst four normal Hilits (representing humors), known as Safra, Kan, Phlegm and Savda (6,8). Quantitative or qualitative changes in any Hilit and the resulting disturbance of dynamic homeostasis of these Hilits may result in the development of corresponding symptoms, including increased quantity of urine, facial edema and weak pulse. Among these, abnormal Savda is the dominant syndrome in disease progression and often develops in conjunction with other abnormal Hilits (8). In traditional Uighur medicine, Savda is the major syndrome responsible for almost all complex diseases, including asthma, type II diabetes mellitus, cancer, and various cardiovascular and neurodegenerative illnesses (8). Previous studies have demonstrated that abnormal Savda is associated with relatively consistent biological changes in complex diseases that are manifested holistically within a population (9–11). Abnormal Savda may be treated with a unique Uighur prescription, comprised of ten herbal ingredients mixed in specific proportions. These are C. dichotoma (10.6%), A. italic (10.6%), G. Uralensis (7.1%), A. capillus-veneris (4.9%), E. humifusa (4.9%), Z. jujuba (4.9%), L. angustifolia (4.9%), F. vulgare (4.9%), M. officinalis (4.9%) and A. pseudoalhagi (42.3%) (7). A number of studies have demonstrated that Uighur prescription may mitigate oxidative stress associated with abnormal Savda, possibly by protecting cells from mitochondrial oxidative damage (12). Uighur medicine may also modulate abnormal changes in the neuroendocrine-immunity network and prevent carcinogenesis in murine models (13). Furthermore, flavonoids isolated from abnormal Savda Munziq are capable of inducing cell-cycle arrest and apoptosis of tumor cells (14). The results of these studies indicate that the biological basis of Savda may change as abnormal Savda develops, but is restored following Uighur prescription treatment. In the disease state, protein expression is dynamically altered in a spatiotemporal manner and modulated by post-translational processing and chemical modifications. Along with the application of emerging proteomics techniques, serum/plasma has become a biomedium for the study of disease etiology, diagnostic biomarkers and drug targets of asthma in contemporary medicine (15). In particular, proteomics analysis has improved knowledge on the allergens of asthma and the effects of clinical therapy, and has even guided personalized therapy (16). In addition, abnormal changes in the whole regulatory network of protein expression are associated with the overall pathological state of patients with asthma (13). This holistic concept of understanding disease etiology through understanding of biological systems is shared by the theories of traditional Uighur medicine. The present study assessed the effect of Uighur prescription of abnormal Savda on the regulatory network of relevant plasma proteins in asthma patients using proteomics. It also identified differentially expressed proteins that are potentially targeted by Uighur prescription. The aim of the present study was to provide evidence for the role of Uighur prescription in treating abnormal Savda at the proteomics level and to contribute to the scientific interpretation and application of Uighur medicine. Materials and methods Uighur prescription Abnormal Savda Munziq is a traditional Uyghur medicinal herbal preparation that consists of the following (10,12,13): 15 g Cordia dichotoma fruit and Ziziphus jujube fruit, 7 g Anchusa italic plant, Dracocephalum moldavica L, Lavandula angustifolia, Adiantum capillus-veneris, Foeniculum vulgare Mill, Euphorbia humifusa Willd, 10 g Glycyrrhiza uralensis Fisch and 15 g Alhagi pseudalhagi. Abnormal Savda Mushil preparations contain: 45 g Alhagi pseudalhagi and Fructus Cassiae fistulae, 15 g Pogonatherum crinitum, Terminalia chebula Retz. and Tenninlia chebula, 12 g Rosa rugosa, 6 g Polypodium vulgare, Glycyrriza Uralensis Fisch. and Foeniculum vuLgare Mill., 10 g Lavandula angustifolia, Adiantum capillus-veneris, Euphorbia humifusa Willd, Anchusa italic, Iberis pectilata L., Viola tianschanica Maxim, Nymphaea L. and Amygdalus communis L., 18 g Raisin, 30 g Cordia dichotoma fruit and Qizil Guliqent and 21 g Cassia angustifolia. All ten herbs were originally grown in Xinjiang, China and collected by the Chi Kang Barbour Pharmaceutical Co. (Xinjiang, China) by professional herbal growers. Patient data In total, 40 patients diagnosed with bronchial asthma between August 2013 and January 2015 were selected for the present study according to the diagnostic criteria of the Guide for Prevention and Treatment of Bronchial Asthma established by the Chinese Medical Association in 2013 (17) and the Global Initiative for Asthma in 2010 (revised in 2012) (18). The present study consisted of 23 males and 17 females, with a mean age of (36.93±12.14) years old (age range, 20 and 60 years old). Abnormal Hilits, including abnormal Kan, Phlegm, Safra or Savda, were identified by an independent diagnosis of two experienced physicians specialized in Uighur medicine. The classification outcome as abnormal Savda was further defined according to the established criteria of Uighur medicine (6,7) and using the assessment of symptom scores for abnormal Savda, including slow pulse (<60 beats/min), bleary eyes, dark purple lips, blue tongue, cool skin temperature (1 month. All enrolled individuals were orally administered with an Uighur prescription of abnormal Savda (patent no. ZL 02130082.8, China), including abnormal Savda Munziq extract powder at a dose of 3–6 g twice daily for 10–15 day, followed by Savda Mushil powder at a dose of 3–6 g twice daily for 3–5 days based upon the specific status of the asthma patients, as described previously (7). The entire clinical therapy involved completion of ≥3 courses of treatment, with each course consisting of treatment with abnormal Savda Munziq at a dose of 3–6 g for 10–15 days, followed by a 3–5 day therapy with abnormal Savda Mushil at a dose of 3–6 g. The study design was approved and monitored by the Ethics Committee of the Xinjiang Medical University (Urumqi, China). All procedures of the study were in accordance with the Helsinki Declaration. Informed consent was obtained from all patients, and patient data were analyzed anonymously throughout the experiment. Sample collection Blood samples (2 ml) were obtained from 40 asthma patients by venipuncture into evacuated blood collection tubes containing EDTA-K2 anticoagulant (Promega Corporation, Madison, WI, USA). Plasma samples were separated by centrifugation at 3,000 × g for 10 min at 4°C and preserved at −80°C. Blood samples were collected twice: Prior to treatment (at baseline level) and once following final treatment with abnormal Savda Mushil. Protein extraction and proteomic analysis Proteomics analysis was performed using 8-plex isobaric tags for relative and absolute quantitation (iTRAQ, Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Plasma samples were collected twice; once at baseline and once following treatment, and were randomly and equally divided into four subgroups (n=4 in each group). Up to five samples from different individuals in each subgroup were mixed in equal volumes to form a pooled sample for further testing. The resulting samples were enriched for low-abundance proteins by depletion of medium- and high-abundance proteins using a pre-packed 1-ml affinity liquid chromatography (LC) column provided with a ProteoMiner™ low abundance protein enrichment kit (catalogue no. 56-2588-44; Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the manufacturer's instructions. Following enrichment, precipitated proteins were dissolved in 300 µl lysis buffer consisting of 6 M urea, 4% 3-cyclohexylamino propanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate, 2 mM EDTA (all from Promega Corporation) and 1 mM phenylmethane sulfonyl fluoride (Sigma-Aldrich; Merk KGaA). Following reduction, 10 mM dithiothreitol and 100 mM NH4HCO3 (Sigma-Aldrich; Merck KGaA) were added at 56°C for 45 min. Subsequently, alkylation was performed with 55 mM iodoacetic acid (Promega Corporation) and 100 mM NH4HCO3 for 45 min. Samples were then precipitated in 80% acetone at −20°C overnight and dissolved in 0.8 M urea and 500 mM tetraethylammonium bromide (TEAB; pH 8.5). Each protein sample (30 µg) was digested by supplementing sequencing-grade modified trypsin (Promega Corporation) at an enzyme/substrate ratio of 1:30 (w/w) in dissociation buffer (0.1% in TEAB; Promega Corporation) at 37°C for 24 h. The tryptic peptides were then labeled with 8-plex iTRAQ reagents (AB Sciex, Foster City, CA, USA) according to the manufacturer's instructions (iTRAQ113, 114, 115 and 116 for samples of the baseline, and 117, 118, 119 and 121 for samples following treatment). The reaction solvents were removed by speed vacuum at 3,000 × g and the labeled peptides were dissolved in 20 mM NH4FA (pH 10.0; Sigma-Aldrich; Merck KGaA) for subsequent analysis. Peptide fractionation by strong cation exchange chromatography and C18 column reversed-phase (RP) chromatography High-resolution strong cationic exchange (SCX) chromatography was performed to remove redundant iTRAQ reagents and any interfering substances that could affect mass spectrometry (MS) analysis. Labeled peptides were loaded onto an SCX column (Luna SCX, 4.6×250 mm; Phenomenex Inc., Torrance, CA, USA), and eluted by a stepwise linear elution program as follows: 0–10 min equilibration in Buffer A at pH 3.0 including 25% acetonitrile (ACN), 20 mM KCl and 10 mM KH2PO4. A 10–15 min fast elution was also completed using 0–5% Buffer B prepared with 25% ACN, 1 M KCl and 10 mM KH2PO4, and a pH 3.0, 15–50 min linear elution with 5–30% Buffer B, and 50–55 min washing elution with 30–80% Buffer B. For desalting and further fractionation, peptide fractions were loaded onto an RP column (Luna C18, 4.6 mm inner diameter ×250 mm length, Phenomenex, Inc.), and eluted by a step linear elution program as follows: 0–10 min equilibration in 100% solution A (2% ACN and 20 mM NH4FA, pH 10.0), 10–15 min