A gain-of-function mutation in STAT6.

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.

Transcriptional activation of HIV by Mycobacterium tuberculosis in human monocytes.

Recently 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 up-regulation 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-alpha) in monocyte cultures. However, neutralizing antibody to TNF-alpha 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.

Repression of IL-4-induced gene expression by IFN-gamma requires Stat1 activation.

IFN-gamma antagonizes many physiological responses mediated by IL-4, including the inhibition of IL-4-induced IgE production. This event is largely mediated at the level of transcription. We observed that the IL-4 response element of the germline epsilon promoter is sufficient to confer IFN-gamma-mediated repression onto a reporter construct. The inhibitory effects were observed in both lymphoid and nonlymphoid cell lines. Stat1, which is activated by IFN-gamma, cannot recognize the Stat6-specific IL-4 response element in the epsilon promoter. Hence, competitive DNA binding does not seem to be the underlying mechanism for the inhibitory effect. This is supported by the observation that inhibition is not seen at early time points, but requires prolonged IFN-gamma treatment. IFN-gamma stimulation results in a loss of IL-4-induced Stat6 tyrosine phosphorylation, nuclear translocation, and DNA binding. Using the fibrosarcoma cell line U3A, which lacks Stat1, we demonstrated that the transcription activation function of Stat1 is required for the IFN-gamma-mediated repression. Repression was restored by overexpression of Stat1alpha, but not Stat1beta, in U3A cells. Treatment with IFN-gamma, but not IL-4, specifically up-regulates the expression of SOCS-1 (silencer of cytokine signaling), a recently characterized inhibitor of cytokine signaling pathways, such as IL-6 and IFN-gamma. Overexpression of SOCS-1 effectively blocks IL-4-induced Stat6 phosphorylation and transcription. This suggests that IFN-gamma-mediated repression of IL-4-induced transcription is at least in part mediated by SOCS-1.

Identification of a STAT4 binding site in the interleukin-12 receptor required for signaling.

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 beta2 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 beta1 and beta2 subunits is sufficient for STAT4 activation but not for STAT1 or STAT3 activation.

A mutant cell line defective in response to double-stranded RNA and in regulating basal expression of interferon-stimulated genes.

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 NFkappaB and IFN regulatory factor 1. Analysis of the pivotal dsRNA-dependent serine/threonine 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.

Non-glycosylated human B7-1(CD80) retains the capacity to bind its counter-receptors.

Though the cell surface-associated costimulator B7-1(CD80) is known to be highly N-glycosylated, the functional significance of this N-glycosylation has not been evaluated. Two experimental approaches were taken to assess the influence of N-glycosylation on human B7-1 function. First, stable K562 transfectants expressing human B7-1 were treated with the N-glycosylation inhibitor tunicamycin. This treatment reduced the levels of B7-1 at the cell surface as judged by both indirect immunofluorescence/flow cytometry and immunoprecipitation analyses. Significantly, the non-glycosylated cell surface-associated B7-1 on tunicamycin-treated cells retained the capacity to bind CTLA-4 x Ig, a soluble derivative of the CTLA-4(CD152) counter-receptor. Second, experiments were performed with bacterially-produced non-glycosylated derivatives of human B7-1, comprising either the complete B7-1 extracellular domain (hB7-1 x ed) or the membrane-proximal IgC-homologue domain of B7-1 in isolation (hB7-1 x IgC). While the hB7-1 x IgC derivative failed to bind to CTLA-4, the larger hB7-1 x ed derivative associated with CTLA-4 x Ig in cell-free binding assays. Futhermore, recombinant hB7-1 x ed effectively blocked B7-1-mediated costimulation in an in vitro T cell proliferation assay, suggesting that this soluble non-glycosylated B7-1 derivative is capable of engaging CD28, the B7 counter-receptor implicated in T cell activation. Taken together, these data indicate that the N-glycosylation of B7-1 is not required for its association with counter-receptors. Moreover, the findings pave the way for the therapeutic use of recombinant bacterial B7-1 derivatives as competitive inhibitors of B7-mediated signals.

