Differential effect by chronic disease risk: A secondary analysis of the ChooseWell 365 randomized controlled trial.

Objective: Whether employees' health status is associated with the effectiveness of workplace health promotion programs is unknown. The objective of this study was to determine if the effect of a workplace healthy eating intervention differed by baseline chronic disease status. Methods: This was a secondary analysis of a randomized controlled trial conducted September 2016 to February 2018 among US hospital employees to test the effect of a 12-month behavioral intervention (personalized feedback, peer comparisons, and financial incentives) on diet and weight. Participants were classified as having chronic disease (yes/no) based on self-reported hypertension, hyperlipidemia, heart disease, stroke, pre-diabetes, diabetes, cancer or another serious illness. BMI was measured at study visits and calories purchased were measured from cafeteria sales data over 24 months. Mixed models with random effects assessed heterogeneity of treatment effects by chronic disease. Results: Participants (N = 548) were mostly female (79.7 %) and white (81.2 %); 224 (40.9 %) had chronic disease. Among those with chronic disease, intervention participants reduced caloric intake by 74.4 [22.3] kcal more than control, with a smaller difference between intervention and control (-1.9 [18.7] kcal) (three-way p-interaction = 0.02). The effect on BMI for those with chronic disease (0.47 [0.21] kg/m2) indicated weight stability among intervention participants and weight gain among controls while the effect (-0.56 [0.18] kg/m2) for those without chronic disease was the opposite (three-way p-interaction < 0.01). Conclusions: Those with chronic diseases had greater reductions in calories purchased and gained less weight. Employers with limited resources for health promotion might consider tailoring programs to employees at highest risk.

Seasonal variability in environmental tobacco smoke exposure in public housing developments.

The risk of tobacco smoking and second-hand smoke (SHS) exposure combined are the leading contributors to disease burden in high-income countries. Recent studies and policies are focusing on reducing exposure to SHS in multiunit housing (MUH), especially public housing. We examined seasonal patterns of SHS levels within indoor common areas located on Boston Housing Authority (BHA) properties. We measured weekly integrated and continuous fine particulate matter (PM2.5) and passive airborne nicotine in six buildings of varying building and occupant characteristics in summer 2012 and winter 2013. The average weekly indoor PM2.5 concentration across all six developments was 9.2 µg/m3, higher during winter monitoring period (10.3 µg/m3) compared with summer (8.0 µg/m3). Airborne nicotine concentrations ranged from no detection to about 5000 ng/m3 (mean 311 ng/m3). Nicotine levels were significantly higher in the winter compared with summer (620 vs. 85 ng/m3; 95% CI: 72-998). Smoking-related exposures within Boston public housing vary by season, building types, and resident smoking policy. Our results represent exposure disparities that may contribute to health disparities in low-income communities and highlight the potential importance of efforts to mitigate SHS exposures during winter when outdoor-indoor exchange rates are low and smokers may tend to stay indoors. Our findings support the use of smoke-free policy as an effective tool to eliminate SHS exposure and protect non-smokers, especially residents of MUH.

STAT1-regulated lung MDSC-like cells produce IL-10 and efferocytose apoptotic neutrophils with relevance in resolution of bacterial pneumonia.

Bacterial pneumonia remains a significant burden worldwide. Although an inflammatory response in the lung is required to fight the causative agent, persistent tissue-resident neutrophils in non-resolving pneumonia can induce collateral tissue damage and precipitate acute lung injury. However, little is known about mechanisms orchestrated in the lung tissue that remove apoptotic neutrophils to restore tissue homeostasis. In mice infected with Klebsiella pneumoniae, a bacterium commonly associated with hospital-acquired pneumonia, we show that interleukin (IL)-10 is essential for resolution of lung inflammation and recovery of mice after infection. Although IL-10(-/-) mice cleared bacteria, they displayed increased morbidity with progressive weight loss and persistent lung inflammation in the later phase after infection. A source of tissue IL-10 was found to be resident CD11b(+)Gr1(int)F4/80(+) cells resembling myeloid-derived suppressor cells (MDSCs) that appeared with a delayed kinetics after infection. These cells efficiently efferocytosed apoptotic neutrophils, which was aided by IL-10. The lung neutrophil burden was attenuated in infected signal transducer and activator of transcription 1 (STAT1)(-/-) mice with concomitant increase in the frequency of the MDSC-like cells and lung IL-10 levels. Thus, inhibiting STAT1 in combination with antibiotics may be a novel therapeutic strategy to address inefficient resolution of bacterial pneumonia.

