Background: HIV/AIDS remains a global concern that, although manageable using anti-retroviral therapy (ART), is still eluded by a cure with paucity of knowledge regarding its acquisition and spread especially through the male genital tract (MGT)1–4. Several authors have shown the human foreskin to be an effective mucosal effector site with heterogenous populations of innate and adaptive immune cells, that are permissive to HIV infection5–8. In support of this, medical male circumcision (MMC), has been reported to confer up to 60 % risk reduction in HIV acquisition9–17. Most studies have focused on investigating blood lymphoid immune cells and their interaction with HIV-1, this study sought to elucidate the myeloid cell composition of the inner and outer foreskin, and to investigate the susceptibility of these cells to ex vivo HIV infection by (i) Isolating migratory and non-migratory Langerhans cells (LCs) and “macrophage-like” cells from the foreskin epidermis (ii) Immunophenotyping and characterising foreskin LCs and “macrophage-like” cells using CD4+CCR5+ as proxy for HIV susceptibility, HLA-DR+CD80/86+ for maturation, and the mannose receptor, DC-SIGN and Siglec-1 as HIV attachment factors and (iii) Investigating the HIV susceptibility of foreskin epidermal cells using an optimised ex vivo pluricellular foreskin infection model of suspension cells. Methodology: Foreskin specimen were obtained from 60 seronegative adult South African men (aged 18-35 years) undergoing voluntary medical male circumcision (vMMC). Migratory and non-migratory foreskin cells were isolated from the inner and outer foreskin using spontaneous migration and enzymatic digestion of remnant epidermal tissue respectively, and subsequently immunophenotyped using multiparameter flow cytometry (n=31). The optimal HIV infection model was determined through assessment of different infection models inclusive of i) epidermal sheets, ii) foreskin explants and iii) pluricellular suspension cells (n=5). Using the ex vivo pluricellular foreskin infection model of suspension cells (n=17), Subtype C transmitted founder (T/F) and chronic infection derived (CC) infectious molecular clones (IMCs) were used alongside Subtype B NL4-3 IMCs with CCR5, CXCR4 and BaL envelopes. The extent of HIV infection was quantified by measurement of p24 in different immune cell subsets over a time-course. The different HIV infected cell subsets were characterized using CD45, CD207, CD1a, CD11c, CD14, CD3, HLA-DR, CD80/86, CD209, CD206, CD169, CD4 and CCR5. Results: Foreskin myeloid cells contained a rare population of LCs (1.11 % ± 1.02 %;) that was predominantly migratory (p = 0.0084) and “macrophage-like” cells (9.87 % ± 9.64 %) that, in addition to being 8-fold more abundant (p

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os autores têm como objetivo, investigar a matriz extra celular, musculatura lisa e densidade vascular do prepúcio de pacientes tabagistas. Espécimes de prepúcio foram obtidas de 20 jovens adultos (média de idade= 27.2) submetidos a postectomia. Dentre os pacientes analisados, um grupo (n=10) possui história prévia de tabagismo (3 to 13 maços/ano, média = 5.8 3.2), e outro grupo (n=10) formam o grupo controle, não fumantes. A coloração de Tricrômico Masson foi utilizada para quantificar tecido conectivo, musculatura lisa e vasos. A coloração Resorcina-fucsina de Weigert foi utilizada para estabelecer as fibras do sistema elástico e a coloração, Vermelho de Picrosirius para o estudo do colágeno. O estudo estereológico foi realizado utilizando o software Image J, para determinar as densidades volumétricas. Para a análise bioquímica o colágeno total foi determinado em &#956;g de hidroxiprolina por MG de tecido seco. O estudo estatístico foi realizado lançando mão do t-teste (p<0,05). Fibras do sistema elástico de fumantes apresentaram-se aumentadas em 42.5% quando comparado ao grupo controle (p=0,002). Em contraste, musculatura lisa (p=0,42) e densidade vascular (p=0,16) não mostraram nenhuma diferença estatística. Foi realizado uma análise quantitativa utilizando Vermelho de Picrosirius sob luz polarizada, que evidenciou a presença de colágeno tipo I e III, sem diferença estatisticamente significativa. A concentração total do colágeno não mostrou diferença entre tabagistas e o grupo controle. (73.1&#956;g/mg 8.0 vs. 69.2&#956;g/mg 5.9, respectivamente, p=0,23). Tabagismo está associado a um significante aumento de fibras do sistema elástico do tecido prepucial. Estes resultados podem, possivelmente, explicar os altos índices de falha na uretroplastia peniana, com uso de flap de prepúcio em fumantes / It has been speculated by McAninch et al. (1), that smokers might experience worse results