Ocular distribution, spectrum of activity, and in vivo viral neutralization of a fully humanized anti-herpes simplex virus IgG Fab fragment following topical application.

Herpes simplex ocular infection is a major cause of corneal blindness. Local antiviral treatments exist but are associated with corneal toxicity, and resistance has become an issue. We evaluated the biodistribution and efficacy of a humanized anti-herpes simplex virus (anti-HSV) IgG FAb fragment (AC-8; 53 kDa) following repeated topical administration. AC-8 was found in the corneal epithelium, anterior stroma, subepithelial stromal cells, and retinal glial cells, with preferential entry through the ocular limbus. AC-8 was active against 13 different strains of HSV-1, with 50% and 90% mean effective concentrations (MEC(50) and MEC(90), respectively) ranging from 0.03 to 0.13 μg/ml, indicating broad-spectrum activity. The in vivo efficacy of AC-8 was evaluated in a mouse model of herpes-induced ocular disease. Treatment with low-dose AC-8 (1 mg/ml) slightly reduced the ocular disease scores. A greater reduction of the disease scores was observed in the 10-mg/ml AC-8-treated group, but not as much as with trifluridine (TFT). AC-8 treatment reduced viral titers but less than trifluridine. AC-8 did not display any toxicity to the cornea or other structures in the eye. In summary, topical instillation of an anti-HSV FAb can be used on both intact and ulcerated corneas. It is well tolerated and does not alter reepithelialization. Further studies to improve the antiviral effect are needed for AC-8 to be considered for therapeutic use.

Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909.

The induction of potent CD8+ T cell responses by vaccines to fight microbes or tumors remains a major challenge, as many candidates for human vaccines have proved to be poorly immunogenic. Deoxycytidyl-deoxyguanosin oligodeoxynucleotides (CpG ODNs) trigger Toll-like receptor 9, resulting in dendritic cell maturation that can enhance immunogenicity of peptide-based vaccines in mice. We tested whether a synthetic ODN, CpG 7909, could improve human tumor antigen-specific CD8+ T cell responses. Eight HLA-A2+ melanoma patients received 4 monthly vaccinations of low-dose CpG 7909 mixed with melanoma antigen A (Melan-A; identical to MART-1) analog peptide and incomplete Freund's adjuvant. All patients exhibited rapid and strong antigen-specific T cell responses: the frequency of Melan-A-specific T cells reached over 3% of circulating CD8+ T cells. This was one order of magnitude higher than the frequency seen in 8 control patients treated similarly but without CpG and 1-3 orders of magnitude higher than that seen in previous studies with synthetic vaccines. The enhanced T cell populations consisted primarily of effector memory cells, which in part secreted IFN- and expressed granzyme B and perforin ex vivo. In vitro, T cell clones recognized and killed melanoma cells in an antigen-specific manner. Thus, CpG 7909 is an efficient vaccine adjuvant that promotes strong antigen-specific CD8+ T cell responses in humans.

Phase I and phase II xenobiotic reactions and metabolism of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in aggregating liver cell cultures.

Aggregating fetal liver cell cultures were tested for their ability to metabolize xenobiotics using ethoxycoumarin-O-deethylase (ECOD), as marker of phase I metabolism, and glutathione S-transferase (GST), as marker for phase II reactions. Significant basal activities, stable over 14 days in culture were measured for both ECOD and GST activities. The prototype cytochrome P450 inducers, 3-methylcholanthrene (3-MC) and phenobarbital (PB), increased ECOD and GST activities reaching an optimum 7 days after culturing, followed by a decline in activity. This decline was partially prevented by 1% dimethyl sulfoxide (DMSO) added chronically to the culture medium. DMSO was also found to induce ECOD activity and to a lesser extent GST activity. Furthermore, it potentiated in a dose-dependent manner the induction of ECOD by PB. The food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is metabolically transformed through a number of pathways in vivo. It was therefore used to examine the metabolic capacity in fetal and adult liver cell aggregates. Metabolism of MeIQx was mainly through N2-conjugation, resulting in formation of the N2-glucuronide and sulfamate conjugates for non-induced fetal liver cells. These metabolites were also found in large amounts in non-induced adult liver cells. Low levels of cytochrome P450-mediated ring-hydroxylated metabolites were detected in both non-induced fetal and adult liver cells. After induction with arochlor (PCB) or 3-MC, the major pathway was ring-hydroxylation (cytochrome P450 dependent), followed by conjugation to beta-glucuronic or sulfuric acid. The presence of the glucuronide conjugate of N-hydroxy-MeIQx, a mutagenic metabolite, suggested an induction of P450 CYP1A2. The metabolism of MeIQx by liver cell aggregates is very similar to that observed in vivo and suggests that aggregating liver cell cultures are a useful model for in vitro metabolic studies in toxicology.