Distinct inflammatory signals have physiologically divergent effects on epigenetic regulation of Foxp3 expression and Treg function.

Foxp3 expression in regulatory T cells (Treg) is required for their development and suppressive function. How different inflammatory signals affect Foxp3 chromatin structure, expression and Tregs plasticity are not completely known. In the present study, the Toll-like receptor 2 (TLR2) ligand peptidoglycan inhibited Foxp3 expression in both natural Treg (nTreg) and TGFß-driven adaptive Treg (aTreg). Inhibition was independent of paracrine Th1, Th2 and Th17 cytokines. PGN-induced T cell-intrinsic TLR2-Myd88-dependent IFR1 expression and induced IRF1 bound to IRF1 response elements (IRF-E) in the Foxp3 promoter and intronic enhancers, and negatively regulated Foxp3 expression. Inflammatory IL-6 and TLR2 signals induced divergent chromatin changes at the Foxp3 locus and regulated Treg suppressor function, and in an islet transplant model resulted in differences in their ability to prolong graft survival. These findings are important for understanding how different inflammatory signals can affect the transplantation tolerance and immunity.

Restricted tissue tropism and acquired resistance to Sindbis viral vector expression in the absence of innate and adaptive immunity.

Our previous studies suggest that replication-defective Sindbis vectors might be promising agents for specific tumor targeting and detection. However, the effects of innate and/or adaptive anti-viral immunity, in particular, the IFN-I/STAT1 signaling pathway, may impact their therapeutic potential. Using a bioluminescent imaging system, we demonstrate that although most normal cells are not permissively transduced by replication-defective Sindbis vector, transduction of liver non-sinusoidal endothelial occurs the first time IFN-I/STAT1 signaling deficient mice are inoculated with the vector. Transduction of some cells is not surprising since STAT1 knockout animals show significant delay in IFN responses such as the production of IFN-alpha/beta and transcriptional activation of several anti-viral genes (IRF7, RIG-I, PKR, TLR3, USP18, ISG15). However, beyond the initial vector transduction, which resolves rapidly, secondary inoculums of Sindbis vectors do not transduce any liver cells, suggesting that an alternative antiviral pathway may protect against further transduction. Other known signaling pathways were examined using mice lacking functional TLR3, tumor necrosis factoralphaR or nuclear factor-kappa B (p50). Surprisingly, none of those pathways seem to play a significant role in anti-Sindbis responses. Thus it appears that in vivo, in contrast to the ready transduction of tumor cells, transduction of normal cells by replication-defective Sindbis vector is limited, possibly by a novel mechanism.

A phase II trial of perifosine in locally advanced, unresectable, or metastatic pancreatic adenocarcinoma.

BACKGROUND: Perifosine, a heterocyclic alkylphosphocholine signal transduction inhibitor, has activity against multiple cell types in vitro. This is a phase II study to determine activity and toxicity of perifosine in pancreatic adenocarcinoma. PATIENTS AND METHODS: Previously untreated patients with locally advanced, unresectable, or metastatic pancreatic adenocarcinoma, performance status Eastern Cooperative Oncology Group 0 or 1, were enrolled. An oral loading dose of 900 mg was followed by 100 mg per day until progression or unacceptable toxicity. Response criteria in solid tumors (RECIST) methodology and a 2-stage design were used. Suspension could occur for inadequate response in the first cohort or for more than 25% grade 3 or greater toxicity. RESULTS: Ten patients were enrolled. Six received 1 month and 4 received 2 months of treatment. Four discontinued therapy as a result of progression and 2 because of clinical deterioration. Three died during treatment. One patient had stable disease but discontinued therapy as a result of unacceptable adverse events (95% confidence interval: 0.3-45%). There were no objective responses and all patients died of progressive disease. Median overall and progression-free survival was 1.85 months (95% confidence interval: 0.9-2.7) and 1.5 months (95% confidence interval: 0.9-1.9) respectively. CONCLUSION: The study was suspended and subsequently terminated as a result of unacceptable adverse events during the first stage. Perifosine does not appear to be worthy of further study in this group of patients.

