Slc17a6–8. VGLUT1 (Slc17a7) and VGLUT2 (Slc17a6) are expressed in glutamatergic neurons, while VGLUT3 (Slc17a8) is expressed in neurons classically defined by their use of another transmitter, such as acetylcholine and serotonin. As glutamate is both a ubiquitous amino acid and the most abundant neurotransmitter in the adult central nervous system, the discovery of the VGLUTs made it possible for the first time to identify and specifically target glutamatergic neurons. By molecular cloning techniques, different VGLUT isoforms have been genetically targeted in mice, creating models with alterations in their glutamatergic signalling. Glutamate signalling is essential for life, and its excitatory function is involved in almost every neuronal circuit. The importance of glutamatergic signalling was very obvious when studying full knockout models of both VGLUT1 and VGLUT2, none of which were compatible with normal life. While VGLUT1 full knockout mice die after weaning, VGLUT2 full knockout mice die immediately after birth. Many neurological diseases have been associated with altered glutamatergic signalling in different brain regions, which is why conditional knockout mice with abolished VGLUT-mediated signalling only in specific circuits may prove helpful in understanding molecular mechanisms behind such pathologies. We review the recent studies in which mouse genetics have been used to characterize the functional role of VGLUT2 in the central nervous system.During the past decade, three proteins that possess the capability of packaging glutamate into presynaptic vesicles have been identified and characterized. These three vesicular glutamate transporters, VGLUT1–3, are encoded by solute carrier genes  Slc17a6–8, act as vesicular glutamate transporters . Their discovery has enabled proper identification of glutamate signalling neurons and studies of glutamatergic neurotransmission   ,10. The ections) .in vivo. For example, the switch from VGLUT2 to VGLUT1 expression was observed in vivo by both Fremeau et al. and Wojcik et al. in 2004, when they genetically deleted VGLUT1 expression in mice using full knockout strategies  and conditional gene targeting  have been utilized. We describe these techniques briefly in et al. produced a mouse model where VGLUT2-dependent glutamate signalling was inhibited by a complete targeted deletion of the Vglut2 gene  showed some residual VGLUT2 mRNA expression. Interestingly, this complete knockout of VGLUT2 was not compensated for by an increase in VGLUT1 levels, also shown by Western blot analysis. As the distribution of VGLUT1 and 2 differs between brain regions, the authors performed electrophysiological studies on cells isolated both from the hippocampus (with high VGLUT1 expression) and the thalamus (with high VGLUT2 expression) from wild-type and Vglut2-/- mice respectively. These studies revealed a 95% reduction in evoked glutamate response in thalamic neurons from Vglut2-/- mice, while hippocampal neurons functioned normally. Since the homozygotes were not viable, behavioural analyses were performed on mice heterozygous for the VGLUT2 deletion (Vglut2+/-). These studies are described in the section for heterozygote studies below.In 2006, the first two studies of VGLUT2 gene targeting in the mouse were published. Our group, as well as that of Moechars and co-workers, both made full knockout studies, although by using different gene targeting strategies. Moechars ut2 gene . This deflox/floxVglut2 mice by insertion of one LoxP site upstream of exon 4 of the mouse Vglut2 genomic sequence and one LoxP site downstream of exon 6. Full knockout mice were obtained by mating of the flox/floxVglut2 mice with phosphoglycerate kinase I promoter (PGK)-Cre mice , wild-type and heterozygote siblings immediately initiated breathing and showed a stable breathing behaviour. In contrast, no respiratory behaviour was observed in the full knockout embryos.In our case, the loss of VGLUT2 was achieved through a conditional approach using the Cre/LoxP system . We geneCre mice . None offlox/flox;PCreVglut2 mice, we were interested in further investigating the respiratory CPG and in particular the pre-Bötzinger complex (PBC) which is a network pacing the respiratory rhythm. The PBC is located in the rostroventrolateral medulla, and its neurons are dependent on activation of glutamate receptors (N-methyl D-asperate (NMDA) and non-NMDA) to depolarize. By the targeted deletion of VGLUT2, we abolished the presynaptic release of glutamate and could show that this release was crucial to initiate the respiratory-related rhythm generation of the PBC. When fetuses were placed in a plethysmographic chamber to record ventilation, none of the flox/flox;PCreVglut2 showed any breathing behaviour, consistent with their malformed, defective lungs, while wild-type mice breathed at a normal frequency. To resolve the mechanisms behind this failed breathing initiation, we performed electrophysiology studies of brainstems from surgically delivered embryos. In contrast to wild-type preparations, recordings on brainstems from mutants showed no spontaneous rhythmic activity of the hypoglossal nerve root (12n). To investigate rhythm generation from the PBC, medullar slices were prepared and analysed through calcium imaging and extracellular electrophysiological recordings. For wild-type slices from E16.5 embryos, a spontaneous rhythmic activity was observed as changes in calcium-induced fluorescence, which as previously observed by others  belongs to the better characterized CPGs, although before the production of VGLUT2 knockout mice it had been somewhat difficult to pin-point the role of glutamate signalling in this circuit. Since no respiratory behaviour was observed in the y others  could bey VGLUT2 –25; howeflox/floxVglut2 mice by flanking exon 2 of the mouse Vglut2 genomic sequence with LoxP sites, which resulted in a null allele as exon 1 only contains about 5% of the coding sequence of Vglut2 and as the reading frame is disrupted. The authors were interested in evaluating the role of VGLUT2 in ventromedial hypothalamic (VMH) neurons, which are predominantly glutamatergic and previously were shown to express VGLUT2 neurons and could show two findings: 1) most of the SF-1 neurons are glutamatergic while a smaller proportion is GABAergic, and 2) glutamate release is disrupted in their flox/flox;SF1-CreVglut2 mice (no excitatory postsynaptic currents (EPSCs) were detected in the knockout neurons). Having confirmed this, the authors analysed features related to feeding and blood glucose levels. Tong et al. found that the knockout mice had normal body-weight when fed a standard chow diet but developed some increased body-weight compared to controls when fed a high-fat, high-sucrose diet. The authors concluded that release of glutamate from the SF-1 neurons plays a small role in regulating energy balance upon fat-feeding but not in standard feeding.The brain contains two kinds of glucose-sensing neurons: ‘glucose-excited’ neurons that increase their firing rate as glucose levels rise, and ‘glucose-inhibited’ neurons that decrease their firing as glucose levels fall. The decreased activity of the ‘glucose-excited’ neurons, the increased activity of the ‘glucose-inhibited’ neurons action, input from peripheral blood glucose sensors, as well as a combination of the three, are suggested as the means by which the brain detects low glucose levels. The glucose-sensing neurons are abundant in parts of the hypothalamus, including the VMH, the lateral hypothalamus, and the arcuate nucleus, and in the hindbrain. The presence of glucose-sensing neurons implicates the VMH in the neurocircuitry behind detection of hypoglycaemia and the adequate counterresponses. Tong and collaborators set out to test directly the hypothesis that the glutamatergic neurons of the VMH play a role in this neurocircuitry by inactivating VGLUT2 specifically in these neurons. For this, they crossed their SF-1-Cre transgenic mice , which eThe mice were then subjected to fasting for 24 hours, whereupon blood glucose, insulin, and glucagon were measured. The findings were quite striking. While insulin levels were similar in the knockout and control groups, blood glucose and glucagon levels were lower in the knockouts. The pancreatic hormone glucagon works on the liver and stimulates break-down of glycogen to glucose (glycogenolysis) as well as synthesis of glucose from other sources (gluconeogenesis). Gluconeogenesis can be detected in liver-derived mRNA as it leads to transcriptional regulation of gluconeogenic enzymes and a co-activator, PGC-1α. While these liver-expressed genes were transcriptionally increased in the controls, no increased gene expression was observed in the knockouts. These experiments showed that the impaired glucose homeostasis in the knockout mice was mediated by failure to increase blood glucagon levels which was followed by failure to induce expression of gluconeogenic genes in the liver.flox/flox;SF1-CreVglut2 mice suffered a more substantial fall in blood glucose than did the controls, a greater degree of hypoglycaemia that was followed by impaired glucagon response. The authors then performed hypoglycaemic clamp studies, where hypoglycaemia was induced by infusion of human regular insulin and sustained at similar levels in a controlled way for 60–90 minutes during which samples for epinephrine and glucagon were taken. This analysis showed that the knockout mice required higher rates of glucose infusion to keep the clamp, a finding consistent with decreased rates of glucagon production, which was also detected. In addition, a hypoglycaemia-induced increase in epinephrine observed in the controls was not detected in the knockout mice. The authors then treated the mice with 2-deoxyglucose (2-DG) by injection into the third ventricle, which in the controls led to brain-mediated increased blood glucose levels, plasma glucagon levels, and increased gene expression of gluconeogenic genes in the liver. None of these responses were observed in the flox/flox;SF1-CreVglut2 mice. Together, these experiments showed that the counterregulatory responses to hypoglycaemia are deficient in the flox/flox;SF1-CreVglut2 mice, and due to the very selective targeting event of Vglut2 in this mouse model the authors were able to confidently suggest that the effects are mediated via loss of glutamate release in the VMH.An acute injection of insulin revealed that the flox/floxVglut2 mice. The CamKII-Cre mouse has been carefully analysed and the Cre recombinase has been shown to be expressed in the cortex, hippocampus, striatum, and amygdala  and found an increased frequency and duration in the open arm, especially in the outer part of this arm, in the knockouts compared to the controls. In addition to suggesting a decreased anxiety-related behaviour, analysis of the total frequency in the different compartments showed that the knockout mice were significantly more active than the controls. We then turned to the more sensitive Multi-Concentric Square Field (MCSF)  , develop-/-Vglut2 mice are models of completely absent VGLUT2-mediated glutamate signalling, either in the whole brain or in specific neuronal circuits. There is evidence that alterations in the number of VGLUT2 molecules also affect the glutamate signalling properties  and chronic constriction injury (CCI). After surgery, mice were tested using the von Frey set-up to assess mechanical sensitivity and the writhing test with acetone exposure to