Hamsteres infectados com Leishmania (L.) chagasi são considerados um dos melhores modelos para estudo de fatores relacionados à imunossupressão que ocorre durante a leishmaniose visceral ativa, pois apresenta manifestações clínicas similares ao que ocorre no homem e não conseguem controlar a infecção. Neste estudo, hamsteres infectados intraperitonealmente com 2x107 parasitos foram utilizados para avaliação de possíveis fatores envolvidos na dinâmica da imunossupressão. Os parâmetros avaliados foram a produção de citocinas de padrão Th1 e Th2, produção de óxido nítrico por células esplênicas e detecção de apoptose em linfócitos esplênicos em tempos precoces (6, 20, 48 e 72 horas), intermediários (7 e 15 dias) e tardios (30 e 60 dias) de infecção. Inicialmente avaliamos a carga parasitária no baço e observamos aumento progressivo dependente do tempo de infecção, ratificando que hamster infectado é um bom modelo para desenvolvimento de doença. A resposta celular frente a mitógeno e antígeno de Leishmania foi o parâmetro utilizado para avaliação de imunossupressão. Observamos resposta preservada à concanavalina A em todos os tempos de infecção e resposta preservada ao antígeno de Leishmania nos tempos precoces, porém com ausência de resposta antígeno específica a partir do tempo de 7 dias de infecção. A quantificação de óxido nítrico foi determinada em sobrenadante de células esplênicas de cultura estimuladas com concanavalina A e antígeno de Leishmania, pelo método de Griess. Observamos inicialmente baixo nível de produção de óxido nítrico em todos os tempos de infecção, no entanto quando as células foram estimuladas com antígeno de Leishmania observamos níveis ainda menores nos tempos de 48 e 72 horas de infecção. O perfil de citocinas produzido foi determinado pela reação em cadeia da polimerase por transcrição reversa, sendo detectado RNA mensageiro tanto de citocinas Th1, quanto Th2 em todos os tempos de infecção (precoce, intermediária e tardia), em células ex-vivo e estimuladas; e também em animais controles não infectados; não havendo um padrão de dicotomização de produção de citocinas neste modelo experimental. Detecção de apoptose em células esplênicas não aderentes (prováveis linfócitos T) ex-vivo e em cultura estimuladas com concanavalina A e antígeno de Leishmania foi avaliada pelos seguintes métodos: Anexina V, caspase 3 clivada e TUNEL por citometria de fluxo. Houve marcação da exposição de fosfatidil serina pelo método da anexina V, nos tempos de 6 horas de infecção, quando as células foram estimuladas com mitogeno ou antígeno de Leishmania; e com 48 horas e 60 dias de infecção somente quando estimuladas com antígeno de Leishmania. Em células esplênicas em cultura estimuladas com concanavalina não houve marcação de caspase 3 clivada, porém em células esplênicas ex-vivo nos tempos 20, 48 e 72 horas de infecção houve detecção de apoptose, definida por marcação de caspase 3 clivada. A marcação de células esplênicas não aderentes para quebra de DNA foi baixa em todos os tempos analisados, porém observamos aumento de marcação em células esplênicas não aderentes, estimuladas com antígeno nos tempos de 20 horas e posteriormente de 7 dias até 60 dias de infecção. Nossos resultados sugerem que a imunossupressão na leishmaniose visceral no modelo de hamsteres infectados com Leishmania (L.) chagasi é antígeno dependente e instala-se nos tempos intermediários de infecção, a partir de 7 dias. Esta imunossupressão parece estar relacionada com aumento de apoptose em linfócitos já nos tempos precoces de infecção, que pode favorecer a progressão da infecção, por ocorrer principalmente em células esplênicas antígenos reativas. Nossos dados sugerem ainda, que a imunossupressão não é decorrente da dicotomização da produção de citocinas Th1 e Th2 / Hamsters infected by Leishmania (L.) chagasi are considered one of the most remarkable models to study several characteristics related to immunosuppression, raised during active visceral leishmaniasis especially because hamsters show similar clinical manifestations as it is found in humans without surmounting the infection. In this study, hamsters infected intraperitoneally with 2x107 parasites were used to evaluate the possible factors involved in the dynamic of immunosuppression that occurs during the disease development. The evaluated parameters were the production of Th1 and Th2 cytokines profile, nitric oxide production of splenic cells and detection of apoptosis in splenic lymphocytes at early (6,20, 48 e 72 hours), intermediate (7 e 15 days) and late times (30 e 60 days) of infection. Initially, we evaluate the parasite load in the spleen and observed a progressive increase dependent on the time of infection, ratifying that infected hamsters are good models to set up the disease development. Cellular response upon mitogen or Leishmania antigen was the parameter used to evaluate the immunosuppression showing a preserved response to concanavalin A at all period of infection and a preserved response to the Leishmania antigen at all early phases however with no specific antigen response from 7 day of infection until the late phase of infection. Nitric oxide quantification was determined in the splenic cells supernatant in culture stimulated upon concanavalin A and Leishmania antigen and we observed initially low level of nitric oxide production, measured by the Griess method. Nonetheless, when the cells were stimulated upon Leishmania antigen we observed a lower level nitric oxide production at 48 and 72 hours of infection. mRNA of Th1 e Th2 cytokines profile was determined by RT-PCR at all period of infection studied in infected or non infected hamsters showing no difference at the cytokine profile in this experimental model. Detection of apoptosis in the non adherent splenic cells (probably T lymphocytes) ex-vivo and in the stimulated culture with concanavalin A and Leishmania antigen was evaluated by the following methods: annexin V-FITC, cleaved caspase 3 and TUNEL by flow cytometry analysis. There was phosphatidilserine staining by annexin method, at 6 hour of infection when cells were stimulated by mitogen or Leishmania antigen at 48 hours and 60 days of infection only when stimulated by Leishmania antigen. Splenic cells in culture stimulated by concanavalin A showed no cleaved caspase 3 staining in all period of infection studied. Otherwise, apoptosis was detected in the ex-vivo splenic cells at 20, 48 and 72 hours of infection by cleaved caspase 3 staining. We observed a low DNA break staining of non adherent splenic cells in all period analyzed, although this staining was increased in non adherent cells stimulated upon Leishmania antigen at 20 hours from 7 day of infection until 60 day of infection. Our results suggest that a visceral leishmaniasis immunosuppression in the hamsters model infected by Leishmania (L.) chagasi is antigen dependent and sets up in the intermediate phases of infection from 7 days on. This immunosuppression seems to be related to the apoptosis increase in lymphocytes already in the early period of infection probably favoring its progression especially because of its occurrence in reactive splenic antigen. Our data also suggest that the immunosuppression is not provided by dichotomization in the Th1 and Th2 cytokines profile