after urethral reconstruction. We decided to investigate the extracellular matrix, smooth muscle and vascular density in the foreskin of smoker patients. Foreskin samples were obtained from 20 young adults (ranging in age from 23 to 36 years; mean SD = 27.2 5.8) submitted to circumcision from July 2008 to October 2009. Of the patients analyzed, one group (n=10) had a previous history of chronic smoking (3 to 13 packs/year, mean = 5.8 3.2) i.e., one pack per day for 3 to 13 years. The control group was composed of 10 non-smoker patients. Foreskin samples were studied by histology and biochemistry. Means were compared using the two-tailed t-test (p<0.05). The elastic system fibers in the foreskin of smoker patients increased in 42.5% when compared to the control group. In contrast, the smooth muscle fibers, vascular density and total collagen concentration did not show any significant variation between smokers and controls. Smoking is associated with a significant increase of elastic system fibers in foreskin tissue. These results could possibly explain the high failure rate of penile urethroplasty in smokers by using foreskin flaps

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os autores têm como objetivo, investigar a matriz extra celular, musculatura lisa e densidade vascular do prepúcio de pacientes tabagistas. Espécimes de prepúcio foram obtidas de 20 jovens adultos (média de idade= 27.2) submetidos a postectomia. Dentre os pacientes analisados, um grupo (n=10) possui história prévia de tabagismo (3 to 13 maços/ano, média = 5.8 3.2), e outro grupo (n=10) formam o grupo controle, não fumantes. A coloração de Tricrômico Masson foi utilizada para quantificar tecido conectivo, musculatura lisa e vasos. A coloração Resorcina-fucsina de Weigert foi utilizada para estabelecer as fibras do sistema elástico e a coloração, Vermelho de Picrosirius para o estudo do colágeno. O estudo estereológico foi realizado utilizando o software Image J, para determinar as densidades volumétricas. Para a análise bioquímica o colágeno total foi determinado em &#956;g de hidroxiprolina por MG de tecido seco. O estudo estatístico foi realizado lançando mão do t-teste (p<0,05). Fibras do sistema elástico de fumantes apresentaram-se aumentadas em 42.5% quando comparado ao grupo controle (p=0,002). Em contraste, musculatura lisa (p=0,42) e densidade vascular (p=0,16) não mostraram nenhuma diferença estatística. Foi realizado uma análise quantitativa utilizando Vermelho de Picrosirius sob luz polarizada, que evidenciou a presença de colágeno tipo I e III, sem diferença estatisticamente significativa. A concentração total do colágeno não mostrou diferença entre tabagistas e o grupo controle. (73.1&#956;g/mg 8.0 vs. 69.2&#956;g/mg 5.9, respectivamente, p=0,23). Tabagismo está associado a um significante aumento de fibras do sistema elástico do tecido prepucial. Estes resultados podem, possivelmente, explicar os altos índices de falha na uretroplastia peniana, com uso de flap de prepúcio em fumantes / It has been speculated by McAninch et al. (1), that smokers might experience worse results after urethral reconstruction. We decided to investigate the extracellular matrix, smooth muscle and vascular density in the foreskin of smoker patients. Foreskin samples were obtained from 20 young adults (ranging in age from 23 to 36 years; mean SD = 27.2 5.8) submitted to circumcision from July 2008 to October 2009. Of the patients analyzed, one group (n=10) had a previous history of chronic smoking (3 to 13 packs/year, mean = 5.8 3.2) i.e., one pack per day for 3 to 13 years. The control group was composed of 10 non-smoker patients. Foreskin samples were studied by histology and biochemistry. Means were compared using the two-tailed t-test (p<0.05). The elastic system fibers in the foreskin of smoker patients increased in 42.5% when compared to the control group. In contrast, the smooth muscle fibers, vascular density and total collagen concentration did not show any significant variation between smokers and controls. Smoking is associated with a significant increase of elastic system fibers in foreskin tissue. These results could possibly explain the high failure rate of penile urethroplasty in smokers by using foreskin flaps