assay for chemical nociception. All mice showed a lowered threshold in von Frey's as a response to the mechanical allodynia caused by the SNI, except the +/-Vglut2 mice which did not respond to the injury. The same was observed after acetone exposure, where +/-Vglut2 mice failed to develop a cold allodynia-like response to injury. For the CCI test of mechanical allodynia, +/-Vglut2 mice did not differ from controls in von Frey's test but did respond to acetone spray, however to a lesser extent than did wild-type mice. Thus, +/-Vglut2 mice fail to display typical signs of neuropathic pain. Both these studies, where different aspects of pain were investigated, converge on VGLUT2-dependent signalling—and not VGLUT1—as being an important player in mediating neuropathic pain responses, a finding of importance not least for drug development in the pain area.The same model was further studied by the same group, and it was compared to heterozygous analyses . Vglut1++/-Vglut2 mice . The neurons of RTN then send inhibitory GABAergic signals back to the thalamocortical neurons. These neurons are also intensively activated by PTZ  in tibialis anterior. In control mice, almost all NMJs were innervated by motor neurons, an innervation that was markedly reduced in the G93ASod1;flox/+Vglut2 mouse. Thus, in the G93ASod1;flox/+Vglut2 model, parts of the reduced NMJ innervation could be rescued. This indicated to us that the rescued motor neuron cell-bodies in the spinal cord were in fact connected to the muscle. Interestingly, cholinergic motor neurons in two cranial nuclei (VII and XII) of the brainstem responded differently to reduced VGLUT2. In the VIIth cranial nucleus, we observed a complete rescue of motor neurons by reducing VGLUT2 levels, while the XIIth cranial nucleus was completely unaffected by the disease and no cell loss was observed.Glutamate is the most abundant excitatory neurotransmitter, and its presence in the extracellular space must be tightly regulated as excessive release of glutamate into the synaptic cleft can induce excitotoxicity and death of the postsynaptic neuron. This excitotoxicity has been suggested to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS), a disease where motor neurons gradually die leading to paralysis of the patient. The only approved drug to treat ALS reduces excitotoxicity by reducing glutamatergic signalling . A decregnalling . Sod1G93flox/+Vglut2 mouse model we were able to evaluate a putative role for VGLUT2 in ALS. This study shows that glutamate excitotoxicity is part of the ALS disease progression and that different motor neuron populations have different susceptibility for this toxicity. Hence, decreased glutamate signalling could be beneficial for motor neuron survival in ALS.By creating this heterozygotic ex utero, as new-born pups die immediately after birth due to dysfunctional respiratory CPG. Using the conditional approach, VGLUT2 has been linked to normal function of the hypothalamic regulation of feeding and maybe somewhat more surprisingly, given the restricted distribution of VGLUT2 in the cerebral cortex, to higher brain function. These two studies, in which the Cre/LoxP system was used, have given novel insights into mechanisms that may be useful for the understanding of human pathologies, in these cases diabetes and schizophrenia-like behaviour. Also heterozygous VGLUT2 mice have been analysed with the purpose of addressing human conditions (both pain and epilepsy have been addressed), and it has been shown that VGLUT2 does play a role in mediating these conditions. Further analyses will reveal more about the mechanisms behind these findings. In a recent study, a connection between VGLUT2 gene expression and ALS was shown, which specified a role for VGLUT2 in motor neuron survival.During the past few years, several laboratories have used gene-targeting techniques in mice to study the functional roles of all three VGLUTs, i.e. VGLUT1–3. Our group has, together with others around the world, focused on VGLUT2, the predominant isoform during embryonic development and in deep brain structures, including the thalamus, in the adult. Full knockout analyses revealed that VGLUT2 is essential for life The research field of mouse genetics of the VGLUTs is fairly new, and we expect that the next few years will see a rapid development in this field, which will certainly give new insights into the role of the different VGLUTs in glutamatergic neurotransmission in the central nervous system.


Various combinations of vectors were studied. The results indicate that the vaccine induces a dose-dependent expression of the HIV-1 transgenes in vitro. Furthermore, the gag, pol, and nef transgenes are expressed differentially in A549 cells in an MOI-dependent and formulation-dependent manner, yielding an unexpected enhancement of protein expression in trivalent vs. monovalent formulations. Our data suggest that the presence of additional virus in multivalent formulations increases individual transgene expression in A549 cells, even when the amount of DNA encoding the gene of interest remains constant. This enhancement appears to be controlled at the transcriptional level and related to both the total amount of virus and the combination of transgenes present in the formulation.Recombinant adenovirus vectors have been extensively used in gene therapy clinical studies. More recently, the capability of inducing potent cell-mediated and humoral immunity has made these vectors equally attractive candidates for prophylactic or therapeutic vaccine applications. Merck and Co., Inc., developed HIV-1 vaccine candidates based on adenovirus serotype 5 (Ad5) vectors in which the E1 gene, a critical component for adenovirus replication, was replaced by the cytomegalovirus immediate/early promoter, followed by mutated versions of the HIV-1 gag, pol or nef genes . Vaccine performance was evaluated  A549  potency , such thA549 cells were infected with monovalent formulations of MRKAd5gag, MRKAd5pol or MRKAd5nef vectors  at multitrivalent formulation with the individual monovalent formulations, we assessed transgene transcription and expression levels in both formulations by ELISA and RT-qPCR  somehow facilitated the overall transgene expression.excessive viral load likely compromises the cellular machinery, possibly leading to accelerated cell death.Interestingly, at high MOI (>700 for ELISA and >1000 for RT-qPCR), more protein and mRNA were detected in cells infected with the monovalent formulations as compared with the trivalent formulations Figure  and 3. STo investigate whether the increase of transgene expression observed with the trivalent formulation was due to the higher amount of virus infecting the host cells or related to the simultaneous presence of one or more transgenes, we infected A549 cells with monovalent formulations of MRKAd5gag, MRKAd5pol or MRKAd5nef, supplemented either with "empty vectors"  or with a combination of EV and another monovalent vector to reach the equivalent number of virus particles that the cells were exposed to with the trivalent formulation. In this way, EV can normalize total non-replicating Ad5 virus concentration while leaving individual transgene concentration constant.Surprisingly, ELISA analysis from cells infected with EV and either monovalent MRKAd5gag or MRKAd5nef revealed that the presence of EV in a given monovalent formulation dramatically boosted transgene expression, even exceeding the transgene expression level of the trivalent formulation Figure . WhereasTo confirm that the enhancement of transgene expression levels observed in the presence of EV was not transgene-dependent and that this enhancement occurs at the mRNA level, we compared transcription levels by RT-qPCR of A549 cells infected by monovalent Ad5 vectors supplemented with 2× EV or 2× of a specific Ad5 construct. The results confirmed that gag and nef transcriptions were maximal in conditions supplemented with EV, especially in the MOI range of 10 to 100, and minimal in the monovalent formulations (Figure in vitro can be reproducibly modulated by the presence of additional replication-incompetent adenovirus. This modulation can occur in circumstances where another transgene-encoding adenovirus is present, as well as with the addition of adenovirus that does not contain the coding region for any transgene of interest. The mere presence of additional "empty" adenovirus particles devoid of transgene appears to enhance transgene expression. Our findings suggest these unexpected results may be worth consideration during dose and potency assay development for adenovirus-based therapeutics. Additional work is required to elucidate the mechanism of the observed transgene expression enhancement by the presence of additional adenoviral vectors. The data presented suggest that the mechanisms responsible for this enhancement are functioning prior to or during the transcription of the vector transgene. We hypothesize that the presence of additional virus results in an increase of cellular stress signals that activate transcription regulators such as MAP kinase and/or NFκB which in turn could up-regulate the CMV promoter activity driving transgene expression [The data suggest that for an experimental adenovirus-based HIV-1 vaccine, transgene expression in non-permissive cells pression ,13.All authors are employees of Merck, which paid for this study in its entirety.MNT carried out the RT-qPCR study, JAR carried out the ELISA study. KEO led the study. MNT, JAR and KEO wrote the manuscript. All authors read and approved the final manuscript.


In contrast to reports of extensive hypoxia in human gliomas in situ measured by pO2 histography, non-invasive methods of assessing glioma oxygenation, including nitroimidazole binding, have yielded surprisingly contradictory results. In order to investigate the relationship of necrosis, hypoxia, nitroreductase activity and cellular respiration in human gliomas, subcutaneous models using the human glioma cell lines M059K, M006 and M010b were developed in the murine SCID host. Intracranial growth of the M006 line was achieved in nude rats. The nitroreductive capacity of glioma cell lines was assessed and found to be similar to transplanted tumours previously reported in the literature. This suggests that if substantial numbers of viable hypoxic cells were present in situ in gliomas, then nitroimidazole-binding techniques should be capable of identifying them. Inter-tumour variability in the amount of necrosis was seen. M006 xenografts growing in subcutaneous and intracranial sites revealed extensive necrotic regions histologically, some of which were surrounded by cells labelled heavily for [3H]misonidazole, while other areas were lightly labelled. Similar binding patterns were seen for subcutaneous M059K tumours, while subcutaneous M010b tumours display necroses of which almost all were surrounded by heavily labelled cells. The oxygen consumption rates of tumour cell lines grown in vivo, in which venous pO2 concentrations are of the order of 2-5%, were two to sevenfold less than those of the same lines grown as monolayers in vitro under oxygen concentrations of 18%. We postulate that glioma cell lines behave as 'oxygen conformers', in that their rate of oxygen consumption appears to vary with the availability of oxygen. Together with the misonidazole-binding data, the results in this glioma tumour model are consistent with coordinate inhibition or down-regulation of respiration under moderate hypoxia.


