Assessment of the risk of introduction of H5N1 HPAI virus from affected countries to the U.K.

The Department for Environment, Food and Rural Affairs (Defra) has monitored epidemiologic developments following outbreaks of H5N1 in Asia since the beginning of 2004 and publishes risk assessments as the situation evolves. The U.K. applies safeguard measures that reflect EU rules to enable imports to continue when they present negligible risk. Defra risk assessments (RA) identify possible pathways by which the H5N1 virus may be introduced to the U.K. These assessments provide a basis for identifying appropriate surveillance activities to ensure early detection, should the virus be introduced, and disease control measures to be taken, should the virus be detected in the U.K. Nevertheless, these assessments have highlighted that many fundamental uncertainties still remain. These uncertainties center on the geographic and species distribution of infection outside Asia and the means of dissemination of the virus. However, the evolving developments demonstrated that regulatory decisions had to be made despite these uncertainties. Improvements in our current RA abilities would greatly benefit from systematic studies to provide more information on the species susceptibility, dynamics of infection, pathogenesis, and ecology of the virus along with possible pathways by which the H5N1 virus may be disseminated. Such an approach would assist in reducing uncertainties and ensuring that regulatory risk management measures are regularly reviewed by taking into account the most recent scientific evidence. The likelihood of the persistence of H5N1 outside Asia in the coming years and the effects of control programs in Asia and other affected regions to reduce the prevalence of infection are also important factors.

Peroxiredoxin genes are not induced in myeloid leukemia cells exposed to ionizing radiation.

Peroxiredoxins (Prx) comprise an extended family of small antioxidant proteins which conserve a thioredoxin-dependent catalytic function that can contribute to cell protection from reactive oxygen species (ROS). ROS generation is one of the deleterious intracellular effects of ionizing radiation, but the role of Prx during radiation treatment has not been extensively explored. Present experiments measure effects of ionizing radiation on expression of human Prx types I (PAGA), II (NKEF-B) and IV (AOE372) in human myeloid leukemia cells (K562). Prx gene transcription was analyzed by amplifying with RT-PCR cDNAs complementary to each Prx-specific coding sequence and by identifying the derived products with Southern blotting procedure. Transcripts of GAPDH were used as the endogenous standard for semi-quantitative comparisons. No consistent increase in Prx gene expression was detected at time intervals up to 72 h after gamma radiation doses that caused cell cycle arrest and nuclear damage (maximum 20 Gy). Immunoblots also were consistent with a prolonged expression or stability of the Prx I/II proteins. Similarly, a cytotoxic concentration of the oxidant hemin, which stimulates rapid hemoglobinization of K562 cells, caused no induction of Prx gene expression. Our results indicate a high Prx stability in human radio-resistant leukemia cells.

Novel shift of Jak/Stat signalling characterizes the protective effect of aurintricarboxylic acid (ATA) from tumor necrosis factor-alpha toxicity in human B lymphocytes.

Previous results demonstrated that the occurrence of death in human peripheral B lymphocytes by TNF-alpha was paralleled by the activation of the cytoplasmic Jak1 and Tyk2 protein kinases, along with the recruitment of transcription factors Stat3 and Stat5b. In this study we demonstrate that the balance of survival signals in the presence of TNF-alpha was altered by the addition of a salicylate compound, the endonuclease inhibitor aurintricarboxylic acid (ATA). Apoptosis effected by TNF-alpha alone was suppressed by ATA and this event was paralleled by phosphorylation and nuclear translocation of Jak2, Stat2, Stat4 and NF-kB, along with inhibition of caspase activation. These results confirm that among the different cellular responses evoked by TNF-alpha in human B cells, recruitment of Jak/Stat proteins and possible related gene modulation represent contributing factors and address the issue of the development of potential therapeutic strategies aimed at the control of systemic or local effects produced by TNF-alpha.

The laboratory role in diagnosis of infections transmitted by arthropods.

This article provides an overview of arbovirus diseases from the perspective of laboratory diagnosis and related responsibilities of health personnel. Although the disease manifestations are very diverse, general lessons relevant to optimal use of laboratory resources can be drawn from medically important examples. This approach is warranted, because under conditions of bioterrorism or emergence of a novel pathogen, ecologic clues that ordinarily facilitate identification of an arbovirus infection most likely will be absent.

