[Male sexuality after external continent urinary diversion type Mitrofanoff]. / Étude de la sexualité masculine après dérivation urinaire externe continente de type Mitrofanoff.

OBJECTIVE: To evaluate the influence of continent external urinary diversion type Mitrofanoff on male sexuality. MATERIAL AND METHODS: Between 1992 and 2011, 140 patients underwent continent urinary diversion type Mitrofanoff at an academic hospital. Among 76 men, 46 were interviewed about their sexuality after this operation. This study was performed using a set of validated questionnaires (IIEF, DAN PSS and Urolife), grouped by the model of the CTMH. Patients were divided according to their marital status: group 1: patients married before surgery (15 cases), group 2: patients married after surgery (7 cases) and group 3: singles (24 cases). RESULTS: In the first group, the functional dimension of sexuality was positive with an overall score of 81%, the sexual discomfort score was assessed at 26 % and the sexual satisfaction score was 77%. In the second group, sexual function was considered conserved in all cases with a satisfaction score estimated at 98%. These patients reported a feeling of well-being following the disappearance of urinary incontinence with integrity of their body images. In contrast, in the last group, relatively impaired sexual function was noted (65%) with a satisfaction score estimated at 59%. These disorders were multifactorial, mainly related to neurological causal pathology. CONCLUSION: To our knowledge, this is the first study about male sexuality in patients with a continent urinary diversion type Mitrofanoff. Marital status has a major role in the sexuality of these patients. A prospective study with pre- and postoperative evaluation will better clarify the factors affecting sexuality in these patients.

Distinct regulatory elements are required for faithful expression of human CD4 in T cells, macrophages, and dendritic cells of transgenic mice.

To identify the regulatory elements controlling expression of the human CD4 (hCD4) gene in different cell types of the immune system, deletion and chimeric (human/murine) reporter genes were constructed and tested in transgenic (Tg) mice. Regulatory elements required for the proper hCD4 expression in the immature double-positive thymic T cells were identified in the enhancer and in the 3' end of intron 1. Expression of hCD4 in macrophages is controlled by at least 2 sets of regulatory elements: one present in front of exon 1 and the second at the 5' end of intron 1. The hCD4 elements required for expression on both myeloid and lymphoid CD8alpha(+) dendritic cells (DCs) from lymph node and thymus were found to be different from those required for macrophage expression. The results indicate that expression of hCD4 in T cells, macrophages, and DCs is controlled by distinct regulatory elements.

Two distinct Notch1 mutant alleles are involved in the induction of T-cell leukemia in c-myc transgenic mice.

We have previously characterized a large panel of provirus insertion Notch1 mutant alleles and their products arising in thymomas of MMTV(D)/myc transgenic mice. Here, we show that these Notch1 mutations represent two clearly distinct classes. In the first class (type I), proviral integrations were clustered just upstream of sequences encoding the transmembrane domain. Type I Notch1 alleles produced two types of mutant Notch1 RNA, one of which encoded the entire Notch1 cytoplasmic domain [N(IC)] and the other of which encoded a soluble ectodomain [N(EC)(Mut)] which, in contrast to the processed wild-type ectodomain [N(EC)(WT)], did not reside at the cell surface and became secreted in a temperature-dependent manner. A second, novel class of mutant Notch1 allele (type II) encoded a Notch1 receptor with the C-terminal PEST motif deleted (DeltaCT). The type II Notch1(DeltaCT) protein was expressed as a normally processed receptor [N(EC)(WT) and N(IC)(DeltaCT)] at the cell surface, and its ectodomain was found to be shed into the extracellular medium in a temperature- and calcium-dependent manner. These data suggest that both type I and type II mutations generate two structurally distinct Notch1 N(EC) and N(IC) proteins that may participate in tumor formation, in collaboration with the c-myc oncogene, through distinct mechanisms. Constitutive type I N(IC) and type II N(IC)(DeltaCT) expression may enhance Notch1 intracellular signaling, while secreted or shed type I N(EC)(Mut) and type II N(EC) proteins may differentially interact in an autocrine or paracrine fashion with ligands of Notch1 and affect their signaling.