Biochemical characterization of serologically detected human melanoma antigens was undertaken for the development of immunodiagnostic assays in melanoma. An antiserum from a human melanoma patient, which detected melanoma antigens expressed on a large proportion of different melanoma cells, was used in leucocyte-dependent cytotoxic antibody (LDA) 51Cr-release assays to monitor the purification of melanoma antigens in urea/acetate extracts of lactoperoxidase 125I-labelled melanoma cell membranes. The separation procedures included affinity chromatography on Concanavalin A, gel filtration on porous polyacrylamide beads and preparative isoelectric focusing. The fractions were also monitored by polyacrylamide electrophoresis in sodium dodecyl sulphate and by measurement of beta 2 microglobulin and carcinoembryonic antigen content. The antigens detected by this antiserum appeared to be acidic (pI 3.5) low-mol.-wt glycoproteins of approximately 15,000 daltons which were resistant to heating at 56 degrees C and digestion with neuraminidase, but susceptible to repeated freeze-thawing and trypsin digestion. They did not appear to be related to HLA antigens, beta 2 microglobulin or known foetal antigens. The nature of the antigens detected in these studies is as yet unknown, but they appear similar to those described in the sera and urine of melanoma patients in previous reports. Thes combined results and the frequent expression of these antigens on melanoma cells from different patients suggest that assays to detect this antigen may provide a valuable immunodiagnostic aid in the management of melanoma.


Glial cells coordinate the differentiation, metabolism, and excitability of neurons; they modulate synaptic transmission and integrate signals emanating from neurons and other glial cells. Several evidences underlying the relation between these pathways and the regulatory mechanisms of ion concentration, supporting the role of Aquaporins (AQPs) in these processes. The goal of this review is to summarize the localization of different isoforms of AQPs in relation to glial cells both in central and peripheral nervous system, underlying AQP involvement in physiological and in pathophysiological conditions such as brain edema, glioma and epilepsy.  In particular, there has been controversy over the localization of AQP1 in the PNS. Nevertheless, Gao and co-workers  reportedThese findings suggest that members of the AQP family are differentially expressed in the peripheral versus CNS.2.2.1AQP1 mediates water transport in several organs where it plays an important role, such as the formation of urine and aqueous humor in the eye , 110 or Increasing evidences indicate that brain astrocytes express AQP1 under pathologic conditions. AQP1 is found in reactive astrocytes accumulating in the lesions of subarachnoid hemorrhage , contusiAfter spinal cord injury (SCI) there was evidence that reactive astrocytes show a significant increase of AQP1 expression surrounding the lesion site, while reactive astrocytes distant from the lesion express markedly less it . So, givet al. [Moreover, AQP1 may play during the intake of water that precedes axonal elongation. Nesic et al.  demonstret al. , 36. GAPet al. , co-loca+ from intracellular to extracellular space may also require movement of H2O in the same direction [In brain tumours AQP1 expression increases with the grade of malignancy , 81. In irection , suggest1)). Glial Fibrillary Acidic Protein (GFAP)-positive Schwann cells are known to express AQP1 too [1)) but high resolution studies using electron microscopy are needed to determine the nature of AQP1-positive cells in nerve bundles and the detailed subcellular localization of AQP1 in both nerve plexuses and bundles in the PNS.In the PNS satellite cells of the trigeminal ganglia of rat show AQP1 expression  , glia limitans (brain-subarachnoid CSF), as well as ependymal cells and subependymal astrocytes (brain-ventricular CSF) , 78.The osmosensitive organs display a unique pattern of AQP4 compartmentation . In thesRecent study using AQP4 knock-out mice indicated that AQP4 is involved in the formation and resolution of brain and spinal cord edemas , 69, 70.In general AQP4 expression is up-regulated in astrocytes associated with brain edema. For exemple, AQP4 over-expression in human astrocytomas correlates with the presence of brain edema on magnetic resonance scans . CorrelaAQP4 also facilitates the elimination of excess brain water. Excess water is eliminated primarily through the glia limiting membranes into the CSF. Greater brain water accumulation and intracranial pressure were found in AQP4 knock-out versus wild-type mice with brain tumour, brain abscess, focal cortical-freeze injury, and after infusion of normal saline directly into brain extracellular space , 69, 70,Changes in astrocyte shape and function are known also to occur in association with human immunodeficiency virus (HIV) dementia (HIVD). Using Western Blot analysis, Hillaire and collaborators  showed t+ and H2O regulation [+ re-uptake into glial cells could be AQP4-dependent, as water influx coupled to K+ influx is thought to underlie activity-induced glial cell swelling [via both electron microscopic and co-immunoprecipitation analyses [-/- mice showed impaired retinal and cochlear physiology presumably due to altered K+ metabolism [+ clearance [+ following neural activity. Eid and collaborates [+ and an increased propensity for seizure. Nevertheless, another study reported that a significant increase in AQP4 is observed in sclerotic, but not in non-sclerotic, hippocampi obtained from patients with medically intractable temporal lobe epilepsy [Recent studies have found also changes in astroglia Kir channels and AQP4 water channels in temporal lobe epilepsy specimens . Alteratgulation . In factswelling , 101. Inanalyses , 55 whiltabolism , 43, 48.aborates  reportedepilepsy . HoweverAQP4 seems to be involved also in autism. In fact, while earlier studies did not show evidence of astrogliosis or microglial activation , more rePardo and co-workers  suggesteAQP4 is abundantly expressed in the basolateral membrane of the ependyma and glia limitans lining subependymal layer and pia. This distribution feature indicates that AQP4 provides a highly efficient pathway to convey the redundant water from parenchyma to ventricle system and subarachnoid space , 30, 63.in vivo. In fact the intranigral injection of lipopolysaccharide, that mimics the BBB disruption in memingoencephalitis [+) and restoration of osmotic equilibrium in absence of astrocytes. It is well known, in fact, that glial cells play an important role in regulating the homeostasis to ensure an appropriate neuronal environment. The astrocytes may help clear excess K+ around active neuron [Tomas-Camardiel and colleagues  providedphalitis  and provphalitis , 90. It e neuron  and in te neuron .2.5In the CNS AQP5 was detected in astrocytes and in neurons . In prim2.6AQP8 expression was initially described in the testis, pancreas, placenta and liver . By Nort2.7AQP9 is a water channel transporting glycerol, mannitol and urea. It was originally identified in human leukocytes , is alsoAQP9 has been hypothesized to play a role in extracellular water homeostasis and edema formation similar to AQP4  and seemAQP9 may play an important role in the malignant progression of brain tumours and it can be used as a biomarker for molecular diagnosis and as a new target for gene therapy, but the molecular mechanisms by which AQP9 affects the proliferation and apoptosis of astrocytic tumours need to be studied further.The discovery of AQPs has provided a molecular basis for understanding water transport in a number of tissues including the nervous system. In fact, emerging evidences suggest that brain AQPs play important roles for the dynamic regulation of brain water homeostasis and for the regulation of CSF. A tissue-specific pattern of AQP expression in the nervous system and in different type of glia cells, supports the idea that water movement in brain tissues is carefully regulated in concert with other transport processes from the micro- to macroscopic levels and that AQPs serve as elements of complex signaling assemblies.Knock-out mice have confirmed the role of AQPs in transepithelial fluid transport, in cell migration, in regulation of glycerol and in forming CSF. Moreover, recent data support roles for AQPs in glial system, both in physiological and in pathophysiological conditions. Glial expression of AQPs is in fact altered in brain edema conditions and in presence of stroke, tumour and infection but also in neurologic and neurodegenerative disease such as epilepsy, HIVD, autism and Alzheimer.


It has already been established that a single intrapleural inoculation of crystalline silica (quartz) produces malignant lymphomas of histiocytic type (MLHT) in Wistar-derived rats. It has now been shown that after treatment with Min-U-Sil, rats of the Alderley Park strain have a tumour incidence of 35%, whereas the incidence in Agus rats is 5% and in PVG 8%. There was also a significant difference in the incidence of MLHT caused by injecting different samples of crystalline silica, particularly of tridymite. There was correlation between cytotoxicity to mouse peritoneal macrophages and tumour incidence, except for one dust (DQ12). Zeta potential, number of particles and their size range were considered, but the incidence does not show a clear correlation with these measurements. The results are discussed.


