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PRL receptor (PRLR) signal transduction supports PRL-induced growth/differentiation processes. While PRL is known to activate Jak2-Stat5 (signal transducer and activator of transcription 5) signaling pathway, the mechanism by which cell proliferation is stimulated is less known. We show that PRL induces proliferation of bovine mammary gland epithelial cells and AP-1 site activation. Using PRLR mutants and the PRLR short form, we have found that both homodimerization of PRLR wild type and the integrity of box-1 and C-distal tyrosine of PRLR intracellular domain are needed in PRL-induced proliferation and AP-1 activation. The effect of PRL has been assayed in the presence of dexamethasone (Dex), insulin, and alone. We found that Dex negatively regulates PRL-induced proliferation and AP-1 site activation. We demonstrate that PRL exerts activation of AP-1 transcriptional complex, and the mechanism by which this activation is produced is also studied. We show that PRL induces an increase in the c-Jun content of AP-1 transcriptional complexes. The PRL-induced c-Jun of AP-1 transcriptional complex diminishes in the presence of Dex in a dose-dependent manner. Dex inhibition was reversed by the higher concentration of PRL added to cells. Despite the fact that the regulation of the AP-1 site is complex, we found that PRL activates the c-Jun amino terminal kinase (JNK), while glucocorticoid prevents this JNK activation. These data support a regulation of cellular growth by PRL-PRLR system by increasing AP-1 transcriptional complex activity via JNK activation. JNK activation can be repressed by glucocorticoid in a DNA-binding-independent manner.

Neuroblastoma cell lines (SK-N-SH and SK-N-MC) were induced to differentiate, as detected by the expression of neurofilament proteins of 68 and 200 kDa, and to express adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule) after stimulation with tumour necrosis factor-alpha (TNF-alpha). This induction was accompanied by the arrest of cell growth. The induction of neuroblastoma adhesion by TNF-alpha could be inhibited by the nitric oxide synthase inhibitors, L-N-monomethyl arginine (L-NMMA) and L-N6-(1-imidoethyl)-lysine (highly specific for the inducible enzyme), but not by the inactive enantiomer D-NMMA. These results indicate that TNF-alpha induces the adhesion of neuroblastoma cells via nitric oxide. This was confirmed by the finding that the adhesion/differentiation of SK-N-SH and SK-N-MC cells can be directly induced by the addition of nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetyl-penicillamine, into the culture medium. The isoform of the nitric oxide synthase induced in human neuroblastoma cells by TNF-alpha treatment was identified enzymatically as isoform II by Western blotting and by the polymerase chain reaction. Thus TNF-alpha induces the in vitro adhesion/differentiation of human neuroblastoma cells through nitric oxide synthesized by a calcium-independent inducible form of nitric oxide synthase, clearly indicating that isoform II of nitric oxide synthase can be expressed in human neuronal cell types.

Immature neural cell lines could be productively infected by HIV-1. Interestingly, this infection was associated with a differentiation to a mature neuronal phenotype, characterized by the expression of mature neurofilaments and cell adhesion molecules, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1. Infection also induced TNF-alpha and IL-1beta mRNA expression, as well as the synthesis of inducible nitric oxide synthase by neuroblastoma cells. Exogenous addition of TNF-alpha, but not of IL-1beta or many other cytokines, including nerve growth factor, mimicked those effects induced by infection. Moreover, blocking endogenous TNF-alpha or NO production in cultures of infected cells with a neutralizing anti-TNF-alpha antibody or inducible nitric oxide synthase inhibitors prevented the expression of the mature cell phenotype as well as expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1. Addition of NO generators and TNF-alpha activated NF-kappaB- and intercellular cell adhesion molecule-1-dependent promoter transcription, whereas inducible nitric oxide synthase inhibitors prevented the transcriptional activation of intercellular cell adhesion molecule-1 promoter that was induced by TNF-alpha. Those results suggest that HIV can infect immature neural cells and this infection induces their neural development via a TNF-alpha- and NO-mediated mechanism.

Polymerase chain reaction (PCR), virus culture and antigen detection assays are useful for early detection of vertically transmitted human immunodeficiency virus type 1 (HIV‐1) infection in infants under 12 months of age. Sixty‐four children born to HIV‐1‐seropositive mothers were evaluated. Thirteen children (20.3%) were repeatedly positive by PCR analysis. There was 100% concordance between the results obtained from PCR and culture assays. Measurement of p24 antigen in serum was, in contrast, a less sensitive marker of HIV infection: only 5/13 infants had positive p24 antigen results. We have investigated the relationship among the HIV‐1 biological phenotype, replicative capacity of viral isolates, HIV RNA copy number in plasma, p24 antigenaemia, CD4 T lymphocyte counts and the clinical status in 13 HIV‐infected infants. Six out of 13 HIV‐1 isolates from these patients were classified as rapid/high and seven as slow/low. We have found a significantly positive correlation between the replication rate of HIV isolates and their capacity to induce syncytia in vitro. The HIV‐1 isolates with rapid/high and syncytium‐inducing phenotype, and isolates with slow/low and non‐syncytium‐inducing phenotype were obtained from infants who had HIV‐1 RNA copy number ml−1 plasma values of 27654–83520 and 1342–34321, respectively. Levels of HIV‐1 RNA were measured in sequential plasma samples from three HIV‐infected infants and their biological properties determined in vitro. Our findings indicate that infants who carried viruses with more cytophatic biological phenotype and who had higher viral RNA copy numbers in blood were more likely to have lower CD4+ T cell counts and more likely to develop full‐blown AIDS.

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