A G1 cell cycle arrest induced by ligands of the reovirus type 3 receptor is secondary to inactivation of p21ras and mitogen-activated protein kinase.

The reovirus type 3 S1 gene product (type 3 hemagglutinin; HA3) is the viral protein responsible for binding to a mammalian cell-surface receptor. It has been shown that HA3 binding to its receptor inhibits cell growth, even in the continuous presence of serum mitogens. Here, receptor-mediated signal transduction leading to growth arrest was studied after binding with synthetic or recombinant ligands in the absence of viral infection. Receptor ligation caused rapid inactivation of p21(ras), a decrease in Raf phosphorylation and in mitogen-activated protein kinase (MAPK) enzymatic activity, and G1 cell cycle arrest. Transfection and expression of constitutively active v-Has-ras prevented the G1 arrest, indicating that inactivation of p21(ras) is causative. Interestingly, v-Has-ras expression also decreased the efficiency of reoviridae replication, suggesting that inactivation of p21(ras) signals is required at some step of the viral cycle. This study may define new mechanisms regulating cell growth and support the approach of using viral proteins to identify and study cellular receptors. Synthetic receptor ligands with antiproliferative properties may be useful in drug development with the aim of blocking mitosis.

Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice.

Transgenic (Tg) mice expressing the complete coding sequences of HIV-1 in CD4+ T cells and in cells of the macrophage/dendritic lineages develop severe AIDS-like pathologies: failure to thrive/weight loss, diarrhea, wasting, premature death, thymus atrophy, loss of CD4+ T cells, interstitial pneumonitis, and tubulo-interstitial nephritis. The generation of Tg mice expressing selected HIV-1 gene(s) revealed that nef harbors a major disease determinant. The latency and progression (fast/slow) of the disease were strongly correlated with the levels of Tg expression. Nef-expressing Tg thymocytes were activated and alpha-CD3 hyperresponsive with respect to tyrosine phosphorylation of several substrates, including LAT and MAPK. The similarity of this mouse model to human AIDS, particularly pediatric AIDS, suggests that Nef may play a critical role in human AIDS, independently of its role in virus replication.

Transgenic mice expressing human immunodeficiency virus type 1 in immune cells develop a severe AIDS-like disease.

We have constructed transgenic (Tg) mice expressing the entire human immunodeficiency virus type 1 (HIV-1) coding sequences in cells targeted by HIV-1 infection in humans. These Tg mice developed a severe AIDS-like disease leading to early death (< 1 month). They developed muscle wasting, severe atrophy and fibrosis of lymphoid organs, tubulointerstitial nephritis, and lymphoid interstitial pneumonitis. In addition the expression of RANTES was increased in various tissues of these Tg mice relative to that in the normal controls. Disease appearance was correlated with the levels of transgene expression. The numerous pathologies observed in these mice are remarkably similar to those observed in human AIDS and, more specifically, in pediatric AIDS.

The murine AIDS virus Gag precursor protein binds to the SH3 domain of c-Abl.

The Pr60gag protein of the murine AIDS (MAIDS) defective virus promotes the proliferation of the infected target B cells and is responsible for inducing a severe immunodeficiency disease. Using the yeast two-hybrid system, we identified the SH3 domain of c-Abl as interacting with the proline-rich p12 domain of Pr60gag. The two proteins were shown to associate in vitro and in vivo in MAIDS virus-infected B cells. Overexpression of Pr60(gag) in these cells led to a detectable increase of the levels of c-Abl protein and to its translocation at the membrane. These results suggest that this viral protein serves as a docking site for signaling molecules and that c-Abl may be involved in the proliferation of infected B cells.