Malignant pleural mesothelioma is considered an almost incurable tumour with increasing incidence worldwide. It usually develops in the parietal pleura, from mesothelial lining or submesothelial cells, subsequently invading the visceral pleura. Chromosomal and genomic aberrations of mesothelioma are diverse and heterogenous. Genome-wide profiling of mesothelioma versus parietal and visceral normal pleural tissue could thus reveal novel genes and pathways explaining its aggressive phenotype.Well-characterised tissue from five mesothelioma patients and normal parietal and visceral pleural samples from six non-cancer patients were profiled by Affymetrix oligoarray of 38 500 genes. The lists of differentially expressed genes tested for overrepresentation in KEGG PATHWAYS (Kyoto Encyclopedia of Genes and Genomes) and GO (gene ontology) terms revealed large differences of expression between visceral and parietal pleura, and both tissues differed from mesothelioma. Cell growth and intrinsic resistance in tumour versus parietal pleura was reflected in highly overexpressed cell cycle, mitosis, replication, DNA repair and anti-apoptosis genes. Several genes of the “salvage pathway” that recycle nucleobases were overexpressed, among them TYMS, encoding thymidylate synthase, the main target of the antifolate drug pemetrexed that is active in mesothelioma. Circadian rhythm genes were expressed in favour of tumour growth. The local invasive, non-metastatic phenotype of mesothelioma, could partly be due to overexpression of the known metastasis suppressors NME1 and NME2. Down-regulation of several tumour suppressor genes could contribute to mesothelioma progression. Genes involved in cell communication were down-regulated, indicating that mesothelioma may shield itself from the immune system. Similarly, in non-cancer parietal versus visceral pleura signal transduction, soluble transporter and adhesion genes were down-regulated. This could represent a genetical platform of the parietal pleura propensity to develop mesothelioma.Genome-wide microarray approach using complex human tissue samples revealed novel expression patterns, reflecting some important features of mesothelioma biology that should be further explored. Malignant mesothelioma is an aggressive and incurable tumour with currently a median survival of 12 monthsMoreover cytogenetic studies have shown that mesotheliomas have highly complex and variable chromosomal aberrationsGene expression analysis of six mesothelioma samples where two were from the same patient, seven parietal pleural samples where two were from the same patient and three visceral pleural samples were accomplished . Mean agPCA  and a PLS  model showed that mesothelioma, parietal and visceral pleural tissues had distinct differential gene expression profilesThere were 392 differentially expressed genes between the normal parietal and visceral pleura, where 341 genes were down-regulated and only 52 genes overexpressed in parietal pleura .No gene ontology (GO) entities were overexpressed in parietal pleura but several entities were down-regulated compared to visceral pleura . Among tSince the parietal pleura is the predilection site for mesothelioma, we further compared the mRNA expression of mesothelioma with normal parietal pleura. There were 828 overexpressed and 1004 down-regulated genes in tumour tissue . Of thesDown-regulated GO entities were related to multicellular organism development and cell communication, defense, cell adhesion and interestingly, several circadian rhythm genes . MoreoveAll samples of tissue adjacent to the tissue subjected to microarray, except control no. 3 where analysed by immunohistochemistry for protein expression of six selected genes. Due to limited biological material (needle biopsies) we had to be very selective in chosing which genes to verify. Overexpression was verified for Thymidylate Synthase, VG5Q, Chk1, NQO1 and RAD21, where tumour cells were positive in most cases. Normal mesothelial cells, that was a minor population of the biopsies  stained Histological pictures of normal parietal samples and biphasic mesothelioma stained with VG5Q, Thymidylate Synthase, and Mesothelin antibodies are shown illustrating the expression in normal pleura and the malignant epithelial and sarcomatous components .Genome-wide profiling of malignant pleural mesothelioma versus normal parietal pleura showed several new and interesting expression patterns highly relevant to the biology of mesothelioma. The gene expression differences between the parietal and visceral pleural tissues described here for the first time were significant and may be important for understanding the parietal pleura propensity for developing mesothelioma. Many of those features have been recognised mainly in epithelial malignant tumours, as will be discussed below, thus showing important genotypic similarities between this tumour of probably mesodermal origin and epithelial cancers. Moreover, 150 differentially expressed genes without known function were identified that may gain importance in the future.When interpreting gene expression data one must also keep in mind that they represent relative values, so that overexpression e.g. in tumour also could reflect down-regulation in the normal tissue.There were significant expression differences between these two pleural membranes. Interestingly the expression of the visceral pleura of case 2, 4 and 5 was much more alike than the parietal pleura of 2, 4 and 5 that formed a separate cluster, showing that tissues with a similar phenotype also share gene expression profile characteristicsImportantly there were more down-regulated than overexpressed genes in tumour versus parietal tissue corresponding with the recent findings of more chromosomal losses than gains in mesotheliomaAs an expression of rapidly dividing cells, polymerases for RNA and DNA synthesis were overexpressed as well as genes of the purine and pyrimidine metabolism, but strikingly this was confined to genes of the so-called “salvage pathways”, where nucleobases are recycled rather than synthesized de novoIt is known that cell cycle deregulation is a general feature of malignancy. Overexpression of the cell cycle, replication and M-phase genes reflect the importance of this also in mesothelioma , Table 5The overexpressed CDC6 encodes a protein essential for the initiation of DNA replication but has recently been shown to possess oncogenic properties by suppression of the INK4/ARFin vitroEssential for the initiation of eukaryotic genome replication are the MCM (mini-chromosome maintenance protein) complex that consist of MCM2-7, proteins possessing DNA helicase activity, and may act as a DNA unwinding enzymes. GMNN (geminin) regulate this complex and ensures genomic stability in cycling cells by preventing firing (or activation) of new replication origins before completion of a mitotic cycle, to ensure that DNA is replicated only once per cell cycle. MCM2, 3 and 6 that were overexpressed in our material  are assoPRKCI (protein kinase C iota) is a serine- threonine kinase involved in cell cycle regulation by controlling the key cell cycle regulator CDK7ORC6L is also overexpressed and is an essential gene that coordinates chromosome replication and segregation with cytokinesis and is overexpressed in colorectal cancer versus normal colon tissuesRecently in a genome-wide study of localised melanomas that did or did not metastasize within four years, DNA replication genes were highly overexpressed in the metastatic group. In our material 10 genes out of their 35 were overexpressed  and several other negative prognostic genes were common with our study Circadian rhythm genes have recently been related to replication, damage responses and carcinogenesis and may play a master role in cell divisionMoreover, we found the positive regulators of circadian rhythms and cell cycle ARNTL/BMAL1 (aryl hydrocarbon receptor nuclear translocator-like) and its heterodimer NPAS2 Apoptotic pathways and genes therein were mainly down-regulated in contrast to anti-apoptotic genes which were overexpressed . Genes eAURKA (Aurora kinase A) was overexpressed, and in mammalian cells overexpression leads to centrosome amplification, genetic instability and transformation, as well as cisplatin resistance. Its activation of the NFkB pathway has been proposed as an important mechanismAngiogenesis is important for tumour progression and survival DNA repair overexpression has recently been implicated in primary tumours with subsequent high metastatic potential, e.g. melanomaβ- family receptor TGFBR2, and chemokine ligands . These are involved in inflammatory responses, chemotaxis of monocytes, activation of natural killer cells, but also in cancer suppression. Anomaly of these functions may be important for tumour progression. Loss of the tumour suppressor TGFBR2 expression is seen in many cancers with microsatellite instability and deleted in large-cell lung carcinomaMalignant tumours are generally known to express factors that modulate their environment, e.g. growth and pro-angiogenic factors, but are generally not responsive to normal control mechanisms of the microenvironment. Interestingly cytokine-cytokine receptor interaction pathways were severely altered by down-regulation of 19/21 genes in KEGG PATHWAYS (not shown), 197 genes of signal transduction and 33 out of 271 inflammatory genes . The dowFinally, mutation and dysfunction of the detoxifier GSTM1 is related to high risk of head and neck and lung cancer in smokersThe study design as a whole was developed to avoid caveats of microarray analysis of complex tissues. Since initiaton and progression to a clinically detected malignant mesothelioma takes 20–60 years there are several unknown steps. We believe that our included control patients  facilitated a true differential expression between malignant and healthy tissue. Lack of appropriate control samples in earlier studies may have been one reason for incongruent resultsThe list of differentially expressed genes are based on a test that the average expression level is up or downregulated, however for as many as 519 of the reported upregulated genes and 542 of the downregulated there is no overlap in gene expression levels between the tumour and reference material. A large number of the genes that are found as differentially expressed represent pathways and biological processes widely known differentially regulated in cancer. The procedure to identify the genes takes both magnitude of change (fold change) and variability within the groups into account, and the p - values are corrected for multiple testing making it very likely that the reported genes are representative of changes even if the number of samples is low.Some overexpressed genes were confirmed by immunohistochemistry and genes encoding proteins overexpressed in mesothelioma were also overexpressed here . The genes FUT4 and ST6GALNAC3 coding for CD15 and Sialyl Transferase that are negative markers of mesothelioma, were down-regulatedMesothelioma arises in the pleura, but from which cell type? The mesothelial cell has been taken for granted as the progeny of mesothelioma, but recent studies showed that stem cells derived from adipose tissue, circulating multipotent fibrocytes and adult bone marrow-derived stem cells are able to transform to both epithelial and mesenchymal cellsDocumentation of cell types and relative amount of each type by visual inspection of two-dimensional slides of adjacent tissue as done here was feasible and easy, but utmost important as the variability of cell content was high. For this reason we suggest that by any technique used to obtain material for comparisons of DNA or RNA from complex tissues, an evaluation of cell-types are should be pursued. This is, to our knowledge, the first mesothelioma microarray study to report the clinical status, histological description of cell types and an estimate of the proportion of cell types in biopsies from cases and controls. Even with a small number of samples with high variability in cell content we could see a differential expression of the three complex systems of cells, the tumour, the parietal and the visceral pleura.In conclusion, we have demonstrated a significant differential gene expression of mesothelioma, visceral and parietal pleura by genome-wide profiling, based on tissue samples that contained all the cell types normally seen. The highly malignant, resistant but non-metastatic phenotype of pleural mesothelioma was reflected in the present gene profile. Significant dysregulation of circadian rhythm genes may be important in driving the malignant process. An introvert and immunologically defensive genotype of mesothelioma was reflected by down-regulation of adhesion, cytokine receptors, ligands and inflammatory response genes. Normal parietal pleura showed downregulation of adhesion, solute transporter and signal tranduction systems that could confer to its susceptibility of transformation by asbestos. The results underscore the vast complexity of mesothelioma biology and that large-scale methods are necessary to reveal new functional pathological aspects, finally aiming at target discovery.The study protocol was approved by the Regional Committee of Research Ethics of Central Norway, the Health Departement and the Norwegian Social Science Data Service. Informed consent was obtained from all participants.Mesothelioma patients diagnosed between 2003–2005 were included. They were all subjected to a clinical examination and answered a patient history questionnaire. Diagnostic biopsies and material for gene expression were taken from adjacent locations with needle by Computer Tomography and/or ultrasound guidance. Diagnostic samples were formalin-fixed and paraffin-embedded. Material for gene expression analysis was snap-frozen in liquid nitrogen within two minutes. Biopsies of morphologically normal pleura were obtained from persons who underwent Video-Assisted Thoracoscopy (VATS) for recurrent pneumothorax, after obtaining patient history and informed consent. Parietal pleura that was stripped from the thoracic wall and visceral pleura dissected from the wedge-resections of the lung, were snap-frozen in liquid nitrogen within two minutes. Mesothelioma diagnosis was carried out by senior pathologists and re-examined by H. Sandeck, by including a standard panel of antibodies for immunohistochemistry as well as supplementary antibodies were used.Biopsies from tumour and control adjacent to the biopsies for microarray analysis, were examined histologically by H. Sandeck to identify which cell types were included in each specimen and also estimate the relative content of cells of each type .Methods used for RNA extraction were optimized to assure a high quality RNA from the small needle biopsies of the tumours. The final technique chosen was homogenization of frozen tissue with MagnaLyser (Roche Diagnostics) following the manufacturer's procedure 2×50 sec, but using 700 µL lysis buffer  as it gave higher RNA yield. The material was then incubated for 30 min at room temperature, centrifuged at 13000G for two minutes. 350 µL of the supernatant was used for further RNA isolation. Manual isolation with High Pure RNA Tissue Kit  according to the producer's protocol was performed. Quality control of RNA was done with NanoDrop  and Bioanalyzer .Microarray experiments were performed at the Norwegian Microarray Consortium (NMC) at NTNU, Trondheim, Norway. Gene expression analysis was performed by the Affymetrix GeneChip system according to the manufacturer's Eukaryote Two-Cycle protocol, starting with 75 ng deep frozen total RNA. Labelled cRNA was hybridized to the Affymetrix Human Genome U133 Plus 2.0 GeneChip , of 38 500 genes and 47 000 trancripts, allowing genome-wide expression on a single array. The GeneChips were scanned using the GeneChip Scanner 3000 (Affymetrix). Quality controls were assessed using the GCOS v1.4 software, according to the manufacturer's manual (Affymetrix). All experiments have been submitted to ArrayExpress registered with accession number E-MTAB-47.The raw probe set intensities were normalised by robust multi array average (RMA). Quality control was done of Benjamini and HochbergCell specific expression of proteins encoded by six selected genes, were validated by immunohistochemistry (respective gene symbols in brackets). The following antibodies were tested on fixed tissues adjacent to samples subjected to microarray. Thymidylate Synthase (TYMS )  dilution 1∶50, VG5Q (AGGF1)  dilution 1∶500, Chk1(CHEK1), dilution 1∶10, overnight incubation at −4°C, NQO1 (NQO1)  dilution 1∶50, RAD21 (RAD21)  dilution 1∶500 and mesothelin (MSLN) dilution 1∶10, overnight incubation at −4°C. Selected positive and negative controls were included for all antibodies.


