The soluble ectodomain of herpes simplex virus gD contains a membrane-proximal pro-fusion domain and suffices to mediate virus entry

Entry of herpes simplex virus (HSV) 1 into cells requires the interaction of HSV gD with herpesvirus entry mediator or nectin1 receptors, and fusion with cell membrane mediated by the fusion glycoproteins gB, gH, and gL. We report that the gD ectodomain in soluble form (amino acids 1-305) was sufficient to rescue the infectivity of a gD-null HSV mutant, indicating that gD does not need to be anchored to the virion envelope to mediate entry. Entry mediated by soluble gD required, in addition to the receptor-binding sites contained within residues 1-250, a discrete downstream portion (amino acids 261-305), located proximal to the transmembrane segment in full-length gD. We named it as profusion domain. The pro-fusion domain was required for entry mediated by virion-bound gD, because its substitution with the corresponding region of CD8 failed to complement the infectivity of gD(-/+) HSV. Furthermore, a receptor-negative gD (gD(Delta6-259)) inhibited virus infectivity when coexpressed with wild-type gD; i.e., it acted as a dominant-negative gD mutant. The pro-fusion domain is proline-rich, which is characteristic of regions involved in protein-protein interactions. P291L-P292A substitutions diminished the gD capacity to complement gD(-/+) HSV infectivity. We propose that gD forms a tripartite complex with its receptor and, by way of the proline-rich pro-fusion domain, with the fusion glycoproteins, or with one of them. The tripartite complex would serve to recruit/activate the fusion glycoproteins and bring them from a fusion-inactive to a fusion-active state, such that they execute fusion of the virion envelope with cell membrane.     

Effects of insulin-sensitising agents in mice with hepatic insulin resistance

Aims/hypothesis The metabolic abnormalities of insulin resistance are ameliorated by insulin sensitisers via different mechanisms. Metformin decreases hepatic glucose output, whereas rosiglitazone (RSG) is an agonist for peroxisome proliferator activated receptor (PPAR), highly expressed in fat. To gain insight into the mechanisms of action of these drugs, we compared their actions in two models of insulin resistance: the obese, hyperglycaemic ob/ob mouse and the liver specific insulin receptor knockout (LIRKO) mouse.Methods Control, ob/ob, and LIRKO mice were divided into three groups that received metformin (300 mg/kg body weight/day), RSG (3 mg/kg body weight/day), or placebo for 3 weeks.Results In the presence of the severe hepatic insulin resistance of the LIRKO mouse, neither metformin nor RSG had any significant effect on glucose or insulin tolerance tests. On the other hand, RSG decreased serum concentrations of total cholesterol, LDL, and HDL in LIRKO mice. Adipocyte PPAR gene and protein expression, and adipocyte size were all increased in LIRKO mice treated with RSG, whereas fat-cell size in control animals was decreased by RSG.Conclusion/interpretation TZDs probably improve some lipid parameters of the dysmetabolic syndrome associated with diabetes mellitus even in the presence of absolute hepatic insulin resistance, but both metformin and TZDs require an operating insulin signalling system in the liver for their effects in glucose homeostasis.Abbreviations TZD Thiazolidinedione - RSG rosiglitazone - LIRKO liver specific insulin receptor knockout     

Epidemiology of Post-Operative Venous Thromboembolismin Asian Countries

Background In Asia, the prevalence of post-operative venous thromboembolism (VTE) is traditionally thought to be low and the routine use of thromboprophylaxis remains controversial.Methods We performed an exhaustive literature search for published studies on VTE in Asia. Predefined data were extracted from individual studies: country involved, number of patients, type of patient population, type, duration and dose regimens of treatments, if any, method used to detect deep-vein thrombosis (DVT) and pulmonary embolism (PE), and duration of follow-up. The main endpoints were the incidences of systematically detected DVT, and symptomatic DVT or PE. Overall adjusted percentages and 95% confidence intervals (CI) were calculated.Results In clinical studies in patients not receiving thromboprophylaxis, the adjusted incidence of total DVT was 13% (95% CI: 10% to 16%) in general surgery, 16% (95% CI: 13% to 20%) after total hip replacement, 50% (95% CI: 44% to 55%) after total knee replacement and 18% (95% CI: 12% to 24%) in hip fracture surgery. The adjusted incidence of PE was 1% (95% CI: 0% to 2%) in general surgery and 1.4% (95% CI: 1% to 3%) after total hip replacement. In autopsy studies, the incidence of fatal PE ranged from 0.2% to 6.0%, increasing consistently over a period of 30 years in Japan and Hong Kong.Conclusions Post-operative VTE is frequent in Asian general and orthopedic surgery patients and the incidence of autopsy-proven fatal PE is increasing over time. The use of routine prophylaxis in Asian patients undergoing high-risk surgical procedures should be considered.Presented in part at the XVIII Congress of The International Society on Thrombosis and Haemostasis, Paris, France (July 6-12, 2001)*SMART: Surgical Multinational Asian Registry in Thrombosis     

Central role of tumour necrosis factor, GM-CSF, and interleukin 1 in the pathogenesis of juvenile chronic myelogenous leukaemia