A phase II study of high-dose bevacizumab in combination with irinotecan, 5-fluorouracil, leucovorin, as initial therapy for advanced colorectal cancer: results from the Eastern Cooperative Oncology Group study E2200.

AIM: Patients with untreated advanced colorectal cancer were enrolled to this single arm phase II multi-center cooperative group trial of bevacizumab combined with IFL. The first 20 patients received irinotecan (125 mg/m(2)), 5-fluorouracil (500 mg/m(2)) and leucovorin (20 mg/m(2)) weekly for four of six weeks and high-dose bevacizumab (10 mg/kg) every other week. Following a toxicity review of other trials using IFL, subsequent patients were enrolled at reduced doses of irinotecan (100 mg/m(2)) and 5-fluorouracil (400 mg/m(2)). RESULTS: Of the 92 patients accrued to the study, toxicity data are available for 87 patients and efficacy data for 81 patients. At a median follow-up of 37.5 months, median overall survival is 26.3 months, median progression free survival is 10.7 months and 1-year survival is 85%. The overall response rate is 49.4% (6.2% complete responses). A reduction in the starting doses of irinotecan and 5-fluorouracil decreased the occurrence of vomiting, diarrhea and neutropenia related complications. Bleeding occurred in 37 patients; all events but two were grade 1 or grade 2. There were nine reports of grade 3 or grade 4 thrombo-embolic events. Hypertension of any grade occurred in 13% of patients and proteinuria was infrequent. CONCLUSION: High-dose bevacizumab added to IFL is a well-tolerated and highly active regimen in patients with previously untreated metastatic colorectal cancer.

Assessment of quality of life in adolescent patients with orthopaedic problems: are adult measures appropriate?

New pressures of accountability brought on by a rapidly evolving system of health care financing have underscored the need for standardized, valid measures of patient outcome that reflect the effect of clinical intervention on all aspects of quality of life. In response, there has been a burgeoning interest in the area of outcomes assessment and measurement of quality of life after orthopaedic intervention in adults, but less attention has been focused on the assessment of broadly defined outcomes in children. In an effort to borrow from the broader adult experience in this area, the authors sought to examine whether the Medical Outcomes Study Short Form 36 (SF-36) or the EuroQol questionnaire, widely accepted adult health status measures, would be valid in this setting. These two measures were administered to 196 adolescent patients (10-18 years old) seeking orthopaedic evaluation. Tests of scale properties and construct validity show that these properties are maintained in this population, but neither instrument reflected known differences in health status among this cohort. Most importantly, both the SF-36 and the EuroQol exhibited serious ceiling effects (most respondents scored at the top of their scales), despite evidence indicating those patients often had suboptimal health status. Thus, neither the SF-36 nor the EuroQol is valid for use in this population. The assessment of pediatric health status demands outcomes measures specifically designed to reflect the unique needs of this population.

Capturing quality of life in pediatric orthopaedics: two recent measures compared.