CNNM4 gene, encoding a putative metal transporter, accounting for the condition in all seven families. Nine mutations are described in all, three missense, three terminations, two large deletions, and a single base insertion. We confirmed expression of Cnnm4 in the neural retina and in ameloblasts in the developing tooth, suggesting a hitherto unknown connection between tooth biomineralization and retinal function. The identification of CNNM4 as the causative gene for Jalili syndrome, characterized by syndromic CRD with AI, has the potential to provide new insights into the roles of metal transport in visual function and biomineralization.The combination of recessively inherited cone-rod dystrophy (CRD) and amelogenesis imperfecta (AI) was first reported by Jalili and Smith in 1988 in a family subsequently linked to a locus on chromosome 2q11, and it has since been reported in a second small family. We have identified five further ethnically diverse families cosegregating CRD and AI. Phenotypic characterization of teeth and visual function in the published and new families reveals a consistent syndrome in all seven families, and all link or are consistent with linkage to 2q11, confirming the existence of a genetically homogenous condition that we now propose to call Jalili syndrome. Using a positional-candidate approach, we have identified mutations in the  These genes encode proteins of diverse functions, including participants in the phototransduction cascade and the visual cycle, structural components of photoreceptors, and photoreceptor-specific transcription factors.Cone-rod dystrophy (CRD [MIM 204700]) or dentine (dentinogenesis imperfecta [DI] [MIM #125490] and dentine dysplasias [MIM %125400]), the two major hard-tissue components of teeth. Nonsyndromic AI has been attributed to mutations in five genes, which fall into three groups and involve the following: enamel-matrix proteins , enzymes controlling postsecretory processing of enamel-matrix proteins , and a gene of unknown function (FAM83H∗611927]). Only a single gene (DSPP [MIM ∗125485]) has been implicated in nonsyndromic DI.In comparison with retinal degeneration, little is understood about the genetic causes of abnormal tooth biomineralization (reviewed by Bailleul-Forestier and colleagues209900), Alstrom syndrome (MIM 203800), spinocerebellar ataxia type 7 (MIM 164500), and selected syndromes characterized by ectodermal abnormalities. AI and DI have been described as part of syndromes involving bone abnormalities, giving insight into different forms of human biomineralization.MSX2 mutations have been described as AI even though there is evidence of dentine involvement.In addition, CRD, AI, or DI can be part of syndromes involving multiple tissues and organs. CRD is a feature of some forms of Bardet Biedl syndrome  was first reported in a large consanguineous Arab kindred living in the Gaza strip (Gaza A),The combination of familial CRD and AI , listed in Phenotypic characterization demonstrated consistent dental and ocular phenotypes between familiesTo determine whether this was indeed a homogeneous syndrome, we undertook genetic analyses in these families. Although the Scottish family consists of a single case and was too small for genetic analysis, Affymetrix 50K SNP array analysis in the Turkish family (data not shown) and microsatellite genotyping in the remaining three new families  containiCNNM4 gene (MIM ∗607805)  . Nine muMMP20 and KLK4 mutations.To characterize the tooth phenotype in greater detail, we obtained two exfoliated deciduous molar teeth from the affected individual from the Scottish family, together with matched control teeth. These were sectioned longitudinally and then subjected to ultrastructural analysis . This anIn addition, we performed immunofluorescence on mouse retina sections and immunohistochemistry on rat incisors and confirmed Cnnm4 expression in both retina and developing teeth . AmeloblCNNM4 was originally identified as a member of a gene family with four protein-encoding members in humans, each sharing a 31 residue repeat present in cyclin boxes.2+ or other metal transport. Mg2+ in particular is necessary for many cellular functions, including its role as a cofactor for enzymes of the phototransduction cascade, and global magnesium deficiency has been shown to have severe effects on the retina, leading to photoreceptor and retina pigment epithelium cell death.2+ ions2+ ions.2+ or Zn2+ transport is likely to lead to abnormal functioning of these receptors and their parent cells. GABA receptors modulate neurotransmitter release in the retina2+ ions.2+ transport could lead to excitotoxic conditions in the retina. Future work will be required to confirm whether CNNM4 shares the magnesium-transport function of CNNM2 or whether it is involved in the transport of other metal ions.Like CNNM2, CNNM4 is predicted to have four transmembrane helices and contains a cyclic nucleotide-binding domain, two cystathionine-beta-synthase (CBS) domains, and a DUF21 domain of unknown function. CBS domains appear to have a role in sensing the energy status of cells by binding to ATP and are present in proteins such as CLC chloride channels and IMPDH1, mutations in which are a known cause of retinal degeneration.CNNM4 have clinical consequences that are limited to retinal function and biomineralization is surprising given the wide tissue distribution of the encoded protein,The finding that mutations in


Objective: Human defensins and cathelicidins are a family of cationic antimicrobial peptides (AMPs), which play multiple roles in both innate and adaptive immune systems. They have direct antimicrobial activity against several microorganisms including burn pathogens. The majority of components of innate and adaptive immunity either express naturally occurring defensins or are otherwise chemoattracted or functionally affected by them. They also enhance adaptive immunity and wound healing and alter antibody production. All mechanisms to explain multiple functions of AMPs are not clearly understood. Prior studies to localize defensins in normal and burned skin using deconvolution fluorescence scanning microscopy indicate localization of defensins in the nucleus, perinuclear regions, and cytoplasm. The objective of this study is to further confirm the identification of HBD-1 in the nucleus by deconvolution microscopic studies involving image reconstruction and wire frame modeling. Results: Our study demonstrated the presence of intranuclear HBD-1 in keratinocytes throughout the stratum spinosum by costaining with the nuclear probe DAPI. In addition, HBD-1 sequence does show some homology with known cationic nuclear localization signal sequences. Conclusion: To our knowledge, this is the first report to localize HBD-1 in the nuclear region, suggesting a role for this peptide in gene expression and providing new data that may help determine mechanisms of defensin functions. Antimicrobial peptides (AMPs), including both defensins and cathelicidins, are part of the innate immune system, which provides the first line of defense against a wide spectrum of common burn and wound pathogens.Human defensins are cationic antimicrobial peptides with molecular weights of 4 to 5 kDa, containing a conserved motif of 6 cysteines linked by 3 disulfide bonds. On the basis of their size and pattern of disulfide bonding, human defensins are classified into α and β categories, and are expressed in various cell types.20In our previous work, we have localized AMPs in burned and normal skin to specific cell types and areas of the epidermis and dermis, using deconvolution fluorescence microscopy.l-lysine, and fixed with 3.7% paraformaldehyde. This was followed by 60-min incubation in goat serum and staining with appropriate probes and antibodies as previously described.22Patients admitted to the Regional Burn Center at Memorial Medical Center, Springfield, IL, or the Johns Hopkins Burn Center with 10% to 40% total body surface area burns were included in the study. Normal skin samples were obtained from remnants of split thickness skin autografts harvested during skin grafting procedures and were frozen in sucrose-based embedding media . Ten-micrometer sections were cut in a microtome, floated onto 18-mm glass cover slips that had been coated with poly-The stained sections were placed under Elvanol , and covered with a cover slip. The samples were scanned with an Applied Precision Delta Vision Scanning Microscope , fitted with an Olympus 1 × 70 microscope . Image scans were acquired in a series at a step-size thickness of 0.2 μ m by a Photometries  PXL CCD camera. Image analysis was performed by transferring each data set to a Silicon Graphics workstation using SoftWoRxTM software . All data sets were subjected to 5 deconvolution iterations and subsequently used for image reconstructions and modeling. Stacking each of the individual sections produces a 3-dimensional (3D) image on a 2-dimensional background, resulting in an image projection.25The Figure The extraction of a number of cells and nuclei from these 3D models and the application of fish-net mapping permitted us to visualize specific probes as distinct areas with no color-mixing  and single color/channel imaging. It is apparent Fig  that theWe attempted to detect nuclear targeting sequences in HBD-1. This was achieved using a database search engine . HBD-1 show some homology with the nuclear targeting sequences (KRX(10-12)KRRK) of the known nuclear translocation proteins, monopartite and bipartite Figure . This maThis study demonstrates nuclear localization of HBD-1. Considering that both defensins and cathelicidins display multiple functions, we hope that current findings may help explain mechanisms for some of these functions. AMPs are able to differentially recognize and disrupt negatively charged bacterial cell membranes,Currently, there are 2 AMPs which have been localized to the nucleus, this study localizing HBD-1 in the nucleus of keratinocytes in skin, and the recent study by Bandholtz et al,The mechanism by which AMPs enter the nucleus has not been studied and is subject to speculation, although several possibilities exist. In eukaryotic cells, signal-mediated macromolecular transport between the nucleus and the cytoplasm is an integral part of many processes, such as gene expression, signal transduction, and cell-cycle progression. Even though molecules can enter the nucleus of actively dividing cells through nuclear membrane breaks during mitosis, the only portal of entry into the nucleus of nondividing cells is the nuclear pore complex (NPC). NPCs allow passive exchange of ions, small molecules, and small proteins (< 20 kDa), but restrict the passage of macromolecules to only those bearing appropriate nuclear localizing signal (NLS). Considering the small size of AMPs (4–5 kDa for defensins), the question arises as to whether NPCs do allow the passive transport of AMPs into the nucleus.Another mechanism by which AMPs could enter the nucleus is based on AMP cationic sequence, a sequence that could constitute a nuclear localization and recognition signal and promote nuclear transportation. The directional transport of thousands of proteins and RNAs through the NPC in the course of normal cellular metabolism is mediated by a family of more than 20 proteins, the karyopherins.16 and poly (Arg)16] promote translocation of plasmid DNA into the nucleus. Many cationic peptides, other than those known to function as nuclear localizing motifs, appear to have a nuclear translocation ability regarding DNA plasmids. The ability of LL-37 to deliver plasmid DNA into mammalian cells has been demonstared.Polymers of basic amino acids [i.e poly (Lys)One possible functional importance of nuclear localization of AMPs may be coupled to affecting gene expression.In summary, our observation, when taken together with other previously reported findings of the actions and mechanisms of AMPs, suggest possibilities for new roles for AMPs in the modulation of gene expression and/or protein synthesis. Further studies are required to elucidate these possibilities.


