Monkeypox virus detection in rodents using real-time 3'-minor groove binder TaqMan assays on the Roche LightCycler

During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture. The rodent tissue samples were appropriately processed and then tested by using an integrated approach involving real-time polymerase chain reaction (PCR) assays, an antigen-detection immunoassay, and virus culture. We designed and extensively tested two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan-orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan-orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. In addition, we attempted to verify the presence of monkeypox virus by culture on multiple cell lines, by immunohistology, and by electron microscopy, with negative results. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses. In light of the recent monkeypox virus transmissions, early detection of the virus is crucial for both natural outbreaks and potential acts of bioterrorism.     

Coping with dental treatment: The potential impact of situational demands

Coping strategies and anxiety responding of dental patients were studied in order to test the generalizability of previous findings based on volunteer blood donors. State and trait coping measures were administered once, and a process coping scale was administered at three points throughout treatment. Self-report, behavioral observation, and psychophysiological measures of anxiety were sampled for the same periods as process coping. Findings included the replication of a negative relationship between avoidant coping and patient anxiety ratings. Fluctuations in coping were evident across periods, and impact of situational demands and constraints was introduced as an explanation for these variations. A method for direct assessment of coping consistency was introduced. On the basis of the replicable associations with anxiety measures, the ability to detect changes in coping within a situation, and the ability to provide direct evidence of coping consistency, the use of process methodology for coping assessment is encouraged.This research was conducted while the first author was supported by funding from the Medical Research Council of Canada.Portions of this research were presented at the annual convention of the Society of Behavioral Medicine, Philadelphia, 1984.     

Postmenopausal estrogen use, cigarette smoking, and cardiovascular morbidity in women over 50. The Framingham Study

We studied the effect of estrogen use on morbidity from cardiovascular disease in 1234 postmenopausal women, aged 50 to 83 years, participating in the Framingham Heart Study's 12th biennial examination (index examination). The medication history recorded at biennial examinations 8 through 12 was used to classify the degree of estrogen exposure before eight years of observation for cardiovascular morbidity and mortality. Despite a favorable cardiovascular risk profile and control for the major known risk factors for heart disease, women reporting postmenopausal estrogen use at one or more examinations had over a 50 per cent elevated risk of cardiovascular morbidity (P less than 0.01) and more than a twofold risk for cerebrovascular disease (P less than 0.01) after the index examination. Increased rates for myocardial infarction (P less than 0.05) were observed particularly among the estrogen users who smoked cigarettes. Conversely, among nonsmokers estrogen use was associated only with an increased incidence of stroke (P less than 0.05). No benefits from estrogen use were observed in the study group; in particular, mortality from all causes and from cardiovascular disease did not differ for estrogen users and nonusers.     

Murine model of pulmonary anthrax: kinetics of dissemination, histopathology, and mouse strain susceptibility

Bioweapons are most often designed for delivery to the lung, although this route is not the usual portal of entry for many of the pathogens in the natural environment. Vaccines and therapeutics that are efficacious for natural routes of infection may not be effective against the pulmonary route. Pulmonary models are needed to investigate the importance of specific bacterial genes in virulence, to identify components of the host immune system that are important in providing innate and acquired protection, and for testing diagnostic and therapeutic strategies. This report describes the characteristics of host and Bacillus anthracis interactions in a murine pulmonary-infection model. The infective dose varied depending on the route and method of inoculation. The germination process in the lung began within 1 h of inoculation into the lung, although growth within the lung was limited. B. anthracis was found in the lung-associated lymph nodes approximately 5 h after infection. Minimal pneumonitis was associated with the lung infection, but significant systemic pathology was noted after dissemination. Infected mice typically succumbed to infection approximately 3 to 4 days after inoculation. The 50% lethal doses differed among inbred strains of mice, but within a given mouse strain, neither the age nor the sex of the mice influenced susceptibility to B. anthracis.     

