Les lymphocytes T CD4 (LT CD4) jouent un rôle central dans la régulation des réponses immunitaires vis-à-vis des agents infectieux et des vaccins. Cependant, leur différenciation in vivo est encore mal comprise et les caractéristiques des LT CD4 capables de persister à long terme tout en assurant une réponse immunitaire protectrice sont mal définies. L’approfondissement de ces connaissances est indispensable pour le développement de nouveaux vaccins. <p>Pour approcher cette question, nous avons utilisé deux approches expérimentales. La première est un suivi de la différenciation des LT CD4 au cours de la réponse immune primaire chez des sujets vaccinés contre l’hépatite B ;la deuxième est la caractérisation phénotypique et fonctionnelle des LT CD4 mémoires antigène(Ag)-spécifiques pendant la phase d’état. Cette analyse a été réalisée au sein des LT CD4 spécifiques d’Ag vaccinaux, l’Ag de surface du virus de l’hépatite B (HBs) et la toxine tétanique (TT), ainsi que ceux spécifiques des Ag du cytomégalovirus (CMV). Les LT CD4 Ag-spécifiques ont été mis en évidence par cytométrie de flux après marquage intracytoplasmique du ligand du CD40 (CD40L) exprimé en réponse à une stimulation de courte durée par l’Ag. Des expériences basées sur la stimulation par la toxine du syndrome du choc toxique et le marquage du segment Vbeta2 du récepteur des LT ont démontré la bonne sensibilité et spécificité de cette méthode.<p>Le suivi de la réponse primaire chez 11 donneurs jusqu’à plus d’un an après immunisation par le vaccin anti-hépatite B a permis d’établir un modèle de différenciation des LT CD4 Ag-spécifiques in vivo chez l’homme. Nous avons mis en évidence des LT CD4 spécifiques d’un nombre limité de peptides immunodominants de la protéine HBs suggérant une réponse de type oligoclonale. Grâce à l’utilisation d’un cytomètre neuf couleurs, nous avons mené une analyse détaillée de l’hétérogénéité de la population mémoire HBs-spécifique. L’expression du CCR7 permet de distinguer des cellules de type mémoire centrale (LTCM, CCR7+) et effectrice (LTEM, CCR7-) se distinguant notamment par leur capacité à migrer vers les ganglions lymphatiques ainsi que par leurs propriétés fonctionnelles. Nous avons montré l’existence de ces deux sous-populations au sein des cellules HBs-spécifiques mais par opposition à leur définition initiale, ces LTCM sont capables de produire des cytokines effectrices. La proportion importante de LTCM exprimant le Ki67 témoigne d’une activité proliférative persistante in vivo et suggère la capacité de ces cellules à s’auto-renouveler et éventuellement à alimenter le pool des LTEM. La proportion importante de LTCM exprimant la chaîne alpha du récepteur à l’IL-7 (CD127) suggère que ces cellules sont sensibles aux signaux émanant de l’IL-7, une cytokine dont le rôle dans le maintien de la mémoire lymphocytaire T est connu. Compte tenu de la relevance potentielle de ces caractéristiques uniques pour le développement de vaccins et de l’accumulation de travaux montrant l’avantage sélectif des LTCM à conférer une immunité protectrice, nous avons focalisé la dernière partie de ces recherches sur cette sous-population. Une étude transversale des LTCM spécifiques de plusieurs types d’Ag (éliminés (HBs et TT) ou persistants (CMV)) a été menée. Nos résultats montrent une hétérogénéité, variable selon l’Ag, de la capacité de ces cellules à produire des cytokines effectrices et de leur phénotype de différenciation. Cette donnée nouvelle soulève la possibilité que les LTCM soient hétérogènes dans leur capacité à conférer une immunité protectrice. L’acquisition du marqueur KLRG1 par une fraction des LTCM s’associe à une capacité accrue à produire des cytokines effectrices et à une expression élevée du CD127. La possibilité que ces cellules soient particulièrement aptes à conférer une immunité protectrice et durable est discutée, tout comme les mécanismes menant à leur génération et l’intérêt de ces connaissances pour la conception de nouveaux vaccins.<p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Costimulation blockade protocols are promising alternatives to the use of chronic immunosuppression for promoting long-term allograft survival. However, the efficacy of costimulation blockade-based protocols is decreased by environmental insults such as viral infections. For example, lymphocytic choriomeningitis virus (LCMV) infection at the time of costimulation blockade treatment abrogates skin allograft survival in mice. In this dissertation, we test the hypothesis that viruses shorten allograft survival by activating the innate immune system through pattern-recognition receptors (PRRs), such as toll-like receptors (TLRs). To investigate the role of innate immunity in shortening allograft survival, costimulation blockade-treated mice were co-injected with TLR2 (Pam3Cys), TLR3 (polyinosinic:polycytidylic acid, poly(I:C)), TLR4 (lipopolysaccharide, LPS), or TLR9 (CpG DNA) agonists, followed by transplantation with skin allografts 7 days later. Costimulation blockade prolonged skin allograft survival that was shortened in mice coinjected with TLR agonists. To investigate the underlying mechanisms of this observation, we used synchimeric mice, which circulate trace populations of anti-H2b transgenic alloreactive CD8+ T cells. In synchimeric mice treated with costimulation blockade, co-administration of all four TLR agonists prevented deletion of alloreactive CD8+ T cells. These alloreactive CD8+ T cells 1) expressed the proliferation marker Ki-67, 2) upregulated CD44, and 3) failed to undergo apoptosis. We also demonstrate that costimulation blockade-treated CD8α-deficient mice exhibit prolonged allograft survival when co-injected with LPS. These data suggest that TLR agonists shorten allograft survival by impairing the apoptosis of alloreactive CD8+T cells. We further delineate the mechanism by which TLR agonists shorten allograft survival by demonstrating that LPS and poly(I:C) fail to shorten allograft survival in IFNRI- deficient mice. Interestingly, the ability of poly(I:C) to more potently induce IFN-α/β than LPS correlates with its superior abilities to shorten islet allograft survival