fast elution with 0–12% solution B (80% ACN and 20 mM NH4FA, pH 10.0), 15–50 min linear elution with 12–56% solution B, and 50–55 min washing elution with 56–80% solution B. All procedures were performed using a prominence high performance liquid chromatography (HPLC) system (Shimadzu Corporation, Kyoto, Japan) with a flow rate of 1.0 ml/min and the peptides were monitored at 214 nm. In addition, the fractions containing the peptides were collected at a rate of 1 tube/min during a linear elution period. Peptide analysis by nano-liquid chromatography coupled with Q-exactive MS Peptide fractions were loaded onto a nano-RP column (5 µm Hypersil C18 phenomenex Luna SCX 100A; 75 µm × 100 mm; Thermo Fisher Scientific, Inc.) mounted in a Prominence Nano HPLC system (Shimadzu Corporation). The peptides were then eluted with an ACN gradient from 5–40% containing 0.1% formic acid (Sigma-Aldrich; Merck KGaA) for 65 min at 400 nl/min. Elutes were then transferred to a Thermo Scientific™ Q-exactive™ mass spectrometer (Thermo Fisher Scientific, Inc.), which was run in positive ion mode and in a data-dependent manner with a full MS scan of 350 to 6,000 m/z, 70,000 resolution, 320°C, 400 nl/min flow rate under a nubuliser pressure of 1,800 V, MS/MS scan with minimum signal threshold 17,500 and isolation at 2 kDa. In order to evaluate the performance of MS on iTRAQ-labeled samples, two MS/MS acquisition modes-higher collision energy dissociation and collision induce dissociation-were employed. In order to optimize the MS/MS acquisition efficiency of higher collision energy dissociation, normalized collision energy was systemically examined from 25 to 70%. Database search and quantitative data analysis Raw MS/MS data were converted into Mascot generic format (MGF) using Proteome Discoverer 1.3 (Thermo Fisher Scientific Inc.). Exported MGF files were searched using Mascot 2.3 (Matrix Science, Inc., Boston, MA, USA) against the Uniprot Human 2009-12 database (www.uniprot.org) with a precursor mass tolerance set at 15 ppm and product ion tolerance of 0.02 kDa. An automatic decoy database search was performed. Carbamidomethylation of cysteines was set as a fixed modification (C), and oxidation of methionines (M), Gln to pyro-Glu (N-term Q) and 8-plex iTRAQ modifications of N-term, K and Y were considered variable modifications. A maximum of one miscleavage was acceptable. A protein with at ≥1 unique peptide and a false discovery rate <0.01 qualified for further quantification analysis. Moreover, the fold change in protein abundance was defined as the median ratio of all significantly matched spectra with tag signals. Based on Proteome Discoverer 1.3 software analysis (Thermo Fisher Scientific, Inc.), the coefficient of variation distribution of all quantified proteins and quantitative results derived from duplicated injections were compared in parallel. The differential expression of all proteins was presented as a fold change in iTRAQ ratios. In addition, the upregulation of a protein was indicated by an increase of ≥1.2-fold, and downregulation by a decrease of ≤0.83-fold (1.0/1.2). Bioinformatics analysis by MetaCore™ software Differentially expressed proteins were further characterized using a the MetaCore™ 6.18 software package (http://thomsonreuters.com/metacore, Thomson Reuters Co., New York, NY, USA) and an online database (https://portal.genego.com; Thomson Reuters Co.) in order to understand the underlying signaling pathways and protein-protein interaction networks, and to evaluate candidate proteins as potential biomarkers. Enzyme-linked immunosorbent assay (ELISA) The plasma level of candidate proteins was verified for plasma samples collected at baseline and following ELISA using commercially available reagents (USCN Life Science Inc., Wuhan, China) according to the manufacturer's instructions. ELISA reagents were used for the following candidate proteins: Perioxiredoxin 2 (PRDX2; catalogue no. SEF757Hu), protein S100A7 (catalogue no. SEC035Hu), transforming growth factor-β1 (TGF-β1; catalogue no. SEA124Hu), myeloperoxidase (MPO; catalogue no. SEA601Hu), carboxypeptidase B2 (CPB2; catalogue no. SEA615Hu) and keratin type II cytoskeletal 6A (KRT6A; catalogue no. SED234Hu). The final data were confirmed by three independent measurements of each protein. Statistical analysis Statistical analysis was performed using SPSS 17.0 statistical software for windows (SPSS, Inc., Chicago, IL USA). All P-values were two-sided, and P<0.05 was used to indicate a statistically significant difference. The data of protein expression derived from the ELISA experiment at baseline and following treatment were statistically compared using a paired samples t-test. Results Identification of proteins differentially expressed by Nano-LC coupled with Q-Exactive MS Therapeutic evaluation models for the treatment of asthma patients with abnormal Savda receiving unique Uighur prescription were established using proteomics analysis. Following preparation of pooled samples at baseline and following treatment by depletion of high-abundance proteins, enzymatic digestion and iTRAQ labeling, resulting peptides were simultaneously analyzed by nano-LC coupled with Q-Exactive MS, leading to the output of peptide spectra with 95% confidence intervals representing the relative and absolute quantitation of each sample. To analyze the effect of Uighur treatment on proteomic profiles, peptide spectra data of pooled samples corresponding to biological replicates at baseline and following treatment were collectively analyzed using a functional module of Proteome Discoverer software. Analysis of peptide spectra set a fold change ≥1.2 or ≤0.83 (1.0/1.2) as the cutoffs for quantitative differences, and 22 proteins were identified as differentially expressed in response to the corresponding treatment (Table I). Among these, 16 proteins were upregulated whereas 6 were downregulated. Table I. Identification of differentially expressed proteins in the plasma of patients with asthma and with an abnormal Savda in response to the treatment with the unique Uighur prescription. Table I. Identification of differentially expressed proteins in the plasma of patients with asthma and with an abnormal Savda in response to the treatment with the unique Uighur prescription. Peptide detection Nos. Protein information Rec. Symbol Uniprot ID MW (kDa) Calc. pI Peptide score Peptide coverage Unique peptides Fold-change^a 1 Keratin, type II cytoskeletal 6A KRT6A P02538 60 8 1,803.12 55.32 3 6.346 2 Calmodulin-like protein 3 CALL3 P27482 16.9 4.42 37.41 8.05 1 4.897 3 Keratin, type II cytoskeletal 1 K2C1 P04264 66 8.12 1,883.75 53.73 34 4.878 4 Keratin, type II cytoskeletal 72 K2C72 Q14CN4 55.8 6.89 177.81 7.24 1 4.511 5 Keratin, type I cytoskeletal 27 K1C27 Q7Z3Y8 49.8 5.05 44.26 6.54 1 3.927 6 Annexin A1 ANXA1 P04083 38.7 7.02 65.14 8.96 2 3.499 7 InaD-like protein INADL Q8NI35 196.2 4.94 18.24 0.33 1 2.748 8 Keratin, type I cytoskeletal 10 KRT10 P13645 58.8 5.21 1,061.74 44.18 21 2.664 9 Thioredoxin THIO P10599 11.7 4.92 56.05 8.57 1 2.065 10 Carboxypeptidase B2 CPB2 Q96IY4 48.4 7.71 54.12 3.55 2 1.841 11 Serum amyloid A-2 protein SAA2 P0DJI9 13.5 9.14 161.27 31.97 1 1.697 12 Peroxiredoxin-2 PRDX2 P32119 21.9 5.97 48.54 9.09 2 1.502 13 Myeloperoxidase MPO P05164 83.8 8.97 30.51 1.61 1 1.501 14 Keratin, type II cytoskeletal 2 epidermal KRT2 P35908 65.4 8 718.83 28.01 10 1.390 15 Zinc finger and BTB domain-containing protein 18 ZBT18 Q99592 58.3 5.69 28.1 1.34 1 1.345 16 Transforming growth factor beta-1 TGFβ1 P01137 44.3 8.53 35.31 7.69 2 1.263 17 Thrombospondin-4 TSP4 P35443 105.8 4.68 142.57 6.35 4 0.764 18 Protein S100-A7 S100A7 P31151 11.5 6.77 50.31 10.89 1 0.701 19 Retinol-binding protein 4 RBP4 P02753 23 6.07 123.03 9.95 2 0.692 20 Platelet factor 4 variant PF4VL P10720 11.5 9.1 193.28 48.08 2 0.679 21 Lysozyme C LYSC P61626 16.5 9.16 115.72 18.24 3 0.642 22 Protein CASC1 CASC1 Q6TDU7 83.1 5.29 24.76 1.40 1 0.581 a For a candidate protein, the fold-change (ratio) of >1.2 (base 2 logarithm values) or <0.80 (−1.2) in the plasma content was considered and reported as upregulated or downregulated following treatment with prescriptions, respectively. Rec. Symbol, recommended symbol; Cacl.PI, theoretically calculated isoelectric point. Bioinformatics analysis of differentially expressed proteins by MetaCore™ software and ontology database The role of the 22 identified proteins during disease development was further evaluated by bioinformatics analysis using MetaCore™ 6.16 software and an online database (http://www.genego.com). Gene Ontology analysis revealed that the majority of these proteins were localized in intermediate and keratin filaments, as well as the cytoskeleton. These proteins acted as peroxidases, oxidoreductases, acceptors of peroxides or carbohydrate binding proteins, and participated in the process of cytoskeleton remodeling, development, impaired lipoxin A4 signaling and stimulating transforming growth factor beta (TGFβ) signaling. Regarding disease pathology, these proteins were largely involved in the defense and inflammatory response and the response to oxidative stress and wound healing (Fig. 1). Biomarker Assessment analysis based on the Disease Ontology database identified myeloperoxidase (MPO) and TGFβ1 as potential biomarkers for asthma and chronic obstructive pulmonary disease (COPD), keratin type II cytoskeletal 6A (KRT6A) as a biomarker for asthma and retinol-binding protein 4 (RBP4) for COPD (Fig. 1). The results of the present study suggested that these proteins, which were potentially targeted by the prescription for abnormal Savda, were most likely associated with dysregulation of overall protein interaction and signaling network during the development and progression of asthma. Figure 1. Gene ontology of annotation of the differentially expressed proteins. This analysis included assessment of the function of 22 proteins as described in Table I. The components for biological processes, cellular components, and molecular function of differentially expressed proteins are presented