Preclinical Studies with Umbilical Cord Mesenchymal Stromal Cells in Different Animal Models for Muscular Dystrophy

Umbilical cord mesenchymal stromal cells (MSC) have been widely investigated for cell-based therapy studies as an alternative source to bone marrow transplantation. Umbilical cord tissue is a rich source of MSCs with potential to derivate at least muscle, cartilage, fat, and bone cells in vitro. The possibility to replace the defective muscle cells using cell therapy is a promising approach for the treatment of progressive muscular dystrophies (PMDs), independently of the specific gene mutation. Therefore, preclinical studies in different models of muscular dystrophies are of utmost importance. The main objective of the present study is to evaluate if umbilical cord MSCs have the potential to reach and differentiate into muscle cells in vivo in two animal models of PMDs. In order to address this question we injected (1) human umbilical cord tissue (hUCT) MSCs into the caudal vein of SJL mice; (2) hUCT and canine umbilical cord vein (cUCV) MSCs intra-arterially in GRMD dogs. Our results here reported support the safety of the procedure and indicate that the injected cells could engraft in the host muscle in both animal models but could not differentiate into muscle cells. These observations may provide important information aiming future therapy for muscular dystrophies.

Systemic chemotherapy-induced microsatellite instability in the mononuclear cell fraction of women with breast cancer can be reproduced in vitro and abrogated by amifostine

Objectives Microsatellite instability (MSI) induction by alkylating agent-based chemotherapy (ACHT) may underlie both tumor resistance to chemotherapy and secondary leukaemias in cancer patients. We investigated if ACHT could induce MSI in tumor-derived plasma-circulating DNA (pfDNA) and in normal peripheral blood mononuclear (PBMN) cells. We also evaluated if amifostine could interfere with this process in an in-vitro model. Methods MSI was determined in pfDNA, PBMN cells and urine cell-free DNA (ufDNA) of 33 breast cancer patients before and after ACHT. MCF-7 cells and PBMN from normal donors were exposed in vitro to melphalan, with or without amifostine. Results We observed at least one MSI event in PBMN cells, pfDNA or ufDNA of 87, 80 and 80% of patients, respectively. In vitro, melphalan induced MSI in both MCF-7 and normal PBMN cells. In PBMN cells, ACHT-induced MSI occurred together with a significant decrease in the expression of the DNA mismatch repair gene hMSH2. Amifostine decreased hMSH2 expression and also prevented MSI induction only in normal PBMN cells. Conclusions ACHT induced MSI in PBMN cells and in tumour-derived pfDNA. Because of its protective effect against ACHT induction of MSI in normal PBMN cells in vitro, amifostine may be a potential agent for preventing secondary leukaemias in patients exposed to ACHT.

Leukotriene B(4) is essential for selective eosinophil recruitment following allergen challenge of CD4(+) cells in a model of chronic eosinophilic inflammation

Subcutaneous heat-coagulated egg white implants (EWI) induce chronic, intense local eosinophilia in mice, followed by asthma-like responses to airway ovalbumin challenge. Our goal was to define the mechanisms of selective eosinophil accumulation in the EWI model. EWI carriers were challenged i.p. with ovalbumin and the contributions of cellular immunity and inflammatory mediators to the resulting leukocyte accumulation were defined through cell transfer and pharmacological inhibition protocols. Eosinophil recruitment required Major Histocompatibility Complex Class It expression, and was abolished by the leukotriene B4 (LTB4) receptor antagonist CP 105.696, the 5-lipoxygenase inhibitor BWA4C and the 5-lipoxygenase activating protein inhibitor MK886. Eosinophil recruitment in EWI carriers followed transfer of: a) CD4(+) (but not CD4(-)) cells, harvested from EWI donors and restimulated ex vivo; b) their cell-free supernatants, containing LTB4. Restimulation in the presence of MK886 was ineffective. CC chemokine receptor ligand (CCL)5 and CCL2 were induced by ovalbumin challenge in vivo. mRNA for CCL17 and CCL11 was induced in ovalbumin-restimulated CD4(+) cells ex vivo. MK886 blocked induction of CCL17 Pretreatment of EWI carriers with MK886 eliminated the effectiveness of exogenously administered CCL11, CCL2 and CCL5. In conclusion, chemokine-producing, ovalburnin-restimulated CD4(+) cells initiate eosinophil recruitment which is strictly dependent on LTB4 production. (C) 2008 Elsevier Inc. All rights reserved.