and induce allo-specific CTL activity as measured by an in vivo cytotoxicity assay. The ability to shorten allograft survival and induce IFN-α/β is a TLR-dependent process for LPS, but is a TLR-independent process for poly(I:C). Strikingly, the injection of IFN-β impairs alloreactive CD8+T cell deletion and shortens allograft survival, similar to LPS and poly(I:C). These data suggest that LPS and poly(I:C) shorten allograft survival by inducing IFN-α/β through two different mechanisms. Finally, we present data showing that viruses (LCMV, Pichinde virus, murine cytomegalovirus and vaccinia virus) impair alloreactive CD8+T cell deletion and shorten allograft survival, in a manner comparable to LPS and poly(I:C). Similar to LPS, LCMV and MCMV exhibit an impaired ability to shorten allograft survival in MyD88-deficient mice. These data suggest that the MyD88 pathway is required for certain viruses and TLR-agonists to shorten allograft survival. In this dissertation, we present data supporting an important role for TLRs and IFN- α/β in shortening allograft induced by costimulation blockade. Our findings suggest that targeting these pathways during the peri-transplant period may enhance the efficacy of costimulation blockade protocols in the clinic.

Bacterial infections are a common and deadly threat to vulnerable patients. Alternative strategies to fight infection are needed. β-Glucan, an immunomodulator derived from the fungal cell wall, provokes resistance to infection by inducing trained immunity, a phenomenon that persists for weeks to months. Given the durability of trained immunity, it is unclear which leukocyte populations sustain this effect. Macrophages have a life span that surpasses the duration of trained immunity. Thus, we sought to define the contribution of differentiated macrophages to trained immunity. Our results show that β-glucan protects mice from infection by augmenting recruitment of innate leukocytes to the site of infection and facilitating local clearance of bacteria, an effect that persists for more than 7 d. Adoptive transfer of macrophages, trained using β-glucan, into naive mice conferred a comparable level of protection. Trained mouse bone marrow-derived macrophages assumed an antimicrobial phenotype characterized by enhanced phagocytosis and reactive oxygen species production in parallel with sustained enhancements in glycolytic and oxidative metabolism, increased mitochondrial mass, and membrane potential. β-Glucan induced broad transcriptomic changes in macrophages consistent with early activation of the inflammatory response, followed by sustained alterations in transcripts associated with metabolism, cellular differentiation, and antimicrobial function. Trained macrophages constitutively secreted CCL chemokines and robustly produced proinflammatory cytokines and chemokines in response to LPS challenge. Induction of the trained phenotype was independent of the classic β-glucan receptors Dectin-1 and TLR-2. These findings provide evidence that β-glucan induces enhanced protection from infection by driving trained immunity in macrophages.

Os doentes transplantados renais têm alto risco para desenvolver queratoses actínicas e câncer de pele. Para verificar o efeito quimioprofilático da acitretina estudamos a evolução de 13 doentes transplantados renais com queratoses actínicas múltiplas e história de carcinomas cutâneos submetidos a tratamento por 12 meses (20mg/dia). Fez-se a avaliação clínica e laboratorial regularmente em todo o período do estudo. Realizou-se exame histopatológico, demonstração imuno-histoquímica de sub-populações de linfócitos T (CD4, CD8), células natural killer e células de Langerhans, sua quantificação e comparação em biopsias de pele, sem lesão, de área exposta e protegida do sol antes, após seis e 12 meses de tratamento. Observou-se melhora das lesões cutâneas e ausência de aparecimento de novos tumores em 12 dos 13 pacientes. Não ocorreram alterações laboratoriais relacionadas a função renal, hepatotoxicicidade e hiperlipidemia. Não houve diferenças significativas histopatológicas e da população de linfócitos T e células natural killer da pele exposta e protegida do sol com o tratamento. Verificou-se aumento numérico de células de Langerhans epidérmicas aos 12 meses quando comparado aos da pele antes e após seis meses de tratamento (p = 0,002 e p = 0,003). Em nossa casuística o uso de acitretina em doses baixas foi útil para melhorar o aspecto cutâneo e prevenir lesões cutâneas pré-cancerosas e carcinomas. O aumento das células de Langerhans epidérmicas estaria relacionado ao efeito imunomodular da acitretina. / Renal transplant recipients have an increased incidence of actinic keratosis and skin cancer. In order to examine the chemoprophylatic effects of low-dose acitretin on skin cancer development we submitted 13 renal transplanted patients to acitretin therapy (20 mg/day) for 12 month. The patients were assessed at monthly intervals during the first 6 months and every two months until the 12th month for new skin lesions and for acitretin toxicity. Normal skin biopsies of sun exposed and sun protected area were taken for histopathological exam and submitted to immunohistochemistry technique to demonstrate CD4+ and CD8+ T lymphocytes, natural killer cells and Langerhans cells wich were counted and compared in the beginning, after 6th month and 12th month of the treatment. There was an improvement of actinic keratosis and all patients but one did not develop new skin cancer. Side-effects were well-tolerated and no significant biochemical effects were observed. Although there were no differences in the microscopic aspects of the skin and in the number of CD4+ and CD8+ T lymphocytes and natural killer cells, there was a significant increase in the number of epidermal Langerhans cells after 12 months of acitretin therapy. The data obtained permit us to conclude that low dose acitretin therapy is safe, well-tolerated and partially effective in chemoprophylaxis of skin cancer in renal transplant recipients. The increase in epidermal Langerhans cells observed may be an expression of the immunomodulatory effect of acitretin.