Intracortical connectivity of layer VI pyramidal cells in the barrel cortex of mice

ABSTRACT : The whisker-to-barrel pathway of rodents is formed by a series of somatotopic projections from the mystacial whisker follicles to the layer IV of the primary somatosensory cortex such that each follicle corresponds to a cluster of cortical neurons called barrel. Barrels are present in layer IV but form part of functional columns that comprise the entire depth of the somatosensory cortex. Interestingly, the cortex of the barrelless mouse strain (BRL) is organized such a manner that thalamocortical afferents do not remodel their projections in layer IV and barrels fail to appear. Nevertheless, functionally, a columnar organization persists, indicating that functional columns are not only provided by thalamocortical projections and layer IV cells. Since in the visual cortex of cats, layer VI cells contribute to the response properties of layer IV neurons, we wonder whether layer VI pyramidal cells could contribute to the columnar organization of the primary somatosensory cortex of mice. To address -this question, we morphologically analyzed the distribution of intracortical axon collaterals of layer VI neurons after in-vivo juxtacellular injections of biocytin in the C2 barrel column. Injected hemispheres were tangentially serial cut and intracortical collaterals of individual layer VI neurons were reconstructed at the light microscopic level. The position of axonal boutons was recorded to evaluate the distribution of presumed synaptic contacts. In normal (NOR) mice, cluster analysis shows that layer VI pyramidal cells can be classified in four statistically different clusters of neurons. Moreover, we assume that two classes are formed by cortico-cortical neurons and two classes are formed by cortico-thalamic neurons. Looking at the direction of the main axon in the white matter, we noticed that its orientation correlates perfectly with the type of neuron: cortico-cortical neurons send main axon medially whereas cortico-thalamic neurons send main axon laterally. Performing the same study in the BRL strain, we showed that the BRL mutation affects layer VI pyramidal cells tangentially and radially: the effects of the mutation are illustrated by a significant decrease of the index of colurnnarization and a significant decrease of percentage of boutons in granular and supragranular layers comparing to NOR neurons. In spite of these differences, the same four classes of layer VI neurons have been found in BRL mice. Using a tangential analysis of the boutons distribution, we showed that putative synapses are distributed mainly in the C2 barrel column. This was observed for each layer, type of neuron, cluster or strain, indicating that layer VI pyramidal cells could participate to the functional columnar organization of the barrel cortex. To determine post-synaptic partners of layer VI neurons in layer IV, we conducted an ultrastructural analysis of layer VI-to-IV contacts. We showed that synapses principally occur on spines and spiny dendritic shafts, supposed to belong to excitatory neurons. We furthermore showed that pre-synaptic elements are significantly different between en passant and terminaux contacts, which support hypothesis that terminaux boutons should show longer duration of facilitation than en passant boutons. RÉSUMÉ : Le «whisker-to-barrel pathway» des rongeurs est caractérisé par une série de projections somatotopiques depuis les follicules des moustaches ('whiskers') jusqu'à la couche IV de l'aire somatosensorielle primaire, de telle façon que chaque follicule corresponde à un groupe de neurones corticaux appelés tonneaux (`barrels'). Les tonneaux sont seulement présents en couche IV mais font partie de colonnes fonctionnelles qui s'étendent sur toute la profondeur du cortex somatosensoriel. Chez les souris mutantes barrelless (BRL), le cortex somatosensoriel est organisé de façon telle que lés afférences thalamocorticales ne remodellent pas leurs projections en couche IV et que les tonneaux n'apparaissent pas. Fonctionnellement, pourtant, une organisation en colonnes persiste, ce qui indique que les colonnes fonctionnelles ne sont pas uniquement produites par les projections thalamocorticales et par les cellules de la couche IV. Puisque les cellules de la couche VI contribuent à influencer les réponses des cellules de la couche IV dans le cortex visuel du chat, nous nous sommes demandé si ces cellules ne pourraient pas aussi contribuer à l'organisation en colonnes du cortex somatosensoriel primaire de la souris. Pour répondre à cette question, nous avons analysé de façon morphologique la distribution intracorticale des collatéraux axonaux de neurones de la couche VI. Suite à des injections juxtacellulaires de biocytine in-vivo dans la colonne C2, les hémisphères cérébraux ont été tangentiellement coupés en série et les collatéraux intracorticaux des neurones de la couche VI ont été reconstruits en microscopie optique. La position des boutons axonaux a aussi été enregistrée pour évaluer la distribution des contacts synpptiques potentiels. Chez les souris NOR, une analyse multivariée montre que les cellules pyramidales de la couche VI sont distribuées en quatre classes. Deux de ces classes sont probablement formées de neurons cortico-corticaux, alors que les deux autres sont probablement formées de neurones corticothalamiques. En observant la direction de l'axone principal dans la matière blanche, nous avons noté que son orientation est parfaitement corrélée avec le type supposé de neurone : les neurones corticocorticaux envoient leurs axones principaux médiallement, alors que les neurons cortico-thalamiques envoient leurs axones principaux latéralement. En menant la même étude chez les souris BRL, nous avons montré que la mutation affecte les cellules pyramidales de la couche VI de façon tangentielle, mais aussi radiaire : les effets de 1a mutation se traduisent par une diminution significative de l'index de « columnarization » et de la connectivité en couches granulaire et supragranulaire. Malgré ces différences, les quatre mêmes classes de neurones ont été retrouvées. En utilisant une analyse tangentielle de la distribution des boutons, nous avons montré que les synapses potentielles sont distribuées principalement dans la colonne C2. Cette observation a été faite dans chaque couche, chaque type de neurones, chaque classe de neurones et chaque souche de souris, indicant que les cellules de la couche VI participent certainement à l'organisation en colonne du cortex somatosensoriel. Pour déterminer les partenaires post-synaptiques des cellules de la couche VI en couche IV, nous avons conduit une analyse ultrastructurelle de ces contacts. Nous avons montré que les synapses interviennent principalement sur les épines et sur les dendrites supposés appartenir à des cellules excitatrices. Nous avons aussi montré que les éléments pré-synaptiques de ces synapses sont significativement differents selon le type de bouton, en passant ou terminal, ce qui supporte l'hypothèse que les boutons terminaux seraient capables d'une plus longue facilitation.