The neuroprotective effect of dental pulp cells in models of Alzheimer`s and Parkinson`s disease

Aim of the present study was to investigate the neuroprotective effect of dental pulp cells (DPCs) in in vitro models of Alzheimer and Parkinson disease. Primary cultures of hippocampal and ventral mesencephalic neurons were treated for 24 h with amyloid beta (A beta(1-42)) peptide 1-42 and 6-OHDA, respectively. DPCs isolated from adult rat incisors were previously cultured in tissue culture inserts and added to the neuron cultures 2 days prior to neurotoxin treatment. Cell viability was assessed by the MTT assay. The co-culture with DPCs significantly attenuated 6-OHDA and A beta(1-42)-induced toxicity in primary cultures of mesencephalic and hippocampal neurons, and lead to an increase in neuronal viability in untreated cultures, suggesting a neurotrophic effect in both models. Furthermore, human dental pulp cells expressed a neuronal phenotype and produced the neurotrophic factors NGF, GDNF, BDNF, and BMP2 shown by microarray screening and antibody staining for the representative proteins. DPCs protected primary neurons in in vitro models of Alzheimer`s and Parkinson`s disease and can be viewed as possible candidates for studies on cell-based therapy.

Prolactin-releasing peptide in ewe: cDNA cloning, mRNA distribution and effects on prolactin secretion in vitro and in vivo

RT-PCR followed by 5'- and 3'- rapid amplification of cDNA ends was used to clone and sequence ovine prolactin-releasing peptide (PrRP). The cDNA was characterised by short 5'- and 3'-untranslated regions and a GC-rich (71%) coding region. The nucleotide and deduced amino acid sequences for the coding region showed 95.6 and 94.9% identity with bovine PrRP but the amino acid sequence of PrRP31 was conserved between these species. Northern blot analysis and RT-PCR showed that, as in the rat, the peptide was more abundantly expressed in the brainstem than the hypothalamus. However, in the ovine hypothalamus, PrRP mRNA expression was more widespread than in the rat, with expression detected in both rostral and caudal parts of the mediobasal hypothalamus. The effects of synthetic ovine PrRP on prolactin secretion both in vitro and in vivo were also examined. In primary cultures of sheep pituitary cells, PrRP significantly (P

SOX8 is expressed during testis differentiation in mice and synergizes with SF1 to activate the Amh promoter in vitro

Sox8 is a member of the Sox family of developmental transcription factor genes and is closely related to Sox9, a key gene in the testis determination pathway in mammals. Like Sox9, Sox8 is expressed in the developing mouse testis around the time of sex determination, suggesting that it might play a role in regulating the expression of testis-specific genes. An early step in male sex differentiation is the expression of anti-Mullerian hormone (AMH) in Sertoli cells. Expression of the Amh gene during sex differentiation requires the interaction of several transcription factors, including SF1, SOX9, GATA4, WT1, and DAX1. Here we show that SOX8 may also be involved in regulating the expression of Amh. Expression of Sox8 begins just prior to that of Amh at 12 days post coitum (dpc) in mouse testes and continues beyond 16 dpc in Sertoli cells. In vitro assays showed that SOX8 binds specifically to SOX binding sites within the Amh minimal promoter and, like SOX9, acts synergistically with SF1 through direct protein-protein interaction to enhance Amh expression, albeit at lower levels compared with SOX9. SOX8 and SOX9 appear to have arisen from a common ancestral gene and may have retained some common functions during sexual development. Our data provide the first evidence that SOX8 may partially compensate for the reduced SOX9 activity in campomelic dysplasia and substitute for Sox9 where Sox9 is either not expressed or expressed too late to be involved in sex determination or regulation of Amh expression.