according to the gene ontology database (http://www.genego.com/; version 6.16). As presented in Table I, 6 out of 22 proteins, namely PRDX2, CBP2, MPO, TGFβ1, S100 A7 and KRT6A, may be molecular targets of Uighur prescription for abnormal Savda. Thus, these proteins served as pivotal candidate biomarkers for abnormal Savda-type asthma. A potential interaction and regulation network is associated with these proteins regarding cellular signaling and gene expression (Fig. 2), which was distributed in the extracellular space, membrane and cytoplasm. These proteins interacted with diverse effectors, including endopeptidases, matrix metalloproteinases and phosphatases, and were primarily regulated by transcription factors of distinct downstream signaling pathways. Figure 2. Network profile of protein interactions associated with eight candidate proteins at the cellular and genetic levels by MetaCore. This network was generated using the shortest path algorithm to map interaction among different proteins. The arrowheads indicated the direction of the interaction. The color of the lines between nodes indicated activating (green), inhibiting (red) and unspecified (black) interactions. Verification of changes in candidate proteins by ELISA To verify the data from the proteomics and bioinformatics analysis, the plasma levels of the 6 candidate proteins were determined using whole blood (plasma) samples collected at baseline (before treatment) and after treatment by ELISA (Table II). The analysis demonstrated a significant upregulation of PRDX2 and CPB2, and downregulation of S100A7 and KRT6A in the plasma of patients in response to treatment (all P0.05). Due to the consistency between the results of iTRAQ proteomics for PRDX2, CPB2 and S100A7 (Table I) and the ELISA data (Table II), these proteins may be the potential targets of Uighur prescription for abnormal Savda. However, the ELISA results for the expression of KRT6A were inconsistent with those of iTRAQ proteomics, which indicated upregulation in response to the corresponding treatment. Table II. ELISA verification of candidate protein expression in response to the treatment of abnormal Savda in asthma patients with Uighur prescription. Table II. ELISA verification of candidate protein expression in response to the treatment of abnormal Savda in asthma patients with Uighur prescription. Plasma protein level (ng/ml)^a Protein Prior to treatment (mean ± SD) Following treatment (mean ± SD) Paired t-test P-value PRDX2 379.170±40.978 419.180±48.579 <0.001b CBP2 1,693.570±114.878 1,783.030±194.455 0.030^b MPO 253.900±46.917 273.606±46.263 0.065 TGFβ1 0.410±0.314 0.445±0.306 0.531 S100A7 1.747±0.115 1.686±0.111 0.026b KRT6A 0.592±0.128 0.557±0.131 0.027^b a Samples from 40 patients b P<0.05 indicating a statistically significant difference. CBP2, carboxypeptidase B2; MPO, myeloperoxidase; TGFβ1, transforming growth factor β1; S100A7, protein S100 A7; KRT6A, keratin type II cytoskeletal 6A; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation. Discussion Recent proteomics studies of asthma have made significant progress in the identification of biomarkers and drug targets for asthma (10,12,13). Proteomics analysis of bronchial epithelium of asthma patients who were prescribed with budesonide (glucocorticoids) identified a number of differentially expressed proteins, including fibronectin 1, secretoglobin 1, KRT6A, interleukin enhancer-binding factor 3, dihydropyrimidinase like 5, cofilin 1, enolase 1 and vimentin as potential drug targets (16). Plasma-based proteomics revealed that heat-shock protein 70 and eotaxin were upregulated whereas vitamin D binding protein 3 was downregulated in patients diagnosed with asthma. These alterations were reversed following treatment with glucocorticoids (19). In a mouse model of acute-phase asthma, serum-based proteomics identified that immunomodulatory proteins were differentially expressed following glucocorticoid therapy (20). In addition, proteomics analysis determined that expression of 78 kDa glucose-regulated protein (GRP78) was upregulated in the lungs of mice with acute-phase asthma, and bronchial perfusion of anti-GRP78 small interfering RNA may modulate the inflammatory response induced by eosinocytes and bronchial hyper-responsiveness (21). Furthermore, increased expression of glycoprotein 39 and intercellular adhesion molecule 1 was detected in bronchoalveolar lavage fluid and lung tissues from asthma models of mice and monkeys, which was reversed following glucocorticoid treatment (22). Although different drugs may have distinct molecular targets, the progression or clinical therapy of asthma may be causally associated with overall changes in the expression of proteins throughout the whole body, which may be integrated into the blood plasma. Therefore, it is necessary to investigate the entire regulatory network of protein expression associated with abnormal Savda, which is potentially targeted by Uighur prescription of abnormal Savda through a holistic concept that is shared by both systems biology and Uighur medicine. In the present study, a therapeutic evaluation model was established and was used to evaluate abnormal Savda in patients with asthma receiving treatment with unique Uighur prescription. In total, proteomics identified 22 differentially expressed proteins in response to the corresponding treatment. Bioinformatics analysis demonstrated that a majority of these proteins were localized in intermediate filaments and the cytoskeleton, indicating that these proteins may act as antioxidant enzymes and binding proteins, and be crucial in the defense and inflammatory response, and the response to oxidative stress or wound healing. A database search identified that MPO, TGFβ1, KRT6A and RBP4 acted as biomarkers for asthma or COPD, as reported previously (19). There was a discrepancy between the iTRAQ and ELISA data collected in the present study, therefore the association of these four proteins with the effect of the prescription was not fully confirmed in the current study. The majority of participants in the current study presented with no severe side effects or adverse events following treatment with Uighur prescription. A total of 4 patients suffered from a slight degree of diarrhea, which may be controlled. Our group plans on analyzing adverse events that occur following administration of Munziq and Mushil of abnormal Savda in patients with asthma. It has been demonstrated that abnormal Savda asthma can induce metabolic disorder, and disrupt gluconeogenesis and host immunity (23). However, abnormal Savda Munziq is capable of counteracting abnormal metabolism and is important in upregulating gluconeogenesis and immune disorders (24). The present study aimed to identify a novel medication target of applying the Munziq and Mushil in treating abnormal Savda asthma from the perspective of protein genomics. The consistency between the iTRAQ and ELISA data for PRDX2, CPB2 and S100A7 indicated that the expression of these proteins may be associated with abnormal Savda in asthma patients and that these proteins may be targeted by Uighur prescription. Of these biomarkers, PRDX2 is one of the non-selenium-dependent peroxidases with anti-oxidative activities and serves a potential role in the modulation of oxidative stress-related signaling and disease processes (25). PRDX2 may participate in a variety of cellular signaling pathways, and may reduce hyper-responsiveness during allergic airway inflammation by eliminating cellular reactive oxygen species (26). CPB2 is a zinc-containing carboxypeptidase that is involved in regulating the coagulation-fibrinolysis balance in a variety of diseases, including cancer and cardiovascular disease (27). It has been demonstrated that CPB2 regulates thrombin-mediated tissue inflammation and other inflammatory responses by inactivating a variety of active inflammatory mediators, including bradykinin, C3a, C5a and thrombin-cleaved osteopontin (28). S100A7/psoriasin is expressed in the airway epithelium, lung epithelial cells and macrophages (29). S100A7 is induced by oxidative stress and is involved in a variety of inflammatory diseases (30). The expression of S100A7/psoriasin is increased in the nasal lavage fluid of allergic patients and the S100A7 gene polymorphism is associated with allergic rhinitis (31). In addition, S100A7 may be important in the development of breast cancer by increasing inflammatory cell infiltration, stimulating the pro-inflammatory response, and promoting oxidative stress and angiogenesis (32,33). These observations are in accordance with the results of the present study, which suggest that treatment of abnormal Savda in asthma patients with Uighur prescription upregulates the expression of PRDX2 and CPB2 and thus the anti-oxidative and anti-inflammatory response capacity of the body, whereas it downregulates the expression of S100A7, which may reduce oxidative stress and inflammatory responses. In conclusion, the results of the current study indicate that the therapeutic effect of Uighur prescription for abnormal Savda in patients with asthma is achieved by predominantly targeting the entire regulatory network of protein expression, particularly of those proteins involved in responses to inflammation and oxidative stress. 