Fetal development in rhesus monkeys exposed prenatally to cocaine.

Using a timed-breeding protocol, one group of female rhesus monkeys was implanted subcutaneously with osmotic minipumps containing 0.3 mg/kg/h cocaine (N=18) or saline (N=18) from day 24 postconception through gestation. Another group received cocaine (N=12) or saline (N=8) from conception through day 42 of gestation. Mean levels of cocaine in maternal serum were approximately 150 ng/ml during pregnancy. A total of 56 pregnancies were documented in 42 adult monkeys, and 39 pregnancies completed full-term. Maternal food consumption and body weight increased during pregnancy, and there were no significant differences among experimental groups. Although both groups with a history of cocaine exposure had lower survival rates compared to pair-fed controls, of the fetuses that survived, fetal heart rate, fetal biparietal diameter, and mean gestational length were in the normal range for all experimental groups. Similarly, body weight, biparietal diameter, body length, and modified Apgar scores at birth did not differ significantly among experimental groups. The results indicate that surviving fetuses exhibited normal growth.

Epithelial defect in prostates of Stat5a-null mice.

The transcription factor Stat5a critically mediates prolactin (PRL)-induced mammary gland development and lactogenesis. PRL also stimulates growth and differentiation of prostate tissue. Specifically, hyperprolactinemia gives rise to prostate hyperplasia, and prostate size is reduced in PRL-deficient mice. We therefore investigated the importance of Stat5a for prostate development and function by examining Stat5a-null mice. The absence of Stat5a in mice was associated with a distinct prostate morphology characterized by an increased prevalence of local disorganization within acinar epithelium of ventral prostates. Affected acini were typically filled with desquamated, granular epithelial cells that had become embedded in dense, coagulated secretory material. These features were reminiscent of acinar cyst formation and degeneration frequently observed in human benign prostate hyperplasia, however, cystic changes in prostate acini of Stat5a-deficient mice were not associated with increased prostate size or morphologic hallmarks of epithelial hyperplasia. Instead, immunohistochemistry of the prostate-specific secretory marker, probasin, suggested that hypersecretory function of the epithelium could underlie local congestion and cyst formation in prostates of Stat5a-null mice. Serum testosterone and PRL levels were normal in Stat5a knockout mice, but prostate PRL receptor expression was reduced as determined by immunohistochemistry. Expression levels or activation states of other PRL signal transduction proteins, including Stat5b, Stat3, Stat1, ERK1, and ERK2 were not altered. The present study offers the first evidence for a direct role of Stat5a in the maintenance of normal tissue architecture and function of the mouse prostate.

Stat5a and Stat5b: fraternal twins of signal transduction and transcriptional activation.

Stat5a and Stat5b are discretely encoded transcription factors that mediate signals for a broad spectrum of cytokines. Their activation is often an integral component of redundant cytokine signal cascades involving complex cross-talk and pleiotropic gene regulation by Stat5 has been implicated in cellular functions of proliferation, differentiation and apoptosis with relevance to processes of hematopoiesis and immunoregulation, reproduction, and lipid metabolism. Although Stat5a and Stat5b show peptide sequence similarities of > 90%, targeted gene disruptions in mice yield distinctive phenotypes. Prolactin-directed mammary gland maturation fails without functional Stat5a, while disruption of Stat5b in males mitigates growth hormone effects on hepatic function and body mass. The molecular basis for this biologic dichotomy is probably multifaceted. Limited structural dissimilarities between the Stat5a and Stat5b transactivation domains, or subtle differences in the DNA-binding affinities of Stat5 dimer pairs undoubtedly influence gene regulation, but cell-dependent asymmetries in availability of phosphorylated Stat5 can be an underlying factor. Differences in serine phosphorylation(s) of Stat5a and Stat5b, or Stat5 associations with adaptor proteins or co-transcription factors are other potential sources of functional disparity and the signal amplitude, frequency or duration also can be significant. In addition to Stat5 signal attenuation by phosphatase actions or classical feedback inhibition, truncated forms of Stat5 lacking in transactivation capacity may compete upstream for activation and diminish access of full length molecules to DNA binding sites.