Ultrastructural study of the TG180 murine sarcoma cell invasion by Toxoplasma gondii: comparison between in vivo and in vitro cell cultures

Infection of non-adherent TG180 murine sarcoma cells with Toxoplasma gondii was compared, at the ultrastructural level, in both in vivo and in vitro conditions. Suspensions of 3.0 x 10(6) TG180 cells infected in vitro with 1.0 x 10(6) parasites of the RH strain were harvested between the first and 6th day post-infection and processed for transmission electron microscopy. In vivo infection was made by intraperitoneal inoculation in mice of 1.0 x 10(6) TG180 cells, that were co-inoculated with a parasite suspension at the same cell concentration. Cells were harvested 10, 20, 30 min and 24, 48 h post-inoculation and processed for transmission electron microscopy at the same conditions of the in vitro culture. It was observed TG180 murine sarcoma cells with intense and equivalent intracellular parasitism in both conditions. Host cells with parasitophorous vacuoles containing up to 16 parasites, as well as parasites undergoing mitoses or presenting a bradyzoite-like morphology, were frequently seen in both culture methods.

Characterization of tumor antigen specific CD8+ T cells and semi-invariant Vα24/Vβ11 NKT cells in tumor invaded liver

Summary: Detailed knowledge on tumor antigen expression and specific immune cells is required for a rational design of immunotherapy for patients with tumor invaded liver. In this study, we confirmed that Cancer/Testis (CT) tumor-associated antigens are frequently expressed in hepatocellular carcinoma (HCC) and searched for the presence of CD8+ T cells specific for these antigens. In 2/10 HLA-A2+ patients with HCC, we found that MAGE-A10 and/or SSX-2 specific CD8+ T cells naturally responded to the disease, since they were enriched in tumor lesions but not in non-tumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, suggesting that these CTL were selected in vivo for high avidity antigen recognition, providing the rational for specific immunotherapy of HCC, based on immunization with CT antigens such as MAGE-Al 0 and SSX-2. Type 1 NKT cells express an invariant TCR α chain (Vα24.1α18, paired with Vβ11 in human) and share a specific reactivity to αGalactosylceramide (αGC) presented by CD1d. These cells can display paradoxical immuno-regulatory properties including strong anti-tumor effects upon αGC administration in murine models. To understand why NKT cells were not sufficiently protective against tumor development in patients with tumor invaded liver, we characterized the diversity of Vα24/Vβ11 NKT cells in healthy donors (HD) and cancer patients: NKT cells from HD and patients were generally diverse in terms of TCR β chain (Vβ11) variability and NKT cells from HD showed a variable recognition of αGC loaded CD 1 d multimers. Vα24/ Vβ11 NKT cells can be divided in 3 populations, the CD4, DN (CD4-/CD8-) and CD8 NKT cell subsets that show distinct ability of cytokine production. In addition, our functional analysis revealed that DN and CD8 subsets displayed a higher cytolytic potential and a weaker IFNγ release than the CD4 NKT cell subset. NKT cell subsets were variably represented in the blood of HD and cancer patients. However, HD with high NKT cell frequencies displayed an enrichment of the DN and CD8 subsets, and few of them were suggestive of an oligoclonal expansion in vivo. Comparable NKT cell frequencies were found between blood, non-tumoral liver and tumor of patients. In