Regulation of nuclear factor-kappa B and its inhibitor I kappa B-alpha/MAD-3 in monocytes by Mycobacterium tuberculosis and during human tuberculosis.

Blood monocytes from patients with active tuberculosis are activated in vivo, as evidenced by an increase in the stimulated release of proinflammatory cytokines, such as TNF-alpha, and the spontaneous expression of IL-2R. Further, monocytes from patients demonstrate an augmented susceptibility to a productive infection with HIV-1 in vitro. Mycobacterium tuberculosis and its components are strong signals to activate monocytes to production of cytokines. In this study we examined the basis of activation of monocytes during active tuberculosis and by M. tuberculosis. We found a constitutive degradation of I kappa B-alpha, the major cytoplasmic inhibitor of nuclear factor kappa B (NF-kappa B), in freshly isolated PBMC and monocytes from patients with tuberculosis. In contrast, I kappa B-alpha levels in PBMC and monocytes from healthy subjects or from patients with nontuberculous pulmonary conditions were intact. Further, by electrophoretic mobility shift assay, NF-kappa B was activated in monocytes from tuberculous patients. The expression of I kappa B-alpha gene, which is responsive to activation by NF-kappa B, was up-regulated in PBMC and monocytes from patients, but not in mononuclear cells from healthy subjects or those with nontuberculous lung diseases. By contrast, the expression of other adherence-associated early genes, such as IL-8 and IL-1 beta, was not up-regulated in PBMC of tuberculous patients. Further, M. tuberculosis and its tuberculin, purified protein derivative, induced the degradation of I kappa B-alpha and the expression of I kappa B-alpha mRNA, and purified protein derivative induced the activation of NF-kappa B in monocytes.

Detection of human T-cell lymphotrophic virus type I in archival tissue specimens.

OBJECTIVE AND DESIGN: To develop a method for the detection of human T-cell lymphotropic virus type I (HTLV-I) in archival biopsy specimens. A polymerase chain reaction-based gene amplification method was developed to detect HTLV-I proviral DNA in paraffin-embedded specimens. The specificity of the polymerase chain reaction products was controlled by Southern blot analysis using a nested oligonucleotide probe and by nucleotide sequencing. The nucleophosmin gene and the T-cell receptor-gamma gene were used as controls for the integrity and adequacy of total DNA and T-cell DNA, respectively. This study was conducted with patients referred to an academic medical center. Biopsy specimens were obtained from lesional skin or lymph node from Japanese patients with HTLV-I seropositive adult T-cell leukemia/lymphoma. The main outcome measure was the ability to detect HTLV-I pX region proviral DNA. RESULTS: Comparative analysis of DNA extracted from fresh samples of the HTLV-I infected MT4T-cell line demonstrated that formalin fixation and paraffin embedding resulted in a 100-fold reduction in sensitivity of the assay. Nevertheless, HTLV-I pX sequences were still readily detectable in paraffin-embedded samples of MT4 T cells and adult T-cell leukemia/lymphoma specimens. Both formalin and B5 fixation were suitable for the assay that was 100% specific for HTLV-I-infected tissues. CONCLUSIONS: The use of this method should greatly facilitate investigation of the role of HTLV-I in human diseases by allowing analysis of a wide variety of archival tissue specimens. In addition, the controls designed for the current study can be used in a variety of other polymerase chain reaction-based studies of T cells to ensure against false-negative results caused by DNA degradation or inadequate T-cell density.

Evidence against a role for human T-cell lymphotrophic virus type I (HTLV-I) in the pathogenesis of American cutaneous T-cell lymphoma.

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.

Productivity monitoring of the non-technical staff.

Productivity monitoring of non-technical staff can be a difficult task for the manager, according to the authors. They suggest one method, work-sampling, which can be used effectively with this group, and describe its application in a radiation oncology department.