In this short review, we summarize what is known about the efficacy of targeting the mtor pathway to treat cancer patients, and we explain the rationale behind promising new inhibitors that could show more potent tumour growth inhibition than did the first generation of these drugs.Cancer cells are characterized by aberrant growth arising from deregulated signalling pathways. The mammalian target of rapamycin (m Signal transduction pathways govern appropriately regulated cell growth, division, and survival in human tissues, but these pathways are often deregulated in aggressive cancers that show seemingly uncontrolled growth. This observation has led to the notion that if the molecular cause of a cancer can be determined, then the aberration on which a particular cancer depends could be directly targetedtor) signalling pathway has proved to be an excellent model system for exploring this notion of personalized medicine, because numerous mutations resulting in hyperactivation of the pathway have been identified, the hyperactivation is itself demonstrated by multiple downstream biomarkers, and a specific potent mtor inhibitor, rapamycin, has a long been safely used to treat humanstor signalling, clinical trials of rapamycin analogues, the rationale behind second-generation non-rapamycin-analogue mtor inhibitors, and the ongoing early-phase clinical trials using these agents.The mammalian target of rapamycin  to control downstream processes, including translation of messenger rna, cell growth, and proliferation  when growth factors are not abundant. Similarly, serine/threonine kinase 11 [Stk11 (Lkb1)] negatively regulates mtor activity when cellular energy (adenosine triphosphate) is in low supply, and the tuberous sclerosis complex (Tsc1/Tsc2) negatively regulates both of those signals to mtor. Consequently, cells lacking functional pten, Stk11 (Lkb1), or Tsc2 exhibit deregulated, constitutive signalling to mtor, resulting in multiple types of cancerThe evolutionarily conserved (yeast to man) mferation . Numeroutor by simultaneously binding to a protein called Fkbp12 and to the Fkbp12 rapamycin-binding (frb) domain of mtor. Because of its ability to inhibit mtor-dependent growth and proliferation of B and T cells, rapamycin has long been used as an immuno-suppressant (sirolimus) to prevent organ rejection, and because of its ability to inhibit the fungal orthologue of mtor, it can also be used as an antifungal agent.Rapamycin, a natural product from soil bacteria, inhibits mIn clinical trials, rapamycin and three rapamycin analogues—CCI-779 [temsirolimus ], RAD001 [everolimus ], and AP23573 —have been assessed for their efficacy as anticancer agentstor inhibitors were predicted to be effective in the treatment of tumours with genetic mutations that lead to overactivation of mtor signalling—for example, loss of pten, Stk11 (Lkb1), or Tsc2. Cells and tumours containing mutations or deletions of these tumour suppressors exhibit uncontrolled mtor signalling and an increase in phosphorylation of the downstream targets Eif4Ebp1 (eukaryotic translation initiation factor 4E binding protein 1) and S6 kinasepten inactivation, have responded favourably to treatment with temsirolimustor inhibitors, likely because of the importance of mtor signalling in the expression of Hif1A , a key player in angiogenesis and the growth of renal tumoursiii clinical trial involving patients with metastatic renal cell carcinoma indicate that everolimus may be an effective option for patients suffering from renal cancertor signalling  may be used to identify patients who would benefit from treatment with rapamycin analogues. However, although rapamycin analogues have been effective in the treatment of some tumours that lack pten (such as those of the endometrium), other cancers have been refractory to the effects of mtor inhibitors despite pten loss or mutationBased on preclinical testing, mtor inhibitors undergoing clinical trials are rapamycin analogues, and they therefore inhibit only a portion of mtor signalling—the portion effected by the rapamycin-sensitive mtor complex 1 (mtorc1)torc1 and the rapamycin-insensitive mtor complex 2 (mtorc2) would be a more effective means of treating such tumourstorc1 with rapamycin actually leads to an upregulation of mtorc2-dependent survival signalling through the PI3K-Akt pathway by suppressing a negative feedback looptor and PI3K–Akt is an effective means to target pten-deficient tumourstor inhibitors that target the kinase domain has been proposed as an effective means to block the activities of both mtorc1 and 2 mtorctorc1 and 2 inhibitors. The agent OSI-027 , for example, is an mtorc1 and 2 inhibitor currently being evaluated (phase i) to determine the maximum tolerated dose in patients with lymphoma or solid tumours. Similarly, BEZ235  is being evaluated (phase i and ii) to determine dosing and effectiveness in treating advanced solid tumours, particularly those in patients with Cowden syndrome (caused by pten loss). A third mtorc1 and 2 inhibitor, XL765  is being tested (phase i) alone and in combination therapies.All of the above-described mtor signalling pathway and its aberrant activation has provided strong proof that the age of personalized medicine based on molecular diagnoses is well underway. Progress to this point indicates that further elucidation of the mechanisms involved in predicting sensitivity to rapamycin and analogues is possible, as is development of new, more potent mtor inhibitors for the treatment of cancer. It is important to keep in mind that the signalling pathways inhibited by rapamycin analogues and by mtorc1 and 2 inhibitors are likely crucial for the growth, proliferation, and survival of possibly all human cells, and therefore such compounds should be effective treatments against most tumours in the same way that traditional chemotherapies are. However, the notion of “oncogene addiction” suggests that, if a particular aberration is driving tumour survival, then it may be possible to selectively target the cancer while merely inducing growth arrest or cytostasis in healthy tissues.As a whole, exploiting the m


Individuals with inactivating Notch1 mutations have a high rate of cardiovascular disorders, including valve stenosis and calcification. This article reviews recent progress in the mechanism of cardiovascular calcification and discusses potential molecular mechanisms involved, focusing on Notch receptors. We propose a calcification model where extreme increases in vascular wall cell density due to inflammation-induced cell proliferation can trigger an osteogenic differentiation program mediated by Notch receptors. Recent increase in human lifespan has shifted the spectrum of aging-related disorders to an unprecedented upsurge in cardiovascular diseases, especially calcific aortic valve stenosis, which has an 80% risk of progression to heart failure and death. A current therapeutic option for calcified valves is surgical replacement, which provides only temporary relief. Recent progress in cardiovascular research has suggested that arterial and valve calcification are the result of an active process of osteogenic differentiation, induced by a pro-atherogenic inflammatory response. At molecular level, the calcification process is regulated by a network of signaling pathways, including Notch, Wnt and TGFbeta/BMP pathways, which control the master regulator of osteogenesis Cbfa1/Runx2. Genetic and  Cardiovascular disorders are the most prevalent cause of death in the US. Atherosclerosis, the most frequent cardiovascular disorder, is the result of a gradual accumulation of atheromatous plaques in the arterial wall. The prognostic is especially unfavorable when such plaques accumulate in the coronary arteries, cerebral arteries or aorta, where they can lead to often-fatal cardiovascular accidents such as stroke, heart attacks or heart failure. Plaque calcification causes an increased risk of rupture due to mechanical stress at the interface between the rigid calcified plaque and neighboring soft “unstable” atheroma -3. In adin vitro studies proposed that calcification is the result of an inflammatory process induced by LDL oxidation and uptake by macrophages, which become foam cells [in vivo results showing that anti- inflammatory drugs such as statins may also inhibit osteogenic pathways in myofibroblasts of hypercholesterolemic rabbits [1).A number of defects and/or polymorphisms of proteins involved in the fat transport pathways, notably the apolipoproteins and the LDL receptor, markedly contribute to increased atheroma and calcification risks. Defects in these proteins are associated with hyperlipoproteinemia or hypercholesterolemia, have a frequency of 1:500 to 1:1000 in the general population and are characterized by a significant increase in coronary heart disease susceptibility. In the past decade however, preclinical and clinical studies have proposed that active osteogenic mechanisms contribute to arterial and valvular calcification, replacing the traditional view of passive lipid accumulation and cellular degeneration. This idea has only recently started to be accepted and investigated, although the underlying information has been available for several decades. Early  rabbits -8. Recen, suggesting that inflammation triggers osteoblastic activity of myofibroblast-like cells thus promoting calcification ) [Our recent studies have used innovative functional imaging strategies to monitor valvular and atherosclerotic lesion initiation and progression. These have provided robust evidence that inflammation correlates with osteoblastic activity preceding the development of advanced calcificationig. (2)) -12. Furt1)). Genetic defects that result in elastin fiber