There is a clear need for standardized measures to assess health status that are valid and appropriate to the needs of children with orthopaedic problems. The Child Health Questionnaire and the American Academy of Orthopaedic Surgeons Pediatric Outcomes Data Collection Instrument, two new pediatric health status measures, were assessed for their ability to detect differences in health states in a pediatric orthopaedic population. The instruments have a range of scales designed to measure various aspects of physical and psychosocial health. Two hundred forty-two patients with wide-ranging diagnoses were enrolled in this cross-sectional study. The instruments exhibited ceiling effects in some domains but generally performed as they were intended in this large cohort. Using secondary factor analysis, it was shown that the domains of the instruments appropriately distinguish physical and psychosocial health. Several domains from each instrument discriminated between diagnosis groups and patients with varying numbers of comorbidities. Both of these measures show significant promise and have an important role in helping define the outcomes of children with orthopaedic problems.

Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response.

Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-DeltaN110), 222 (NS5A-DeltaN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-DeltaN110 and NS5A-DeltaN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-kappaB and AP-1 were important in NS5A-DeltaN222 transactivation in the presence of tumor necrosis factor alpha and that NF-IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.

The virus battles: IFN induction of the antiviral state and mechanisms of viral evasion.

Response to IFN involves a rapid and direct signal transduction mechanism that quickly reports that presence of extracellular cytokine to the cell nucleus, preserving the specificity inherent in cytokine-receptor interactions to transcriptionally induce expression of a set of genes encoding important antiviral proteins. Establishment of the resulting antiviral state provides a crucial initial line of defense against viral infection. Studies of IFN-deficient cells and animals derived by gene targeting have demonstrated the essential nature of IFN-mediated innate immunity. The long co-evolutionary history of viruses with their hosts has seen the development of a variety of evasive adaptations that allow viruses to circumvent or inactivate host antiviral mechanisms. Further understanding of both host and viral components of this battle may provide important new strategies for vaccine development and creation of novel antiviral compounds.

Specificity of signaling by STAT1 depends on SH2 and C-terminal domains that regulate Ser727 phosphorylation, differentially affecting specific target gene expression.

Complete activation of signal transducer and activator of transcription 1 (STAT1) requires phosphorylation at both Y701 and a conserved PMS(727)P sequence. S727 phosphorylation of STAT1 in interferon-gamma (IFN-gamma)-treated mouse fibroblasts occurred without a need for p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 or c-Jun kinases, and required both an intact SH2 domain and phosphorylation of Y701. In contrast, UV irradiation-induced STAT1 phosphorylation on S727 required p38MAPK, but no SH2 domain- phosphotyrosine interactions. Mutation of S727 differentially affected IFN-gamma target genes, at the level of both basal and induced expression. Particularly strong effects were noted for the GBP1 and TAP1 genes. The PMS(727)P motif of STAT3 was phosphorylated by stimuli and signaling pathways different from those for STAT1 S727. Transfer of the STAT3 C-terminus to STAT1 changed the stimulus and pathway specificity of STAT1 S727 phosphorylation to that of STAT3. Our data suggest that STAT C-termini contribute to the specificity of cellular responses by linking individual STATs to different serine kinase pathways and through an intrinsically different requirement for serine phosphorylation at different target gene promoters.

IRF3 and IRF7 phosphorylation in virus-infected cells does not require double-stranded RNA-dependent protein kinase R or Ikappa B kinase but is blocked by Vaccinia virus E3L protein.

Induction of interferon-alpha (IFNalpha) gene expression in virus-infected cells requires phosphorylation-induced activation of the transcription factors IRF3 and IRF7. However, the kinase(s) that targets these proteins has not been identified. Using a combined pharmacological and genetic approach, we found that none of the kinases tested was responsible for IRF phosphorylation in cells infected with Newcastle disease virus (NDV). Although the broad-spectrum kinase inhibitor staurosporine potently blocked IRF3 and -7 phosphorylation, inhibitors for protein kinase C, protein kinase A, MEK, SAPK, IKK, and protein kinase R (PKR) were without effect. Both IkappaB kinase and PKR have been implicated in IFN induction, but cells genetically deficient in IkappaB kinase, PKR, or the PKR-related genes PERK, IRE1, or GCN2 retained the ability to phosphorylate IRF7 and induce IFNalpha. Interestingly, PKR mutant cells were defective for response to double-stranded (ds) RNA but not to virus infection, suggesting that dsRNA is not the only activating viral component. Consistent with this notion, protein synthesis was required for IRF7 phosphorylation in virus-infected cells, and the kinetics of phosphorylation and viral protein production were similar. Despite evidence for a lack of involvement of dsRNA and PKR, vaccinia virus E3L protein, a dsRNA-binding protein capable of inhibiting PKR, was an effective IRF3 and -7 phosphorylation inhibitor. These results suggest that a novel cellular protein that is activated by viral products in addition to dsRNA and is sensitive to E3L inhibition is responsible for IRF activation and reveal a novel mechanism for the anti-IFN effect of E3L distinct from its inhibition of PKR.