Master of Science / Department of Clinical Sciences / Michael W. Sanderson / Escherichia coli O157:H7 has emerged as a significant human food-borne pathogen over the past two decades. While cattle have been identified as the major reservoir of the pathogen, the dynamics of shedding are still largely unknown. The role immunosuppression may play on fecal shedding in cattle is explored. The first study determined whether immunosuppression induced by dexamethasone injections affects the level and duration of fecal shedding of E. coli O157. Six one week old Holstein bull calves were injected intramuscularly with dexamethasone and orally inoculated with 109 CFU of a mixture of three nalidixic-acid resistant strains of E. coli O157. Another five one week old Holstein bull calves, only inoculated with the E. coli O157, served as controls. All calves were necropsied and samples from the gastrointestinal tract were cultured. Dexamethasone treated calves shed at higher levels on days four and seven post-inoculation, but not thereafter. The data from this study suggest that there may be a time dependent correlation between dexamethasone immunosuppression and the concentration of E. coli O157 an animal will shed in the feces and that transient immunosuppression does not result in prolonged shedding of E. coli O157. The goal of the second study was to determine whether calves immunosuppressed by persistent infection with bovine viral diarrhea virus (BVDV) will shed E. coli O157 at a higher level and for a longer duration than a normal animal. Nine six to eight week old calves persistently infected with non-cytopathic BVDV and eight normal calves obtained from separate cow-calf operations were orally inoculated with 109 CFU of a mixture five nalidixic-acid resistant strains of E. coli O157. All calves were necropsied and samples from the gastrointestinal tract cultured. There was no statistical difference in the concentration of E. coli O157 shed or the duration of shedding between the persistently infected BVDV calves and the control calves throughout the length of the study. The data suggest that immunosuppression caused by persistent infection with non-cytopathic BVDV infection does not play a role in the level or duration of shedding of E. coli O157:H7 in calves.