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Exosomes have gained increased research focus due to their key roles as messengers. The components of exosomes include proteins and RNAs that may be horizontally transferred between adjacent or distant cells. Hepatocellular carcinoma (HCC) is among the most malignant types of cancer worldwide, with exosomes implicated to play a crucial role in its regulation; however, the possible function of exosomes in modulating the motile ability of tumor cells and key molecules in HCC remain largely unknown. To investigate the regulatory effect of exosomes on the motile ability of HCC cells, exosomes from the culture medium of different HCC origins (high metastatic MHCC97‑H and low metastatic MHCC97‑L cells) were isolated for in vitro migration and invasion assays. The results indicated that the motile ability of MHCC97‑L cells was significantly increased by pretreatment with MHCC97‑H‑derived exosomes when compared with MHCC97‑L‑exosome pretreatment (P Introduction Exosomes are small endosome-derived vesicles that range between 30 and 100 nm in size, and are actively secreted through the exocytosis pathway (1). The major roles of exosomes are intercellular cross talk and receptor discharge (1–3), and they are typically released from high viability cells, including cancer cells (1). Previous studies have indicated that exosomes are capable of modulating intercellular communication and tumor progression through the transfer of proteins and RNA to adjacent and distant cells (1,4,5). Additionally, the functions of exosomes may vary depending on cell type and intracellular contents (2). In cancer, exosomes are considered to serve essential roles in tumor metastasis by regulating complex interactions between tumor cells and their microenvironment (6,7). However, the regulatory mechanisms of exosomes in tumor metastasis remain to be elucidated. As exosomes are carriers of multiple proteins and RNA molecules, the molecules contained within exosomes may themselves serve key roles in cell-cell communication (1). Thus, proteomics profiling and sequencing are promising platforms for systematically studying exosome components, which may ultimately improve understanding of exosome function. At present, hepatocellular carcinoma (HCC) is a fatal primary malignancy of hepatocytes (8). Emerging diagnostic tools and novel therapeutic strategies for HCC have substantially improved the clinical outcomes; however, the long-term survival of patients with HCC remains relatively poor due to the high possibility of metastasis and/or recurrence (9). Whether a tumor is likely to undergo local or distant metastasis is principally determined by the metastatic potential of tumor cells and the corresponding microenvironment (10,11). As a major component of the cellular microenvironment, exosomes secreted by different tumor cell types are capable of inducing apoptosis of activated T cells by promoting the expression of cell death ligands (12–14), inhibiting natural killer cell functions (15,16), and promoting the generation of suppressor cells derived from myeloid precursors (13). Additionally, various signaling pathways and genes are involved in the communication between tumor cells and their microenvironment (17). For example, a previous study demonstrated that tumor-activated hepatocytes were capable of altering the expression profiles of colon cancer cells in order to support hepatic metastasis (18). However, despite progress in research regarding the role of exosomes in cell communication, the mechanism by which exosomes alter the metastatic potentials of different cell types, particularly liver cancer cells, still requires further investigation. To investigate whether exosomes may alter the metastatic potential of cancer cells, the present study used two HCC cell lines with high and low metastatic potential, MHCC97-H and MHCC97-L, for exosome isolation and characterization. To evaluate the regulatory effect of exosomes on the mobility of HCC cells, exosomes from the culture medium of different HCC origins were isolated for in vitro migration and invasion assays. Additionally, protein profiling was performed on the exosomes from different origins to systematically characterize the content of the exosomes, in order to investigate the regulatory role of exosomes at the molecular level. Materials and methods Cell lines and cell culture The in-house preserved MHCC97-H and MHCC97-L cell lines were provided by The Second Military Medical University of China (Shanghai, China). All cells were cultured in Dulbecco's modified Eagle medium (DMEM; cat no. C11995500BT; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% FBS (cat no. 10100-147-FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a 5% CO2 incubator for 24 h. Exosome purification Exosomes were isolated using a total exosome isolation kit (cat no. 4478359; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. In brief, 5×106 cells were seeded in a volume of 15 ml culture medium at 37°C for 24 h, prior to harvesting the cell culture medium. The culture medium was centrifuged at 2,000 × g at 4°C for 30 min to remove cells and debris. Subsequently, the supernatant containing the cell-free culture medium was transferred to a new tube, and then 15 ml of cell-free culture medium was mixed with 7.5 ml of total exosome isolation reagent. The culture medium/reagent was mixed by vortexing until homogenous, and the samples were incubated at 4°C overnight. Following incubation, the samples were centrifuged at 10,000 × g for 1 h at 4°C. The supernatant was aspirated and discarded, and the pelleted exosomes were resuspended in 1X phosphate-buffered saline (PBS). The exosomes were then washed with 1X PBS, ultra-filtrated with a molecular weight cut-off (MWCO) of 100,000 Da, and finally dissolved in 1X PBS. Transmission electron microscopy (TEM) Exosomes isolated from MHCC97-H and MHCC97-L cells were identified for morphology by transmission electron microscopy (TEM) as previously described (19). In brief, exosomes were transferred to a copper grid coated with 0.125% Formvar in chloroform immediately after isolation. Then the grids were stained with 1% (v/v) uranyl acetate in double-distilled water right before examination. A Hitachi 7100 transmission electron microscope was applied for imaging. Evaluation of HCC cell motile ability following exosome incubation MHCC97-H and MHCC97-L cells were freshly cultured in DMEM supplemented with 10% FBS and incubated with 5% CO2 in air at 37°C for 24 h. Exosomes were isolated from high metastatic MHCC97-H and low metastatic MHCC97-L cells as described above. A total of 10 µg pelleted exosomes from each of the MHCC97-H and MHCC97-L cell lines were individually resuspended in 1 ml culture medium. The MHCC97-L cells were mixed with the MHCC97-H- or MHCC97-L-derived exosomes, and the cells were cultured with 5% CO2 in air at 37°C for 6 h prior to migration and invasion assays. Migration and invasion assay Cell migration was evaluated with a Transwell migration assay, while the invasion assays were performed using the Transwell units (Corning Incorporated, Corning, NY, USA) coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocols. A total of 1×105 MHCC97-H and MHCC97-L cells, and MHCC97-L cells pretreated with exosomes, were seeded onto the upper chamber of the insert in serum-free DMEM (cat no. C11995500BT; Gibco; Thermo Fisher Scientific, Inc.). After 6 h of incubation at 37°C, the membrane of the insert was fixed in 100% methanol at room temperature for 20 min and stained with crystal violet at room temperature for 20 min. After washing twice with PBS, tumor cells on the upper surface of the filters were removed by wiping with cotton swabs. The number of migrated or invaded cells that had passed through the filter to the lower surface were counted under an inverted microscope (Axiovert A1; Zeiss GmbH, Jena, Germany) in ~30 fields of view at ×200 magnification. Mean values were determined from three independent experiments run in duplicate. Statistical analysis of in vitro data Data are presented as the mean ± standard deviation. Student's t test and analysis of variance (ANOVA) were used to determine whether the MHCC97-L and MHCC94-H groups were statistically significantly different in migratory and invasive ability. Following ANOVA results, Dunnett's test was used as a post hoc test. All data were analyzed with GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA) and P<0.05 was considered to indicate a statistically significant difference. Protein preparation and isobaric tags for relative and absolute quantitation (iTRAQ) For each sample, proteins were precipitated by ice-cold acetone, and subsequently centrifuged at 10,000 × g at 4°C for 15 min. A total of 200 µl lysis buffer containing 8 M urea, 2% SDS and 1X protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) was added to resuspend the precipitate. The protein concentration of the samples was determined with a bicinchoninic acid assay (Beijing Transgen Biotech Co., Ltd., Beijing, China) following the manufacturer's protocol. A total of 5 µl DTT (200 mM) was then added to the protein samples, and the samples were incubated at 55°C for 1 h, after which 10 µl iodoacetamide (500 mM) was added to each sample for 30 min in the dark at room temperature to alkylate the proteins. For each sample, the proteins were ultra-filtrated (MWCO, 10,000 Da) and dissolved in 100 µl triethylammonium bicarbonate (100 mM). The proteins were then digested with sequence-grade modified trypsin (Promega Corporation, Madison, WI, USA), and the resultant peptide mixture was labeled using an iTRAQ reagent kit (Shanghai AB SCIEX, Analytical Instrument Trading Co., Shanghai, China). The peptides were labeled with iTRAQ 8-plex reagent as follows: MHCC97-H and MHCC97-L were labeled with 113 and 114 isobaric tags, respectively; while the peptides from biological repetitions were labeled with 115 and 117, respectively (MHCC97-H), or 116 and 118, respectively (MHCC97-L). Equal amounts of labeled samples (100 µg) were then desalted with the Sep-Pak Vac C18 cartridges and dried in a vacuum centrifuge at 4°C for 2 h. High pH reverse-phase separation A total of 400 µg peptide mixture was dissolved in solution A (5% acetonitrile and 0.1% formic acid in water; pH adjusted to 10.0 with ammonium hydroxide), and then fractionated by high pH separation using an Agilent 1260 Infinity System (Agilent Technologies GmbH, Waldbronn, Germany) connected to a reverse phase column (Durashell C18, 5 µm, 4.6×250 mm; Bonna-Agela Technologies, Inc., Tianjin, China). High pH separation was performed using a linear gradient of solution B [0.1% formic acid in 90% acetonitrile (ACN); pH adjusted to 10.0 with ammonium hydroxide] from 2 to 40% over 60 min. The column flow rate was maintained at 700 