The objectives of the study were (1) to quantify adherence rate among the study participants in the ART unit and (2) to identify factors that contribute to non-adherence. This cross sectional study was carried out at Nekemete referral clinic. Data was collected using a self-developed structured questionnaire where a total of 338 participants grouped into adherent and non-adherent based on a score derived from an adherence assessment were interviewed. Data was analysed using the Statistical Package for Social Sciences (SPSS) version 17.0. By using multivariate analysis of variables identified as correlates of adherence, non-adherence was common among those; with age between 18-30 yrs, with no education, who were not married, who had no pipe water supply, those with no electricity in the house, who perceived had no access to assistance from providers, who perceived the health care providers (HCPs) did not keep information confidentially, who had a language barrier with providers, and who were treated with a psychiatric illness. The study concludes that adherence is multi-factorial and varies significantly by individual and care setting. Psychosocial factors were found to impact adherence and should be analysed in more detail by further studies. Three psychosocial factors were independently associated with poor adherence: the study found that patients perceiving poor access; those perceiving problems in information confidentiality (and possibly experiencing stigmatisation); and having psychiatric morbidity (and possibly with less social support) are more likely to be non-adherent. Furthermore, individuals without electricity and those without piped water supply, implying low income, are at risk for non-adherence / Health Studies / M.A. (Public Health)

<p>UVOD: Glavni problem u lečenju karcinoma dojke je kako na osnovu kliničke klasifikacije i morfolo&scaron;kih osobina tumora predvideti njegovo dalje pona&scaron;anje. Vrlo često ni kombinacija standardnih prognostičkih faktora ne daje odgovor o potrebi davanja adjuvantne hemioterapije. U cilju sprovođenja adekvatne dalje terapije karcinoma dojke i otkrivanja agresivnih tipova tumora, a nakon hirur&scaron;kog lečenja, postoji stalna potreba za pronalaženjem novih pokazatelja pomoću kojih bi se identifikovale bolesnice koje imaju povećan rizik od razvoja relapsa bolesti. CILJEVI: Ciljevi su bili da se utvrdi prisustvo, lokalizacija i distribucija tumor infiltri&scaron;ućih limfocita, kako ukupnih, tako i CD4+ i CD8+ T limfocita u tumoru dojke, njihova povezanost sa standardnim prognostičkim parametrima, kao i njihov prognostički značaj tj. razlike u nivou infiltracije limfocita u tumoru u odnosu na pojavu relapsa bolesti i dužinu preživljavanja. METOD: Istraživanjem je obuhvaćeno 120 bolesnica sa invazivnim duktalnim karcinomom, sa tumorom lokalizovanim samo u dojci, bez zahvatanja kože i grudnog mi&scaron;ića, sa veličinom tumora do 5 cm, bez udaljenih visceralnih i ko&scaron;tanih metastaza, koje su operisane u Institutu za onkologiju Vojvodine. U istraživanje su uključene bolesnice bez metastaza u limfnim čvorovima pazu&scaron;ne jame i bolesnice sa metastazama u limfnim čvorovima pazu&scaron;ne jame. Istraživanjem nisu obuhvaćene bolesnice koje su primale neoadjuvantnu (preoperativnu) hemioterapiju, kao ni bolesnice sa multifokalnim i multicentričnim tumorima. REZULTATI: Gust limfocitni infiltrat uočen je u 14% tumora dojke, umeren limfocitni infiltrat uočen je u 38%, a oskudan u 43% tumora dojke. Limfocitni infiltrat nije uočen u 5% tumora. Gust infiltrat CD4+ limfocita uočen je u 8% tumora dojke, umeren u 44%, a oskudan u 43% tumora dojke. CD4+ limfociti nisu uočeni u 5% tumora. Gust infiltrat CD8+ limfocita uočen je u 1% tumora dojke, umeren u 23%, a oskudan u 66% tumora dojke. CD8+ limfociti nisu uočeni u 10% tumora. Utvrđena je pozitivna povezanost između nivoa TIL-a i CD4+ limfocita i veličine tumora, histolo&scaron;kog stepena diferentovanosti tumora, prisustva metastaza u limfnim čvorovima pazu&scaron;ne jame, HER-2 statusa, tripl negativnog tumora i relapsa bolesti. Utvrđena je negativna povezanost između nivoa TIL-a i CD4+ limfocita i estrogen i progesteron receptora, kao i godina starosti. Utvrđena je pozitivna povezanost između nivoa CD8+ limfocita i histolo&scaron;kog gradusa tumora, kao i HER-2 statusa. Utvrđena je negativna povezanost između nivoa CD8+ limfocita i estrogen i progesteron receptora, kao i godina starosti. ZAKLJUČAK:Rezultati ovog istraživanja pokazuju povezanost tumor infiltri&scaron;ućih limfocita i CD4+ limfocita sa brojnim negativnim prognostičkim faktorima, te kraćim vremenom slobodnog intervala bez bolesti, &scaron;to sve ukazuje na to da su tumor infiltri&scaron;ući limfociti kao i CD4+ limfociti lo&scaron; prognostički faktor kod bolesnica sa rakom dojke.