Stimulation of Stat5 by granulocyte colony-stimulating factor (G-CSF) is modulated by two distinct cytoplasmic regions of the G-CSF receptor.

In a manner similar to many other cytokines, treatment of cells with granulocyte CSF (G-CSF) has been shown to induce the tyrosine phosphorylation of the STAT proteins. Activation of Stat1 and Stat5 by G-CSF requires the membrane-proximal cytoplasmic domain of the receptor, including box1 and box2, while G-CSF-stimulated tyrosine phosphorylation of Stat3 also requires a region distal to box 2. In this study, we show that although the membrane-proximal 55 amino acids of the G-CSF receptor are sufficient for activation of Stat5, the maximal rate of Stat5 activation requires an additional 30 amino acids of the cytoplasmic domain. In contrast, the distal carboxyl-terminal region of the receptor appears to down-regulate Stat5 activation in that deletion of this carboxyl terminus results in increased amplitude and prolonged duration of Stat5 activation by G-CSF. Significantly, expression of a truncated dominant-negative Stat5 protein in hemopoietic cells not only inhibits G-CSF-dependent cell proliferation, but also suppresses cell survival upon G-CSF withdrawal. We further show that a potential protein tyrosine phosphatase may play a critical role in the down-regulation of G-CSF-stimulated Stat5 activation. These results demonstrate that two distinct cytoplasmic regions of the G-CSF receptor are involved in the regulation of the intensity and duration of Stat5 activation, and that Stat5 may be an important player in G-CSF-mediated cell proliferation and survival.

Prolonged STAT1 activation related to the growth arrest of malignant lymphoma cells by interferon-alpha.

Multiple biologic effects of interferon-alpha (IFN-alpha), including cell growth inhibition and antiviral protection, are initiated by tyrosine phosphorylation of STAT proteins. Although this signal pathway has been intensively investigated, the relevance of STAT signal persistence has received scant attention. Using paired isogenic lymphoma cells (Daudi), which either are sensitive or resistant to growth inhibition by IFN-alpha, we found comparable initial tyrosine phosphorylation of multiple STAT proteins; however, the phosphorylation durations and associated DNA-binding activities diverged. Phosphorylation and DNA-binding capacity of STAT1 decreased after 4 to 8 hours in resistant cells, as compared with 24 to 32 hours in sensitive cells, whereas phosphorylation of STAT3 and STAT5b was briefer in both lines. Functional significance of the prolonged STAT1 signal, therefore, was explored by experimental interruption of tyrosine phosphorylation, either by premature withdrawal of the IFN-alpha or deferred addition of pharmacologically diverse antagonists: staurosporine (protein kinase inhibitor), phorbol 12-myristate 13-acetate (growth promoter), or aurintricarboxylic acid (ligand competitor). Results indicated that an approximately 18-hour period of continued STAT1 phosphorylation was associated with growth arrest, but that antiviral protection developed earlier. These differences provide novel evidence of a temporal dimension to IFN-alpha signal specificity and show that duration of STAT1 activation may be a critical variable in malignant cell responsiveness to antiproliferative therapy.

Prolactin activates Stat1 but does not antagonize Stat1 activation and growth inhibition by type I interferons in human breast cancer cells.