CD8 beta increases CD8 coreceptor function and participation in TCR-ligand binding.

To study the role of CD8 beta in T cell function, we derived a CD8 alpha/beta-(CD8-/-) T cell hybridoma of the H-2Kd-restricted N9 cytotoxic T lymphocyte clone specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260. This hybridoma was transfected either with CD8 alpha alone or together with CD8 beta. All three hybridomas released interleukin 2 upon incubation with L cells expressing Kd-peptide derivative complexes, though CD8 alpha/beta cells did so more efficiently than CD8 alpha/alpha and especially CD8-/- cells. More strikingly, only CD8 alpha/beta cells were able to recognize a weak agonist peptide derivative variant. This recognition was abolished by Fab' fragments of the anti-Kd alpha 3 monoclonal antibody SF1-1.1.1 or substitution of Kd D-227 with K, both conditions known to impair CD8 coreceptor function. T cell receptor (TCR) photoaffinity labeling indicated that TCR-ligand binding on CD8 alpha/beta cells was approximately 5- and 20-fold more avid than on CD8 alpha/a and CD8-/- cells, respectively. SF1-1.1.1 Fab' or Kd mutation D227K reduced the TCR photoaffinity labeling on CD8 alpha/beta cells to approximately the same low levels observed on CD8-/- cells. These results indicate that CD8 alpha/beta is a more efficient coreceptor than CD8alpha/alpha, because it more avidly strengthens TCR-ligand binding.