In previous studies on patients with juvenile chronic myelogenous leukaemia (JCML), we found excessive proliferation of malignant monocyte-macrophage elements in the absence of exogenous growth factor, and impaired growth of normal haematopoietic progenitors. In the current study, six newly-diagnosed JCML patients were investigated to characterize the disease further. In co-cultures, JCML cell culture supernatant as well as patient plasma obtained at diagnosis produced a striking reduction in numbers of control marrow BFU-E, CFU-GM, CFU-Meg and CFU-GEMM colonies. Monoclonal anti-tumour necrosis factor alpha neutralizing antibodies (anti-TNF-alpha Ab) abolished these inhibitory properties. In sharp contrast, JCML supernatants exerted a marked growth-promoting effect on autologous JCML cells cultured in clonogenic assays. Anti-TNF-alpha Ab and anti-granulocyte-macrophage colony-stimulating factor neutralizing antibodies (anti-GM-CSF Ab) both reversed the stimulating effect. Recombinant GM-CSF and recombinant TNF alpha produced a profound increase in JCML colonies when tested individually and anti-GM-CSF Ab reversed the TNF-alpha effect. Expression studies of TNF-alpha and TNF-alpha receptor genes of cultured JCML cells demonstrated mRNAs for both. Further, TNF-alpha activity was assayed in a wide variety of cell culture supernatants and in normal and patients' plasma, and only the JCML specimens showed increased TNF-alpha values. Recombinant interleukin-1 alpha (IL-1 alpha) also stimulated JCML colony growth, but polyclonal anti-IL-1 neutralizing antibodies did not suppress JCML colony numbers nor did it reverse the effects of TNF-alpha or GM-CSF. The evidence indicated that the JCML monokine which inhibits normal haematopoiesis is TNF-alpha and that the endogenously-produced TNF-alpha and GM-CSF from JCML cells play an important role in the pathogenesis of the disease by acting as autocrine growth factors. IL-1 alpha also stimulates JCML cell proliferation as an accessory factor and augments the effect of GM-CSF, TNF-alpha or both.     

Clinical investigation of human alpha interferon in chronic myelogenous leukemia

Fifty-one patients with previously untreated or minimally treated chronic myelogenous leukemia in chronic phase received human alpha interferon 3 to 9 X 10(6) units intramuscularly (IM) daily until complete hematologic remission, then at doses ranging from 3 X 10(6) units every other day to 9 X 10(6) units daily. Forty-one (80%) patients achieved a hematologic response, 36 (71%) of them attaining a complete hematologic remission with normal peripheral WBC and differential counts. Responding patients showed continuous but slow normalization of several other blood and marrow parameters including platelet counts, serum lactic dehydrogenase and B12 levels, and marrow cellularity and maturation index. Suppression of the Philadelphia chromosome on serial cytogenetic studies of marrow metaphases was documented in 20 of the 36 patients who achieved complete hematologic remission (56%; 39% of total group), eight of whom (22%) had a decrease of the Philadelphia chromosome-positive metaphases to less than 35%. These changes were persistent for 6 months or longer in 18 patients, seven of whom had continuous suppression of the Philadelphia chromosome to less than 90% for a median of 30+ months (range 21+ to 39+ months). After a median follow-up period of 37 months, 25 patients remain in continued disease control with interferon therapy. The projected 3-year survival rate is 76%, with a yearly death rate of 6%, 9%, and 9% in the first 3 years. Response, Philadelphia chromosome suppression, and survival were significantly better among patients in the low-risk category compared to intermediate- and high-risk categories, as defined by a multivariate analysis-derived prognostic model. The projected 3- year survival rate was 94% for patients who achieved a complete hematologic remission on interferon therapy and 45% for those who did not. Thirteen patients have developed blastic crisis, six with lymphoid and three with undifferentiated morphology. We conclude that human leukocyte alpha interferon effectively controls chronic myeloid leukemia and allows reappearance of diploid hemopoietic cells in some patients.     

Phenotypic and functional characterization of T-BAM (CD40 ligand)+ T- cell non-Hodgkin's lymphoma

The precise mechanisms regulating T-helper function have been intensively investigated. We and others have recently identified a new T-cell-B-cell-activating molecule called T-BAM that directs B-cell differentiation by interacting with the CD40 molecule on B cells. Using a specific monoclonal antibody against T-BAM (5C8), we have previously shown that T-BAM expressing T cells are predominantly CD4+CD8- and in normal lymphoid tissue have a unique distribution. However, no information has been obtained regarding the phenotype and functional properties of human neoplastic T cells. Therefore, we investigated T- BAM expression immunohistochemically in 87 well-characterized T-cell non-Hodgkin's lymphomas and lymphoid leukemias (LL). We found that 21/81 neoplasms expressed detectable T-BAM and these positive tumors belong almost exclusively to the CD4+CD8- subtype. In addition, to determine whether T-BAM expression could be induced on T-BAM-LL cells, we activated T-BAM-LLs in vitro and showed that T-BAM could be upregulated only in CD4+CD8- tumors. Our studies clearly show that T- BAM is constitutively expressed in a large number of T-cell neoplasms with a relative mature phenotype (CD4+CD8-) and that only CD4+ neoplastic T cells can be induced in vitro to express this molecule. Additional studies are necessary to identify the biologic significance of T-BAM expression and its potential and clinical implications.     