Type I interferons (IFN alpha and IFN beta) are presently used in the adjuvant treatment of several human cancers. However, these cytokines have demonstrated only modest success in breast cancer therapy, and research efforts have focused on improving their efficacy. Recent progress in understanding the molecular mechanisms underlying the antiproliferative effects of IFNs has identified the cytoplasmic transcription factor Stat1 as a critical mediator. It is, therefore, possible that IFN-induced growth inhibition of mammary epithelial cells is counteracted by other cytokines that also use Stat1. One such candidate IFN-antagonist with particular relevance to breast cancer is the mammotropic hormone prolactin (PRL). The main goal of this study was to examine whether PRL would interfere with type I IFN (IFN alpha/beta) signal transduction by competing for limited cytoplasmic Stat factors. A second aim was to test whether pretreatment of mammary tumor cell lines with IFN gamma could enhance the effect of IFN alpha/beta. By analyzing the effect of PRL on IFN alpha/beta-induced tyrosine phosphorylation of Stat proteins and their binding to IFN-regulated genes, we now report that costimulation of PRL receptors did not interfere with IFN alpha/beta signals in several human breast cancer cell lines, including T47D, MCF-7, and BT-20. Specifically, PRL did not affect IFN alpha/beta-induced tyrosine phosphorylation or heterodimerization of Stat1 and Stat2 in any cell line. Instead, IFN alpha/beta- and PRL-induced tyrosine phosphorylation of Stat1 was additive and occurred without evidence of competition for limited concentrations of cytoplasmic Stat1. A similar additive relationship was observed on IFN alpha/beta- and PRL-induced Stat3 tyrosine phosphorylation. Furthermore, electrophoretic mobility shift assays showed that type I IFNs induced predominantly Stat1-Stat2 or Stat1-Stat3 heteromeric complexes with various IFN-response elements of IFN-stimulated genes, whereas PRL induced Stat1 homodimers. Despite significant mutual use of Stats by IFNs and PRL, these results indicated a high degree of signaling specificity in the two receptor systems, and that cytoplasmic levels of Stat proteins were not limiting. Similarly, PRL did not interfere with the growth-inhibitory effect of IFN beta. On the other hand, the study indicated that pretreatment of human breast cancer cell lines with IFN gamma enhanced the growth-inhibitory action of type I IFNs, suggesting a possible avenue for improving the effect of type I IFNs in the treatment of breast cancer patients.

Activation of the Jak2-Stat5 signaling pathway in Nb2 lymphoma cells by an anti-apoptotic agent, aurintricarboxylic acid.

Biological effects of many hormones and cytokines are mediated through receptor-associated Jak tyrosine kinases and cytoplasmic Stat transcription factors, including critical physiological processes such as immunity, reproduction, and cell growth and differentiation. Pharmaceuticals that control Jak-Stat pathways are therefore of considerable interest. Here we demonstrate that a single Jak-Stat pathway can be activated by aurintricarboxylic acid (ATA), a negatively charged triphenylmethane derivative (475 Da) with anti-apoptotic properties. In prolactin (PRL)-dependent Nb2 lymphocytes, ATA sustained cell growth in the absence of hormone and mimicked rapid PRL-induced tyrosine phosphorylation of Jak2 and activation of Stat5a and Stat5b with tyrosine phosphorylation, heterodimerization, DNA binding, and induction of the Stat5-regulated pim-1 protooncogene. ATA also mimicked PRL activation of serine kinases ERK1 and ERK2. However, unlike PRL, ATA did not regulate Stat1 or Stat3. ATA also did not affect Jak3, which is activated in these cells by interleukin-2 family cytokines. Although the mechanism and specificity by which ATA activates Jak2, Stat5, and ERKs in Nb2 cells are still unclear, the present study demonstrates that certain hormone or cytokine effects on Jak-Stat pathways can be discretely imitated by a low molecular weight, non-peptide pharmaceutical. The results are also consistent with Stat5 involvement in lymphocyte growth and survival.

Antiproliferative action of interferon-alpha requires components of T-cell-receptor signalling.

Signal transduction through both cytokine and lymphocyte antigen receptors shares some common pathways by which they initiate cellular responses, such as activation of mitogen-activated protein kinase(s). However, other signalling components appear to be uniquely coupled to each receptor. For example, the interferon receptors transduce regulatory signals through the JAK/STAT pathway, resulting in an inhibition of growth and of antiviral effects, whereas this pathway apparently plays no role in T-cell-receptor (TCR)-dependent gene expression. Conversely, signal transduction through the TCR requires the tyrosine kinases Lck and ZAP-70 and the tyrosine phosphatase CD45. Here we show that, unexpectedly, transmission of growth-inhibitory signals by interferon-alpha (IFN-alpha) in T cells requires the expression and association of CD45, Lck and ZAP-70 with the IFN-alpha-receptor signalling complex.

Simian virus-40 large-T antigen binds p53 in human mesotheliomas.