µl/min and the column temperature was maintained at 45°C. Following separation, the column was re-equilibrated at the initial conditions for 15 min. A total of 40 fractions were collected, and any two fractions with the same time interval (including, 1 and 21, 2 and 22) were pooled to reduce the fraction numbers. In total, 20 fractions were obtained and dried in a vacuum concentrator at 4°C for 2 h. Low pH nano-liquid chromatography-mass spectrometry (nano-LC-MS)/MS analysis The fractions were resuspended with 80 µl solution C (0.1% formic acid in water), separated by nano-LC and analyzed by electrospray tandem mass spectrometry. The experiments were performed on a Nano LC1000 system (Thermo Fisher Scientific, Inc.) connected to a quadrupole-Orbitrap mass spectrometer (Q-Exactive Plus; Thermo Fisher Scientific, Inc.), equipped with an online nano-electrospray ion source. A total of 2 µl peptide sample was loaded onto the trap column (Thermo Fisher Scientific Inc., Acclaim PepMap C18, 100 µm × 2 cm) with a flow rate of 10 µl/min, and subsequently separated on the analytical column (Acclaim PepMap C18, 75 µm × 15 cm; Thermo Fisher Scientific, Inc.), with a linear gradient of solution D (0.1% formic acid in ACN) between 3 and 35%. The column flow rate was maintained at 300 nl/min, the column temperature was maintained at 40°C, the nebulizer pressure of ~15 MPa and an electrospray voltage of 2.8 kV at the inlet of the mass spectrometer was used. Following the nano-LC separation, the column was re-equilibrated at the initial conditions for 15 min. The Q-Exactive Plus mass spectrometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra (m/z 300–1,500) were acquired with a mass resolution of 70 K, followed by 10 sequential high-energy collisional dissociation MS/MS scans with a resolution of 17.5 K. In all cases, one microscan was recorded using a dynamic exclusion of 30 sec. Mass spectrometry data analysis The raw files from the Q-Exactive instrument were searched against the human database provided by the Universal Protein Resource (http://www.uniprot.org/uniprot, released on 10 April 2014, with 20,264 entries) using Proteome Discoverer (PD) 1.4 (Thermo Fisher Scientific, Inc.). The enzyme specificity of trypsin and a maximum of two missed cleavages were selected for protease digestion. PD was used with a parent ion tolerance of 10 parts per million and a fragment ion mass tolerance of 0.05 Da. Carbamidomethylation of cysteine, as well as iTRAQ modification of the peptide N-terminus and lysine residues, were set as a fixed modification; oxidation of methionine and iTRAQ 8-plex labeling of tyrosine were specified as variable modifications. A decoy database search strategy was adopted to estimate the false discovery rate (FDR) for peptide identification. Scaffold (version 4.3.2, Proteome Software, Inc., Portland, OR, USA) was used to validate the MS/MS based peptide and protein identifications. The proteins were assembled using the parsimony method and accepted if the peptide FDR was 99.0%. Proteins containing similar peptides that could not be distinguished based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Differentially expressed protein filtering and gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway analyses Proteins with expression fold change >2 and Student's t-tests, P<0.05 were filtered as differentially expressed proteins between exosomes isolated from MHCC97-H and MHCC97-L cells. GO and KEGG pathway analyses were conducted using the R packages GO.db (version 3.4.1), KEGG.db (version 3.23) and KEGGREST (version 1.16.1). The P-value threshold was set at 0.01 to filter significantly enriched biological processes and KEGG pathways. Identification of significantly altered subnetworks Subnetwork identification was conducted with a heat-diffusion model based on the HotNet2 algorithm. The expression profile change was used as a heat signal input. The networks used for this analysis were obtained from the Human Protein Reference Database (HPRD) (20), iRefIndex (21) and Multinet (22) as recommended by the algorithm authors. A subnetwork identification result with P<0.05 and minimum edge weight threshold δ≥0.0003 were selected for subsequent consensus subnetwork construction. The consensus subnetworks were derived by the following steps: Initially, a complete weighted graph combining all the subnetworks identified from each interaction network was generated. In the weighted graph, proteins served as vertices and the edge between any pair of proteins were weighted by the number of networks, in which HotNet2 reports them in the same subnetwork. Then, the consensus subnetworks were identified by i) initializing the consensus subnetworks with connected components with edge weights ≥2 (connected components were defined as core genes of the consensus subnetworks), and ii) extending the subnetworks by adding similar genes to a given subnetwork until all weight one edges ended in the consensus subnetworks. The construction of the consensus networks was performed using customized Python scripts (version 2.7.13, Python Software Foundation, Wilmington, DE, USA). Results Electron microscopy of isolated exosomes Exosomes were isolated from the culture media of MHCC97-H and MHCC97-L cells using a total exosome isolation kit. To verify that the isolated structures were exosomes, the isolates were examined by electron microscopy (Fig. 1). The electron images depicted rounded structures with a size range of 50–100 nm in diameter and a cup-shaped morphology, which confirmed the successful isolation of exosomes according to previously described exosome characteristics (5,23,24). Figure 1. Transmission electron microscopy images of exosomes released from MHCC97-H and MHCC97-L cells. Scale bar, 100 nm. Exosomes from different origins significantly alter the migratory and invasive abilities of cancer cells The HCC cell lines, MHCC97-H and MHCC97-L, were used in the present study to assess the potential tumor regulatory role of exosomes. MHCC97-H cells exhibited a higher motile ability compared with that of MHCC97-L cells when analyzed by in vitro migration and invasion assays (Fig. 2A). Statistical analyses confirmed that, the migratory and invasive capacity of MHCC97-H cells was ~1.5 times higher than that of the MHCC97-L cells (Fig. 2B; P<0.0001 and P<0.005), respectively, which is in accordance with a previous study (25). a-receptor interaction 11 5.38×10⁻⁸ hsa04610 Complement and coagulation 10 7.51×10⁻⁸ a ECM, extracellular matrix. Subnetwork identification based on a heat-diffusion-like model To elucidate the potential influences of differentially expressed proteins, a heat-diffusion-like model was constructed based on the HotNet2 algorithm (27). This model used the different expression profiles between exosomes from MHCC97-H and MHCC97-L cells as stimulated directed heat signals radiated from the corresponding protein to its interacting partner. Subsequently, diffusion was evaluated in the genome-scale interaction networks to identify significantly altered gene subnetworks, which revealed a combination of proteins across different pathways and complexes. The three interaction networks were extracted from the HPRD (20), iRefIndex (21) and Multinet (22). A single subnetwork was significantly altered in the exosomes of MHCC97-H cells when compared with MHCC97-L cells (P=0.0001), as depicted in Fig. 6. This subnetwork contained 38 proteins (Presented in Table III), of which the core components principally belonged to three different groups according to their interactions. The first group contained apolipoprotein E (APOE), low density lipoprotein receptor-related protein 1 (LRP1), talin 1 (TLN1), filamin A (FLNA), lamin A, β-actin (ACTB) and CAP1. These core components were indicated to directly interact and were thus associated. Furthermore, it has been reported that these proteins are associated with cell-cell interactions in cell migration indicating their relevance to cancer metastasis (28). Proteins with close interactions to the core components of the subnetwork (PYGL, G6PD, LUM and TKT) were associated with glucose metabolization, which likely provides the energy required for cell transformation and migration. CAP1, the expression of which was significantly altered between MHCC97-H and MHCC97-L exosomes, was a core component and placed centrally in the subnetwork, implicating it as a key linker gene. The other two highly interactive groups contained laminin subunit γ1, nidogen 1 and fibulin 1 (FBLN1), and actinin α1 (ACTN1) and glycogen phosphorylase B, respectively. Figure 6. Interactions between core proteins in the subnetwork. Interactions between proteins in the subnetwork determined from three different databases. Each type of protein is depicted as a different color node (blue represents lipoprotein-associated proteins; green represents cell microenvironment and extracellular matrix formation-associated proteins; red represents oncogene encoded proteins). CAP1, adenylyl cyclase-associated protein 1; ACTN1/4, actinin α1/4; PYGB, glycogen phosphorylase B; ACTB, β-actin; LMNA, lamin A; FLNA, filamin A; TLN1, talin 1; LRP1, low density lipoprotein receptor-related protein 1; APOB, apolipoprotein B; APOE, apolipoprotein E; LAMC, laminin subunit γ1; NID1, nidogen 1; FBLN1, fibulin 1; RAN, ras-related nuclear protein. Table III. Consensus subnetworks identified by HotNet2 algorithm. Table III. Consensus subnetworks identified by HotNet2 algorithm. Group Component Size 1 APOB, APOE, FLNA, LRP1, PYGL, TLN1, UBA1, FBLN1, LAMC1, NID1, ACTN1, ACTN4, PYGB, LMNA, ACTB, ANXA2, CAP1, WDR1, G6PD, LUM, PGK1, PPIA, TKT, MET, RAN 25 2 C1S, ITIH4, ITIH3, ALDOA, LDHB, MYH9, NRP2, NAMPT, PSMD3, UGDH, SDC4, MYL6, PLEC 13 All the core components were classified based on function, as the majority were either associated with lipoprotein activity (APOB, APOE and LRP1, depicted as blue nodes in Fig. 6), or with the cell microenvironment and extracellular matrix formation (including FLNA, TLN1, FBLN1, ACTN1/4 and ACTB, depicted as green nodes in Fig. 6). The other