</p> / <p>INTRODUCTION: The main problem in the treatment of breast cancer that based on clinical classification and morphological characteristics of the tumor to predict its future behavior. Very often, not a combination of standard prognostic factors does not answer the need of giving adjuvant chemotherapy. In order to implement adequate further treatment of breast cancer and detection aggressive types of tumor, after surgical treatment, there is a constant need to find new indicators by which we can identify patients who have an increased risk of relapse. OBJECTIVES: The objectives were to determine the presence, localization and distribution of tumor infiltrating lymphocytes, in total, as well as CD4+ and CD8+ T lymphocytes in breast cancer, their correlation with standard prognostic parameters, as well as their prognostic value i.e. differences in the level of infiltration of lymphocytes in a tumor in relation to the occurrence of disease relapse and survival. METHOD: The study included 120 patients with invasive ductal carcinoma, tumor localized only in the breast without involvement of the skin and pectoral muscle, the size of tumors up to 5 cm without distant visceral and bone metastases, which are operated at the Institute of Oncology. The study included patients without metastases in axillary lymph nodes and patients with metastases in axillary lymph nodes. The research not covered by patients receiving neoadjuvant chemotherapy, or patients with multifocal and multicentric tumors. RESULTS: The high amount of lymphocytic infiltrate was observed in the 14% a breast tumor, a moderate amount of lymphocytic infiltrate was observed in 38%, and the low in 43% breast tumors. Lymphocytic infiltrate was not observed in 5% of the tumor. High CD4+ lymphocyte infiltration was observed in 8% of breast, moderate in 44%, and the low in 43% of breast tumors. CD4+ lymphocytes were not observed in 5% tumors. High infiltration of CD8+ lymphocytes was observed in 1% of breast, moderate in 23%, and the low 66% breast tumors. CD8+ lymphocytes have not been observed in 10% tumors. There is a positive correlation between the level of TIL and CD4+ lymphocytes and tumor size, histological grade of tumor differentiation, presence of metastases in axillary lymph nodes, HER-2 status, triple negative tumors and relapses of disease. There was a negative correlation between the level of TIL and CD4+ cell counts and estrogen and progesterone receptors, as well as age. There is a positive correlation between the level of CD8+ cells and histological grade of the tumor, and HER-2 status. There was a negative correlation between the level of CD8+ lymphocytes and estrogen and progesterone receptors, as well as age. CONCLUSION: The results of this study demonstrate the association between tumor infiltrating lymphocytes and CD4+ lymphocytes with a number of negative prognostic factors, and shorter free interval without the disease, all of which indicates that the tumor infiltrating lymphocytes and CD4+ lymphocytes bad prognostic factor in patients with breast cancer.</p>

<p>Uobičajeni pristup i testiranju biolo&scaron;kog pona&scaron;anja materijala je da se počne sa jednostavnim in vitro testovima baziranim na ćelijskim kulturama. In vitro testovi citotoksičnosti su danas jedan od osnovnih načina za procenu biolo&scaron;kog odgovora na materijal jer su brži, lak&scaron;i za ponavljanje, ocenjivanje i jeftiniji u odnosu na eksperimente koji se izvode na životinjama i ljudima. Koriste se kao neka vrsta skrining testova za procenu biolo&scaron;ke sigurnosti materijala. Za razliku od ćelijskih kultura, istraživanja koja uključuju eksperimentalne životinje pružaju bolji uvid u biokompatibilnost materijala, zbog mogućnosti praćenja kompleksnog imunolo&scaron;kog odgovora živog organizma. Smatraju se neophodnim za ocenu bilo&scaron;kih odgovora na novi materijal, pre nego &scaron;to se on upotrebi na ljudima. Mnogi aspekti biolo&scaron;kog odgovora ne mogu biti reprodukovani in vitro testovima uključujući krvne interakcije, zarastanje rana, reakcije preosetljivosti, karcinogenezu, hroničnu inflamaciju. Eksperimenti na životinjama pružaju informacije o ovim tipovima efekata bez izlaganja ljudi riziku. Cilj ovog istraživanja bio je da se oceni biokompatibilnost objekata od polimera na bazi epoksi smole Accura&reg; ClearVue&trade; (hemijski sastav: 4,4&rsquo;- izopropilidendicikloheksanol, produkti oligomerne reakcije sa 1-hlor-2,3- epoksipropanom(40-65%), sme&scaron;а triaril-sulfonijum soli (50% propilen-karbonata i 50% triaril-sulfonijum heksafluoroantimonatnih soli) (1-10%) i 3-etil-3hidroksimetil-oksetan(10-20%). U oceni citotoksičnosti materijala Accura&reg; ClearVue&trade; kori&scaron;ćeni su agar diguzioni i MTT test. Oba testa rađena sun a ćelijskim kulturama L929 (mi&scaron;iji fibroblasti) i MRC-5 (humani fibroblasti). Ocena biokompatibilnosti testiranog materijala vr&scaron;ena je na osnovu urađenog testa iritacije oralne mukoze na modelu bukalne kesice hrčka, &scaron;to je definisano standardom ISO 10993-10:2010. Biokompatibilnost materijala ispitana je i implantacijom uzoraka u potkožno tkivo dorzuma pacova soja Wistar.