Human mesenchymal stem cell responses to hydrostatic pressure and shear stress.

The effects of mechanical stimuli to which cells are exposed in vivo are, at best, incompletely understood; in this respect, gene-level information regarding cell functions which are pertinent to new tissue formation is of special interest and importance in applications such as tissue engineering and tissue regeneration. Motivated by this need, the present study investigated the early responses of human mesenchymal stem cells (hMSCs) to intermittent shear stress (ISS) and to cyclic hydrostatic pressure (CHP) simulating some aspects of the biological milieu in which these cells exist in vivo. Production of nitric oxide (NO) and mRNA expression of several known mechanosensitive genes as well as ERK1/2 activation in the hMSC response to the two mechanical stimuli tested were monitored and compared. NO production depended on the type of the mechanical stimulus to which the hMSCs were exposed and was significantly higher after exposure to ISS than to CHP. At the conditions of NO peak release (i.e., at 0.7 Pa for ISS and 50,000 Pa for CHP), ISS was more effective than CHP in up-regulating mechanosensitive genes. ERK1/2 was activated by ISS but not by CHP. The present study is the first to report that PGTS2, IER3, EGR1, IGF1, IGFBP1, ITGB1, VEGFA and FGF2 are involved in the response of hMSCs to ISS. These findings establish that, of the two mechanical stimuli tested, ISS is more effective than CHP in triggering expression of genes from hMSCs which are bioactive and pertinent to several cell functions (such as cell differentiation and release of specific growth factors and cytokines) and also to tissue-related processes such as wound healing.

Cervicovaginal secretory antibodies to human immunodeficiency virus type 1 (HIV-1) that block viral transcytosis through tight epithelial barriers in highly exposed HIV-1-seronegative African women.

Antibodies to human immunodeficiency virus (HIV) of the IgA, IgG, and IgM isotypes and high levels of the HIV suppressive beta-chemokine RANTES (regulated on activation, normally T cell expressed and secreted) were found in the cervicovaginal secretions (CVSs) of 7.5% of 342 multiply and repeatedly exposed African HIV-seronegative female sex workers. The antibodies are part of a local compartmentalized secretory immune response to HIV, since they are present in vaginal fluids that are free of contaminating semen. Cervicovaginal antibodies showed a reproducible pattern of reactivity restricted to gp160 and p24. Locally produced anti-env antibodies exhibit reactivity toward the neutralizing ELDKWA epitope of gp41. Study results show that antibodies purified from CVSs block the transcytosis of cell-associated HIV through a tight epithelial monolayer in vitro. These findings suggest that genital resistance to HIV may involve HIV-specific cervicovaginal antibody responses in a minority of highly exposed HIV-seronegative women in association with other protecting factors, such as local production of HIV-suppressive chemokines.

Detection of Y chromosome DNA as evidence of semen in cervicovaginal secretions of sexually active women.

The detection of traces of semen in cervicovaginal secretions (CVS) from sexually active women practicing unprotected sex is a prerequisite for the accurate study of cervicovaginal immunity. Two semen markers, the prostatic-specific antigen (PSA) and the Y chromosome, were detected in parallel in CVS obtained by a standardized vaginal washing of consecutive women attending the principal medical center for sexually transmitted diseases of Bangui, Central African Republic. PSA was detected by immunoenzymatic capture assay in the cell-free fraction of CVS, and the Y chromosome was detected by a single PCR assay of DNA extracted by silica from the cell fraction (Y PCR). Fifty (19%) cell-free fractions of the 264 beta-globin-positive CVS samples were positive for PSA, and 100 (38%) cell fractions of the CVS samples were positive for the Y chromosome. All the 50 (19%) PSA-containing CVS samples were also positive for the Y chromosome. Fifty (19%) CVS samples were positive only for the Y chromosome, with no detectable PSA. The remaining 164 (62%) CVS samples were both PSA and Y chromosome negative. These findings demonstrate that CVS from sexually active women may contain cell-associated semen residues unrecognized by conventional immunoenzymatic assays used to detect semen components. The detection of cell-associated male DNA with a highly sensitive and specific procedure such as Y PCR constitutes a method of choice to detect semen traces in female genital secretions.

Active and selective transcytosis of cell-free human immunodeficiency virus through a tight polarized monolayer of human endometrial cells.

We report that both primary and laboratory-adapted infectious human immunodeficiency virus type 1 (HIV-1) isolates in a cell-free form are capable of transcytosis through a tight and polarized monolayer of human endometrial cells. Trancytosis of cell-free HIV occurs in a strain-selective fashion and appears to be dependent on interactions between HIV envelope glycoproteins and lectins on the apical membrane of the epithelial cells. These findings provide new insights into the initial events occurring during heterosexual transmission of the virus.

Secretory leukocyte protease inhibitor inhibits infection of monocytes and lymphocytes with human immunodeficiency virus type 1 but does not interfere with transcytosis of cell-associated virus across tight epithelial barriers.

In the present study, we demonstrate that recombinant human secretory leukocyte protease inhibitor (rhSLPI) inhibits infection of lymphocyte- and monocyte-derived tumor cell lines and peripheral blood lymphocytes with laboratory-adapted isolates and with the primary isolate, NDK, of free human immunodeficiency virus type 1 (HIV-1). In contrast, rhSLPI did not exhibit inhibitory activity toward transcytosis of cell-associated HIV-1 through a tight monolayer of endometrial epithelial cells. These observations indicate that the inhibitory effect of SLPI is restricted to free HIV-1 in corporal fluids.

Presence of integrated DNA sequences of adeno-associated virus type 2 in four cell lines of human embryonic origin.