Chemotherapy induces tumor metastasis and dormancy

Chemotherapy is widely used as a systemic treatment modality in cancer patients and provides survival benefits for a significant fraction of treated patients H However, some patients suffer from cancer relapse and rapidly progress to metastasis, suggesting that following chemotherapy their residual tumor developed a more aggressive phenotype 4 5. Although some molecular mechanisms involved in chemo-resistance and chemotherapy-induced metastatic relapse have been reported, more investigations and understanding of these processes are necessary before any translation into the clinic might be considered. By using the syngeneic metastatic 4T1 murine breast cancer model, we observed that chemotherapy treatment and selection of chemotherapy-resistant cancer cells in vitro can induces two opposite phenotypes: a dormant one and a relapsing-metastatic one. Previous studies in our laboratory demonstrated that irradiation of mammary gland promotes tumor metastasis, at least in part, by inducing the recruitment of CD11b+ cells to both the primary tumor and the lungs at a pre-metastatic stage. In this study we found that CD11b+ cells may also play important roles in chemotherapy-induced tumor metastasis and dormancy in vivo. Tumor cells expressing the stem cell marker Sca-1 were enriched by chemotherapy treatment in vitro, as well as in tumor metastasis in vivo. Furthermore, tumor-derived CD11b+ cells were capable to maintain and expand this population in vitro. These results suggest that the expansion of a tumor cell population with stem cell features might be a mechanism by which chemotherapy induces metastasis. On the other hand, the same drug treatment in vitro generated resistant cells with a dormant phenotype. Dormant tumor cells were able to induce an in vivo immune- inflammatory response in the draining lymph node, which is normally absent due to the immunosuppressive effects of tumor-recruited myeloid derived- suppressor cells (MDSCs). Genome-wide gene expression analysis revealed the enrichment of invasion and metastasis-related genes in the relapsing metastatic tumor cells and immune response-related genes in the dormant tumor cells. Interestingly, CD11b+ cells derived from the microenvironment of growing-metastatic tumors, but not CD11b+ cells derived from the spleen of tumor-free mice, were able to instigate outgrowth of dormant tumor cells in vivo. Also, dormant cells formed growing and metastatic tumors when injected into immune-compromised NGS mice. These results point to a role of chemotherapy in enabling treated tumor cells to acquire immune response-inducing capabilities, while impairing the recruitment of CD11b+ cells and their differentiation into an immune-suppressive cell. The molecular mechanisms underneath these effects are being further investigated. In conclusion, results obtained in this model indicate that chemotherapy can induce a dormant phenotype in cancer cells and that this state of dormancy can be broken by MDSCs educated by relapsing tumors. Understanding the mechanism beyond these effects, in particular unraveling the genetic or epigenetic determinants of dormancy vs relapse, might open the way to therapies aimed and maintaining residual cells escaping chemotherapy in a state of sustained dormancy. - La chimiothérapie est un traitement systémique largement utilisé chez les patients cancéreux qui donne un avantage de survie significatif pour une bonne partie de patients traités (1-3). Cependant, certains patients souffrent d'une rechute et progressent ensuite vers la métastase. Ceci suggère que leur tumeur résiduelle a développé un phénotype agressif suite à la chimiothérapie (4-5). Bien que certains mécanismes moléculaires impliqués dans la chimiorésistance et la rechute métastatique ont été identifiés, d'avantage d'études sont nécessaires afin de mieux comprendre ce phénomène et de développer des nouvelles thérapies cliniques. En utilisant un modèle syngénique de cancer du sein métastatique chez la sourie (4T1), nous avons observé que la sélection des cellules cancéreuses résistantes à la chimiothérapie in vitro peut induire deux phénotypes opposés: un phénotype de dormance et un phénotype de progression métastatique. Une étude précédente issue de notre laboratoire a démontré que l'irradiation de la glande mammaire favorise la métastase de tumeurs recourants suite au recrutement de cellules CD11b+ dans la tumeur primaire et dans les poumons pré-métastatiques. Dans notre étude nous avons constaté que les cellules CD11b+ peuvent également jouer un rôle important dans la formation de métastases induites par la chimiothérapie ainsi que dans le maintien de la dormance in vivo. Nous avons également observé un enrichissement de cellules tumorales exprimant le marqueur de cellule souche Sca-1 parmi les cellules tumorales résistantes à la chimiothérapie et dans les cellules qui on formé des métastases in vivo. Des cellules CD11b+ dérivées du microenvironnement tumorale favorisent l'expansion de la population de cellules tumorales Sca-1+ in vitro. Ces résultats suggèrent que l'expansion d'une population de cellules tumorales avec des caractéristiques de cellules souches pourrait constituer un mécanisme par lequel la chimiothérapie induit des métastases dans des tumeurs récurrentes. D'autre part le même traitement de chimiothérapie peut générer des cellules résistantes avec un phénotype dormant. Les expériences in vivo indiquent que les cellules tumorales dormantes induisent une réponse immunitaire inflammatoire dans le ganglion lymphatique de drainage, qui est normalement réprimée par des cellules myéloïdes suppressives de tumeur (MDSC). Une analyse d'expression de gènes a révélé l'enrichissement de gènes liés à l'invasion et à la métastase dans les cellules tumorales récurrentes et des gènes liés à la réponse immunitaire dans les cellules tumorales dormantes. Les cellules CD11b+ issues du microenvironnement des tumeurs récurrents ont incité la croissance des cellules tumorales dormantes in vivo, tandis que les cellules CD11b+ dérivées de la rate de souris non porteuses de tumeur ne l'étaient pas. Les mécanismes moléculaires sous-jacents restent à découvrir. En conclusion, les résultats obtenus dans ce modèle indiquent que la chimiothérapie pourrait favoriser non seulement l'induction d'une dormance cellulaire, mais également que les cellules dormantes seraient adroits de induire une réponse immunitaire capable les maintenir dans un état de dormance prolongé. Un déséquilibre dans cette réponse immunitaire pourrait des lors briser cet état de dormance et induire une progression tumorale. Comprendre les mécanismes responsables de ces effets, en particulier l'identification des déterminants génétiques ou épigénétiques liés à la dormance vs la rechute, pourraient ouvrir la voie à des nouvelles thérapies visant le maintien d'un état de dormance permanente des cellules résiduelles après chimiothérapie.