Phosphorylation-induced dimerization of interferon regulatory factor 7 unmasks DNA binding and a bipartite transactivation domain.

Interferon regulatory factor 7 (IRF7) is an interferon (IFN)-inducible transcription factor required for activation of a subset of IFN-alpha genes that are expressed with delayed kinetics following viral infection. IRF7 is synthesized as a latent protein and is posttranslationally modified by protein phosphorylation in infected cells. Phosphorylation required a carboxyl-terminal regulatory domain that controlled the retention of the active protein exclusively in the nucleus, as well as its binding to specific DNA target sequences, multimerization, and ability to induce target gene expression. Transcriptional activation by IRF7 mapped to two distinct regions, both of which were required for full activity, while all functions were masked in latent IRF7 by an autoinhibitory domain mapping to an internal region. A conditionally active form of IRF7 was constructed by fusing IRF7 with the ligand-binding and dimerization domain of estrogen receptor (ER). Hormone-dependent dimerization of chimeric IRF7-ER stimulated DNA binding and transcriptional transactivation of endogenous target genes. These studies demonstrate the regulation of IRF7 activity by phosphorylation-dependent allosteric changes that result in dimerization and that facilitate nuclear retention, derepress transactivation, and allow specific DNA binding.

Distinct requirements for IFNs and STAT1 in NK cell function.

NK cell functions were examined in mice with a targeted mutation of the STAT1 gene, an essential mediator of IFN signaling. Mice deficient in STAT1 displayed impaired basal NK cytolytic activity in vitro and were unable to reject transplanted tumors in vivo, despite the presence of normal numbers of NK cells. IL-12 enhanced NK-mediated cytolysis, but poly(I:C) did not, and a similar phenotype occurred in mice lacking IFNalpha receptors. Molecules involved in activation and lytic function of NK cells (granzyme A, granzyme B, perforin, DAP10, and DAP12) were expressed at comparable levels in both wild-type and STAT1(-/-) mice, and serine esterase activity necessary for CTL function was normal, showing that the lytic machinery was intact. NK cells with normal cytolytic activity could be derived from STAT1(-/-) bone marrow progenitors in response to IL-15 in vitro, and enhanced NK lytic activity and normal levels of IFN-gamma were produced in response to IL-12 treatment in vivo. Despite these normal responses to cytokines, STAT1(-/-) mice could not reject the NK-sensitive tumor RMA-S, even following IL-12 treatment in vivo. Whereas in vitro NK cytolysis was also reduced in mice lacking both type I and type II IFN receptors, these mice resisted tumor challenge. These results demonstrate that both IFN-alpha and IFN-gamma are required to maintain NK cell function and define a STAT1-dependent but partially IFN-independent pathway required for NK-mediated antitumor activity.

Stat5 is essential for the myelo- and lymphoproliferative disease induced by TEL/JAK2.

STAT5 is activated in a broad spectrum of human hematologic malignancies. We addressed whether STAT5 activation is necessary for the myelo- and lymphoproliferative disease induced by TEL/JAK2 using a genetic approach. Whereas mice transplanted with bone marrow transduced with retrovirus expressing TEL/JAK2 develop a rapidly fatal myelo- and lymphoproliferative syndrome, reconstitution with bone marrow derived from Stat5ab-deficient mice expressing TEL/JAK2 did not induce disease. Disease induction in the Stat5a/b-deficient background was rescued with a bicistronic retrovirus encoding TEL/JAK2 and Stat5a. Furthermore, myeloproliferative disease was induced by reconstitution with bone marrow cells expressing a constitutively active mutant, Stat5a, or a single Stat5a target, murine oncostatin M (mOSM). These data define a critical role for Stat5a/b and mOSM in the pathogenesis of TEL/JAK2 disease.