core components that did not belong to an interactive group were MET and ras-related nuclear protein (RAN; depicted as red nodes in Fig. 6), indicating that these genes exert a distinct influence in the subnetwork while remaining indirectly associated with the CAP1-centered core. MET and RAN are established cancer-associated genes, particularly regarding cancer development and migration (29,30). Thus, taken together these results indicated an altered fraction of genes that may serve crucial functions in cancer cell-derived exosomes. Discussion The interactions between cancer cells and their microenvironment are important in tumor development (16). In recent studies on HCC, it has been demonstrated that exosomes secreted by cancer cells serve as messengers in cell-cell communication by conveying molecular information between tumor and adjacent cells (31,32). However, while exosomes are a key component of the cellular microenvironment, whether they regulate the migration and invasion of HCC cells remains unresolved. Additionally, the molecular contents of exosomes derived from HCC cells remain largely unclear, and the regulatory role of exosomes regarding metastatic potential requires further elucidation. The present study has indicated that horizontal transfer of exosomes between cells of different origin may lead to changes in the receiver cell metastatic potential, which thus provides insight into how exosomes may be involved in cancer development. This alteration in metastatic ability is unlikely to result from a change in the direct expression of several individual proteins, but rather from changes in complex mechanisms consisting of multiple biological pathways (33). A number of proteins are enriched in exosomes, including membrane trafficking proteins (Rab proteins and Annexins), adhesion molecules (lactadherin) and signal transduction proteins (protein kinases) (1,26,34). Following validation of the regulatory role of exosomes regarding cell mobility, the present study conducted protein profiling of exosomes from different origins to systematically investigate the differentially expressed proteins within the exosomes. Among the differentially expressed proteins, CAP1 was revealed to be significantly upregulated within exosomes from high metastatic HCC cells. CAP1 has been demonstrated to be significantly overexpressed in human HCCs and correlated with HCC metastasis, and thus was suggested to be a potential independent prognostic factor in patients with liver cancer (35). Loss of CAP1 expression may lead to cell polarity defects and altered distributions of actin filaments and mRNA determinants during development (36). Meanwhile, a previous study demonstrated that CAP1 was overexpressed in pancreatic cancer, and indicated an involvement of CAP1 in aggressive pancreatic cancer cell behavior (37). The present study identified that CAP1 expression was relatively higher in the exosomes of MHCC97-H cells compared with those of MHCC97-L cells, which indicates a regulatory role of CAP1 in cell mobility alterations associated with secreted exosomes. However, a limitation of the present study is that RNA-sequencing screening was not performed to compare the genetic components within exosomes of different origins. It has previously been indicated that the exchange of genetic materials, including mRNA and microRNA, is an alternative way of exosome-mediated intercellular communication that may reprogram the recipient cells (5,38). Thus, future studies should focus on the potential regulatory functions of genetic materials within exosomes. Additionally, the level of CAP1 within exosomes and its association with the regulation of the motile ability of cancer cells requires further characterization. By constructing a heat-diffusion-like model, a fraction of genes were identified that was significantly altered between exosomes isolated from MHCC97-H and MHCC97-L cells. Genes within this group were divided into several classes. A major part of this subnetwork consisted of lipoprotein genes, which is consistent with the origin of exosomes, as cell-derived vesicles with membranous structure. These lipoproteins are frequently present in different classes of extracellular vesicles, including microvesicles (39), microparticles (40,41) and exosomes (42,43). Lipoproteins are key components of exosome structure and participate in exosome synthesis, transport and interactions with cells. It has also been reported that APOE and APOB were associated with hepatocyte-derived exosomes (44), while other proteins of this group (including CLIC1 and NRP2) have been associated with the cell microenvironment and extracellular structure (45,46), thus indicating that these proteins may serve key roles in the exosomal regulation of cell metastatic ability. Additionally, it has been reported that tumor cells may modulate the surrounding microenvironment to enhance their metastatic potential through the secretion of exosomes (47). In the present study, the altered expression of exosome lipoproteins may have resulted from differences in the status of the origin cell, as the cell lines possessed different motile abilities. Indeed, several of these proteins (including APOB and APOE) have previously been identified to be associated with cancer (48,49). FLNA, which crosslinks actin into dynamic extracellular networks and interacts with multiple binding proteins with different biological functions, has been associated with human cancer proliferation, migration and invasion (50), and thus is correlated with cancer development (51,52). Furthermore, TLN1, which encodes a cytoskeletal protein important in the assembly of actin filaments, is considered as a diagnostic and prognostic marker in human HCC (53,54). FBLN1, another component of the fibrillary extracellular matrix, has also been associated with cancer (55). As these cell microenvironment-associated proteins have been associated with cancer migration and metastasis, afforded by their biological functions, alterations in the expression of these proteins in exosomes may impact on the target cell micro environment, and ultimately cause a change in the motile ability of HCC cells, as observed in the present study. Indeed, the results of the present study were consistent with previous observations, indicating that exosomes may not only affect cells but also the surrounding environment (56). Also noteworthy was the identification of two established oncogenes (MET and RAN) in the differentially expressed subnetwork. The subnetwork structure indicated that these oncogenes were indirectly associated with the extracellular microenvironment and membrane structure, since they are highly interactive with each other (57). This result suggests that oncogenes may affect HCC cell status and migration by changing the extracellular environment, thus leading to a change in the motile ability of cells. MET, which is frequently identified as a key gene in the process of cancer cell migration, serves a role in epithelial-to-mesenchymal transition and contributes to the tumor microenvironment (58). Meanwhile, RAN is associated with the formation and organization of the microtubule network, and previous results have demonstrated that microtubule perturbation may regulate remodeling of the tumor microenvironment to alter cell migratory potential (57). The tumor cell secretome has also been implicated in this mechanism (59). The identification of MET and RAN oncogenes within HCC exosomes indicates that these genes may not only influence the origin cells, but also have an impact on adjacent cells through exosome transportation. Thus, the identification of these genes provides insight into the complex mechanisms of exosome-associated processes and pathways, particularly regarding cancer development and migration. Further studies of these genes may provide more novel perspectives on the association between exosomes and cancer. In conclusion, the results from the present study indicated that exosomes may alter the metastatic potential of cancer cells. To the best of our knowledge, the present study was the first to conduct protein profiling of exosomes from different HCC cell origins, which identified protein candidates associated with metastasis and recurrence. Collectively, these data may provide the foundation for further studies into the regulatory role of exosomes in cell-cell communication in HCC and other cancers. Acknowledgements The present study was supported by the National Natural Science Foundation of China (grant nos. 31201008 and 31400634), the Specialized Science and Technology Key Project of Fujian Province (grant no. 2013YZ0002-3), the Science and Technology Infrastructure Construction Program of Fujian Province (grant no. 2014Y2005), the Scientific Foundation of Fuzhou City (grant nos. 2015-S-143-1 and 2015-S-143-21), the Natural Science Foundation of Fujian Province (grant no. 2015-J-05173) and the Scientific Research Project of the Health and Family Planning Commission of Fujian province (grant no. 2015-1-96). References 1 Thery C, Zitvogel L and Amigorena S: Exosomes: Composition, biogenesis and function. Nat Rev Immunol. 2:569–579. 2002.PubMed/NCBI 2 Stoorvogel W, Kleijmeer MJ, Geuze HJ and Raposo G: The biogenesis and functions of exosomes. Traffic. 3:321–330. 2002. 