HLA photoaffinity labeling reveals overlapping binding of homologous melanoma-associated gene peptides by HLA-A1, HLA-A29, and HLA-B44.

Melanoma-associated genes (MAGEs) encode tumor-specific antigens that can be recognized by CD8+ cytotoxic T lymphocytes. To investigate the interaction of the HLA-A1-restricted MAGE-1 peptide 161-169 (EADPT-GHSY) with HLA class I molecules, photoreactive derivatives were prepared by single amino acid substitution with N beta-[iodo-4-azidosalicyloyl]-L-2,3-diaminopropionic acid. These derivatives were tested for their ability to bind to, and to photoaffinity-label, HLA-A1 on C1R.A1 cells. Only the derivatives containing the photoreactive amino acid in position 1 or 7 fulfilled both criteria. Testing the former derivative on 14 lymphoid cell lines expressing over 44 different HLA class I molecules indicated that it efficiently photoaffinity-labeled not only HLA-A1, but possibility also HLA-A29 and HLA-B44. MAGE peptide binding by HLA-A29 and HLA-B44 was confirmed by photoaffinity labeling with photoreactive MAGE-3 peptide derivatives on C1R.A29 and C1R.B44 cells, respectively. The different photoaffinity labeling systems were used to access the ability of the homologous peptides derived from MAGE-1, -2, -3, -4a, -4b, -6, and -12 to bind to HLA-A1, HLA-A29, and HLA-B44. All but the MAGE-2 and MAGE-12 nonapeptides efficiently inhibited photoaffinity labeling of HLA-A1, which is in agreement with the known HLA-A1 peptide-binding motif (acidic residue in P3 and C-terminal tyrosine). In contrast, photoaffinity labeling of HLA-A29 was efficiently inhibited by these as well as by the MAGE-3 and MAGE-6 nonapeptides. Finally, the HLA-B44 photoaffinity labeling, unlike the HLA-A1 and HLA-A29 labeling, was inhibited more efficiently by the corresponding MAGE decapeptides, which is consistent with the reported HLA-B44 peptide-binding motif (glutamic acid in P2, and C-terminal tyrosine or phenylalanine). The overlapping binding of homologous MAGE peptides by HLA-A1, A29, and B44 is based on different binding principles and may have implications for immunotherapy of MAGE-positive tumors.

Selective absence of CD8+ TCRalpha beta+ intestinal epithelial cells in transgenic mice expressing beta2-microglobulin-associated ligands exclusively on thymic cortical epithelium.

Whereas interactions between the TCRalpha beta and self MHC:peptide complexes are clearly required for positive selection of mature CD4(+) and CD8(+) T cells during intrathymic development, the role of self or foreign ligands in maintaining the peripheral T cell repertoire is still controversial. In this report we have utilized keratin 14-beta2-microglobulin (K14-beta2m)-transgenic mice expressing beta2m-associated ligands exclusively on thymic cortical epithelial cells to address the possible influence of TCR:ligand interactions in peripheral CD8(+) T cell homeostasis. Our data indicate that CD8(+) T cells in peripheral lymphoid tissues are present in normal numbers in the absence of self MHC class I:peptide ligands. Surprisingly, however, steady state homeostasis of CD8(+) T cells in the intestinal epithelium is severely affected by the absence of beta2m-associated ligands. Indeed TCRalpha beta(+) IEL subsets expressing CD8alpha beta or CD8alpha alpha are both dramatically reduced in K14-beta2m mice, suggesting that the development, survival or expansion of CD8(+) IEL depends upon interaction of the TCR with MHC class I:peptide or other beta2m-associated ligands elsewhere than on thymic cortical epithelium. Collectively, our data reveal an unexpected difference in the regulation of CD8(+) T cell homeostasis by beta2m-associated ligands in the intestine as compared to peripheral lymphoid organs.

Differential distribution of thyroid hormone receptor isoform in rat dorsal root ganglia and sciatic nerve in vivo and in vitro.