Differentiation of monocytes to macrophages switches the Mycobacterium tuberculosis effect on HIV-1 replication from stimulation to inhibition: modulation of interferon response and CCAAT/enhancer binding protein beta expression.

HIV-1 replication is inhibited in uninflamed lung macrophages and is stimulated during tuberculosis. Attempts to recapitulate activation of HIV-1 replication in primary monocytes and macrophages ex vivo and in the untreated and PMA-treated THP-1 cell line model in vitro have produced opposite results depending on the state of differentiation of the cells. After infection with Mycobacterium tuberculosis, monocytes enhanced HIV-1 replication and produced a stimulatory 37-kDa CCAAT/enhancer binding protein beta (C/EBPbeta) transcription factor, whereas macrophages suppressed HIV-1 replication and produced an inhibitory 16-kDa C/EBPbeta transcription factor. IFN-beta induced inhibitory 16-kDa C/EBPbeta in macrophages, but had no effect on C/EBPbeta expression in monocytes. Macrophages, but not monocytes, were able to activate IFN-stimulated gene factor-3 (ISGF-3), a transcription factor composed of STAT-1, STAT-2, and IFN regulatory factor (IRF)-9, after infection with M. tuberculosis or stimulation with type I IFN. Macrophages expressed IRF-9 DNA-binding activity, but monocytes did not, and addition of the IRF-9 component reconstituted ISGF-3 in extracts of IFN-treated monocytes. Modulation of IFN responsiveness upon differentiation occurred at least in part through a post-transcriptionally regulated increase in IRF-9 expression. Both monocytes and macrophages maintained IFN responsiveness, activating STAT-1 homodimer formation and transcription of the STAT-1 gene after IFN stimulation. In addition, both monocytes and macrophages were able to activate NF-kappaB upon infection with M. tuberculosis. These results show that induction of ISGF-3, expression of the inhibitory 16-kDa C/EBPbeta, and suppression of HIV-1 replication via a transcriptional mechanism are macrophage-specific responses to infection with M. tuberculosis.

Divergent roles of STAT1 and STAT5 in malignancy as revealed by gene disruptions in mice.

Stat proteins are latent transcription factors activated by tyrosine phosphorylation downstream of cytokine and growth factor receptors and have been implicated in a variety of cell growth regulatory pathways. Constitutive phosphorylation has also been observed in various transformed cell line and in primary malignant tissue, suggesting that Stat protein activation may contribute to the transformed phenotype. One method to distinguish between a causative role in malignancy as opposed to bystander phosphorylation from the increased tyrosine phosphorylation that accompanies transformation is to investigate cell growth and malignancy in the absence of particular Stat proteins using targeted gene disruptions in transgenic mice. Such studies show that Stat1 primarily mediates growth inhibitory signals and contributes to the host rejection of tumors, and that its activation in transformed cells is not necessary for malignancy. Activation of Stat5 can be both necessary and sufficient for malignant transformation, and single Stat5-target genes have been identified that are critical for heightened proliferation. Nonetheless, some malignancies that are characterized by constitutively phosphorylated Stat5 are not altered by the loss of Stat5 protein. Its role in these cases may be redundant with other transforming events that are in themselves sufficient to cause disease, rendering tyrosine phosphorylation of Stat5 unnecessary in these transformed cells. Oncogene (2000).

Intravenous ancrod for treatment of acute ischemic stroke: the STAT study: a randomized controlled trial. Stroke Treatment with Ancrod Trial.

