La voie PI3K/AKT est impliquée dans la progression de divers cancers humains, et semble jouer un rôle majeur dans le développement de tumeurs lymphoïdes. Elle pourrait être impliquée dans la pathogénie du lymphome folliculaire (LF) par certains mécanismes non identifiés. Les travaux de thèse portent sur l'étude des anomalies de la voie PI3K/AKT dans le LF, dans le but d'apporter une nouvelle cible thérapeutique. 38 biopsies tissulaires de LF humain ont été étudiées pour une analyse mutationnelle du gène PIK3CA dans les exons 9 et 20 par séquençage. Les mêmes échantillons ont été analysés par western blot et immunohistochimie pour détecter l'expression des protéines AKT, AKT phosphorylée (pAKT), et PTEN. Deux cas de lymphadénite ont été utilisés comme témoins.Les résultats obtenus montrent que l'expression d'AKT était présente dans tous les cas de LF et lymphadénite, et 14/38 (37%) échantillons de LF et 2/2 cas de lymphadénite exprimaient pAKT. 9/38 (24%) échantillons de LF ont montré un niveau élevé d'expression de pAKT, alors que 5/38 (13%) cas de LF, et 2/2 échantillons de lymphadénite montraient un faible niveau d'expression de pAKT. L'expression de PTEN a été observée dans 30/38 (79%) cas de LF et 2/2 cas de lymphadénite, tandis que 8/38 (21%) cas ont montré une perte d'expression de PTEN. En outre, 3 cas qui expriment pAKT montrent une perte d'expression de PTEN. Aucune mutation du gène PIK3CA n'a été détectée dans les échantillons étudiés. Ces données suggèrent que la voie PI3K/AKT peut être activée dans certains cas de LF, soit en raison de la phosphorylation d'AKT, soit en raison d'une perte d'expression de PTEN, en absence de mutations de PIK3CA. / The phosphoinositide 3- kinase (PI3K) pathway is involved in the growth of various human cancers, including lymphoid malignancies. However its role in the pathogenesis of follicular lymphoma (FL) has not been yet described.The PhD work focuses on the study of alterations in the PI3K/AKT pathway in follicular lymphoma, in order to provide a new therapeutic target.To clarify this point, biopsy tissue samples from 38 human FL cases were investigated for PIK3CA somatic mutations in exons 9 and 20 using Sanger sequencing. The same samples were analyzed using western blotting and immunohistochemistry to detect expression of AKT, phosphorylated AKT (pAKT), and PTEN proteins. Two cases of benign lymphadenitis were used as controls. AKT expression was present in all FL and lymphadenitis cases. 14/38 (37%) FL and 2/2 lymphadenitis cases expressed pAKT. 9/38 (24%) FL samples showed high level of pAKT, whereas 5/38 (13%) FL cases and 2/2 benign lymphadenitis samples expressed pAKT at low level. PTEN expression was observed in 30/38 (79%) FL and 2/2 benign lymphadenitis cases, whereas 8/38 (21%) of FL cases showed loss of PTEN expression. In addition, 3 cases with positive pAKT did not express PTEN. PIK3CA mutations were not detected in any sample. These data suggest that the PI3K/AKT signaling pathway could be activated in a subset of FL cases, due to either AKT phosphorylation or PTEN downregulation, in the absence of PIK3CA mutations.

No description available.

