Interleukin-4 (IL-4) is a cytokine that plays a crucial role in the pathophysiology of asthma and allergic diseases. IL-4-induced gene expression is largely mediated through the activation of the latent transcription factor STAT6. We identified a STAT6 mutant (STAT6VT)) that is activated independently of IL-4 stimulation. STAT6VT carries two amino acid changes in the SH2 domain that affect the overall structure and stability of the monomeric and dimeric protein. When overexpressed in mammalian cells, STAT6VT undergoes tyrosine phosphorylation, binds DNA, and activates transcription in the absence of IL-4 stimulation. Using the Jak1-and Jak3-deficient fibroblast line U4A, we demonstrate that phosphorylation is mediated by an IL-4-independent tyrosine kinase that is not able to activate the wild-type STAT6 protein. These results suggest that small changes in STAT6 could result in hyperactivation of the protein and constitutive expression of STAT6-dependent genes. Such a mutation, if found in vivo, could cause genetic predisposition for atopic diseases.

The specificity of the various STAT SH2 domains for different tyrosine-containing peptides enables cytokines to activate different signaling pathways and to induce distinct patterns of gene expression. We show that STAT4 has a unique peptide specificity and binds to the peptide sequence pYLPSNID (where pY represents phosphotyrosine). This motif is found at tyrosine residue 800 in the ␤2 subunit of the interleukin-12 receptor and is required for DNA binding and transcriptional activity of STAT4. Our data demonstrate that transfection of interleukin-12 receptor ␤1 and ␤2 subunits is sufficient for STAT4 activation but not for STAT1 or STAT3 activation.IL-12 1 is a heterodimeric cytokine secreted from antigen presenting cells in response to bacteria and intracellular parasites and thus plays an important role in host defense against bacterial pathogens (1). IL-12 promotes the proliferation of T cells and NK cells and is required for the differentiation of T cells into the Th1 subset of T helper cells. Th1 cells are critical for cell-mediated immune responses, because they secrete IFN-␥, which enhances the activity of cytotoxic T cells and NK cells (1-3). In addition to these essential functions, Th1-dominated responses are associated with pathologic autoimmune and inflammatory conditions such as rheumatoid arthritis and inflammatory bowel disease (4). Given this potential for immunopathology, it is important to understand the mechanisms that control the Th1 response to develop possible therapeutic interventions.IL-12 signals through the IL-12 receptor, which is composed of at least two subunits designated ␤1 and ␤2 (5, 6). Both IL-12 receptor subunits are members of the hemapoietin receptor superfamily and have strong homology to the gp130 receptor (5). The ␤1 receptor, although a low affinity binder of IL-12, is not capable of transducing an IL-12-mediated signal (6). A second subunit of the IL-12 receptor was subsequently identified that when coexpressed with the ␤1 subunit forms a high affinity receptor for IL-12 and confers IL-12 signaling (6). IL-12R ␤2 expression is differentially regulated in Th1 versus Th2 cells (7). Th1 cells but not Th2 cells express the ␤2 subunit of the IL-12 receptor (8). Following T cell activation, IL-12 and IFN-␥ treatment induces ␤2 expression, whereas IL-4, a cytokine produced by Th2 cells, inhibits ␤2 expression resulting in loss of IL-12 signaling (7,8). Therefore, expression of the ␤2 subunit of the IL-12 receptor is a crucial determinant of Th1 versus Th2 development.Cytokine binding to its receptor leads to activation of JAK kinases that phosphorylate the receptor on tyrosines located in the intracellular domain. The phosphorylated regions are binding sites for signal transduction molecules called STATs that are rapidly recruited to the receptor and tyrosine phosphorylated by JAK kinases. Tyrosine phosphorylation of STAT proteins induces their dimerization and translocation to the nucleus where they bind to specific DNA sequences and regulate transcription. IL-12 stimulation ...