Ultrastructural Changes Suggestive of Focal Segmental Glomerulosclerosis in Atypical Minimal Change Nephrotic Syndrome

In an attempt to recognize early stages of focal segmental glomerulosclerosis (FSGS) in patients with a clinical course suggesting a diagnosis other than minimal change disease (MCD) and normal histology, or minor, nondiagnostic changes on light microscopy (LM), we used a protocol for systematic and extensive electron microscopy (EM) examination of kidney biopsies obtained from such patients. By this method ultrastructural pathology was found in 8 patients. These changes were localized, involving only portions of single glomerular segments. The findings included mild to moderate increase of the mesangial matrix, focal wrinkling of the capillary basement membrane, and early obliteration of the normal architecture of individual capillary loops, as well as electron-dense deposits in a mesangial and subendothelial distribution. Of these 8 patients, 2 are at present in remission without therapy (in 1, following therapy with cyclophosphamide); 3 are in remission on steroid therapy; 1 developed massive proteinuria during pregnancy, after a spontaneous remission lasting almost 2 years; 1 patient advanced to terminal renal failure 3 1/2 years after biopsy; and 1 died of sepsis 1 month after biopsy. We believe that the ultrastructural changes found may represent early or mild FSGS and that the protocol described can add valuable information in clinically worrisome patients in whom renal histology appears normal.     

Discriminating adaptive attributions: Agreement between self-report and objective ratings

According to the reformulated learned helplessness model of depression, causal attributions are an important mediator of the effects on mood of positive and negative experiences. Adaptive attributions for negative events are assumed to be external, unstable, and specific. In the present study, subjects exposed to one of two attribution training procedures or a control condition made attributions for hypothetical events under neutral and adaptive instructional sets. Attributions were rated by subjects and coders blind to the purpose of the study. Results indicated that subjects' views of adaptive causal attributions were congruent with predictions from the learned helplessness model. The ratings of the objective coders indicated that subjects' attributions really did change in response to the adaptive instructions in the predicted direction. Implications of these results for the reformulated learned helplessness model and depression therapies that include an attribution retraining component are discussed.The authors would like to thank Dan Russell for his very helpful comments on earlier drafts of this paper.     

Nonlinear Binding of Valproic Acid (VPA) and E-Δ2-Valproic Acid to Rat Plasma Proteins

The binding characteristics of valproic acid (VPA) and its pharmacologically active monounsaturated metabolite, E-²-VPA, to rat plasma proteins were compared. The plasma free fraction was determined by a rapid equilibrium procedure, which minimizes the interfering effects of nonesterified fatty acids liberated by in vitro lipolysis. Nonlinear binding behavior was observed with both compounds over their respective pharmacologic concentration range. Multiple binding-site models were invoked to explain the binding isotherm. The 2-unsaturated compound has a much higher affinity for the rat plasma proteins (mainly albumin) than its saturated precursor. The equilibrium association constants for the high- and intermediate-affinity sites were more than an order of magnitude higher with E-²-VPA than with VPA (10⁴–10⁶ versus 10³ M ^–1). This difference in binding affinity was also reflected by a lower plasma free fraction for E-²-VPA compared with VPA (<<10 versus >20% at total concentrations of less than 100 µg/ml). A more pronounced dose- and concentration-dependent variation in the distribution and clearance kinetics is predicted for the 2-unsaturated analogue compared to VPA. Also, the structural dependency in plasma protein binding observed with these branched-chain fatty acids may provide insights into the mechanism of interaction between fatty acyl molecules and albumin.     

Regioselectivity and Enantioselectivity of Metoprolol Oxidation by Two Variants of cDNA-Expressed P4502D6