In situ follicular neoplasia (ISFN) is defined as a monoclonal proliferation of B cells with immunophenotypic and genetic features of follicular lymphoma (FL) but confined to germinal centers of lymph nodes or other organs. It may not be associated with underlying overt lymphoma. It can be associated with lymphoproliferative disorders other than FL. A fifty-seven-year-old caucasian male initially presented with atypical chest pain, which led to cardiology evaluation. Patient underwent a coronary CT angiogram, which revealed a calcium score of 0, however also incidentally revealed mediastinal lymphadenopathy. Patient had a bronchoscopy which revealed no endobronchial lesions bilaterally. Using endo-bronchial ultrasound, right carinal lymph node was visualized, and trans-bronchial fine needle aspiration was performed. Cytology was positive for necrotic lesion with atypical cells. Patient had a dedicated CT scan of chest which showed enlarged sub-carinal lymph node measuring 3.3 x 3.0 cm. PET/CT scan showed increased uptake in the sub-carinal lymph nodes, also increased uptake of mid para-esophageal lymph nodes. It also showed some low-grade lymphadenopathy in right lower paratracheal region as well as mesenteric lymphadenopathy with misty appearance. Small pulmonary nodules were also noted in right middle and lower lobes with no associated uptake. Patient was scheduled for a mediastinoscopy and lymph node dissection. Patient proceeded with mediastinoscopy and a total of 4 lymph node specimens were removed from level 4R and level 7. Pathology from one of the lymph nodes revealed necrotizing granulomatous inflammation with staining consistent with histoplasmosis. Interestingly, two other lymph nodes showed in situ follicular neoplasia. Immunohistochemical stains demonstrated rare secondary lymphoid follicles with unremarkable morphology, showing strong germinal center staining with BCL2. FISH analysis was normal indicating absence of t(14;18). Pathology showed morphologically unremarkable B-cell nodules, concentrated in the cortical area which were CD20 positive and BCL2-positive. Patient underwent subsequent treatment with anti-fungal agents for the Histoplasmosis and is currently under surveillance for in-situ follicular lymphoma. In-situ follicular neoplasia is considered a premalignant lesion and a precursor of follicular lymphoma. Incidence of ISFN is difficult to ascertain, as it is usually a subclinical diagnosis. Incidence of FL is 3.18 per 100,000 population in the United States and findings suggest that ISFN is likely more frequent than that. Also, similar to FL, ISFN is seen in middle-aged and older individuals, mean age being around the fifth decade of life.Incidentally found ISFN without prior or simultaneous lymphoma is associated with a very low rate of clinical progression. Because some cases of ISFN are associated with prior or concurrent lymphoma, screening studies including computed tomography (CT) scan and bone marrow biopsy should be conducted after the diagnosis of ISFN is made. In the absence of overt lymphoma, it has been recommended that patients with ISFN be observed without chemotherapy, based on the very low incidence of progression into overt FL. The clinical significance of ISFN is not fully understood, however studies have demonstrated that incidentally found ISFN without prior or simultaneous lymphoma is associated with a very low rate of clinical progression. (

Orientador: Eunice Oba / Resumo: As substâncias desreguladores endócrinas de diversas origens e propriedades químicas acomentem uma diversidade de mamíferos, animais silvestres e seres humanos, que podem interagir com o sistema reprodutivo feminino e prejudicar a homeostase hormonal. Dentro dessa classificação, os parabenos podem ser utilizados como conservantes em diversos produtos comerciais. Seus efeitos se tornam cada vez mais complexos em relação à idade, história reprodutiva, ambiente endócrino da espécime no momento da exposição, como também não é sabido todos os reais mecanismos desses compostos quando associados. Sendo assim, o objetivo deste estudo foi avaliar os efeitos dos parabenos em ratas Wistar durante um período de 90 dias consecutivos de exposição. Assim, as ratas foram distribuídas em quatro grupos experimentais: controle, que recebeu somente óleo de milho como veículo, e tratados com propil e buti-parabeno nas doses de 10 mg/kg, 100 mg/kg, ou 200 mg/kg e expostas via subcutânea. Foi utilizada a técnica de citologia vaginal para investigar a ciclicidade das fêmeas, bem como a caracterização das fases do ciclo estral pelo método Shorr. Além desses parâmetros, foram analisados aspectos morfológicos de ovários e útero, quantificação das estruturas ovarianas e avaliação ultraestutural de folículos pré-ovulatórios. Foram avaliados a concentração plasmática de estrógeno, peso corporal bem como o peso dos órgãos reprodutivos. Os resultados revelaram que o método Shorr, foi eficaz para identificar... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The endocrine disruption substances of many origins and chemistry properties affect a diversity of mammals, wild animals and human beings. These substances can interact with the female reproductive system and damage the hormonal homeostasis. In this classification, the paraben could be used as preservatives in different commercial products. Its effects get even worst and complex with the age, reproductive history, and endocrine environment of the specie in the moment of exposure of the drug. In complement, it is not known the actual mechanisms of those compounds when they are associated. Thus, the goal of this experiment was evaluate the effects of parabens in females Wistar during 90 consecutive days of exposure. Therefore, the females were divided in four experimental groups: Control group – Corn oil; and a mixture of propil and butyl paraben with 10 mg/kg, 100 mg/kg and 200 mg/kg and exposed through the subcutaneous pathway. The vaginal cytology was performed to investigate the cyclicality as well as the characterization of the estrous phases by the Shor method. Besides that, morphological aspects of the ovary and uterus, counting of ovaries structure and ultrastructural of pre-ovulatory follicle. To complete the evaluation, the plasmatic estrogen, body weight and reproductive organ weight were assessed. The Shor method was effective to characterize and identify the cells types of the vaginal epithelium and identify the estrous cycle phase. Moreover, the relative and absol... (Complete abstract click electronic access below) / Mestre