degradation  have been associated with fibroblast and smooth muscle cell proliferation, cardiovascular calcification as well as with a chronic inflammatory state. Although having different etiologies, both intimal and medial calcification involve the activation of a pro-inflammatory mechanism and the initiation of smooth muscle cell proliferation, which likely continue through similar calcification paradigms. A very similar thickening and calcification process induced by inflammation affects heart valves [While intimal calcification is connected to the atherosclerotic process and lipid processing, type II diabetes and chronic kidney disease often lead to the calcification of the medial layer of the vascular wall. This process is connected to elastin degradation by proteolytic enzymes rather than to lipid transport and oxidation and can take place independently of atherosclerosis. Elastin degradation peptides have pro-inflammatory properties similar to the oxidized cholesterol and can act as macrophage chemoattractants   de-differentiation and proliferation , from ci disease , 21. Ests Stro-1 , CD105, s Stro-1 , 18.1)). This hormone promotes VSMC calcium influx and calcification by interacting with the Wnt pathway [1)). Similarly to the origin of osteoblast and smooth muscle cell precursors in the neointima, the mechanism by which the inflammatory process triggers the proliferation of these precursors remains unclear as well. Macrophages can induce osteogenic differentiation and calcification both through direct cell contact with precursor cells or by releasing signaling molecules such as 1,25-dihydroxyvitamin D  ), a transcription factor that promotes cell survival in hypoxic conditions typically associated with accelerated cell proliferation, for example in cancer, atheromas or proliferating stem cells (reviewed in [3)). A concurrent mechanism of calcification following the proliferation of a large number of smooth muscle cells and reduced nutrient and oxygen access to cells due to tighter cellular packing may be mediated by hypoxia and nutrient deprivation. This can explain why calcification nodules usually appear inside the neointima, in highly proliferative areas, and then extend towards the arterial lumen . In factiewed in ). HIF-1 iewed in  and in oiewed in , all of iewed in , providiThe list of proinflammatory factors that regulate osteogenic differentiation of vascular cells is rapidly growing and includes: osteopontin, BMPs, Msx-2, oxidized lipoproteins, RANKL, PTH, matrix GLA protein, fibrillin, pyrophosphate, tumor necrosis factor-alpha, oxysterols, osteoprotegerin, insulin-like growth factor, interleukins, 1,25-di-hydroxyvitamin D, transforming growth factor-beta, estradiol, decorin, fetuin, and many others. Following the initial cardiovascular injury and inflammation, which may be caused by a number of factors such as high blood cholesterol, infections or genetic disorders, calcification likely proceeds through a unique molecular mechanism that involves the concerted action of a complex signaling network which is highly similar to normal skeletal bone development.1)). Wnt signaling includes LDL receptor related proteins Lrp5/6, which are co-receptors for Wnt binding but can also bind ApoE [1)). Wnt implication in calcification is part of a larger signaling network that also includes BMP [One signaling pathway directly connected to the lipid transport system with a well-established role in osteoblast differentiation and calcification is the Wnt pathway ). In turn, Notch signaling induces the expression of Dkk2 (Dickkopf), a Wnt signaling inhibitor thought to have osteoclastogenic and tumor suppressor properties [While Wnt signaling is thought to promote osteoblast differentiation and calcification, Notch is known to promote cell proliferation and activation. Wnt and Notch cellular signals complement each other in an oscillating network of signals associated with a segmentation clock that regulates embryonic somite development . The twooperties , 42. Heyoperties . The Notoperties , but onloperties , has amp3)). Notch ligands include “classical” ligands Jagged – Jag1 and Jag2 - and Delta-like - DLL1, DLL3 and DLL4 - as well as several atypical ligands DNER [3). Notch receptor selectivity for either DLL or Jag ligands is regulated through glycosylation by Fringe glycosyltransferases [hairy/enhancer of split (HES1-HES7) and HES-related (Hey1-2 and HeyL) genes, which are members of the bHLH Orange family of transcriptional repressors, thus inhibiting a number of genes.Notch signaling is a complex juxtacrine signaling mechanism initiated by the interaction of Notch transmembrane receptors (Notch1-4) with their ligands, which are also transmembrane proteins  family members act in the same direction. In some cases, hairy/E(spl) family members may even have opposing actions: Runx2 activity and osteogenesis are stimulated by HES1 [Transgenic mouse models and human genetic disorders suggest that the four Notch receptors and their ligands have different functions. Logically, they should then be expected to induce different HES/Hey transcription patterns, since different HES/Hey isoforms appear to have vastly different roles in cellular specification. For example HES3 is selectively expressed in the cerebellum, HES5 is strongly upregulated in the embryo around E10 and Hey2 is preferentially expressed in the heart. It is also important to note that not all  by HES1 , but rep3)).Atypical Notch ligands contain IgC2 and fibronectin domains instead of EGF-like repeats and induce a different set of genes by binding to different transcription regulators, the Deltex family of transcriptional repressors. These are mostly involved in nervous system gene expression and development. Both canonical and atypical Notch ligands generate the same NICD fragments, which then go on to initiate divergent transcription mechanisms through CSL and Deltex, respectively. Canonical Notch ligands inhibit Deltex-dependent gene expression, thus inhibiting neuronal differentiation and promoting cell proliferation through the CSL/HES pathway . Mice d1)) and reduces contact inhibition [The epithelial-mesenchymal transition, mentioned as a potential source of mesenchymal stem cells in the adult vasculature and cardiac valves, may occur as a result of Notch activation by Jag1, which represses the activation of Wnt pathway . Preferehibition  and cellhibition . This suhibition . In conthibition .A number of human genetic disorders resulting from mutations in Notch signaling have demonstrated the implication of Notch in the development and maintenance of the cardiovascular system. For example the Alagille syndrome is an autosomal dominant disorder associated with mutations or deletions of Jag1 gene or, in less than 1% of cases, with Notch2 mutations. Clinical symptoms are variable but typically include congenital heart defects, cholestasis, butterfly vertebrae and skeletal and facial abnormalities. The cardiovascular problems are amplified by frequent kidney abnormalities. In mouse models however, both Jag1 and Notch2 heterozygous mutations are necessary to reproduce the human Alagille phenotype . The AllCADASIL , a common form of stroke, is induced by Notch3 mutations that prevent its glycosylation and internalization , 60 and 1)).Finally, the strongest connection to date between Notch signaling and cardiovascular calcification was made by identifying inactivating Notch1 mutations in 2 families with a multi-generation history of bicuspid aortic valve and aortic valve calcification . Wild-ty1)). Juxtacrine communication between macrophages appears to be regulated by increased DLL4 and Notch3 expression and DLL4/ Notch binding, which induces a pro-inflammatory response and macrophage activation and proliferation [A review on the role of Notch signaling in cardiovascular calcification cannot be complete without analyzing the role of Notch in macrophages, which trigger the inflammatory response and subsequent plaque formation in atherosclerosis and valve calcification ). Notch and TNFalpha/ NFkappaB pathways cooperate in several ways to induce cell proliferation . NFkappiewed in , whereasiewed in  and alsoiewed in , 68. NFkiewed in . 1)). Moderate NO quantities are normally released with a vasodilatatory role by endothelial cells lining the vascular wall, but the larger amounts generated by activated macrophages can be a key factor in the mechanism of atherosclerosis [Another paracrine molecule released by activated macrophages is nitric oxide . NO andThe increased risk of mortality and morbidity associated with cardiovascular calcification has lead to the design of new therapeutic strategies to prevent and even reverse this process. For example, in the case of statins, although recent clinical trials have failed to show a reduction in advanced calcific aortic stenosis, a growing body of research indicates that early use of statins may have therapeutic advantages in preventing cardiovascular calcification. A large number of recent publications confirm what was until recently just a hypothesis: that arterial and valve calcifications are active inflammatory diseases triggered by pro-atherogenic factors , 12, 73.Despite the complexity of cardiovascular calcification, rapid progress has been made over the past few years in understanding its intricacies and the pathways and signaling mechanisms of molecules involved. This generates the expectation that, besides preventive measures currently available such as non-specific anti-inflammatory medication and life-style changes, a new generation of mechanism-specific drugs, which can target genetic risk factors and actually reverse atheromatous plaques and calcification, will become available in the near future.