Covalent assembly of a soluble T cell receptor-peptide-major histocompatibility class I complex.

We used stepwise photochemical cross-linking for specifically assembling soluble and covalent complexes made of a T-cell antigen receptor (TCR) and a class I molecule of the major histocompatibility complex (MHC) bound to an antigenic peptide. For that purpose, we have produced in myeloma cells a single-chain Fv construct of a TCR specific for a photoreactive H-2Kd-peptide complex. Photochemical cross-linking of this TCR single-chain Fv with a soluble form of the photoreactive H-2Kd-peptide ligand resulted in the formation of a ternary covalent complex. We have characterized the soluble ternary complex and showed that it reacted with antibodies specific for epitopes located either on the native TCR or on the Kd molecules. By preventing the fast dissociation kinetics observed with most T cell receptors, this approach provides a means of preparing soluble TCR-peptide-MHC complexes on large-scale levels.

A p65/p95 neural surface receptor is expressed at the S-G2 phase of the cell cycle and defines distinct populations.

A surface receptor complex of Mr approximately 65 000 (p65) and approximately 95 000 (p95) is expressed in cells of the central nervous system of mice. This receptor is recognized by monoclonal antibody 87.92.6 or by reovirus type 3 haemagglutinin as unnatural ligands. The p65/p95 receptor is expressed mostly in neural embryonic precursors undergoing proliferation, especially those in the S-G2 phase of the cell cycle. Receptor expression decreases progressively throughout embryogenesis to low but detectable levels in the adult brain. Biochemical characterization revealed that the neural p65/p95 receptor complex is indistinguishable from the p65/p95 receptor expressed in T cells, where receptor ligation leads to a mitogenic block. In neural and lymphoid tissues the p65/p95 receptor (or an associated protein) possesses a tyrosine kinase enzymatic activity. Receptor ligation in neural cells resulted in the rapid tyrosine phosphorylation of cellular proteins which are different from substrates phosphorylated in T cells. Differential substrate coupling to the receptor may account for differences in signal transduction and biology between neural cells and T cells. Further study of this receptor complex may help define important features of neural proliferation, differentiation and survival.

Analysis of the T-cell receptor beta-chain variable-region (V beta) repertoire in monozygotic twins discordant for human immunodeficiency virus: evidence for perturbations of specific V beta segments in CD4+ T cells of the virus-positive twins.

We analyzed the T-cell receptor (TCR) V beta repertoire in human immunodeficiency virus type 1 (HIV-1)-infected individuals at different stages of disease. To circumvent the effect of HLA and other loci on the expressed TCR repertoire, we compared the TCR repertoire in nine pairs of monozygotic twins who were discordant for HIV infection. A semiquantitative polymerase chain reaction (PCR) assay and flow cytometry enabled us to show distinct differences in the V beta repertoire in the HIV-positive twin compared with the HIV-negative twin. By combining PCR and cytofluorometry, these differences were restricted to a specific set of TCR V beta segments, with members of the V beta 13 family perturbed in six out of seven cases and those of the V beta 21 family perturbed in four out of seven cases studied. Most of the other V beta families remained unchanged. Our results provide direct evidence for a skewed TCR repertoire in HIV infection.

The T cell receptor V beta repertoire in HIV-1 infection and disease.

Viral superantigens (SAg) were shown in mice to induce anergy and deletion of T cells bearing specific T cell receptor V beta subsets, these perturbations being mainly restricted to CD4+ T cells. In accordance with this model, a putative HIV-associated SAg could contribute to the pathogenesis of HIV-1 infection and AIDS. To reveal the presence of this putative molecule, three study protocols were designed that relied on the fact that similarity of the expressed V beta repertoire of a given pair of individuals is proportional to the relative likeness of their MHC background: (1) by using a quantitative PCR technique that allows simultaneous typing of 24 V beta families, the V beta repertoires of HIV-discordant monozygotic twins were compared; (2) the V beta repertoire found in lymph nodes of HIV-infected subjects was contrasted to that found in peripheral blood of the same individuals; (3) the V beta repertoire of a cohort of HIV-infected mothers was compared with that of their HIV-infected and uninfected children. Results from these approaches revealed that significant perturbations of the TCR V beta repertoire were taking place in HIV-infected subjects, and that these alterations were restricted to T cells expressing specific V beta s. These results are consistent with the presence of an HIV-associated SAg in HIV-1 infection.