Purpose. The oxidative metabolism of metoprolol was investigated in two human lymphoblastoma cell-lines transfected with variants of cDNA for cytochrome P4502D6. Methods. The regioselective and enantioselective features of the oxidations of deuterium-labeled pseudoracemic metoprolol were characterized by GC/MS analysis of the substrate and products. Results. There were significant differences between the two P4502D6 variants in the formation kinetics of O-demethylmetoprolol and -hydroxymetoprolol. The h2D6-Val microsomes highly favored the formation of the O-demethylmetoprolol regioisomer 6.3:1 and 2.8:1, respectively from (R)-metoprolol-d₀ and (S)-metoprolol-d₂, while the corresponding ratios for h2D6v2 microsomes were much lower. For both variants, O-demethylmetoprolol formation favored the (R)-substrate 1.5 to 2-fold, while -hydroxymetoprolol formation was non-enantioselective. Similar Km values of metoprolol oxidation, 10-20 µM, were observed for the two microsomal preparations. Conclusions. The regioselectivity, enantioselectivity, and Km values for the h2D6-Val microsomes resemble those observed for the native P4502D6 in human liver microsomes, whereas the h2D6v2 microsomes deviated remarkably in regioselectivity.     

Complement activation mediates intestinal injury after resuscitation from hemorrhagic shock

BACKGROUND: Endothelial cell injury after hemorrhage and resuscitation (HEM/RES) might contribute to intestinal hypoperfusion and mucosal ischemia. Our recent work suggests that the injury might be the result of complement activation. We hypothesized that HEM/RES causes complement-mediated endothelial cell dysfunction in the small intestine. METHODS: Male Sprague-Dawley rats (195-230 g) were anesthetized and HEM to 50% of baseline mean arterial pressure for 60 minutes. Just before RES, animals received either soluble complement receptor-1 (sCR1, 15 mg/kg) to inhibit complement activation or saline vehicle. Resuscitation was with shed blood and an equal volume of saline. Two hours after RES, the small bowel was harvested to evaluate intestinal nitric oxide synthase activity (NOS), neutrophil influx, histology, and oxidant injury. RESULTS: HEM/RES induced tissue injury, increased neutrophil influx, and reduced NOS activity by 50% (vs. SHAM), all of which were completely prevented by sCR1 administration. There were no observed differences in oxidant injury between the groups. CONCLUSION: Histologic tissue injury, increased neutrophil influx, and impaired NOS activity after HEM/RES were all prevented by complement inhibition. Direct oxidant injury did not seem to be a major contributor to these alterations. Complement inhibition after HEM might ameliorate reperfusion injury in the small intestine by protecting the endothelial cell, reducing neutrophil influx and preserving NOS function.     

Interferon-β inhibits proliferation and progression through S phase of the cell cycle in five glioma cell lines

Summary The growth inhibitory effect of IFN- was evaluated in 5 human glioma cell lines (AO2V4, GJC, GJR, NN and NNR) and in normal astrocyte cultures (SC and TM). All 5 glioma cell lines showed an anti-proliferative response to IFN- whereas normal glial cells were non-responsive. IFN- at 10, 100 and 500 U/ml lead to a 30%,70% and 80% relative decrease in cell number after 12 days, respectively in AO2V4 cells. GJC and GJR cell lines also responded significantly to the lowest concentration of IFN- tested and at 500 U/ml the relative cell number decreased 55%. The NN and NNR cells were the least responsive to IFN- with maximum growth inhibition of 30% at 500 U IFN-/ml. Following treatment with IFN-, AO2V4, GJC, GJR and normal astrocytes all expressed mRNA encoding the anti-viral protein, 2-5A synthetase demonstrating that IFN- bound to receptors on all four cell lines and activated signal transduction pathways required for induction of an anti-viral protein. A determination of the relative number of viable cells showed that none of these cells exhibited a significant decrease in cell viability. Since the antiproliferative response to IFN- was not primarily due to cell death, the effect of IFN- on cell cycle progression was evaluated by flow cytometry. All treated glioma cell lines showed a relative increase in proportion of cells in S phase. AO2V4 cells had a 50%–80% increase in the percentage of cells in S phase, whereas GJC, GJR and NNR had percentage increases of 20%–40%. IFN- treatment of normal astrocytes did not significantly alter their cell cycle profile. These data suggest that IFN- exerts its antiproliferative effect on glioma cells by arresting the ordered progression through S phase or decreasing entry into G₂/M phase of the cell cycle.