Inhibition and complexation of activated protein C by two major inhibitors in plasma

To determine the major physiologic inhibitors of activated protein C (APC), plasma was incubated with APC or with Protac C and subjected to immunoblotting. APC:inhibitor complexes gave two major bands reacting with antiprotein C antibodies when immunoblotted on nondenaturing gels, and additional minor bands that varied between serum and plasma. Formation of one of the two major bands of APC:inhibitor complex, but not the other, was stimulated by heparin and only this band reacted with antibodies to the previously described APC inhibitor that is here designated PCI-1. Plasma immunodepleted of PCI-1 formed complexes with APC as visualized with antiprotein C but not anti-PCI-1 antibodies, and exhibited heparin-independent inhibition of APC activity, providing evidence for the existence of a second major physiologic APC inhibitor, PCI-2. Formation of APC:PCI-2 complexes in PCI-1-depleted plasma paralleled inhibition of APC amidolytic activity. PCI-2 was separated from PCI-1 and partially purified using column chromatography. PCI-2 formed inactive complexes of approximately 110,000 molecular weight (mol wt) with APC suggesting PCI-2 has an approximate mol wt of 50,000. Thus, inhibition of APC in plasma involves two major distinct 50,000 mol wt inhibitors, the heparin-dependent PCI-1 and the heparin- independent PCI-2.     

Epstein-Barr virus nuclear protein 2 is a key determinant of lymphocyte transformation.

Epstein-Barr virus (EBV) efficiently transforms B lymphocytes to perpetual proliferation. The EBV laboratory strain P3HR-1 is transformation-incompetent and lacks a DNA segment that includes the EBV nuclear antigen 2 (EBNA-2) gene and a portion of the EBNA leader protein (EBNA-LP) gene. These two genes are expressed in transformed B lymphocytes. Recombinant transformation-competent EBVs were produced by transfecting P3HR-1-infected cells with a cosmid containing the DNA deleted in P3HR-1. Deletion of 105 nucleotides from the middle of the EBNA-2 gene had no discernible affect on transformation. Two larger EBNA-2 deletions abolished transformation but did not affect EBNA-2 nuclear localization. Two naturally occurring EBV variants (EBV types 1 and 2) differ extensively in their growth-transformation phenotype and in their EBNA-LP, EBNA-2, and EBNA-3A, -3B, and -3C genes. Recombinant P3HR-1 carrying EBV-1 EBNA-2 has many of the EBV-1 in vitro growth-transforming effects; recombinant P3HR-1, isogenic except for EBV-2 EBNA-2, has many of the EBV-2 growth-transforming effects including slow emergence of transformants, growth in tight clumps with few surrounding viable cells, and early sensitivity to dilution with fresh medium. Thus, EBNA-2 is an essential molecule in lymphocyte growth transformation by EBV and a major determinant of the differences between EBV-1 and EBV-2 in lymphocyte growth transformation.     

Diabetes association with self-reported health,resource utilization,and prognosis post-myocardial infarction

BackgroundDiabetes mellitus (DM) is associated with increased cardiovascular (CV) risk. We compared health‐related quality of life (HRQoL), healthcare resource utilization (HRU), and clinical outcomes of stable post‐myocardial infarction (MI) patients with and without DM.HypothesisIn post‐MI patients, DM is associated with worse HRQoL, increased HRU, and worse clinical outcomes.MethodsThe prospective, observational long‐term risk, clinical management, and healthcare Resource utilization of stable coronary artery disease study obtained data from 8968 patients aged ≥50 years 1 to 3 years post‐MI (369 centers; 25 countries). Patients with ≥1 of the following risk factors were included: age ≥65 years, history of a second MI >1 year before enrollment, multivessel coronary artery disease, creatinine clearance ≥15 and <60 mL/min, and DM treated with medication. Self‐reported health status was assessed at baseline, 1 and 2 years and converted to EQ‐5D scores. The main outcome measures were baseline HRQoL and HRU during follow‐up.ResultsDM at enrollment was 33% (2959 patients, 869 insulin treated). Mean baseline EQ‐5D score (0.86 vs 0.82; P < .0001) was higher; mean number of hospitalizations (0.38 vs 0.50, P < .0001) and mean length of stay (LoS; 9.3 vs 11.5; P = .001) were lower in patients without vs with DM. All‐cause death and the composite of CV death, MI, and stroke were significantly higher in DM patients, with adjusted 2‐year rate ratios of 1.43 (P < .01) and 1.55 (P < .001), respectively.ConclusionsStable post‐MI patients with DM (especially insulin treated) had poorer EQ‐5D scores, higher hospitalization rates and LoS, and worse clinical outcomes vs those without DM. Strategies focusing specifically on this high‐risk population should be developed to improve outcomes.Trial registration ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT01866904","term_id":"NCT01866904"}}NCT01866904 (https://clinicaltrials.gov).