CONTEXT: Approved treatment options for acute ischemic stroke in the United States and Canada are limited at present to intravenous tissue-type plasminogen activator, but bleeding complications, including intracranial hemorrhage, are a recognized complication. OBJECTIVE: To evaluate the efficacy and safety of the defibrinogenating agent ancrod in patients with acute ischemic stroke. DESIGN: The Stroke Treatment with Ancrod Trial (STAT), a randomized, parallel-group, double-blind, placebo-controlled trial conducted between August 1993 and January 1998. SETTING: Forty-eight centers, primarily community hospitals, in the United States and Canada. PATIENTS: A total of 500 patients with an acute or progressing ischemic neurological deficit were enrolled and included in the intent-to-treat analysis. INTERVENTIONS: Patients were randomly assigned to receive ancrod (n=248) or placebo (n =252) as a continuous 72-hour intravenous infusion beginning within 3 hours of stroke onset, followed by infusions lasting approximately 1 hour at 96 and 120 hours. The ancrod regimen was designed to decrease plasma fibrinogen levels to 1.18 to 2.03 micromol/L. MAIN OUTCOME MEASURES: The primary efficacy end point was functional status, with favorable functional status defined as survival to day 90 with a Barthel Index of 95 or more or at least the prestroke value, compared by treatment group. Primary safety variables included symptomatic intracranial hemorrhage and mortality. RESULTS: Favorable functional status was achieved by more patients in the ancrod group (42.2%) than in the placebo group (34.4%; P=.04) by the prespecified covariate-adjusted analysis. Mortality was not different between treatment groups (at 90 days, 25.4% for the ancrod group and 23% for the placebo group; P=.62), and the proportion of severely disabled patients was less in the ancrod group than in the placebo group (11.8% vs 19.8%; P=.01). The favorable functional status observed with ancrod vs placebo was consistent in all subgroups defined for age, stroke severity, sex, prestroke disability, and time to treatment (< or = 3 or > 3 hours after stroke onset). There was a trend toward more symptomatic intracranial hemorrhages in the ancrod group vs placebo (5.2% vs 2.0%; P=.06), as well as a significant increase in asymptomatic intracranial hemorrhages (19.0% vs 10.7%; P=.01). CONCLUSION: In this study, ancrod had a favorable benefit-risk profile for patients with acute ischemic stroke.

Mechanism of STAT3 activation by insulin-like growth factor I receptor.

Recent evidence indicates that STAT proteins can be activated by a variety of receptor and non-receptor protein-tyrosine kinases. Unlike cytokine-induced activation of STATs, where JAKs are known to play a pivotal role in phosphorylating STATs, the mechanism for receptor protein-tyrosine kinase-mediated activation of STATs remains elusive. In this study, we investigated the activation of STAT proteins by the insulin-like growth factor I receptor (IGF-IR) in vitro and in vivo and assessed the role of JAKs in the process of activation. We found that STAT3, but not STAT5, was activated in response to IGF-I in 293T cells cotransfected with IGF-IR and STAT expression vectors. Moreover, tyrosine phosphorylation of STAT3, JAK1, and JAK2 was increased upon IGF-I stimulation of endogenous IGF-IR in 293T cells transfected with the respective STAT or JAK expression vector. Supporting the observation in 293T cells, endogenous STAT3 was tyrosine-phosphorylated upon IGF-I stimulation in the muscle cell line C2C12 as well as in various embryonic and adult mouse organs during different stages of development. Dominant-negative JAK1 or JAK2 was able to block the IGF-IR-mediated tyrosine phosphorylation of STAT3 in 293T cells. A newly identified family of proteins called SOCS (suppressor of cytokine signaling), including SOCS1, SOCS2, SOCS3 and CIS, was able to inhibit the IGF-I-induced STAT3 activation as well with varying degrees of potency, in which SOCS1 and SOCS3 appeared to have the higher inhibitory ability. Inhibition of STAT3 activation by SOCS could be overcome by overexpression of native JAK1 and JAK2. We conclude that IGF-I/IGF-IR is able to mediate activation of STAT3 in vitro and in vivo and that JAKs are essential for the process of activation.