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-03-29T18:02:18Z No. of bitstreams: 1 andrenettobastos.pdf: 5066328 bytes, checksum: 5fa19bf9587471962ff6d52065ab0f21 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-03-30T11:23:45Z (GMT) No. of bitstreams: 1 andrenettobastos.pdf: 5066328 bytes, checksum: 5fa19bf9587471962ff6d52065ab0f21 (MD5) / Made available in DSpace on 2017-03-30T11:23:45Z (GMT). No. of bitstreams: 1 andrenettobastos.pdf: 5066328 bytes, checksum: 5fa19bf9587471962ff6d52065ab0f21 (MD5) Previous issue date: 2009-09-09 / O objetivo do presente estudo é analisar e comparar os diferentes aspectos histológicos do prepúcio de crianças com hipospádia, com e sem aplicação prévia de testosterona, comparando com o de crianças postectomizados. O prepúcio de nove crianças postectomizadas (G1), 13 crianças com hipospádia sem uso de testosterona (G2) e 13 com hipospádia que fizeram aplicação tópica de propionato de testosterona a 1% por 30 dias (G3) foram incluídas no estudo. Avaliação histológica dos prepúcios para fibras colágenas foi feita com picrosírius, usado para avaliar densidade dessas fibras. Com o Tricrômio de Masson foi avaliada a homogeneidade das fibras colágenas e sua quantificação através do sistema de arcos ciclóides sobreposto a um sistema de vídeomicroscopia, onde obtive-se a densidade de superfície. Resorcina-fucsina de Weigert foi usada para avaliar a densidade e homogeneidade das fibras elásticas. Com o receptor de andrógeno avaliou-se o número e intensidade das células coradas. Fator de Von Willebrand foi usado para avaliar o número e densidade de volume dos vasos sanguíneos. O picrosírius, não mostrou diferenças entre os grupos estudados (p= 0,905). Com o Tricrômio de Masson, observamos que os pacientes tratados com testosterona (G3) apresentaram uma menor homogeneidade (p= 0,001) e menor densidade de superfície de fibras colágenas (0,3 ± 0,1 fibras colágenas) que aqueles não tratados (0,4 ± 0,1 fibras colágenas) (p< 0,001). As crianças postectomizadas apresentaram um padrão de distribuição de fibras elásticas mais denso (p= 0,003) e menos homogêneo (p= 0,008) que os demais grupos. A marcação para receptor de andrógeno foi maior nas crianças do G1 quando comparadas ao G3 (p=0,011). O prepúcios tratados com testosterona (G3) apresentaram aumento do valor numérico absoluto de vasos (8,5 ± 1,3 vasos/campo) (p< 0,001) e aumento da densidade de volume destes vasos (50,5% ± 7,8 vasos/ponto) quando comparados com o grupo não tratado (G2) (24,8% ± 8,6 vasos/ponto) (p< 0,001). O uso tópico de propionato de testosterona a 1% foi capaz de provocar neovascularização, em número absoluto e densidade de volume, e reduzir o tecido fibroso. Crianças tratadas apresentaram padrão de distribuição de fibras colágenas menos homogêneo próxima aos vasos o que sugere menor maturidade destas fibras com menos deposição de colágeno. / The goal of this study is to analyze the different histological features of the foreskins of children with hypospadia, that has and has not recieved application of testosterone cream, and compare them with circumcised patients. The foreskin of 09 circumcised children (G1), 13 children with hypospadia, who did not receive testosterone (G2), and 13 children who received topical 1% testosterone propionate (G3) were included in this study. The histological evaluation of the collagen fiber was done with Picrosirius staining to analyze the density of these fibers. Masson`s trichrome was used evaluate the homogeneity of the collagen fibers and its quantification as their surface density, which was obtained through a videomicroscopy system superimposed with a cycloid arch test system. Elastic fiber density and homogeneity was evaluated with resorcin-fucsin of Weigert. Androgen receptor histochemistry was used to stain intensity and the number of stained cells. With the von Willebrand factor and the same video-microscopy system superimposed with a cycloid arch test system, the number and volume density of the blood vessels was obtained. Picrosirius histochemistry did not show differences among the study groups (p= 0.905). With Masson`s trichrome, testosterone-treated patients (G3) had lower homogeneity (p=0.001) and surface density of the collagen fibers (0.3 ± 0.1 collagen fibers) than those untreated (G2) (0.4 ± 0.1 collagen fibers) (p<0.001). Circumcised children had a denser pattern of elastic fiber distribution (p=0.003), and the elastic fibers were also less homogeneous (p=0.008) than the other groups. The histochemistry for androgen receptor showed a greater staining pattern in children of G1 compared to G3 (p=0.011). Testosterone-treated foreskins (G3) had an increased absolute numbers of blood vessels (8.5 ± 1.3 vessels/field) (p<0.001) and an increased blood vessel volume density (50.5% ± 7.8 vessel/points) compared to untreated subjects (G2) (24.8% ± 8.6 vessel/points) (p<0.001). Topical 1% testosterone propionate produced neovascularization, in absolute numbers and volume density, and reduced fibrous tissue. Treated children had less homogeneous distribution patterns of vessel-surrounding collagen fibers, suggesting a lower maturity of these fibers, with less collagen deposition.