Amyloid precursor protein in peripheral mononuclear cells is up-regulated with cell activation.

The deposition of beta/A4 protein in extraneural organs of patients with Alzheimer disease suggests that this peptide may in part be derived from a peripheral precursor. We studied expression of amyloid precursor protein (APP) in PBMC. APP expression was detectable in resting PBMC by northern blot analysis, immunoblotting studies, and immunohistochemistry. By reverse transcription-polymerase chain reaction, the 751 and 770 APP transcripts containing the Kunitz protease inhibitor (KPI) domain were approximately 10-fold more abundant than the 695 transcript lacking the KPI domain. Activation of PBMC with the lectin PHA-P was associated with an increase in apparent intracellular APP content by cytofluorometry, and an increase in the proportion of the 695 APP transcript lacking the KPI domain. We conclude that resting and activated PBMC express APP and could contribute to a circulating pool of this protein. In addition, PBMC APP is up-regulated with mitogenic stimulation and may participate in the regulation of activation of these cells.

Preferential positive selection of V alpha 2+ CD8+ T cells in mouse strains expressing both H-2k and T cell receptor V alpha a haplotypes: determination with a V alpha 2-specific monoclonal antibody.

A monoclonal antibody, B20.1, was generated by fusing spleen cells from a Lou rat immunized with a soluble alpha/beta T cell receptor (TcR; V alpha 2/V beta 2) to mouse myeloma cells. Analysis of a panel of V alpha 2 mRNA-expressing T cell lines, hybridomas and transfectants revealed that the B20.1 antibody was specific for murine TcR V alpha 2 chains. The V alpha 2+ T cell population was examined in various inbred strains by two-color immunofluorescence using B20.1 and CD4- and CD8-specific antibodies with the following results: (a) the B20.1 antibody detected most members of the TcR V alpha 2 subfamily in the four TcR V alpha haplotypes tested; (b) in most strains examined, TcR V alpha 2 expression was biased to the CD4 subset (7.4%-17.4% V alpha 2+ T cells) as compared to the CD8 compartment (3.8%-13.3%); (c) TcR V alpha 2 expression was not influenced by Mls gene products and (d) increased positive selection of V alpha 2+ CD8+ T cells by H-2k major histocompatibility complex molecules occurred in all murine strains tested of the TcR V alpha a, but not in those bearing the TcR V alpha b haplotype.

Monoclonal antibodies raised against engineered soluble mouse T cell receptors and specific for V alpha 8-, V beta 2- or V beta 10-bearing T cells.

We have characterized a panel of monoclonal antibodies (mAb) produced by immunizing rats with two distinct soluble mouse alpha/beta T cell receptor (TcR). Fifty mAb were found to react with the corresponding surface-bound TcR. Such observations suggest that the soluble TcR molecules used as immunogen are folded in a conformation similar to the native structure. Furthermore, the binding to T cells of four antibodies was found to correlate with the expression of the V alpha 8, V beta 2 or V beta 10 gene segments. Finally, staining of T lymphocytes from various mouse strains suggests that (a) the two anti-V alpha 8 antibodies recognize different epitopes, and each on only a fraction of V alpha 8+ cells; (b) the anti-V beta 10 mAb identifies a V beta 10 polymorphism among mouse strains, and (c) T cells expressing the V beta 2 or V beta 10 gene segments are not subject to major clonal deletion events induced by the major histocompatibility complex class II and Mls products which were tested.