contrast, we identified a gradual enrichment of CD4 NKT cells from blood to the liver and to the tumor, together with a decrease of DN and CD8 NKT cell subsets. Most patient derived NKT cells were unresponsive upon αGalactosylceramide stimulation ex vivo; NKT cells from few patients displayed a weak responsiveness with different cytokine polarization. The NKT cell repertoire was thus different in tumor tissue, suggesting that CD4 NKT cells infiltrating tumors may be detrimental for protection against tumors and instead may favour the tumor growth/recurrence as recently reported in mice. Résumé en français scientifique : Afin de développer le traitement des patients porteurs d'une tumeur dans le foie par immunothérapie, de nouvelles connaissances sont requises concernant l'expression d'antigènes par les tumeurs et les cellules immunitaires spécifiques de ces antigènes. Nous avons vérifié que des antigènes associés aux tumeurs, tels que les antigènes « Cancer-Testis » (CT), sont fréquemment exprimés par le carcinome hepatocéllulaire (CHC). La recherche de lymphocytes T CD8+ spécifiques (CTL) de ces antigènes a révélé que des CTL spécifiques de MAGE-A10 et/ou SSX-2 ont répondu naturellement à la tumeur chez 2/10 patients étudiés. Ces cellules étaient présentes dans les lésions tumorales mais pas dans le foie adjacent. De plus, ces CTL ont démontré une activité cytolytique forte et spécifique contre les cellules tumorales in vitro, ce qui suggère que ces CTL ont été sélectionnés pour une haute avidité de reconnaissance de l'antigène in vivo. Ces données fournissent une base pour l'immunothérapie spécifique du CHC, en proposant de cibler les antigènes CT tels que MAGE-A10 ou SSX-2. Les cellules NKT de type 1 ont une chaîne α de TCR qui est invariante (chez l'homme, Vα24Jα18, apparié avec Vβ11) et reconnaissent spécifiquement l'αGalactosylceramide (αGC) présenté par CD1d. Ces cellules ont des propriétés immuno¬régulatrices qui peuvent être parfois contradictoires et leur activation par l'αGC induit une forte protection anti-tumorale chez la souris: Afin de comprendre pourquoi ces cellules ne sont pas assez protectrices contre le développement des tumeurs dans le foie chez l'homme, nous avons étudié la diversité des cellules NKT Vα24/Vβ11 d'individus sains (IS) et de patients cancéreux. Les cellules NKT peuvent être sous-divisées en 3 populations : Les CD4, DN (CD4- /CD8-) ou CDS, qui ont la capacité de produire des cytokines différentes. Nos analyses fonctionnelles ont aussi révélé que les sous-populations DN et CD8 ont un potentiel cytolytique plus élevé et une production d'IFNγ plus faible que la sous-population CD4. Ces sous-populations sont représentées de manière variable dans le sang des IS ou des patients. Cependant, les IS avec un taux élevé de cellules NKT ont un enrichissement des sous- populations DN ou CDS, et certains suggèrent qu'il s'agit d'une expansion oligo-clonale in vivo. Les patients avaient des fréquences comparables de cellules NKT entre le sang, le foie et la tumeur. Par contre, la sous-population CD4 était progressivement enrichie du sang vers le foie et la tumeur, tandis que les sous-populations DN ou CD8 était perdues. La plupart des cellules NKT des patients ne réagissaient pas lors de stimulation avec l'αGC ex vivo et les cellules NKT de quelques patients répondaient faiblement et avec des polarisations de cytokines différentes. Ces données suggèrent que les cellules NKT CD4, prédominantes dans les tumeurs, sont inefficaces pour la