</p> / <p>Uobičajeni pristup i testiranju biolo&scaron;kog pona&scaron;anja materijala je da se počne sa jednostavnim in vitro testovima baziranim na ćelijskim kulturama. In vitro testovi citotoksičnosti su danas jedan od osnovnih načina za procenu biolo&scaron;kog odgovora na materijal jer su brži, lak&scaron;i za ponavljanje, ocenjivanje i jeftiniji u odnosu na eksperimente koji se izvode na životinjama i ljudima. Koriste se kao neka vrsta skrining testova za procenu biolo&scaron;ke sigurnosti materijala. Za razliku od ćelijskih kultura, istraživanja koja uključuju eksperimentalne životinje pružaju bolji uvid u biokompatibilnost materijala, zbog mogućnosti praćenja kompleksnog imunolo&scaron;kog odgovora živog organizma. Smatraju se neophodnim za ocenu bilo&scaron;kih odgovora na novi materijal, pre nego &scaron;to se on upotrebi na ljudima. Mnogi aspekti biolo&scaron;kog odgovora ne mogu biti reprodukovani in vitro testovima uključujući krvne interakcije, zarastanje rana, reakcije preosetljivosti, karcinogenezu, hroničnu inflamaciju. Eksperimenti na životinjama pružaju informacije o ovim tipovima efekata bez izlaganja ljudi riziku. Cilj ovog istraživanja bio je da se oceni biokompatibilnost objekata od polimera na bazi epoksi smole Accura&reg; ClearVue&trade; (hemijski sastav: 4,4&rsquo;- izopropilidendicikloheksanol, produkti oligomerne reakcije sa 1-hlor-2,3- epoksipropanom(40-65%), sme&scaron;a triaril-sulfonijum soli (50% propilen-karbonata i 50% triaril-sulfonijum heksafluoroantimonatnih soli) (1-10%) i 3-etil-3hidroksimetil-oksetan(10-20%). U oceni citotoksičnosti materijala Accura&reg; ClearVue&trade; kori&scaron;ćeni su agar diguzioni i MTT test. Oba testa rađena sun a ćelijskim kulturama L929 (mi&scaron;iji fibroblasti) i MRC-5 (humani fibroblasti). Ocena biokompatibilnosti testiranog materijala vr&scaron;ena je na osnovu urađenog testa iritacije oralne mukoze na modelu bukalne kesice hrčka, &scaron;to je definisano standardom ISO 10993-10:2010. Biokompatibilnost materijala ispitana je i implantacijom uzoraka u potkožno tkivo dorzuma pacova soja Wistar.</p> / <p>The usual approach in testing biological behavior of materials is to start with simple in vitro tests based on cell cultures. In vitro cytotoxicity tests are one of the basic methods of assessing the biological response to material because they are faster, cheaper, easier for repeating and evaluating compared to experiments carried out on animals and humans. They are used as a kind of screening test for evaluating the biosafety of materials. Unlike cell culture, studies involving experimental animals provide better insight into the biocompatibility of materials due to the possibility of monitoring the complex immune response of a living organism. They are considered necessary for assessing the biological response to new material before it is used on humans. Many aspects of a biological response cannot be reproduced with in vitro tests, including blood interaction, wound healing, hypersensitivity reactions, carcinogenesis, chronic inflammation. Animal experiments provide information about these types of effects without exposing humans to risk.&nbsp; The aim of this study was to evaluate the biocompatibility of polymer objects on the basis of epoxy resins Accura&reg; ClearVue &trade; (chemical composition: 4.4&#39; Isopropylidenedicyclohexanol, oligomeric reaction products with 1-chloro-2.3-epoxypropane (40-65%), a mixture of triaryl sulfonium salt (50% propylene carbonate and 50% of a triarylsulfonium hexafluoroantimonate salt) (1- 10%) and 3-ethyl-3-hydroxymethyl-oxetane (10-20%). In the assessment of the cytotoxicity of materials Accura&reg; ClearVue &trade; agar diffusion and MTT tests were used. Both tests were conducted on cell cultures L929 (mouse fibroblasts) and MRC-5 (human fibroblasts). An assessment of the biocompatibility of the tested material was done on the basis of an oral mucosa irritation test on a hamster cheek pouch as defined by ISO 10993-10: 2010. The biocompatibility of the material was also tested with the implantation of a samples into the dorsal subcutaneous tissue of a Wistar rats. The subcutaneous implantation test, as one of the most reliable methods for assessing the biocompatibility of dental materials, is defined by ISO 10993-6: 2010. The study was conducted on 30 rats which were sacrificed in groups</p>