We found that simian virus 40 (SV40) induces mesotheliomas in hamsters and that 60% of human mesotheliomas contain and express SV40 sequences, results now confirmed by others [ref. 3-5, and presentations by D. Griffiths & R. Weiss, F. Galateau-SallE, and H.I.P. at "Simian virus 40: A possible human polyoma virus," NIH workshop, 27-28 January 1997, Bethesda, MD (transcript available through SAG Corp., Washington, DC 20008)]. Mesothelioma, an aggressive malignancy resistant to therapy, originates from the serosal lining of the pleural, pericardial and peritoneal cavities. The incidence of mesothelioma continues to increase worldwide because of exposure to crocidolite asbestos. However, at least 20% of mesotheliomas in the United States are not associated with asbestos exposure, and only a minority of people exposed to high concentrations of asbestos develop mesothelioma. Thus, other carcinogens may induce mesothelioma in individuals not exposed to asbestos, and/or may render particular individuals more susceptible to the carcinogenic effect of asbestos. We investigated whether the expression of the SV40 large T-antigen (Tag) interferes with the normal expression of the tumor suppressor gene p53 in human mesotheliomas. We found that SV40 Tag retains its ability to bind and to inactivate p53, a cellular protein that when normally expressed plays an important role in suppressing tumor growth and in inducing sensitivity to therapy. Our findings do not establish a cause-and-effect relation, but indicate that the possibility that SV40 contributes to the development of human mesotheliomas should be carefully investigated.

Prolactin stimulates serine/tyrosine phosphorylation and formation of heterocomplexes of multiple Stat5 isoforms in Nb2 lymphocytes.

Transcription factors of the Stat gene family are selectively activated by many hormones and cytokines. Stat5 originally was cloned as a prolactin-stimulated DNA-binding protein, but is also activated by non-lactogenic cytokines in many cell types. The recent identification of two distinct Stat5 genes, which encode a 94-kDa Stat5a and a 92-kDa Stat5b as well as several lower molecular weight isoforms, suggests additional complexity and combinatorial possibilities for transcriptional regulation. We now report a biochemical analysis of prolactin activation of Stat proteins in Nb2 lymphocytes, which was associated with: 1) rapid tyrosine phosphorylation of Stat5a, Stat5b, a COOH-terminally truncated 80-kDa Stat5 form, Stat1alpha, and Stat3; 2) rapid and selective formation of Stat5a/b heterodimers, without involvement of Stat1alpha or Stat3; 3) marked serine, but not threonine phosphorylation of Stat5a and Stat5b; and 4) the appearance of two qualitatively distinct Stat5 protein complexes, which discriminated between oligonucleotides corresponding to the prolactin response elements of the beta-casein and interferon regulatory factor-1 gene promoters. Collectively, our analyses showed that Stat5a and Stat5b respond similarly to prolactin receptor activation, but also suggested that the two genes have evolved unique properties that may contribute to the specificity of receptors that utilize Stat5 signaling proteins.

Biological effects of the interferons and other cytokines.

There were seven workshops that primarily concerned the biological effects of the interferons and the other cytokines. These were: Workshop 6, The refractory state in the response to interferons (IFNs) and antibodies in treated patients; Workshop 7, IFNs, multiple sclerosis, and the nervous system; Workshop 9, Viral inhibition of the response to IFNs and other cytokines; Workshop 10, Cell growth inhibition by IFNs and other cytokines; Workshop 12, Cytokines and cell death; Workshop 13, Interactions between cytokines; and, Workshop 14, Cytokine gene therapy. Summaries of each of these sessions follow.

Human cancer cell lines express a negative transcriptional regulator of the interferon regulatory factor family of DNA binding proteins.

Members of the interferon regulatory factor (IRF) family of DNA binding transcription factors have roles in growth regulation, antiviral responses, and transcriptional induction of interferon (IFN)-activated early response genes. The IRF family member ISGF3 gamma is the DNA binding component of IFN-stimulated gene factor 3 (ISGF3), a multicomponent complex responsible for the stimulation of IFN-alpha-responsive genes. IFN-alpha-stimulated formation of ISGF3 and subsequent gene expression can be inhibited by phorbol esters or expression of the adenovirus E1A protein. We have investigated IFN signaling in human malignant tumor cell lines of the lung, colon, ovary, cervix, and hematopoietic organs and found some of these cells to be defective for IFN-alpha-induced formation of ISGF3. In many cases, an inhibitory activity termed transcriptional knockout (TKO) correlated with nonresponsiveness. TKO purified from a human papillomavirus-negative cervical carcinoma cell line has a molecular size of 19 kDa. The purified protein interacted with the ISGF3 gamma component of ISGF3, preventing binding of ISGF3 to DNA. Purified TKO displaced ISGF3 from its DNA binding site in vitro and prevented ISGF3 gamma, IRF-1, and IRF-2 from interacting with the IFN-stimulated response element. Partially purified TKO can also directly interact with ISGF3 gamma in the absence of DNA. This protein may be involved with the development of malignancies and the inability of IFN to exert its antiproliferative and antiviral effects.