Using autoradiographic techniques carried out under precise conditions we previously demonstrated that both sensory neurons and peripheral glial cells in dorsal root ganglia (DRG) or sciatic nerve, possess specific [125I]-labeled T3 binding sites. Thyroid hormone receptors (TR) include several isoforms (TR alpha(1), TR alpha(2), TR beta(1), TR beta(2...)) The present study demonstrates that while sensory neurons and peripheral glial cells both possess functional TR, they express a differential expression of TR isoforms. Using a panel of antisera to specific for the TR alpha-common (alpha(1) and alpha(2)), TR alpha-1 or TR beta-1 isoforms, we detected TRs isoform localization at the cellular level during DRG and sciatic nerve development and regeneration. Immunohistochemical analysis revealed that during embryonic life, sensory neurons express TR alpha-common and TR beta-1 rather than TR alpha-1. The number of TR alpha-common and TR beta-1 positive neurons as well as the intensity of labeling increased during the first two postnatal weeks and remained more or less stable in adult life. TR alpha-1 immunoreactivity, which was undetectable in embryonic sensory neurons, became discreetly visible in neurons after birth. In developing DRG and sciatic nerves, Schwann cells exhibited TR alpha-common and TR alpha-1 rather than TR beta-1 immunolabeling. The appearance of TR alpha-common and alpha-1 isoform immunoreactivity in the sciatic nerve was restricted to a short period ranging from E17 up to two postnatal weeks. By comparing TR alpha-common and TR alpha-1 immunostaining we can deduce that Schwann cells primarily express TR alpha-1. Afterwards, in adult rat sciatic nerve TR alpha isoforms was no more detected. However transection of sciatic nerve caused a reexpression of TR alpha isoforms in degenerating nerve. The prevalence of TR alpha in Schwann cells in vivo was correlated with in vitro results. The differential expression of TR alpha and beta by sensory neurons and Schwann cells indicates that the feedback regulation of circulating thyroid hormone could occur by binding to either the alpha or beta TR isoforms. Moreover, the presence of multiple receptor isoforms in developing sensory neurons suggests that thyroid hormone uses multiple signaling pathways to regulate DRG and sciatic nerve development.

I. Identification and characterisation of epigenetically altered tumor supressor genes by proteomic and genomic approches / II. Studies of genes regualted by MeCP2 union to its promoter regions by proteomic and genmomic approches

DNA methylation has an important impact on normal cell physiology, thus any defects in this mechanism may be related to the development of various diseases In this project we are interested in identifying epigeneticaliy modified genes, in general controlled by processes related to the DNA methylation, by means of a new strategy combining protomic and genomic analyses. First, the two Dimensional-Difference Gel Electrophoresis (2-DIGE) protein analyses of extracts obtained from HCT-116 wt and double knockout for DNMT1 and DNMT3b (DKO) cells revealed 34 proteins overexpressed in the condition of DNMTs depletion. From five genes with higher transcript lavels in DKO cells, comparing with HCT-116 wt. oniy AKR1B1, UCHLl and VIM are melhylated in HCT-116. As expected. the DNA methvlation 1s lost in DKO cells. The rneth,vl ation of VIM and UCHLl promoters in some cancer samples has already been repaired, thus further studies has been focused on AKRlBI. AKR1B1 expression due lo DNA methyiaton of promoter region seems to occur specilfically in the colon cancer cell Iines. which was confirmed in the DNA rnethylation status and expression analyses. performed on 32 different cancer cell lines (including colon, breast, lymphoma, leukemia, neuroblastoma, glioma and lung cancer cell Iines) as well as normal colon and normal lymphocytes samples. AKRIBI expression after treatments with DNA demethvlating agent (AZA) was rescued in 5 coloncancer cell lines (including genetic regulation of the candidate gene. The methylation status of the rest of the genes identified in proteomic analysis was checked by methylation specific PCR (MSP) experiment and all appeared to be unmethylated. The similar research has been done also bv means of Mecp2-null mouse model For 14 selected candidate genes the analyses of expression leveis, methylation Status and MeCP2 interaction with promoters are currently being performed.

Neurotensin is a regulator of insulin secretion in pancreatic beta-cells.

Neurotensin (NT) is secreted from neurons and gastrointestinal endocrine cells. We previously reported that the three NT receptors (NTSRs) are expressed in pancreatic islets and beta cell lines on which we observed a protective effect of NT against cytotoxic agents. In this study, we explored the role of NT on insulin secretion in the endocrine pancreatic beta cells. We observed that NT stimulates insulin secretion at low glucose level and has a small inhibiting effect on stimulated insulin secretion from isolated islets or INS-1E cells. We studied the mechanisms by which NT elicited calcium concentration changes using fura-2 loaded islets or INS-1E cells. NT increases calcium influx through the opening of cationic channels. Similar calcium influxes were observed after treatment with NTSR selective ligands. NT-evoked calcium regulation involves PKC and the translocation of PKCalpha and PKCepsilon to the plasma membrane. Part of NT effects appears to be also mediated by PKA but not via the Erk pathway. Taken together, these data provide evidence for an important endocrine role of NT in the regulation of the secretory function of beta cells.

Reduction in the expression of glucose transporter protein GLUT 2 in preneoplastic and neoplastic hepatic lesions and reexpression of GLUT 1 in late stages of hepatocarcinogenesis.