A aspergilose invasiva (AI) é a infecção por fungos filamentosos mais comum em pacientes imunodeprimidos, especialmente em transplantes de células tronco hematopoiético e neoplasias hematológicas. Objetivo: Geral: Estabelecer a comparação entre a dosagem de Galactomanana (GM), 1,3betaD-glucana (BDG) e dados tomográficos no diagnóstico da AI bem como seu papel no desfecho clínico. Específicos: 1. Verificar a sensibilidade e especificidade dos ensaios de Galactomanana e de 1,3betaD-glucana no soro e lavado broncoalveolar. 2. Comparar os resultados da galatomanana e 1,3betaD-glucana com os dados de imagem em pacientes com suspeita de AI. 3. Verificar a relação entre a evolução dos níveis de GM e desfecho clínico (óbito e sobrevida). Casuística, Materiais e Métodos: Realizou-se um estudo tipo coorte prospectiva, incluindo 398 sujeitos das diversas enfermarias de pacientes imunodeprimidos do HCFMUSP, sendo incluídos dois grupos de pacientes: 202(51%) AI e 198(49%) controles. Resultados: Dos casos, 18 (8,8%) tinham aspergilose provada, 28 (13,7%) provável e 158 possível (77,5%), de acordo a classificação de 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer / Mycoses Study Group). Os sujeitos submetidos ao TCTH eram 42,7%, com neoplasias hematológicas 37%, TOS 9% e outras doenças 11,3%. Os fatores de risco associados ao desenvolvimento da AI foram neutropenia, monocitopenia, uso de corticóide, presença de doença pelo citomegalovírus e rejeição ou doença do enxerto contra o hospedeiro. O fator de risco associado à evolução para o óbito foi a presença de AI. Foram observados bons desempenhos para a GM tanto no soro como no LBA com LR menores que os registrados na literatura. O melhor desempenho da GM no soro para aspergilose+provável ocorreu com LR de 0,35 com sensibilidade-S, especificidade-E, valor preditivo positivo- VPP), valor preditivo negativo-VPN) e área sob a curva-ASC de 54,4%, 73,4%, 50,8%, 76,2% e 0,64, sendo os valores superiores para aspergilose provada tanto na S, como E, VPN. No LBA os valores de S-E-VPP-VPN-ASC para GM para LR de 0,65 para aspergilose provável + provada foram 58,3%, 92,6%, 87,5%,71,4% e 0,75, sendo na aspergilose provada os valores de S, e VPN superiores. Nesta casuística, o melhor desempenho para BDG no soro apontou para uma LR de 100 pg/mL na aspergilose provável+provada, com 54,5%, 73,4%, 50,8% e 76,2%, 0,64 respectivamente para S-E-VPP-VPN-ASC. Para BDG no LBA, a LR na aspergilose provável + provada foi de 140 pg/mL, com os mesmos valores de 46,7%, 76,7%, 70%, 55,6% e 0.62, respectivamente. Conclusão: A GM no LBA e no soro foram úteis no diagnóstico da aspergilose mediante emprego de LR menores, sendo mais sensível na LBA, principalmente em estágios iniciais da forma angioinvasiva. A persistência de GM sérica foi relacionada ao óbito em relação à negativação da mesma. A proporção de concordância entre a TC e os biomarcadores no soro e no LBA variou de 0,5 a 0,6, com pequena concordância na estatística kappa. Excelente concordância foi observada entre dois radiologistas independentes, que analisaram de maneira cega as TC de sujeitos com aspergilose provada. Nesta casuística com inclusão de doenças sistêmicas e endêmicas, a BDG teve baixo desempenho diagnóstico / Invasive aspergillosis (IA) has become the leading infectious cause of death in immunocompromised hosts, particularly in subjects under SCTH and hematologic neoplasias. Objectives: General: To compare the performance of GM and BG tests in serum and bronchoalveolar lavage fluid (BAL) and computer tomography (CT) scans in the diagnosis of IA in immunocompromised hosts as well as their role in the patient outcome. Specific: 1. To analyse the sensitivity and specificity of Galactomannan and 1,3 betaD-glucan assays in the serum and bronchoalveolar lavage. 2. To compare the results of Galactomannan and 1,3betaD-glucan assays with CT scans in patients with invasive aspergilosis. 3. To analyse the relationship between the evolution of galactomannan levels and clinical outcome (death or survival). Patients, Materials and Methods: From December 2008 to March 2013, a prospective cohort of 398 patients from several wards of immunocompromised patients of Hospital das Clínicas, Faculdade de Medicina, University of São Paulo was included classified in two groups of patients: 202 (51%) with invasive aspergillosis (IA) and 198 (49%) control patients. Results: Considering 202 cases, 18(8.8%) were subjects with proven, 28(13.7%) with probable aspergillosis and 156(77.5%), with possible aspergillosis, according to 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) criteria. The most common underlying disease were: HSCT (42.7%), hematologic malignancy (37%), SOT (9%), or other diseases (11.3%). The main risk factors associated with IA were neutropenia, monocytopenia, patients under corticosterois, presence of CMV disease, and rejection or graft versus host disease. The risk factor associated with death was the presence of invasive aspergillosis. Good performances for serum and BAL GM were registered with lower cutoffs in the present workin relationship to those found in the literature. The best cutoff for proven + probable aspergillosis for serum GM was observed at 0.35 vallue with Sensitivity-S, Specificity-Sp, Positive Predictive value-PPV), Negative Predictive Value-NPV) and AUC of 54.4%, 73.4%, 50.8%, 76.2% and 0.64; the values for proven aspergillosis alone were higher for S, Sp and NPV. On BAL tests for GM (cutoff value of 0.65) in proven+probable aspergillosis we observed 58.3%, 92.6%, 87.5%,71.4%, 0.75, respectively as S-Sp-PPV-NPVAUC; the sensitivity and VPN were higher in proven