Les épithéliums muqueux se présentent comme porte d’entrée majeure du virus de l’immunodéficience humaine (VIH) et jouent un rôle critique dans la transmission sexuelle du virus. Les cellules épithéliales et les cellules dendritiques dans les muqueuses pluristratifiés, sont des cibles initiales pour la transmission virale. Il a été déjà montré, sur les muqueuses génitales chez l’homme, que la circoncision réduit plus de 60% acquisition du virus chez l’homme. Les mécanismes d’entrée du VIH au niveau du tractus génital chez l’homme sont tres mal connus et pratiquement pas étudiés. Nous avons émis l’hypothèse selon laquelle le prépuce, qui est enlevé lors de la circoncision, pourrait être une porte d’entrée majeure du virus. Nous avons d’abord montré que le VIH peut pénétrer effectivement au niveau du prépuce ex vivo, la partie interne du prépuce présente plus permissive que la partie externe. De plus, le virus pénètre uniquement dans le prépuce après formation d’une synapse virologique entre la cellule infectée, présente dans les secrétions génitales et la surface muqueuse du prépuce. La formation de la synapse virologique induit le bourgeonnement massif et polarisé de virions dans la fente synaptique; ceux ci pénètrent dans l’épiderme du prépuce. À l’inverse, l’entrée du VIH sous forme de virus libre dans le prépuce est inefficace. Nous avons ensuite caractérisé la séquence des événements initiaux mis en place lors de l’entrée du VIH dans le prépuce interne. La première cellule cible immune est la cellule de Langerhans (LCs), dont le rôle physiologique est de protéger la muqueuse contre les pathogènes qui l’envahissent. Après la capture du virus les LCs migrent vers le derme pour former des conjugués cellulaires avec les cellules T CD4+ du derme. La dynamique de migration de LCs est régulée par de sécrétion de cytokines et chimiokines (RANTEs et MIP- 3alpha) induites par la pénétration du virus dans le tissu. Nous avons ensuite caractérisé les mécanismes de l’immunité mis en jeu au niveau des kératinocytes de l’épiderme lors de la formation de la synapse virologique et évalué comment ces signaux contribuent à l’entrée efficace du virus dans la muqueuse du prépuce interne chez l’homme, ex vivo. À l’aide d’un modèle cellulaire de muqueuse simplifiée basé sur des kératinocytes primaires humains ex vivo, nous avons montré que les protéines d’enveloppe du VIH , gp120 mais pas gp41, et les molécules d’adhésion (intégrines LFA-1, leurs ligands ICAM-1 and -3) contrôlent la formation de la synapse virologique. La synapse virologique induit au niveau des kératinocytes l’activation de la voie NF- B indépendentmmment de MyD88 après activation du Toll-like-receptor 4 (TLR4) exprimé par les kératinocytes. En recherchant quelle cytokine pouvait être induite par cette signalisation, nous avons montré que la synapse virologique induit la sécrétion par les kératinocytes d’une cytokine produite par les cellules non-hématopoïétiques, la thymic stromal lymphopoietin (TSLP). La TSLP est un chemoattracteur des cellules myéloïdes comme les cellules LCs et peut induire leur maturation. Nous avons ensuite montré que, sur des explants de prépuce humain ex vivo, la synapse virologique induit bien la sécrétion de TSLP, laquelle en premier lieu attire les LCs qui expriment le TSLP-récepteur vers la surface du tissus induisant leur maturation. Les LCs peuvent ensuite migrer dans l’autre sens vers le derme. La sécrétion des cytokines comme RANTEs, MIP-3alpha, qui sont impliquées lors de la pénétration effective du virus dans les tissus n’a pas de rôle dans la signalisation induite par la formation de la synapse virologique proprement dite. Les kératinocytes de l’épiderme du prépuce interne, grâce à leur réponse innée TSLP induite par la synapse virologique ont un rôle déterminant dans l’entrée du VIH dans le