Expression of two important glucose transporter proteins, GLUT 2 (which is the typical glucose transporter in hepatocytes of adult liver) and the erythroid/brain type glucose transporter GLUT 1 (representing the typical glucose transporter in fetal liver parenchyma), was studied immunocytochemically during hepatocarcinogenesis in rats at different time points between 7 and 65 wk after cessation of 7-wk administration of 12 mg/kg of body weight of N-nitrosomorpholine p.o. (stop model). Foci of altered hepatocytes excessively storing glycogen (GSF) and mixed cell foci (MCF) composed of both glycogenotic and glycogen-poor cells were present at all time points studied. Seven wk after withdrawal of the carcinogen, GSF were the predominant type of focus of altered hepatocytes. Morphometrical evaluation of the focal lesions revealed that the number and volume fraction of GSF increased steadily until Wk 65. MCF were rare at 7 wk, increased slightly in number and size until Wk 37, but showed a pronounced elevation in their number and volume fraction from Wk 37 to Wk 65. In both GSF and MCF, GLUT 2 was generally decreased or partially absent at all time points. Consequently, foci of decreased GLUT 2 expression showed a steady increase in number and volume fraction from Wk 7 to Wk 65. GLUT 1 was lacking in GSF but occurred in some MCF from Wk 50 onward. The liver type glucose transporter GLUT 2 was decreased in all adenomas and hepatocellular carcinomas (HCC). In three of seven adenomas and 10 of 12 carcinomas, expression of GLUT 1 was increased compared with normal liver parenchyma. In two cases of adenoid HCC, cells of ductular formations coexpressed GLUT 2 and GLUT 1. In contrast, normal bile ducts, bile duct proliferations, and cystic cholangiomas expressed only GLUT 1. Seven of 12 HCC contained many microvessels intensely stained for GLUT 1, a phenomenon never observed in normal liver. Whenever adenoid tumor formations occurred, GLUT 1-positive microvessels were located in the immediate vicinity of these formations. Only in one HCC were such microvessels found in the absence of adenoid formations. Our studies indicate that a reduction of GLUT 2 expression occurs already in early preneoplastic hepatic foci and is maintained throughout hepatocarcinogenesis, including benign and malignant neoplasms. Reexpression of GLUT 1, however, appears in a few MCF and in the majority of adenomas and carcinomas.

Implication of DNA methylation and PAX5 factor in the transcriptional regulation of hTERT, the human telomerase reverse transcriptase gene