aspergillosis alone. In this work, the best performance in proven+probable aspergillosis for serum BDG showed 100 pg/ML as cutoff value, with 54.5%, 73.4%, 50.8%,76.2%, 0.64 for S-Sp-PPVNPV- AUC, respectively. For BAL- BDG, the cut off for proven+probable aspergillosis was 140 pg/mL, and we observed 46.7%, 76.7%, 70.0%, 55.,6%, 0.62, respectively for for S-Sp-PPV-NPV-AUC. Conclusion: The serum and BAL GM are useful tests for diagnosis in early stages of angioinvasive form at lower cutoffs; BAL GM is more sensitive. Agreement proportion between CT scan and each biomarker in the serum or BAL ranged from 0.5-0.6, with low ? index. Perfect ? statistic was observed for analysis of CT scan of subjects in proven aspergillosis by two independent radiologists, blinded for diagnosis. Persistence of serum GM was associated to death in relationship with its negativation. BDG test showed low performance in this work, where systemic and endemic diseases were included

A formulação HIVBr18, previamente desenvolvida e testada, é uma vacina de DNA que codifica 18 epítopos CD4, promíscuos e conservados do HIV-1, e que após imunização de camundongos transgênicos para diversas moléculas de HLA de classe II humanas, observou-se proliferação de linfócitos T CD4+ e CD8+ e produção de IFN-? direcionadas a múltiplos epítopos codificados pela vacina. Abordamos aqui estratégias baseadas na fusão ou combinação dos epítopos codificados pela vacina HIVBr18 à glicoproteína D (gD) do HSV-1, e também na coadministração de plasmídeos que codificam citocinas (IL-2, -12, -15 e GM-CSF) visando aumentar a imunogenicidade de HIVBr18. A sequencia de DNA que codifica os 18 peptídeos da vacina HIVBr18 foi amplificada por PCR e clonada em um plasmídeo que abrigava a sequencia da gD do HSV-1. dando origem ao plasmídeo pVAX-gDh-HIVBr18. Animais imunizados com gDh-HIVBr18 apresentaram resposta imunológica similar ao grupo que recebeu somente HIVBr18, não sendo diferente também daqueles que receberam plasmídeos gDh-HIVBr18 que sofreram alterações nas sequências para melhorar o padrão de distribuição hidrofóbica e permitir a migração da proteína de fusão para o meio extracelular. Construímos e testamos um plasmídeo bicistrônico que expressa gDh e HIVBr18 isoladamente, mas também não observamos aumento na resposta imune induzida. A coadministração com o plasmídeo HIVBr18 e plasmídeos que codificam as citocinas IL-12, IL-15 e GM-CSF, proporciona um aumento na magnitude da resposta imunológica induzida contra o pool de peptídeos codificados pela vacina, entretanto sem alteração da amplitude da resposta. Além disso, o plasmídeo de GM-CSF induziu maior número de células T CD4+ polifuncionais. Demonstramos também que a coadministração do plasmídeo que codifica GM-CSF, induz uma resposta imune celular de maior magnitude mesmo em uma condição de dose reduzida. Entretanto, observamos que esta citocina não é um bom adjuvante quando utilizamos como vetor de imunização um adenovírus que expressa os 18 epítopos / The formulation HIVBr18, previously developed and tested, is based on a DNA vaccine encoding 18 conserved and promiscuous HIV-1 CD4 epitopes and after immunization of transgenic mice for many human HLA class II molecules using this DNA vaccine, could be observed proliferation of CD4+ and CD8+ T cells and IFN-y production directed to multiple epitopes encoded by the vaccine. We intend to explore here, strategies based on fusion or combination of epitopes encoded by HIVBr18 vaccine with glycoprotein D (gD) of HSV- 1 and also the coadministration of cytokine-encoding plasmids (pIL-2, -12, -15 and pGM -CSF) aiming to enhance immunogenicity of HIVBr18. The DNA sequence of epitopes encoded by HIVBr18 vaccine was amplified by PCR and cloned into a plasmid that contained the sequence of gD, giving rise to plasmid pVAX-gDh-HIVBr18. After mice immunization, animals immunized with this construct showed similar immune response to the group that received HIVBr18, and also the group of animals that received gDh-HIVBr18 plasmid that had been modified by exchange in peptides order to assure to the molecule a better hydrophobic distribution and allow translocation to the extracellular face of cell membrane. We constructed and injected mice with a bicistronic plasmid expressing gDh and HIVBr18, simultaneously and isolated, but no increase in the magnitude of the immune response was observed. HIVBr18 coadministration with cytokine-encoding plasmids pIL-12, pIL-15 and pGM-CSF, provides an increase in the magnitude of immune response induced against the peptides encoded by the vaccine, and similar breadth. In addition, co-immunization with pGM-CSF induced greater number of polyfunctional CD4 + T cells. We also demonstrate that, even in a low dose approach coadministration of pGM-CSF induced a higher immune response than HIVBr18 alone in the same dose. However, we observed that this cytokine is not a good adjuvant when used in combination with an adenovirus that expresses the 18 HIV-1 epitopes.