Les cancers représentent une cause importante de morbidité et de mortalité chez les personnes vivant avec le VIH. Ce travail s’est inscrit dans l’étude des déterminants immunologiques et virologiques du risque de cancer au sein de la Cohorte ANRS CO3 Aquitaine. Ces facteurs de risque ont été étudiés à l’aide de modèles de Cox à entrée retardée et variables dépendantes du temps dans deux études de cohorte. Une étude cas-témoins nichée dans la Cohorte Aquitaine a recherché à l’aide de modèles de régression logistique conditionnelle un rôle du cytomégalovirus (CMV) dans le risque de cancer.Entre 1998 et 2006, 4 194 patients ont présenté 251 cancers. Une charge virale >500 copies/mL était un déterminant du risque de cancer classant sida (Risque Relatif [RR] = 3.3, Intervalle de Confiance à 95% [IC] : 2.1–5.2, p <0.001) ainsi qu’un taux de lymphocytes CD4 (CD4) <200 cellules/mm3 (RR=6.3, IC : 4.2–9.4, p <0.001). Un taux de CD4 <500 était associé à un risque augmenté de présenter un cancer non classant sida (RR=2.1, IC : 1.4–3.1, p <0.001). Parmi 2 864 patients ayant présenté 16 hépatocarcinomes, un taux courant de CD4 <500 était un facteur de risque de cancer (RR=10.3, IC : 1.3–82.8, p=0.03), mais pas la durée cumulée d’exposition antérieure à des CD4 <500 (p=0.8).Une ADNémie plasmatique à CMV positive n’était pas associée au risque de cancer (p=0.54) chez 143 cas et 284 témoins.Ces résultats sont en faveur du maintien chez les personnes vivant avec le VIH d’une charge virale plasmatique indétectable et d’un taux de CD4 >500 cellules/mm3, si besoin à l’aide des traitements antirétroviraux, afin de prévenir le risque de cancer en plus des mesures préventives traditionnelles. / Persons living with HIV are at higher risk of malignancies. In addition to traditional determinants, deleterious effects of immunological or virological parameters are speculated in this population. The objective of this thesis was to study immunological and virological risk factors for cancer in the ANRS CO3 Aquitaine Cohort of persons living with HIV. Extended Cox proportional hazard models with delayed entry and time-updated variables were used in two cohort studies, and the role of the cytomegalovirus (CMV) in the risk of cancer was assessed in a nested case control study with conditional logistic regression models.Between 1998 and 2006, 4,194 patients presented 251 cancers. Plasma HIV RNA >500 copies/mL and a CD4 cell count (CD4) <200 cells/mm3 were associated with a higher risk of AIDS-defining cancer: (Hazard Ratio [HR]=3.3, 95% Confidence Interval [CI]: 2.1–5.2, p <0.001) and (HR=6.3, CI: 4.2–9.4, p <0.001), respectively. CD4 <500 was associated with a higher risk of non-AIDS-defining cancer (HR=2.1, CI: 1.4–3.1, p <0.001). Among 2,864 patients who presented 16 hepatocarcinomas, current CD4 <500 was a risk factor for cancer (HR=10.3, CI: 1.3–82.8, p=0.03), but not the cumulative exposure to CD4 <500 preceding the current measurement (p=0.8). Positive plasma CMV DNAemia was not associated with the risk of cancer among 143 cancer cases and 284 control patients (p=0.54).These results suggest that maintaining an undetectable plasma HIV RNA viral load and a CD4 cell count above 500 cells/mm3, if needed by prescribing antiretroviral treatment, should prevent cancer occurrence among HIV-infected patients, additionally to the traditional prevention policies

The progressive growth of tumors in human cancer patients and experimental animals has frequently been associated with a generalized depression of immunological responsiveness. Suppressor cells have been implicated as mediators of tumor-associated immunosuppression, but the identities of the cells causing suppression and the mechanisms by which they act have been unclear. The object of this thesis was thus to determine: if suppressor cells capable of non specifically suppressing immune responses were present in anergic mice bearing methylcholanthrene-induced sarcomas; the cell types responsible for suppression; and the mechanisms by which suppression occurs. The spleens of mice with large tumors were found to contain two distinct populations of non specific suppressor cells. One population inhibited the proliferative responses of normal lymphocytes to the T cell mitogen, Concanavalin A (Con A) and the B cell mitogen, lipopolysaccharide (LPS). These cells also inhibited the generation of antibody forming cells by normal lymphoid cells stimulated in vitro with the T cell dependent antigen, sheep red blood cells (SRBC) and the T cell independent antigen, dinitrophenylated-lipopolysaccharide (DNP-LPS). These suppressor cells appeared to be from the macrophage/monocyte line since they adhered to plastic and nylon wool, were removed by carbonyl iron and magnet, and were inactivated by carragheenan treatment, but were not removed by anti-Thy-1 or anti-mouse immunoglobulin sera plus complement. They were among the less dense spleen cells since they were retained in the light fraction after centrifugation on hypaque-ficoll of specific gravity 1.08 and did not appear to require cell division in order to suppress- since mitomycin C treatment did not inactivate them. Cell-cell contact appeared to be essential for suppression. The second population of suppressor cells, which pelleted to the bottom of a hypaque-ficoll gradient, inhibited only the generation of plaque forming cells to the T cell dependent antigen, SRBC. These cells appeared to be T cells since they were non adherent to plastic or nylon wool, were not removed by carbonyl iron and magnet, but were removed by anti-Thy-1 serum plus complement. Cell division was necessary since suppressive activity was totally removed by mitomycin C treatment. Suppression by this cell type appeared to be mediated by a soluble factor with a molecular weight of about 3,500 to 12,000. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