RESUME La télomérase est une enzyme dite "d'immortalité" qui permet aux cellules de maintenir la longueur de leurs télomères, ce qui confère une capacité de réplication illimitée aux cellules reproductrices et cancéreuses. A l'inverse, les cellules somatiques normales, qui n'expriment pas la télomérase, ont une capacité de réplication limitée. La sous-unité catalytique de la télomérase, hTERT, est définie comme le facteur limitant l'activité télomérasique. Entre activateurs et répresseurs, le rôle de la méthylation de l'ADN et de l'acétylation des histones, de nombreux modèles ont été suggérés. La découverte de l'implication de CTCF dans la régulation transcriptionnelle de hTERT explique en partie le mécanisme de répression de la télomérase dans la plupart des cellules somatiques et sa réactivation dans les cellules tumorales. Dans les cellules télomérase-positives, l'activité inhibitrice de CTCF est bloquée par un mécanisme dépendent ou non de la méthylation. Dans la plupart des carcinomes, une hyperméthylation de la région 5' de hTERT bloque l'effet inhibiteur de CTCF, alors qu'une petite région hypométhylée permet un faible niveau de transcription du gène. Nous avons démontré que la protéine MBD2 se lie spécifiquement sur la région 5' méthylée de hTERT dans différentes lignées cellulaires et qu'elle est impliquée dans la répression partielle de la transcription de hTERT dans les cellules tumorales méthylées. Par contre, nous avons montré que dans les lymphocytes B normaux et néoplasiques, la régulation de hTERT est indépendante de la méthylation. Dans ces cellules, le facteur PAX5 se lie sur la région 5' de hTERT en aval du site d'initiation de la traduction (ATG). L'expression exogène de PAX5 dans les cellules télomérase-négatives active la transcription de hTERT, alors que la répression de PAX5 dans les cellules lymphomateuses inhibe la transcription du gène. PAX5 est donc directement impliqué dans l'activation de l'expression de hTERT dans les lymphocytes B exprimant la télomérase. Ces résultats révèlent des différences entre les niveaux de méthylation de hTERT dans les cellules de carcinomes et les lymphocytes B exprimant la télomérase. La méthylation de hTERT en tant que biomarqueur de cancer a été évaluée, puis appliquée à la détection de métastases. Nous avons ainsi montré que la méthylation de hTERT est positivement corrélée au diagnostic cytologique dans les liquides céphalorachidiens. Nos résultats conduisent à un modèle de régulation de hTERT, qui aide à comprendre comment la transcription de ce gène est régulée par CTCF, avec un mécanisme lié ou non à la méthylation du gène hTERT. La méthylation de hTERT s'est aussi révélée être un nouveau et prometteur biomarqueur de cancer. SUMMARY Human telomerase is an "immortalizing" enzyme that enables cells to maintain telomere length, allowing unlimited replicative capacity to reproductive and cancer cells. Conversely, normal somatic cells that do not express telomerase have a finite replicative capacity. The catalytic subunit of telomerase, hTERT, is defined as the limiting factor for telomerase activity. Between activators and repressors, and the role of DNA methylation and histone acetylation, an abundance of hTERT regulatory models have been suggested. The discovery of the implication of CTCF in the transcriptional regulation of hTERT in part explained the mechanism of silencing of telomerase in most somatic cells and its reactivation in neoplastic cells. In telomerase-positive cells, the inhibitory activity of CTCF is blocked by methylation-dependent and -independent mechanisms. In most carcinoma cells, hypermethylation of the hTERT 5' region has been shown to block the inhibitory effect of CTCF, while a short hypomethylated region allows a low transcription level of the gene. We have demonstrated that MBD2 protein specifically binds the methylated 5' region of hTERT in different cell lines and is therefore involved in the partial repression of hTERT transcription in methylated tumor cells. In contrast, we have shown that in normal and neoplastic B cells, hTERT regulation is methylation-independent. The PAX5 factor has been shown to bind to the hTERT 5'region downstream of the ATG translational start site. Ectopic expression of PAX5 in telomerase-negative cells or repression of PAX5 expression in B lymphoma cells respectively activated and repressed hTERT transcription. Thus, PAX5 is strongly implicated in hTERT expression activation in telomerase-positive B cells. These results reveal differences between the hTERT methylation patterns in telomerase-positive carcinoma cells and telomerase-positive normal B cells. The potential of hTERT methylation as a cancer biomarker was evaluated and applied to the detection of metastasis. We have shown that hTERT methylation correlates with the cytological diagnosis in cerebrospinal fluids. Our results suggest a model of hTERT gene regulation, which helps us to better understand how hTERT transcription is regulated by CTCF in methylation-dependant and independent mechanisms. Our data also indicate that hTERT methylation is a promising new cancer biomarker.

On the presence of hepatic stellate cells in portal spaces

Previous studies in mice with hypervitaminosis A have demonstrated that fat-storing cells (hepatic stellate cells-HSCs) participate in schistosomal granuloma fibrogenesis. The origin of such cells in portal areas, away from the Disse spaces, was herein investigated. HSCs were identified in frozen sections of the liver by means of Sudan III staining. They appeared as red-stained cells disposed along the sinusoids of normal mice, but were never found within portal spaces. However, in the chronically inflamed portal spaces of Capillaria hepatica-infected mice, Sudan III-positive cells were frequently present among leukocytes and fibroblast-like cells. Thus, there are no resident HSCs in portal spaces, but their presence there in chronic inflammatory processes indicates that they are able to migrate from peri-sinusoidal areas in order to reach the portal areas.

Fluid flow regulates stromal cell organization and CCL21 expression in a tissue-engineered lymph node microenvironment.

In the paracortex of the lymph node (LN), T zone fibroblastic reticular cells (TRCs) orchestrate an immune response by guiding lymphocyte migration both physically, by creating three-dimensional (3D) cell networks, and chemically, by secreting the chemokines CCL19 and CCL21 that direct interactions between CCR7-expressing cells, including mature dendritic cells and naive T cells. TRCs also enwrap matrix-based conduits that transport fluid from the subcapsular sinus to high endothelial venules, and fluid flow through the draining LN rapidly increases upon tissue injury or inflammation. To determine whether fluid flow affects TRC organization or function within a 3D network, we regenerated the 3D LN T zone stromal network by culturing murine TRC clones within a macroporous polyurethane scaffold containing type I collagen and Matrigel and applying slow interstitial flow (1-23 microm/min). We show that the 3D environment and slow interstitial flow are important regulators of TRC morphology, organization, and CCL21 secretion. Without flow, CCL21 expression could not be detected. Furthermore, when flow through the LN was blocked in mice in vivo, CCL21 gene expression was down-regulated within 2 h. These results highlight the importance of lymph flow as a homeostatic regulator of constitutive TRC activity and introduce the concept that increased lymph flow may act as an early inflammatory cue to enhance CCL21 expression by TRCs, thereby ensuring efficient immune cell trafficking, lymph sampling, and immune response induction.