lutte anti-tumorale et pourraient même favoriser la croissance ou la récurrence tumorale. Donc, une mobilisation spécifique des cellules NKT CD4 négatives par immunothérapie pourrait favoriser l'immunité contre des tumeurs chez l'homme. Résumé en français pour un large public Au sein des globules blancs, les lymphocytes T expriment un récepteur (le TCR), qui est propre à chacun d'entre eux et leur permet d'accrocher de manière très spécifique une molécule appelée antigène. Ce TCR est employé par les lymphocytes pour inspecter les antigènes associés avec des molécules présentatrices à la surface des autres cellules. Les lymphocytes T CD8 reconnaissent un fragment de protéine (ou peptide), qui est présenté par une des molécules du Complexe Majeur d'Histocompatibilité de classe I et tuent la cellule qui présente ce peptide. Ils sont ainsi bien adaptés pour éliminer les cellules qui présentent un peptide issu d'un virus quand la cellule est infectée. D'autres cellules T CD8 reconnaissent des peptides comme les antigènes CT, qui sont produits anormalement par les cellules cancéreuses. Nous avons confirmé que les antigènes CT sont fréquemment exprimés par le cancer du foie. Nous avons également identifié des cellules T CD8 spécifiques d'antigènes CT dans la tumeur, mais pas dans le foie normal de 2 patients sur 10. Cela signifie que ces lymphocytes peuvent être naturellement activés contre la tumeur et sont capables de la trouver. De plus les lymphocytes issus d'un patient ont démontré une forte sensibilité pour reconnaître l'antigène et tuent spécifiquement les cellules tumorales. Les antigènes CT représentent donc des cibles intéressantes qui pourront être intégrés dans des vaccins thérapeutiques du cancer du foie. De cette manière, les cellules T CD8 du patient lui-même pourront être induites à détruire de manière spécifique les cellules cancéreuses. Un nouveau type de lymphocytes T a été récemment découvert: les lymphocytes NKT. Quand ils reconnaissent un glycolipide présenté par la molécule CD1d, ils sont capables, de manière encore incomprise, d'initier, d'augmenter, ou à l'inverse d'inhiber la défense immunitaire. Ces cellules NKT ont démontré qu'elles jouent un rôle important dans la défense contre les tumeurs et particulièrement dans le foie des souris. Nous avons étudié les cellules NKT de patients atteints d'une tumeur dans le foie, afin de comprendre pourquoi elles ne sont pas assez protectrice chez l'homme. Les lymphocytes NKT peuvent être sous-divisés en 3 populations: Les CD4, les DN (CD4-/CD8-) et les CD8. Ces 3 classes de NKT peuvent produire différents signaux chimiques appelés cytokines. Contrairement aux cellules NKT DN ou CDS, seules les cellules NKT CD4 sont capables de produire des cytokines qui sont défavorables pour la défense anti-tumorale. Par ailleurs nous avons trouvé que les cellules NKT CD4 tuent moins bien les cellules cancéreuses que les cellules NKT DN ou CD8. L'analyse des cellules NKT, fraîchement extraites du sang, du foie et de la tumeur de patients a révélé que les cellules NKT CD4 sont progressivement enrichies du sang vers le foie et la tumeur. La large prédominance des NKT CD4 à l'intérieur des tumeurs suggère que, chez l'homme, ces cellules sont inappropriées pour la lutte anti-tumorale. Par ailleurs, la plupart des cellules NKT de patients n'étaient pas capables de produire des cytokines après stimulation avec un antigène. Cela explique également pourquoi ces cellules ne protègent pas contre les tumeurs dans le foie.