The histologic distinction of follicular patterned lesions of thyroid, that is follicular adenoma, follicular carcinoma, and the follicular variant of papillary thyroid carcinoma can be extremely difficult. The differential diagnostic criteria regarding nuclear features of papillary thyroid carcinoma are subjective, resulting in high interobserver variability. Although papillary thyroid carcinoma metastasizes mainly via lymphatic vessels, whereas follicular carcinoma spreads mostly hematogenously, there are no data regarding utility of objective quantitative criteria such as lymphatic and general blood vessel density for the differential diagnosis of these lesions. In this study, 35 follicular patterned lesions of thyroid (14 follicular adenomas, 10 follicular carcinomas, and 11 of the follicular variant of papillary thyroid carcinomas) were evaluated immunohistochemically. An assessment of intra- and peritumoral lymphatic vessel density was performed using novel lymphatic endothelium-specific marker D2-40, and the intra- and peritumoral general vessel density was determined by the panendothelial marker CD31. There were no significant differences in the intra- and/or peritumoral general vessel densities, and peritumoral lymphatic vessel densities among follicular adenoma, follicular carcinoma and the follicular variant of papillary thyroid carcinoma. In contrast, the intratumoral lymphatic vessel density was significantly higher in the follicular variant of papillary thyroid carcinoma than in either follicular adenoma or follicular carcinoma (34.63, 15.04, and 0.11 respectively; P<0.0001). The results of the study show that intratumoral lymphatic vessel density may serve as a useful tool in the differential diagnosis of follicular patterned lesions of thyroid. © 2005 USCAP, Inc All rights reserved.

Indiana University-Purdue University Indianapolis (IUPUI) / Human immunodeficiency virus (HIV) primarily infects T helper (Th) cells. Decrease in the number of Th cells is the hallmark of HIV infection. Latent reservoirs of human immunodeficiency virus (HIV) are the leading barrier towards eradication of HIV infection. T Follicular helper (Tfh) cells are a subset of Th cells that function to provide aid to B cells for their maturation, affinity selection and antibody class switch. Several studies have shown that Tfh cells are a major reservoir of latent as well as productive hiv infection. But in contrast to the fate of other Th cell subsets, the frequency of Tfh cells was shown to have increased during HIV infection which could not be attributed to their reduced susceptibility to HIV infection. The hypothesis was that Tfh cells possess a unique metabolic phenotype that protects them from HIV induced cell death. Transcriptome analysis of Th subsets from human donors and showed that Tfh cells rely less on glycolysis for their energetic requirements and instead have increased transcription of fatty acid synthesis genes. This finding was corroborated by seahorse extracellular flux assay. The results shoId that glycolysis was not essential for Tfh cell differentiation in-vitro. The observed increase in Tfh cell frequency could not be attributed to increased Tfh differentiation upon HIV infection since HIV infection inhibited the differentiation of both non-Tfh and Tfh cells. The results found that bypassing the glycolytic pathway by providing Tfh cells with Galactose in the medium protected ex-vivo infected primary tonsillar cells from HIV induced cell death. This protection could be partly explained by the induction of Baculovirus IAP repeat containing 5 (BIRC5) when the cells utilized Galactose instead of Glucose. The studies together show that Tfh cells have an oxidative metabolic phenotype which protects them from HIV induced cell death in part by induction of BIRC5 expression.