A aspergilose invasiva (AI) é a infecção por fungos filamentosos mais comum em pacientes imunodeprimidos, especialmente em transplantes de células tronco hematopoiético e neoplasias hematológicas. Objetivo: Geral: Estabelecer a comparação entre a dosagem de Galactomanana (GM), 1,3betaD-glucana (BDG) e dados tomográficos no diagnóstico da AI bem como seu papel no desfecho clínico. Específicos: 1. Verificar a sensibilidade e especificidade dos ensaios de Galactomanana e de 1,3betaD-glucana no soro e lavado broncoalveolar. 2. Comparar os resultados da galatomanana e 1,3betaD-glucana com os dados de imagem em pacientes com suspeita de AI. 3. Verificar a relação entre a evolução dos níveis de GM e desfecho clínico (óbito e sobrevida). Casuística, Materiais e Métodos: Realizou-se um estudo tipo coorte prospectiva, incluindo 398 sujeitos das diversas enfermarias de pacientes imunodeprimidos do HCFMUSP, sendo incluídos dois grupos de pacientes: 202(51%) AI e 198(49%) controles. Resultados: Dos casos, 18 (8,8%) tinham aspergilose provada, 28 (13,7%) provável e 158 possível (77,5%), de acordo a classificação de 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer / Mycoses Study Group). Os sujeitos submetidos ao TCTH eram 42,7%, com neoplasias hematológicas 37%, TOS 9% e outras doenças 11,3%. Os fatores de risco associados ao desenvolvimento da AI foram neutropenia, monocitopenia, uso de corticóide, presença de doença pelo citomegalovírus e rejeição ou doença do enxerto contra o hospedeiro. O fator de risco associado à evolução para o óbito foi a presença de AI. Foram observados bons desempenhos para a GM tanto no soro como no LBA com LR menores que os registrados na literatura. O melhor desempenho da GM no soro para aspergilose+provável ocorreu com LR de 0,35 com sensibilidade-S, especificidade-E, valor preditivo positivo- VPP), valor preditivo negativo-VPN) e área sob a curva-ASC de 54,4%, 73,4%, 50,8%, 76,2% e 0,64, sendo os valores superiores para aspergilose provada tanto na S, como E, VPN. No LBA os valores de S-E-VPP-VPN-ASC para GM para LR de 0,65 para aspergilose provável + provada foram 58,3%, 92,6%, 87,5%,71,4% e 0,75, sendo na aspergilose provada os valores de S, e VPN superiores. Nesta casuística, o melhor desempenho para BDG no soro apontou para uma LR de 100 pg/mL na aspergilose provável+provada, com 54,5%, 73,4%, 50,8% e 76,2%, 0,64 respectivamente para S-E-VPP-VPN-ASC. Para BDG no LBA, a LR na aspergilose provável + provada foi de 140 pg/mL, com os mesmos valores de 46,7%, 76,7%, 70%, 55,6% e 0.62, respectivamente. Conclusão: A GM no LBA e no soro foram úteis no diagnóstico da aspergilose mediante emprego de LR menores, sendo mais sensível na LBA, principalmente em estágios iniciais da forma angioinvasiva. A persistência de GM sérica foi relacionada ao óbito em relação à negativação da mesma. A proporção de concordância entre a TC e os biomarcadores no soro e no LBA variou de 0,5 a 0,6, com pequena concordância na estatística kappa. Excelente concordância foi observada entre dois radiologistas independentes, que analisaram de maneira cega as TC de sujeitos com aspergilose provada. Nesta casuística com inclusão de doenças sistêmicas e endêmicas, a BDG teve baixo desempenho diagnóstico / Invasive aspergillosis (IA) has become the leading infectious cause of death in immunocompromised hosts, particularly in subjects under SCTH and hematologic neoplasias. Objectives: General: To compare the performance of GM and BG tests in serum and bronchoalveolar lavage fluid (BAL) and computer tomography (CT) scans in the diagnosis of IA in immunocompromised hosts as well as their role in the patient outcome. Specific: 1. To analyse the sensitivity and specificity of Galactomannan and 1,3 betaD-glucan assays in the serum and bronchoalveolar lavage. 2. To compare the results of Galactomannan and 1,3betaD-glucan assays with CT scans in patients with invasive aspergilosis. 3. To analyse the relationship between the evolution of galactomannan levels and clinical outcome (death or survival). Patients, Materials and Methods: From December 2008 to March 2013, a prospective cohort of 398 patients from several wards of immunocompromised patients of Hospital das Clínicas, Faculdade de Medicina, University of São Paulo was included classified in two groups of patients: 202 (51%) with invasive aspergillosis (IA) and 198 (49%) control patients. Results: Considering 202 cases, 18(8.8%) were subjects with proven, 28(13.7%) with probable aspergillosis and 156(77.5%), with possible aspergillosis, according to 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) criteria. The most common underlying disease were: HSCT (42.7%), hematologic malignancy (37%), SOT (9%), or other diseases (11.3%). The main risk factors associated with IA were neutropenia, monocytopenia, patients under corticosterois, presence of CMV disease, and rejection or graft versus host disease. The risk factor associated with death was the presence of invasive aspergillosis. Good performances for serum and BAL GM were registered with lower cutoffs in the present workin relationship to those found in the literature. The best cutoff for proven + probable aspergillosis for serum GM was observed at 0.35 vallue with Sensitivity-S, Specificity-Sp, Positive Predictive value-PPV), Negative Predictive Value-NPV) and AUC of 54.4%, 73.4%, 50.8%, 76.2% and 0.64; the values for proven aspergillosis alone were higher for S, Sp and NPV. On BAL tests for GM (cutoff value of 0.65) in proven+probable aspergillosis we observed 58.3%, 92.6%, 87.5%,71.4%, 0.75, respectively as S-Sp-PPV-NPVAUC; the sensitivity and VPN were higher in proven aspergillosis alone. In this work, the best performance in proven+probable aspergillosis for serum BDG showed 100 pg/ML as cutoff value, with 54.5%, 73.4%, 50.8%,76.2%, 0.64 for S-Sp-PPVNPV- AUC, respectively. For BAL- BDG, the cut off for proven+probable aspergillosis was 140 pg/mL, and we observed 46.7%, 76.7%, 70.0%, 55.,6%, 0.62, respectively for for S-Sp-PPV-NPV-AUC. Conclusion: The serum and BAL GM are useful tests for diagnosis in early stages of angioinvasive form at lower cutoffs; BAL GM is more sensitive. Agreement proportion between CT scan and each biomarker in the serum or BAL ranged from 0.5-0.6, with low ? index. Perfect ? statistic was observed for analysis of CT scan of subjects in proven aspergillosis by two independent radiologists, blinded for diagnosis. Persistence of serum GM was associated to death in relationship with its negativation. BDG test showed low performance in this work, where systemic and endemic diseases were included