KRAS gene are associated with poor response to this treatment. Therefore, KRAS mutation analysis is employed in the selection of patients for EGFR-targeted therapy and various studies have shown a high concordance between the mutation status in primary CRC and corresponding metastases. However, although development of therapy related resistance occurs also in the context of novel drugs such as tyrosine kinase-inhibitors the effect of the anti-EGFR treatment on the KRAS/BRAF mutation status itself in recurrent mCRC has not yet been clarified. Therefore, we analyzed 21 mCRCs before/after anti-EGFR therapy and found a pre-/posttherapeutic concordance of the KRAS/BRAF mutation status in 20 of the 21 cases examined. In the one discordant case, further analyses revealed that a tumor mosaicism or multiple primary tumors were present, indicating that anti-EGFR therapy has no influence on KRAS/BRAF mutation status in mCRC. Moreover, as the preselection of patients with a KRASwt genotype for anti-EGFR therapy has become a standard procedure, sample sets such ours might be the basis for future studies addressing the identification of potential anti-EGFR therapy induced genetic alterations apart from KRAS/BRAF mutations. Anti-EGFR targeted therapy is a potent strategy in the treatment of metastatic colorectal cancer (mCRC) but activating mutations in the  KRAS) and the V-raf murine sarcoma viral oncogene homolog B1 (BRAF) wes established as two of these mechanisms and preselection of CRC patients with a KRASwt genotype have been shown to increase the therapeutic efficacy of anti-EGFR therapy [KRASwt mutation status [KRAS and BRAF mutation status in primary CRC and corresponding metastases. Although the results of these studies appear contradictory in part, the majority of authors report high rates of concordance between the mutation status of KRAS in primary tumors and corresponding metastases [KRAS mutation status was monitored in the primary tumors and corresponding metastases of 106 cases of mCRC [KRAS in 105 of 106 cases  is one of the most common forms of malignant neoplasia and frequently takes a fatal course following metastasis . CRC is  therapy , 8. Thern status , 10. To tastases , 11–16.  of mCRC . Here, w06 cases  and were06 cases , a fact  regimen . KRAS exon 2, Gl12/13; BRAF exon 15, V600E) as well as allele-specific PCR [49 individual specimens of 21 metastatic CRCs and corresponding metastases collected before and after combined therapy with cetuximab were examined using CGH, certified PCR/DNA sequencing protocols . The majority of samples analyzed in this approach derived from the sample pool of 106 mCRC with 270 syn-/metachronic metastases used in our earlier study on the concordance of tastases . Biopsy μg) of tumor and reference DNA  were cohybridized on metaphase slides . Signals were visualized with a Zeiss Axiophot fluorescence microscope and analyzed with the ISIS digital image analysis system . CGH was performed as previously described . BrieflyKRAS and BRAF mutation analyses were performed on two independent samples from all primary tumors/metastases using protocols described previously [eviously . Brieflyeviously .n = 270) using PCR/DNA sequencing protocols certified by the German Society for Pathology  as well as allele-specific PCR approaches. As shown in KRAS mutation status between primary CRCs and all corresponding metastases in 105 of 106 patients. However in one case , a mutated KRAS gene (Exon 2 G12D) was found in the primary CRC, while no KRAS mutation was observed in a liver metastasis sample obtained after combined cetuximab therapy  was observed in the primary CRC  include both de novo and, more frequently, acquired resistance, which may occur after several months of drug administration and most often is based upon an acquired second mutation in the c-kit and PDGFRα genes [Development of therapy-related resistance is a frequent phenomenon in cancer and also occurs in the context of novel drugs such as monoclonal antibodies and tyrosine kinase inhibitors. In gastrointestinal stromal tumors (GISTs), the most common mesenchymal neoplasm of the gastrointestinal tract, mechanisms of resistance to imatinib mesylate  are responsible for therapy-related resistance in mCRC. Moreover, as the preselection of patients with a KRASwt genotype for cetuximab therapy has become a standard procedure, sample sets such as the one used in this study will have to be conserved carefully for use in future studies particularly with respect to analyses addressing the identification of anti-EGFR therapy-induced genetic alterations apart from KRAS/BRAF mutations.The results of this study provide first evidence that secondary


More than 70 years after its first description, experimental autoimmune encephalomyelitis (EAE) remains an important tool to understand concepts of T cell mediated autoimmunity as well as the roles of the innate and the humoral immune systems. Some EAE models also well reflect mechanisms of tissue damage including demyelination, axonal injury and also cortical changes. A limitation of the classical EAE model is a neglect of CD8 T cell mediated immune mechanisms. Moreover, well characterized models for primary progressive MS or demyelination patterns involving primary oligodendrocyte dystrophy are still not available. Yet many current therapeutic concepts including glatiramer acetate or natalizumab stem from their successful first application in EAE models. New strategies include the widespread use of conditional knockout mice to understand the cell-type specific function of single genes, innovative approaches to establish models on the roles of B cells and CD8 T cells as well as on the relation of inflammation to primary degeneration. In summary, EAE models continue to play an important role in neuroimmunology thereby also stimulating research in other fields of the neurosciences and immunobiology.Multiple sclerosis (MS) is the most common neurologic disease of young adults. In the recent years, our understanding on disease pathomechanisms has considerably improved and new therapies have emerged. Yet a cure for this devastating disorder is still a far cry away and human resources on  The model of experimental autoimmune encephalomyelitis (EAE) stems back from an attempt to understand the pathogenesis of post vaccinal encephalomyelitis, e.g., after rabies vaccination during the 1920s and 1930s . At that time, first immunization experiments with repeated inoculations of human spinal cord homogenate in rabbits and rhesus monkeys resulted in the first models of experimentally induced encephalomyelitis. Over the next 70-80 years, EAE was studied in many different species, including primates and rodents. Improved protocols included the application of a mineral oil-based adjuvant (Freund's adjuvant), and later on the addition of heat-inactivated mycobacteria  that facilitate disease induction after a single inoculation. Some models, especially in mice, also require the intraperitoneal injection of pertussis toxin which may deplete regulatory T cells and thus enhance autoimmune reactions [After World War II, EAE models in rats and guinea pigs dominated neuroimmunological research for many decades. In these species, active immunization used either spinal cord homogenate, myelin or, as first purified antigen in the 1960s, myelin basic protein (MBP) in CFA. Such protocols resulted in a very reproducible disease with an onset 9-12 days after immunization and an acute monophasic or chronic relapsing-remitting/progressive disease course depending on the species or rodent strain . In the The induction of EAE is not restricted to myelin antigens, as the disease can also be elicited by astroglial antigens like S-100 or glial fibrillary acidic protein (for review see ) and neuOver the last 20-30 years, further encephalitogenic myelin antigens were identified, the first of which was proteolipid protein (PLP). Other myelin autoantigens in rodents include myelin oligodendrocyte glycoprotein  research was marked by the characterization of MOG as an autoantigen. Although only a minor component of CNS myelin, MOG is part of the outer myelin layer thus being of special interest as target of a first autoimmune attack before antigen spreading may occur. Immunization with MOG induces not only encephalitogenic T cells, but also a demyelinating autoantibody response in susceptible species. Moreover, demyelinating anti-MOG antibodies enhance disease severity causing extensive demyelination after T cell-mediated CNS inflammation in rat and primate EAE models ,12. SuchIn mice, MOG protein or MOG35-55 peptide can induce EAE in C57BL/6 mice or Biozzi ABH mice resulting in a relapsing-remitting or chronic disease course. In the C57BL/6 mouse strain, the H-2b MHC haplotype governs the ability to induce encephalitogenicity and to develop demyelinating autoantibodies in response to mouse or rat MOG. Transfer of these demyelinating anti-MOG antibodies also enhances demyelination and exacerbates disease severity in mouse models of EAE . Over thIn summary, EAE models mimic many of the clinical, neuropathological and immunological aspects of MS but none is able to reproduce the whole spectrum of the human disease. In particular, many of the classical EAE models mainly affect the spinal cord (and not the brain) and are especially based on CD4 positive T cells, while the recently recognized importance of CD8 positive T cells in autoimmune inflammation of the CNS remains largely scotomized. So far, a model for the primary progressive course of the disease is still lacking and induction of EAE results in extensive tissue damage (including axons and neurons) rather than in primary demyelination. Nevertheless, the data from EAE studies still provide the most important cue for the notion of MS as an autoimmune disease where myelin specific cells initiate an inflammatory reaction in the CNS which ultimately leads to demyelination, gliosis and axonal injury.Research in EAE first focused on the adaptive immune system including T and B cell responses. Recently, components of the innate immune system, in particular macrophages, microglia, Toll-like receptors and even mast cells, have also been recognized as important parts of disease pathogenesis ,21. In pThe pivotal role of T lymphocytes in disease induction was confirmed by adoptive transfer of autoantigen specific T cells . Later oDuring T cell activation, the production of cytokines and chemokines influences the local microenvironment and finally determines the functional outcome of the immune response. In a classical paradigm, IFN-γ results in the differentiation of naive CD4 positive T cells into a "proinflammatory Th1" effector T cell subset which may initiate autoimmunity in the CNS . In contThe invasion of all these immune cells into the CNS requires transmigration over the blood brain barrier. Here, a complex molecular interaction takes place between adhesion molecules on the surface of immune cells and the cerebral endothelial cells forming the blood brain barrier ,46. ThesWhile Th17 cells promote autoimmune tissue inflammation, several regulatory cell types contribute to the delicate balance between autoimmunity and immunoregulation. In EAE, a regulatory role was first established for CD8 positive effector cells  and lateClassical histopathological changes in MS lesions comprise perivascular inflammation, demyelination and gliosis in the white matter. Over the past 10 years, this concept has been refined and axonal injury, cortical demyelination as well as neuronal changes have come into the focus of interest. In acute demyelinating MS lesions, distinct patterns have been described pointing to patient-specific mechanisms of demyelination. In this classification, pattern I is characterized by the presence of T cells and macrophages and pattern II by antibody and complement deposition. In contrast, patterns III and IV are mediated by primary OL damage. In pattern III, an ischemic mechanism leading to loss of MAG, but not of MOG immunoreactivity, was postulated . So far,To a large extent, inflammatory demyelination in MOG-EAE is paralleled by apoptotic loss of OL in the lesions . Thus, oIn recent years, further research focused on the presence of grey matter demyelination in the cortex. Although this specific feature is not predominant in murine MOG-EAE, cortical demyelination was well characterized in a focal model of rat MOG-EAE  and in s2+ and Na+ permeable acid-sensing ion channel-1 [With the avenue of confocal and magnetic resonance imaging techniques, the importance of axonal damage re-entered the stage in neuroimmunological research . In rat hannel-1 . Finallyhannel-1 . Recent hannel-1  while sohannel-1 . Moreovehannel-1 ), althouEAE models may not only serve for the characterization of immunological and neurobiological mechanisms, but can also be valuable for the pre-clinical testing of new diagnostic or treatment modalities. Different magnetic resonance imaging (MRI) techniques have been employed to depict EAE lesions in rat brains, including contrast enhancement with gadolinium or ultra small iron particles , which aRegarding treatment approaches, many established present-day MS therapies like glatiramer acetate as well as natalizumab stem from their first successful application in EAE models. Yet a plethora of new therapeutic approaches are effective in animal models, but have failed in human trials. Well known failures include anti-TNF therapies in MS patients  as well Future directions include EAE studies in conditional knockout mice to carve out the contribution of single molecules to the induction or effector phase of EAE. First studies used the Cre loxP system to conditionally delete genes involved in nuclear factor-κB (NF-κB) signalling pathways . TogethMoreover, the characterization of neurofascin as a novel autoantigen on axons at the node of Ranvier  and the CD8 T cell restricted autoimmune reactions can also be studied after conditional expression of MHC-I as shown in the muscle  or in doSo far, EAE models cover the spectrum of demyelination patterns I and II, but they do not well reflect patterns III and IV with primary OL dystrophy. Yet, this obstacle may be overcome by the analysis of mutants with primary demyelination due to a genetic defect. This was recently suggested in a study of mice with peroxisome-deficient OL. Here, a metabolic defect in myelin producing cells led to demyelination and subsequently also CNS inflammation with infiltration of B cells and activated CD8 positive T cells , similarFinally, all these data from EAE models may also promote knowledge on immune reactions in other neurologic diseases, like infectious disorders of the CNS  or post-The authors declare that they have no competing interests.RAL and DHL performed the literature search and wrote the manuscript.