prépuce. Ainsi, en réponse de la synapse virologique, les kératinocytes secrètent du TSLP après activation de la voie de NF- B dépendante de MyD88. (...) / The mucosal epitheliums are presented as a major portal of HIV-1, and play a critcal role for the sexual transmission of HIV-1. Epithelial cells, and dendritic cells in the pluristratified mucosa, are the initial targets of viral transmission. It has already been shown, in the genital mucosa in men, circumcision reduces by more than 60 % of virus acqusition by men. The entry mechanisms of HIV-1 in the male urogenital tracts are poorly understood and not pratically studied. We suggested that foreskin, removal by circumcision surgery, could be a major portal of HIV-1 entry. We first showed that HIV-1 could enter efficiently ex vivo inner foreskin mucosa, the inner part of foreskin which is more permissive than the outer part. More over, virus penetrates only after the viral synapse (VS) formation between HIV-1-infected cells and foreskin epidermis, in the presence of genital secretion on the surface of foreskin mucosa. The formation of VS induces massive and polarized budding of HIV-1 particles within the synaptic cleft. The virions therefore penetrate into the foreskin epidermis. Inversely, cell-free virus entry is inefficient in the foreskin. Next, we characterized the initial events of HIV-1 VS formation in the inner foreskin. The first cell type that HIV-1 encounters is Langerhans cells (LCs), whose physiological role is to protect the mucosa against invasive pathogens. After capture of virus, LC migrates towards the dermis to form intercellular conjugates with dermal CD4+ T cells. The dynamic of LC migraiton is regulated by the secretion of cytokines in the mucosa, induced by HIV-1 entry, including RANTEs and MIP-3alpha. We then characterized the mechanisms of foreskin epidermal keratinocyte (KC), activated innate immunity during the VS formation and the signaling pathway contributed to the efficient entry of HIV-1 via inner foreskin mucosa. Using a simplified mucosal model based on human primary foreskin keratinocytes, we demonstrated that HIV-1 envelope protein, gp120, but not gp41 and the adhesion molecules (integrins LFA-1, ICAM-1/-3), contribute to the VS formation. VS induces at the KC level the activation of NF- B pathway by I B and p65 molecules, after activation of TLR4 expressed on KCs. By searching for which cytokine could be induced by this signalisation, we then showed that VS induced the secretion by KC of one cytokine, which is produced by the non-hematopoetic cells, thymic stromal lymphopoietin (TSLP). TSLP is an chemoattractant for myeloid cells like LCs, and could induce LC maturation. We then showed, using the foreskin explants that, VS induces the secretion of TSLP, which first attracts LC expressing constitutively TSLP receptors to the apical surface of foreskin, inducing its maturation. These matured LCs then migrate to another direction towards the dermis. The secretion of cytokines such as RANTEs and MIP-3alpha, involved in the HIV-1 efficient entry has no roles in the signaling induced by VS formation as mentioned above. The Inner foreskin keratinocytes, due to the innate response of TSLP induced by VS, have a determinant role in the HIV-1 entry into the foreskin. Likewise, in response to VS, KCs secrete TSLP after NF- B activation dependent on regulator MyD88. The secreted TSLP, in addition to four different proinflammatory cytokines and chemokines (IL-6, IL-8, MIG, and MMP-9) allows virus to attract LCs to the foreskin surface, to capture the virus present in the synaptic cleft in order to facilite efficient transmission at the level of foreskin mucosa. Likely to the conventional immune cells, KCs, in one hand protects the foreskin mucosa and in another, is hijacked to facilitate HIV-1 transmission.