Effector function of human tumor-specific CD8 T cells in melanoma lesions: a state of local functional tolerance.

Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differentiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART-1-specific CD8 T cells that circulate in the blood display robust inflammatory and cytotoxic functions, those that reside in tumor lesions (particularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression.

Inhibition of Trypanosoma cruzi proline racemase affects host-parasite interactions and the outcome of in vitro infection

Proline racemase is an important enzyme of Trypanosoma cruzi and has been shown to be an effective mitogen for B cells, thus contributing to the parasite's immune evasion and persistence in the human host. Recombinant epimastigote parasites overexpressing TcPRAC genes coding for proline racemase present an augmented ability to differentiate into metacyclic infective forms and subsequently penetrate host-cells in vitro. Here we demonstrate that both anti T. cruzi proline racemase antibodies and the specific proline racemase inhibitor pyrrole-2-carboxylic acid significantly affect parasite infection of Vero cells in vitro. This inhibitor also hampers T. cruzi intracellular differentiation.

REPETIDO_A new extract of the plant calendula officinalis produces a dual in vitro effect: cytotoxic anti-tumor activity and lymphocyte activation

BACKGROUND Phytopharmacological studies of different Calendula extracts have shown anti-inflammatory, anti-viral and anti-genotoxic properties of therapeutic interest. In this study, we evaluated the in vitro cytotoxic anti-tumor and immunomodulatory activities and in vivo anti-tumor effect of Laser Activated Calendula Extract (LACE), a novel extract of the plant Calendula Officinalis (Asteraceae). METHODS An aqueous extract of Calendula Officinalis was obtained by a novel extraction method in order to measure its anti-tumor and immunomodulatory activities in vitro. Tumor cell lines derived from leukemias, melanomas, fibrosarcomas and cancers of breast, prostate, cervix, lung, pancreas and colorectal were used and tumor cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of LACE on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in LACE-treated cells. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously human Ando-2 melanoma cells. RESULTS The LACE extract showed a potent in vitro inhibition of tumor cell proliferation when tested on a wide variety of human and murine tumor cell lines. The inhibition ranged from 70 to 100%. Mechanisms of inhibition were identified as cell cycle arrest in G0/G1 phase and Caspase-3-induced apoptosis. Interestingly, the same extract showed an opposite effect when tested on PBLs and NKL cell line, in which in vitro induction of proliferation and activation of these cells was observed. The intraperitoneal injection or oral administration of LACE extract in nude mice inhibits in vivo tumor growth of Ando-2 melanoma cells and prolongs the survival day of the mice. CONCLUSION These results indicate that LACE aqueous extract has two complementary activities in vitro with potential anti-tumor therapeutic effect: cytotoxic tumor cell activity and lymphocyte activation. The LACE extract presented in vivo anti-tumoral activity in nude mice against tumor growth of Ando-2 melanoma cells.

Doublecortin-positive cells in the adult primate cerebral cortex and possible role in brain plasticity and development.

We have demonstrated that cortical cell autografts might be a useful therapy in two monkey models of neurological disease: motor cortex lesion and Parkinson's disease. However, the origin of the useful transplanted cells obtained from cortical biopsies is not clear. In this report we describe the expression of doublecortin (DCX) in these cells based on reverse-transcription polymerase chain reaction (RT-PCR) and immunodetection in the adult primate cortex and cell cultures. The results showed that DCX-positive cells were present in the whole primate cerebral cortex and also expressed glial and/or neuronal markers such as glial fibrillary protein (GFAP) or neuronal nuclei (NeuN). We also demonstrated that only DCX/GFAP positive cells were able to proliferate and originate progenitor cells in vitro. We hypothesize that these DCX-positive cells in vivo have a role in cortical plasticity and brain reaction to injury. Moreover, in vitro these DCX-positive cells have the potential to reacquire progenitor characteristics that confirm their potential for brain repair.

Cytotoxic activity of the dichloromethane fraction from Vernonia scorpioides (Lam.) Pers. (Asteraceae) against Ehrlich's tumor cells in mice

Vernonia scorpioides has been widely used in Brazil to treat skin problems and chronic wounds, such as ulcers of the lower limbs and diabetic lesions. In the present study, we investigated the effect of a dichloromethane (DCM) fraction of V. scorpioides leaf extract on Ehrlich ascitic and solid tumor-bearing mice. The animals were treated once a day with the DCM fraction at a concentration of 5 mg/kg, administered ip during and after the development of the tumor. The lifespan, weight, number and type of leukocytes, number of tumor cells, volume of solid and ascitic tumors were measured. The development of the tumor with pre-treated tumor cells in vitro with the DCM fraction (5 mg/kg) was analyzed and the animals were sacrificed after 7 days. The DCM fraction (5 mg/kg) totally inhibited tumor development when in direct contact with tumor cells, and also ascitic tumor development with in vitro treatment or when administered ip, in loco (after 7 days). Animals treated with the DCM fraction increased their lifespan ca. 2 weeks and maintained their body weight for 30 days. When applied immediately after the inoculation of the tumor cells in vivo, it totally abolished tumor development, with tumor development only decreasing when treatment was started 3 days after the tumor challenge. These data suggest an antineoplastic activity of the fraction. Oral or ip administration of DCM fraction (5 mg/kg) for 7 days did not reduce the solid tumor volume. The cytotoxic activity described here differs from the conventional immune suppressing profile of standard chemotherapy because it increases neutrophil influx to the peritoneal cavity. These results show that, besides exhibiting a tumoricidal activity, the DCM fraction also exhibits inflammatory activity.