The human helper virus-dependent parvovirus adeno-associated virus (AAV) has been found in human female genital tissues including material from first trimester miscarriage. In the latter case, AAV type 2 (AAV-2) DNA and viral proteins were detected mainly in the trophoblast cell layer of placenta. In this report, we present evidence that AAV DNA is also present in established human trophoblast cell lines (JEG-3, JAr, BeWo) and in the human amnion cell line FL. In cells of these lines, AAV-2 DNA could be detected both by PCR and Southern blot analysis. Restriction enzyme analysis indicated that AAV DNA was integrated into the host cell genome. Although the cell lines supported AAV replication when infected with AAV-2 and adenovirus type 2 (Ad2) as a helper virus, superinfection with Ad2 alone did not induce replication of AAV DNA, i.e. it failed to rescue AAV from its integrated state. This is probably due to rearrangements within the integrated AAV genome. The presence of AAV DNA in cells derived from human embryonic tissue corroborates the suggestion that human embryonic tissue may be one of the targets of AAV infection.

High-level ability of secretory IgA to block HIV type 1 transcytosis: contrasting secretory IgA and IgG responses to glycoprotein 160.

The IgG and secretory IgA (S-IgA) responses to the HIV-1 envelope (gp160 antigen) were analyzed in the colostrum (Col) and in the cervicovaginal fluid (CVF) of HIV-l-infected women. We show IgG antibodies (Abs) to the recombinant gp160 to be predominant as compared with the corresponding S-IgA isotype. The low level of the S-IgA response cannot be related to a general disturbance of the mucosal-associated Iymphoid tissue (MALT) because the level of a current Ab to a caries-associated antigen from Streptococcus sobrinus was in the normal range in these secretions. The major subclass of IgA to gp160 was of the alpha1 isotype both in Col and in CVF. However, the specific activities of S-IgA1 and S-IgA2 were different when expressed as the ratio of the anti-gp160 related to total Ig of each subclass. Indeed, the specific activity of the S-IgA2 was predominant over S-IgA1 in the Col, whereas the reciprocal results were found in CVF, showing a subcompartmentalization of these secretions. The ability of S-IgA and IgG to block one of the pathways involved in the HIV-1 penetration across mucosa, i.e., transcytosis through epithelial cells, was evaluated using a functional in vitro assay. Both S-IgA and IgG Abs impaired virus transcytosis, irrespective of the level of antigp160 specific activities. However, specific S-IgA was more efficient than IgG. These features suggest that mucosal specific S-IgA to HIV-1 could be relevant in decreasing infectivity of HIV-1 in corporal fluids.

Dilution assessment of cervicovaginal secretions collected by vaginal washing to evaluate mucosal shedding of free human immunodeficiency virus.

A 10 mM concentration of lithium does not interfere with reverse transcription (RT) or PCR. Sampling of cervicovaginal fluid by vaginal washing, with lithium (10 mM) in the washing buffer as a marker of dilution, may be utilized to accurately determine in HIV-infected women, by quantitative RT-PCR, the genital shedding of acellular HIV RNA at the level of the mucosa itself.

Expression of the non-structural proteins of parvovirus MVMp from recombinant retroviruses: predominant role of the parvoviral NS-1 product in host cell disturbance.

Stable Psi-2 cell transformants were selected for their resistance to neomycin after transfection with a retroviral pZipNeo-SVX vector carrying sequences encoding for the non-structural proteins of parvovirus minute virus of mice (prototype strain, MVMp). Cells producing both NS-1 and NS-2 proteins (PsiNS) or only the NS-2 polypeptide (PsiNS2) were obtained. PsiNS cells exhibited morphological abnormalities and had a reduced clone-forming ability, whereas PsiNS2 cells were indistinguishable from the parental line. These cellular systems produced recombinant retroviral particles which transduced the NS gene(s) into mouse A9 cells. As in the case of Psi-2 cells, A9 transformants expressing both NS-1 and NS-2 proteins were impaired in their cloning efficiency. These results provided a direct confirmation of the predominant role of protein NS-1 in the cytopathic effect of parvoviruses.

Transformation of human fibroblasts by ionizing radiation, a chemical carcinogen, or simian virus 40 correlates with an increase in susceptibility to the autonomous parvoviruses H-1 virus and minute virus of mice.

Morphologically altered and established human fibroblasts, obtained either by 60Co gamma irradiation, treatment with the carcinogen 4-nitroquinoline 1-oxide, or simian virus 40 (SV40) infection, were compared with their normal finite-life parental strains for susceptibility to the autonomous parvoviruses H-1 virus and the prototype strain of minute virus of mice (MVMp). All transformed cells suffered greater virus-induced killing than their untransformed progenitors. The cytotoxic effect of H-1 virus was more severe than that of MVMp. Moreover, the level of viral DNA replication was much (10- to 85-fold) enhanced in the transformants compared with their untransformed parent cells. Thus, in this system, cell transformation appears to correlate with an increase in both DNA amplification and cytotoxicity of the parvoviruses. However, the accumulation of parvovirus DNA in the transformants was not always accompanied by the production of infectious virus. Like in vitro-transformed fibroblasts, a fibrosarcoma-derived cell line was sensitive to the killing effect of both H-1 virus and MVMp and amplified viral DNA to high extents. The results indicate that oncogenic transformation can be included among cellular states which modulate permissiveness to parvoviruses under defined growth conditions.

[Disseminated cutaneous osteomatosis in an infant (author's transl)]. / L'ostéomatose cutanée diffuse du nourrisson.

An infant with disseminated cutaneous osteomatosis as described by Worret and Burgdorf is described. The diagnosis was confirmed by histology which distinguished the bone formation from sub-cutaneous calcification. The prognosis of disseminated cutaneous osteomatosis is guarded because there may be extensive skin involvement and because of the absence of effective treatment.