Although much progress has been made in identifying the signaling pathways that mediate the initial responses to interferons (IFNs), much less is known about how IFN-stimulated genes (ISGs) are kept quiescent in untreated cells, how the response is sustained after the initial induction, and how ISG expression is down-regulated, even in the continued presence of IFN. We have used the cell sorter to isolate mutant cells with constitutively high ISG expression. A recessive mutant, P2.1, has higher constitutive ISG levels than the parental U4C cells, which do not respond to any IFN. Unexpectedly, P2.1 cells also are deficient in the expression of ISGs in response to double-stranded RNA (dsRNA). Electrophoretic mobility-shift assays revealed that the defect is upstream of the activation of the transcription factors NFB and IFN regulatory factor 1. Analysis of the pivotal dsRNAdependent serine͞threonine kinase PKR revealed that the wild-type kinase is present and is activated normally in response to dsRNA in P2.1 cells. Together, these data suggest that the defect in P2.1 cells is either downstream of PKR or in a component of a distinct pathway that is involved both in activating multiple transcription factors in response to dsRNA and in regulating the basal expression of ISGs.

SUMMARYRecently it has been shown that infection with Mycobacterium tuberculosis increases the replication of HIV in mononuclear cells. The objective of this study was to investigate the mechanism(s) of upregulation of HIV in primary human monocytes. Monocytes from healthy subjects were infected with HIV in vitro and then cultured with purified protein derivative (PPD) of M. tuberculosis. Culture supernatants were assessed for HIV p24 and cytokines. HIV expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). PPD induced HIV-infected monocytes to increased expression of HIV RNA and production of HIV p24. This effect correlated with production of tumour necrosis factor-alpha (TNF-a) in monocyte cultures. However, neutralizing antibody to TNF-a only partly abrogated the PPD-induced HIV p24 in these cultures. Also, PPD and culture filtrate of M. tuberculosis induced HIV mRNA expression. Further, using an adenovirus infection system containing an HIV long-terminal repeat (LTR) reporter plasmid, we showed that M. tuberculosis and its PPD induced HIV LTR. Therefore, the effect of M. tuberculosis and its PPD on HIV replication in monocytes is primarily one of transcriptional activation.

We used a standard polymerase chain reaction (PCR)/Southern blot assay (sensitivity > 10(-5)) to detect human T-cell lymphotrophic virus type I (HTLV-I) proviral pX, pol, and env genes in the lesional skin of 42 American patients with cutaneous T-cell lymphoma (CTCL). As in some prior reports using similar methods, a variable proportion of PCR tests were positive (seven of 42 for pX, three of 42 for pol, and two of 37 for env), resulting in an overall positive test rate of 12 of 121 (10%). To determine the significance of these positive test results, we performed several additional studies. D1S80 polymorphism analysis of CTCL cases and HTLV-I PCR analysis of non-CTCL dermatosis controls showed no evidence that positive PCR tests resulted from sample mislabeling, gross HTLV-I contamination, or human endogenous retroviruses. We then modified the standard PCR assay to incorporate ultraviolet (UV) light to destroy low-level PCR contamination. With this modified assay (sensitivity > 10(-5)), only three of 12 previously positive cases were still positive, suggesting that the earlier positives were due to trace contamination of PCR reagents or trace contamination of sample DNA. This interpretation was also supported by: (i) a match between pX and pol sequences cloned from one PCR-positive specimen and the MT4-positive control, (ii) our inability to confirm HTLV-I in any PCR-positive case using genomic dot blotting (sensitivity > 10(-2)), and (iii) negative PCR results when new samples from two of the remaining positive cases were analyzed. Finally, we used our modified UV/ PCR/Southern blot assay to test an additional 28 cases of American CTCL for pX. All of them were negative. Although these studies of 70 cases of American CTCL do not exclude the possibility that another virus is involved in the pathogenesis of this disease, they provide strong evidence against a role for HTLV-I. Furthermore, they emphasize the need for special strategies to control for false-positive PCR tests that can result from even trace levels of contamination with viral DNA. As a consequence, associations between diseases and viruses should be viewed skeptically if they are based primarily on conventional PCR data.