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No description available.

Virus-infected cells can regulate non-permissive bystander cells, but the precise mechanisms remain incompletely understood. Here we report that this process can be mediated by transfer of viral RNA-loaded exosomes shed from infected cells to myeloid-derived suppressor cells (MDSCs), which in turn regulate the differentiation and function of T cells during viral infection. Specifically, we demonstrated that patients with chronic hepatitis C virus (HCV) infection exhibited significant increases in T follicular regulatory (TFR) cells and decreases in T follicular helper (TFH) cells. These MDSC-mediated T-cell dysregulations resulted in an increased ratio of TFR/TFH and IL-10 production in peripheral blood. Specifically, co-culture of MDSCs derived from HCV patients with healthy peripheral blood mononuclear cells (PBMCs) induced expansion of TFR, whereas depletion of MDSCs from PBMCs of HCV patients reduced the increases in TFR frequency and IL-10 production, and promoted the differentiation of IFN-γ-producing TFH cells. Importantly, we found that exosomes isolated from the plasma of HCV patients and supernatant of HCV-infected hepatocytes could drive monocytic myeloid cell differentiation into MDSCs. These exosomes were enriched in tetraspanins, such as CD63 and CD81, and contained HCV RNA, but exosomes isolated from patients with antiviral treatment contained no HCV RNA and could not induce MDSC differentiation. Notably, these HCV RNA-containing exosomes (HCV-Exo) were sufficient to induce MDSCs. Furthermore, incubation of healthy myeloid cells with these HCV-Exo inhibited the expression of miR−124, whereas reconstitution of PBMCs with miR−124 abolished the effects of HCV−Exo on MDSC induction. Taken together, these results indicate that HCV-associated exosomes can transfer immunomodulatory viral RNA from infected cells to neighboring immune cells and trigger MDSC expansion, which subsequently promotes TFR differentiation and inhibits TFH function. This study reveals a previously unrecognized path that represents a novel mechanism of immune dysregulation during chronic viral infection.

Follicular dendritic cells (FDCs) reside in the lymphoid follicles of the secondary lymphoid tissues (sLTs). Following the infection of an individual with human immunodeficiency virus type 1 (HIV-1), viral particles are trapped in massive quantities on the surfaces of FDCs. HIV-1 viral compartments are cell types or tissues between which there is a restriction of virus flow. Compartmentalization of HIV-1 creates numerous sites within the body in which the virus can undergo independent evolution, giving rise to a more diverse total viral population. Given the sessile nature of the FDC, I hypothesized that contrary to common assumptions, FDC-trapped HIV-1 is compartmentalized between different sLTs. Furthermore, given that FDC-trapped HIV-1 represents the major source of virus in the host, I postulated that this compartmentalization would likely impact the diversity of HIV-1 associated with the sLTs. I isolated FDCs, macrophages, and T cells from various sLTs, and sequenced cloned HIV-1 associated with these three cell populations. I subjected the resulting DNA and cDNA sequence data to phylogenetic and other statistical analyses. In support of my hypothesis, I demonstrate that both HIV-1 gp120 and pol sequences cloned from FDCs are compartmentalized between different sLTs. This compartmentalization is even apparent between lymph nodes taken from the same lymph node chain. One of the apparent effects of this compartmentalization is to significantly increase the viral genetic diversity in multiple sLTs when compared with diversity in a single sLT. It also appears that the selective pressures on HIV-1 differ among the sLTs. In addition, when proviruses isolated from macrophages from different sLTs were compared, it was also evident that there is compartmentalization of HIV-1 associated with this cell type as well. Finally, I demonstrate that HIV-1 isolated from an unfractionated population of cells from a single sLT, may be an inadequate representation of the total viral population in that sLT. Taken together, my data suggest that the nature of HIV-1 in the sLTs may be more complex than currently appreciated.