Function of the immunoregulatory CD4-CD8- T cells in the context of autoimmune diabetes

La tolérance immunitaire dépend de la distinction entre le soi et le non soi par le système immunitaire. Un bris dans la tolérance immunitaire mène à l'auto-immunité, qui peut provoquer la destruction des organes, des glandes, des articulations ou du système nerveux central. Le diabète auto-immun, également connu sous le nom diabète juvénile et diabète de type 1, résulte d'une attaque auto-immune sur les cellules β pancréatiques sécrétrices d’insuline, localisées au niveau des îlots de Langerhans du pancréas. Bien que le diabète auto-immun soit traitable par une combinaison d’injections quotidiennes d’insuline d’origine exogène, de régime et d'exercices, beaucoup de complications chroniques peuvent se manifester chez les patients, y compris, mais non limitées à, la cécité, les maladies cardiovasculaires, l’insuffisance rénale et l'amputation. En raison des nombreuses complications liées au diabète auto-immun à long terme, la recherche continue afin de mieux comprendre tous les facteurs impliqués dans la progression de la maladie dans le but de développer de nouvelles thérapies qui empêcheront, renverseront et/ou traiteront cette maladie. Un rôle primordial dans la génération et l'entretien de la tolérance immunitaire a été attribué au nombre et à la fonction des sous-populations de cellules régulatrices. Une de ces populations est constituée de cellules T CD4-CD8- (double négatives, DN), qui ont été étudiées chez la souris et l'humain pour leur contribution à la tolérance périphérique, à la prévention des maladies et pour leur potentiel associé à la thérapie cellulaire. En effet, les cellules de T DN sont d'intérêt thérapeutique parce qu'elles montrent un potentiel immunorégulateur antigène-spécifique dans divers cadres expérimentaux, y compris la prévention du diabète auto-immun. D’ailleurs, en utilisant un système transgénique, nous avons démontré que les souris prédisposées au diabète auto-immun présentent peu de cellules T DN, et que ce phénotype contribue à la susceptibilité au diabète auto-immun. En outre, un transfert des cellules T DN est suffisant pour empêcher la progression vers le diabète chez les souris prédisposées au diabète auto-immun. Ces résultats suggèrent que les cellules T DN puissent présenter un intérêt thérapeutique pour les patients diabétiques. Cependant, nous devons d'abord valider ces résultats en utilisant un modèle non-transgénique, qui est plus physiologiquement comparable à l'humain. L'objectif principal de cette thèse est de définir la fonction immunorégulatrice des cellules T DN, ainsi que le potentiel thérapeutique de celles-ci dans la prévention du diabète auto-immun chez un modèle non-transgénique. Dans cette thèse, on démontre que les souris résistantes au diabète auto-immun présentent une proportion et nombre absolu plus élevés de cellules T DN non-transgéniques, lorsque comparées aux souris susceptibles. Cela confirme une association entre le faible nombre de cellules T DN et la susceptibilité à la maladie. On observe que les cellules T DN éliminent les cellules B activées in vitro par une voie dépendante de la voie perforine et granzyme, où la fonction des cellules T DN est équivalente entre les souris résistantes et prédisposées au diabète auto-immun. Ces résultats confirment que l'association au diabète auto-immun est due à une insuffisance en terme du nombre de cellules T DN, plutôt qu’à une déficience fonctionnelle. On démontre que les cellules T DN non-transgéniques éliminent des cellules B chargées avec des antigènes d'îlots, mais pas des cellules B chargées avec un antigène non reconnu, in vitro. Par ailleurs, on établit que le transfert des cellules T DN activées peut empêcher le développement du diabète auto-immun dans un modèle de souris non-transgénique. De plus, nous observons que les cellules T DN migrent aux îlots pancréatiques, et subissent une activation et une prolifération préférentielles au niveau des ganglions pancréatiques. D'ailleurs, le transfert des cellules T DN entraîne une diminution d'auto-anticorps spécifiques de l'insuline et de cellules B de centres germinatifs directement dans les îlots, ce qui corrèle avec les résultats décrits ci-dessus. Les résultats présentés dans cette thèse permettent de démontrer la fonction des cellules T DN in vitro et in vivo, ainsi que leur potentiel lié à la thérapie cellulaire pour le diabète auto-immun.

A novel medium for the development of in vitro cell culture system from Penaeus monodon

Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 % salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8–7.2. Osmolality was adjusted to 720 ± 10 mOsm kg-1 and temperature of incubation was 25 8C. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (29), modified L-15 and Grace’s insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation ‘‘cocktail’’.