It is well known that too much sugar uptake causes many health problems, including diabetes and obesity (Lustig et al. in Nature 482:27-29, 2012). However, a team of researchers led by Dr. Wanjun Chen of the National Institute of Dental and Craniofacial Research (NIDCR), National Institutes of Health (NIH), USA, have recently shown that d-mannose, a naturally occurring C-2 epimer of glucose is likely beneficial to human health. Their studies have revealed that supraphysiological levels of d-mannose that are safely achievable via drinking-water supplementation can be preventive and therapeutic to experimental autoimmune diabetes and asthmatic lung inflammation (Zhang et al. in Nat Med 23:1036-1045, 2017).

Serum lactate levels are traditionally interpreted as a marker of tissue hypoxia and often used clinically as an indicator of severity and outcome of sepsis/septic shock. Interestingly, recent studies involving the effects of tumor-derived lactate suggest that lactate itself may have an immunosuppressive effect in its local environment. This finding adds to the recent advances in immunometabolism that shed light on the importance of metabolism and metabolic intermediates in the regulation of innate immune and inflammatory responses in sepsis. In this article, we summarize recent studies, showing that the activation of immune cells requires aerobic glycolytic metabolism and that lactate produced by aerobic glycolysis may play an immunosuppressive role in sepsis.

Myeloid progenitor-derived suppressor cells (MDSCs) arise from myeloid progenitors and suppress both innate and adaptive immunity. MDSCs expand during the later phases of sepsis in mice, promote immunosuppression, and reduce survival. Here, we report that the myeloid differentiation-related transcription factor nuclear factor I-A (NFI-A) controls MDSC expansion during sepsis and impacts survival. Unlike MDSCs, myeloid cells with conditional deletion of the Nfia gene normally differentiated into effector cells during sepsis, cleared infecting bacteria, and did not express immunosuppressive mediators. In contrast, ectopic expression of NFI-A in myeloid progenitors from NFI-A myeloid cell-deficient mice impeded myeloid cell maturation and promoted immune repressor function. Importantly, surviving septic mice with conditionally deficient NFI-A myeloid cells were able to respond to challenge with bacterial endotoxin by mounting an acute inflammatory response. Together, these results support the concept of NFI-A as a master molecular transcriptome switch that controls myeloid cell differentiation and maturation and that malfunction of this switch during sepsis promotes MDSC expansion that adversely impacts sepsis outcome.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19593号 / 医博第4100号 / 新制||医||1014(附属図書館) / 32629 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松岡 雅雄, 教授 朝長 啓造, 教授 西渕 光昭 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Background: Cyclosporine is an immunosuppressive agent used to treat immune-mediated and inflammatory diseases in dogs. We have developed a pharmacodynamic (PD) assay that measures interleukin-2 (IL-2) produced by activated T cells to measure the immunosuppressive effects of cyclosporine. Hypothesis/objectives: Our retrospective study extracted data from samples submitted to our laboratory to obtain descriptive statistics, to determine whether assay results predicted treatment effectiveness, and to determine whether cyclosporine formulation or breed affected PD responses. Animals: 1,110 samples were analyzed over 4 years. Methods: Extracted data was analyzed to determine whether there was a relationship between assay results and clinical control, and whether either formulation or breed affected results. Results: We found no relationship between assay results and control of signs, and found that breed did not affect results. At comparable doses, proprietary modified cyclosporine was more immunosuppressive than proprietary non-modified cyclosporine, and both proprietary and generic modified formulations had similar efficacy.

One of the major focuses for our lab involves examining the immunosuppressive properties of various agents and receptors in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). This dissertation encompasses an investigation of cannabidiol in the EAE model, the endocannabinoid CB1 receptor in the EAE model, staphylococcal superantigens (SAgs) as immunosuppressive agents, and various aspects of the EAE model. The first chapter covers the existing literature pertinent to these studies, the second and third chapters cover the material, methods, and results from the studies, and the fourth chapter is a discussion of how those results fit into the existing body of literature. A fifth chapter has also been included which covers two additional studies designed to develop alternative EAE models for our lab; however, both studies turned out differently than expected. One of the most interesting developments from this final chapter was the discovery of an age dependent difference in the memory T cell response of older mice, which allows for more robust disease to be induced when cells from 6 month old mice are used in the passive EAE (P-EAE) model as opposed to mice 10 weeks of age.