Antonio Toniolo

Productive HIV-1 infection of normal human mammary epithelial cells.

Objective, and designTo determine the susceptibility of mammary epithelial cells (MEC) to HIV-1 as breastfeeding is an established route of HIV transmission, although the origin of virus in breastmilk is unclear. MethodsPrimary epithelial cell cultures were derived from the mammary glands of healthy donors; immortalized MEC lines were also used. HIV infection was followed by detection of infectious particle production, p24 antigen, and viral sequences. ResultsSeven out of 11 primary MEC cultures, and two out of three MEC lines were productively infected by HIV-1. Virus replication significantly reduced cell proliferation, although cell viability was only slightly affected. Cytopathic changes were not observed. MEC cultures expressed low levels of surface CD4, galactosylceramide, and CD26, but essentially no human leukocyte antigen (HLA)-DR. Infection of HIV-permissive MEC cells was associated with the upregulation of surface HLA-DR, and CD26. In contrast, the expression of CD4, tissue-specific markers, adhesion molecules, and growth-factor receptors was downregulated. To a lesser extent, similar effects were also observed in non-permissive cells. Hormones (triiodothyronine plus β-estradiol, and prolactin) enhanced HIV replication, possibly through the stimulation of cellular DNA synthesis. ConclusionsWe concluded that HIV-1 replication in ductal/alveolar MEC may be, in part, responsible for the presence of HIV-1 in milk; that hormones may stimulate virus replication;, and that infection reduces the growth of epithelial cells. Although in vitro HIV is produced by MEC to a lesser extent than lymphoid cells, MEC-derived HIV might have selective advantages for the infection of mucosal epithelial cells during breastfeeding.

Heparin-binding domain of human fibronectin binds HIV-1 gp120/160 and reduces virus infectivity

In vitro experiments indicate that components of the host present in body fluids may prevent the attachment of human immunodeficiency virus type 1 (HIV‐1) to target cells. Fibronectin (Fn), a dimeric 440‐kDa extracellular matrix adhesion protein, is secreted by mesenchymal cells and assembled into insoluble matrices. Fn exerts important effects on cell growth and differentiation through a number of discrete functional domains. Several microorganisms are known to bind Fn. We show that, under physiological conditions, HIV‐1 gp120 and gp160 are capable of binding plasma and cellular Fn as well as laminin and vitronectin. Experiments were set up to analyze in detail the binding of HIV gp120 and gp160 to Fn. The gp120 and gp160 specifically recognize the C‐terminal heparin‐binding domain of Fn (Fn‐CTHBD) with a calculated KD of 2.8 × 10−7 M for gp160. Binding of gp160 to Fn‐CTHBD is a saturable and specific process that is blocked by antibodies to Fn‐CTHBD and by heparin and is inhibited to a minor extent by heparan sulfate and dextran sulfate. These observations suggest that gp120/160 specifically recognize the III15 repeat within Fn‐CTHBD. Intact Fn and Fn‐CTHBD strongly inhibit the interaction of gp120/160 with soluble CD4 and, under low serum conditions, are capable of neutralizing the infectivity of HIV‐1 for CD4‐positive T cells. Thus, Fn that is present in plasma and mucinous secretions may well affect HIV infectivity and virus distribution in vivo. J. Med. Virol. 54:44–53, 1998.

Potentiation of the malignant phenotype of the undifferentiated ARO thyroid cell line by insertion of the bcl-2 gene.

We have reported that bcl‐2 is expressed in normal human thyroid epithelium and that its expression is down‐regulated in undifferentiated thyroid tumors. Production of IL‐6 was concomitantly down‐regulated in these forms. Based on these observations, we analyzed whether insertion of bcl‐2 would reverse the highly malignant phenotype of a thyroid cell line (ARO) derived from an undifferentiated carcinoma. This cell line fails to produce Bcl‐2 and IL‐6. By infection with a bcl‐2 retroviral vector, ARO cells expressing bcl‐2 (ARObcl‐2) were obtained. Compared with parental cells, expression of bcl‐2 was associated with enhancement of growth potential (DNA synthesis, in vitro proliferation rate, anchorage‐independent growth in semi‐solid media). Chemotaxis and invasive potential in Boyden chambers were also increased. bcl‐2‐expressing cells showed a reduced response to apoptotic stimuli (low‐serum conditions or anti‐neoplastic drugs). Large branched colonies were formed in Matrigel from ARObcl‐2 cells but not from parental cells. Finally, ARObcl‐2 cells showed a decreased latency of tumor appearance when injected into immunodeficient mice. Potentiation of the malignant phenotype of ARO cells by bcl‐2 was not ascribed to altered expression of (i) cytokine/growth factors (IL‐4, IL‐6, IL‐8, IL‐10, IL‐12, TGF‐α, TGF‐β), (ii) thyroid‐specific transcripts (TG, TPO, TSH‐R, PIGF, PAX‐8) or (iii) genes influencing tumor aggressiveness [VEGF, HMGI (Y), HMGI‐C]. Our data indicate that bcl‐2 potentiates the malignant phenotype of ARO cells not only by limiting the response to apoptotic stimuli but also by enhancing proliferation and tumor aggressiveness. Int. J. Cancer81:956–962, 1999.

Defective interleukin six expression and responsiveness in human mammary cells transformed by an adeno 5/SV40 hybrid virus.

Mammary epithelial cells (MECs) were isolated and cultured from mammary glands of healthy women undergoing reduction mammoplasty. Normal MECs were infected with the transforming hybrid virus adeno-5/SV40. Two transformed epithelial cell lines, M1 and M2, were obtained, characterised phenotypically and studied for the production of and the response to cytokines and growth regulators. In both cell lines, expression of the SV40 large T antigen was associated with loss of interleukin 6 (IL-6) production and responsiveness as well as with down-regulation of IL-8 and transforming growth factor (TGF)-alpha production. Both M1 and M2 cell lines were capable of forming colonies in semisolid media, but upon injection into severe combined immunodeficient (SCID) mice only M2 cells were tumorigenic. DNA synthesis in M1 cells was partially inhibited by serum or TNF-alpha and weakly stimulated by hydrocortisone (HC) and IL-8. In contrast, M2 cells were totally unresponsive to a variety of growth regulators. Both lines overexpressed the p53 protein at levels about 20-fold higher than those observed in primary MEC cultures, but no mutations of the p53 gene could be detected. The date confirm the view that the expression in human mammary cells of different oncogenes - including the SV40 T antigen - is frequently associated with alterations of cytokine production and responsiveness.