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The Covid-19 pandemic has swept the world in fewer than 3 months, and there remains no end in sight. Approximately 6.1% of Covid-19 cases were classified as critical—defined as respiratory failure, shock, and multiple organ dysfunction or failure (1). Among the critically ill Covid-19 patients, ~6–47% of them were intubated in China (2–7), 71–75% were intubated in the United States (8, 9), and 88% were intubated in Italy (10). The sheer volume of patients who require invasive mechanical ventilation support entails that anesthesia professionals have been put under significant pressure during this pandemic. This pressure is exacerbated by the fact that many urgent and emergent surgeries must proceed, even in situations in which patients have confirmed or suspected Covid-19. Clearly, anesthesia providers are playing a fundamental role in the frontline efforts to fight against this formidable pandemic. This paper discusses the impact Covid-19 is having on contemporary anesthesia practice through different phases and highlights some of the lessons we can learn to inform future practice (Figure 1). Figure 1. Impact of the Covid-19 pandemic on anesthesia practice through different phases. Be it as a measure of precaution, resource-saving, better manpower allocation, or ensuring availability of hospital beds, many hospitals throughout the world have canceled or postponed elective and semi-elective surgeries amid the current pandemic. While the reduction in the volume of surgical procedures being performed varies across different hospitals, it can be as high as 70–90%. This move suddenly relieves most anesthesia providers from perioperative care, with only a small portion being deployed to provide anesthesia for urgent or emergent surgeries. At the same time, as a result of the rapidly expanding number of patients admitted to hospitals and intensive care units (ICUs), anesthesiologists are being mobilized and re-deployed to serve outside the perioperative setting. During this pandemic, anesthesia providers are typically being asked to provide the following services: (1) to intubate critically ill patients who require invasive mechanical ventilatory support; (2) to work in the ICU in the roles of intensivists, respiratory therapists, or nurses; (3) to place intra-arterial catheters and peripheral or central intravenous catheters; and (4) to work in the emergency departments or fever clinics to ensure the gaps in resources created by the sudden increase in symptomatic patients are filled (11). This is the overall global picture; however, the type and load of the work assigned to anesthesia providers outside the perioperative environment primarily depend on the number of cases encountered by individual hospitals and vary by country. Various issues that directly impact anesthesia providers have arisen in the midst of providing care to critically ill Covid-19 patients. These issues are related to self-protection, best practices of intubation and ventilation, and professional liability in delivering care to patients outside any specialist scope of practice. In mid-March 2020, an article was published documenting the intubation and ventilation experiences in one of the epicenters—Wuhan, China (11). In this paper, the authors described the personal protective equipment (PPE) used by the Chinese healthcare workers. Of note, when performing invasive procedures in Covid-19 patients, including intubation and ventilation, all healthcare workers in China were required to follow Level III protective measures. Put simply, this mandates coverage of the entire body (11). This practice has caused a wide-range discussion outside China. In comparison, in the United States, standard protective practice does not involve covering the neck or leg below the knee. Although we agree that neither under-protection nor over-protection are warranted, the most ideal approach to self-protection is unclear. We hope this information will come to light with future analyses of worldwide practice data. Regardless of what level of protection is most efficient, the shortage of PPE has caused some significant concerns. Especially at the early stage of the pandemic, there is a global shortage of almost every piece of PPE that is deemed necessary when performing invasive procedures in Covid-19 patients. Many medical practitioners are scrambling to identify methods of sterilizing and reusing N95 masks and/or making their own face shields. Reports of doctors and nurses using unconventional self-protection innovations, such as transparent plastic bags to cover the head and neck, have flooded social media and newspapers. The shortage of PPE and the difference in the availability of self-protection resources across different hospitals, regions, and countries have caused concern and confusion, and this has even resulted in some providers refraining from attending work (12). Moving forward, ensuring adequate PPE supply at all times with a robust production and supply chain capability is a priority. In regards to the best practice when intubating and ventilating Covid-19 patients, there is no universal agreement, but the experiences of different countries should be considered (11, 13–16). Most anesthesia providers typically perform the following steps during intubation: (1) maintain the oxygenation and ventilatory support that has already been used in the patient; (2) avoid bag-mask ventilation if possible; (3) use 100% oxygen for 5 min during pre-oxygenation; (4) cover the patient's nose, mouth, and face; (5) perform rapid sequence induction; (6) aim for complete muscle relaxation; (7) avoid coughing and bucking; (8) perform video laryngoscope guided intubation; and (9) avoid chest auscultation. When delivering ventilatory support, most providers adhere to the following processes. They should avoid non-invasive ventilation, including continuous positive airway pressure and bilevel positive airway pressure, if there are enough ventilators and manpower for invasive mechanical ventilation. This is supported by reports from Lombardy region, Italy, where 11% of cases received non-invasive ventilation and 88% invasive mechanical ventilation during the first 24 h of ICU admission;(10) and in California, United States, 4% of ICU cases received high-flow nasal cannula, 1% non-invasive ventilation, and 91% invasive mechanical ventilation (17). They should adopt lung-protective ventilation strategies; set an ideal oxygenation goal; deliver early prone position ventilation; ensure adequate sedation and analgesia; and provide muscle relaxation when needed (11, 18). Lastly, the best approach to extubation is equally important as it may generate infectious aerosols as a result of patient coughing, and agitation (11). Most anesthesia providers are not credentialled to work outside the perioperative environment, especially in the United States. Although it appears that the Covid-19 crisis is a scenario in which the Good Samaritan principle would apply, there is still a requirement to rapidly authorize anesthesia providers to care for patients in the ICUs, emergency departments, and clinics. Depending on the local policy and practice, credentialing committees should quickly facilitate the process to legally authorize anesthesia providers to deliver necessary services in settings outside the perioperative environment as appropriate. The Covid-19 pandemic presents some unprecedented challenges to anesthesiology departments. The environment is sporadic, chaotic, and unpredictable, with the situation changing daily, if not hourly, especially at the early stage of the pandemic. While every effort is made to ensure all practitioners are updated on the current status via timely communications, confusion and anxiety are commonplace. While it is understandable that almost all practitioners are witnessing a crisis of this severity for the first time in their lives, it is imperative that efforts are invested in streamlining the communication process so things proceed in the most smooth and effective fashion (19). Most anesthesiology departments have quickly established a task force that is specifically responsible for dealing with the Covid-19 crisis. Organized, centralized, clear, and timely communication is essential. The leader of this task force or the individual to whom the leader delegates responsibility needs be in charge of the departmental communication. The message needs be as clear and transparent as possible to avoid any confusion. Reports from front-line staff go to the task force, not the entire department, for collection, summary, and dissemination. Daily conference calls with clinical leadership serve to keep everyone informed and delivering a consistent message to their teams. Every effort needs be made to protect frontline providers (19). The anesthesiology department needs work aggressively with hospital partners to seek alternative sources of supplies when facing a shortage of critical PPE and medications. Counseling for mental health and wellbeing needs be provided to department members (20). Lodging can be considered for individuals who are particularly concerned about risks of contamination of their home environment. Departmental leaders are role models for the team members by offering courage, acting as a source of inspiration, and encouraging a spirit of caring for each other. As of mid-April 2020, the current pandemic appears starting to head into a transition phase, with the progress varying from country to country. The transition phase is characterized by a dramatic decrease, but not complete elimination, of cases and risks of infection. During this phase, regular work order is gradually resumed while continuing to care for varying numbers of Covid-19 patients. During the transition phase, it may be tempting to maximize the capacity of the operating rooms to address the cases that were postponed or rescheduled at the height of the pandemic. However, it is prudent to open the operating rooms more gradually for several reasons. First, the infection risk is lower but still lingers. Infection control requires time and energy and consumes resources. The need to ensure adequate protection and maintain control over the virus should be treated as a higher priority than maximizing caseload, given the potential for severe unintended consequences. Second, perioperative personnel, including anesthesia providers, have relearned and redesigned their approach to patient care to emphasize caution over throughput. Short of a vaccine that abolishes the future risk of Covid-19, there cannot be an immediate return to business as usual. Practitioners likely cannot achieve the necessary level of caution from an infection prevention standpoint, while achieving high throughput surgical volume, without neglecting other aspects