A nuclear tyrosine phosphatase downregulates interferon-induced gene expression.

Alpha and gamma interferons rapidly induce several early response genes in primary human diploid fibroblasts. The transcription rates of these genes are maximal after 1 h of interferon treatment and return to basal levels within 8 h. Three different interferon-activated DNA-binding complexes (ISGF3, GAF, and FcRF gamma) that are responsible for transcriptional activation of cellular genes have been characterized. Assembly of these complexes requires tyrosine phosphorylation of one or more of the protein components. In this report, we demonstrate that a nuclear tyrosine phosphatase is responsible for the deactivation of these interferon-regulated transcription factors and the subsequent transcriptional downregulation of the corresponding genes. Furthermore, tyrosine phosphorylation is required for nuclear localization of the 91-kDa protein that is part of all three interferon-induced transcription complexes. These results provide the first evidence for a nuclear tyrosine phosphatase activity as a mechanism of transcriptional regulation.

Modulation of the anti-proliferative signal of interferon-alpha by tamoxifen in U937 cells.

Several clinical protocols are attempting to utilize the combined anti-proliferative signal of interferon-alpha (INF-alpha) and tamoxifen on cancer cells. We demonstrated here that the effect of these two agents on the growth of the premacrophage U937 cells is antagonistic. This antagonistic effect is paralleled by the ability of tamoxifen to modulate the INF-alpha-induced hyperpolarization in these cells. INF-alpha-induced hyperpolarization was shown before to be an integral part of the anti-proliferative signal of this agent. Tamoxifen liberates Ca2+ from intracellular stores of U937 cells but this effect of the drug is not the cause of its antagonistic effect with the anti-proliferative signal of IFN-alpha. We suggest therefore, that the combined effect of these two anti-cancer drugs could also be advantageous for macrophage proliferation.

Interferon-alpha-induced gene expression: evidence for a selective effect of ouabain on activation of the ISGF3 transcription complex.

Binding of interferons (IFNs) to their cell surface receptors stimulates rapid translocation of cytoplasmic proteins to the nucleus and the expression of a variety of cellular genes within minutes. Translocated proteins subsequently bind to the interferon-stimulated response element (ISRE) located in the promoters of all IFN-activated cellular genes. We report here that ouabain, a specific inhibitor of the Na/K ATPase, selectively inhibited transcription of several IFN-alpha-induced cellular RNAs under conditions in which some other well-described signal transduction pathways remained intact. The latter included induction of human metallothionein 2A (HMT2A) by phorbol ester and induction of IP-10 RNA by IFN-gamma. Ouabain itself induced RNA of the protooncogene c-fos which conversely was inhibited by IFN-alpha. Specificity of the ouabain effects on IFN alpha-induced RNAs with respect to a direct action on the Na/K ATPase was shown with a transfected monkey CV-1 cell line which expresses the ouabain-insensitive rat alpha 1 subunit. Electrophoretic mobility shift assays (EMSAs) using nuclear extracts from ouabain-treated cells demonstrated that ouabain decreased IFN alpha-induced binding of the ISGF3 complex to the ISRE. Reconstitution experiments showed that this effect of ouabain is not due to the inhibition of IFN alpha activation of the ISGF3 alpha subcomponent, which occurs in the cytoplasm, but a selective depletion of the ISGF3 gamma factor which in concert with activated ISGF3 alpha induces interferon-stimulated gene (54 kDa) transcription. These findings imply that intracellular ion balance can selectively regulate the half-life of the ISGF3 gamma protein or the ability of this protein to complex with ISGF3 alpha to activate IFN alpha-regulated cellular genes.