of patient care and safety. Third, as we have learned now, some critically ill patients will continue to occupy the ICU beds, even weeks into the transition phase. Therefore, if a surgical case would typically require ICU admission after surgery, there will be a need to coordinate resource management with the hospital bed flow management team. Finally, the spread of the disease (hopefully through community spread and not at-work exposure/infection) will reduce the available workforce unpredictably. Contact tracing and temporary quarantine further reduce the numbers of available workers. Vigilance is needed as the risk of infection still exists during the transition phase. It has been suggested that all surgical patients undergo SARS-CoV-2 nucleic acid (typically a PCR test) and antibody tests and chest x-rays or CTs even if they are clinically asymptomatic. False negative rates are non-zero but poorly defined (2–29% estimated based on current data June 2020) (21). The preoperative preparation during the transition phase requires standardized approaches and policies. At a minimum, anesthesia providers need to be cautious during preoperative patient preparation. It is prudent to do the following: (1) wear a surgical mask and eye protection (goggles or face shield) when visiting, interviewing, and examining patients; (2) wash hands before and after each visit; (3) wear gloves when touching and examining patients; (4) consider avoiding chest auscultation if not clinically indicated; (4) be vigilant for the signs of infection; (5) follow up on pertinent labs; (6) follow up on chest x-ray or computed tomography results if ordered; (7) always remember to screen the patient for a history of Covid-19 and/or close contact with confirmed cases; and (8) consider testing for Covid-19 and the presence of an antibody response. Moreover, data regarding the protection provided by an immune response to prior Covid-19 infection and the duration of immunity are desperately needed. In the operating rooms, full self-protection including N95 masks or power air purifying respirators (PAPRs), goggles or face shields, and waterproof gowns needs be worn if the patient has confirmed or suspected Covid-19; otherwise, wearing a surgical mask with a face shield should be the minimum for patients without evidence of Covid-19. If a Covid-19 patient is undergoing surgery, the following recommendations are advised: 1) perform the surgery in a dedicated Covid-19 negative-pressure operating room; (2) follow the consensus PPE guidelines during intubation and ventilation; (3) ensure smooth emergence and extubation; (4) use filters that are capable of preventing virus transmission/contamination to the anesthesia machine; (5) try to use disposable supplies when possible; and (6) thoroughly clean/sterilize any non-disposable equipment after surgery. Even after the pandemic has been officially declared over, things will not go back to how they used to be (even if there is an effective vaccine). The impact of Covid-19 on anesthesia practice will be deeply embedded. As the adage goes: what does not kill us makes us stronger. The lessons that can be learned from this pandemic are summarized below. The most effective methods of protecting providers against virus transmission need to be identified (22–24). Different hospitals, regions, and countries have adopted different approaches. Evidence regarding the relationship between the various self-protection mandates that are available and the risk of cross-contamination is needed; neither under-protection nor over-protection is warranted. Different viruses have different behaviors, virulence, and modes of transmission; therefore, preparedness to adjust the approach to self-protection when confronting a novel virus and a new outbreak will be needed. A related issue concerns the adequacy of PPE supplies. Regular stockpile checking needs to be mandated. Methods of sterilizing and reusing different components of PPE need to be investigated and established. The supply chain needs to be bolstered, with contacts regularly maintained. All providers should be trained on the appropriate use of PPE, including the donning and doffing processes. The best practices regarding intubation and ventilation need to be elucidated. Although there is some consensus, most of the actions that have been taken thus far amid this pandemic are opinion-based. Evidence to support or revise these is needed. One example is the non-invasive ventilatory support in critically ill Covid-19 patients. Bilevel positive airway pressure ventilation support was popularly used in the epicenter in Wuhan, China (11). Continuous positive airway pressure ventilation support has been used in the United Kingdom (25). However, non-invasive ventilation support has not been widely recommended for use in both Italy and the United States (10, 17). The three primary factors that determine which one to choose are clinical effectiveness, risk of cross-contamination, and the availability of resources. Clearly, the best practices concerning care for critically ill patients in situations like this pandemic need to be further investigated and discussed. The scopes of the clinical skills that future anesthesia providers should possess need to be clarified. This crisis has taught us that, during pandemics of this nature, anesthetists are not only needed for surgical procedures and airway management but also for work in the ICUs, emergency departments, and clinics. It is plausible to quickly teach practitioners immediately before and during the required activities; however, it would be better if the potential need in any future situation similar to this Covid-19 pandemic is anticipated and our providers are proactively trained so that they possess the skills they may need in an emergency situation. The good news is that the skills required outside the perioperative environment (e.g., ventilatory support) are not something unfamiliar to anesthesia providers, as critical care training is a component of anesthesiology residency in most countries. Therefore, regularly updating knowledge and practicing essential skills can be sufficient to ensure preparedness. A mechanism is needed to rally the team when situations similar to this pandemic occur again. This mechanism includes the ability to quickly assemble a task force, identify the available resources, establish a channel for efficient and clear communication, allocate jobs based on the strength and talent of individual team members, deliver counseling to ensure mental health and well-being, and closely collaborate with colleagues from other departments. The goal of this mechanism is to help practitioners efficiently join forces during the fight against a hidden enemy. The success in this great fight against SARS-CoV-2 resides in the resilience of all professionals related to the care for the Covid-19 patients, including nurses and physicians at different levels of training and practices and across different specialties. As we applaud this unprecedented all-out effort, we should also plan further team building to better prepare for any future outbreaks or pandemics. In a crisis like the Covid-19 pandemic, the traditional conduct of education and research are not permissible due to concerns surrounding virus transmission. Many trainees and research personnel have to stay at home for weeks. Instead of staying passive, anesthesia providers should use this period of time to effectively enhance education and research. Doing so also promotes a feeling of enrichment and satisfaction, which is a positive way of promoting well-being. The widely available remote conferencing platforms revolutionize how people are connected with each other in the modern era, making virtual academic activities possible. Contemporary technologies also allow people to gather online, see each other, talk to each other, reconnect with each other, help each other, exchange information, and move forward together as a team. Finally, the coming months during which people are awaiting for a Covid-19 vaccine will hopefully see a true tipping point in the transition to distance learning, expansion of telemedicine, and remote conferencing that will replace destination continuous medical education, non-essential face-to-face patient encounters, and convention center society meetings. The impact of the Covid-19 pandemic on anesthesia practice varies dynamically with the various phases of the pandemic. As we respond, recuperate, and move forward from the Covid-19 pandemic, the impact on anesthesia practice and the lessons learned should be summarized and addressed to ensure better preparedness and results in the future. The areas in which improvements are needed center on self-protection, best practices, scope of practice, organized response, and remote education, research, and gathering. Preparedness may use certain resources and cause financial concern, especially when a crisis is not observed for many years. Therefore, it would be wise to use the process of preparedness to promote a higher quality of patient care, education, research, and culture building. Simulation and quality assurance activities will facilitate “maintenance of preparedness.” Vigilance is the motto of the North American anesthesiology community, and it appears to be more appropriate now than ever. LM helped with the concept and design, administrative and material support, data interpretation, manuscript drafting, and critical revision of the manuscript for important intellectual content. DM helped with the data interpretation and critical revision of the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version. This work was supported by institutional and departmental sources with which the authors are affiliated. 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Med. 7:452. doi: 10.3389/fmed.2020.00452 Received: 07 May 2020; Accepted: 08 July 2020; Published: 21 July 2020. Edited by: Reviewed by: Copyright © 2020 Meng and McDonagh. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Lingzhong Meng, [email protected]