Animal models of orthopoxvirus infection.

Smallpox was one of the most devastating diseases known to humanity. Although smallpox was eradicated through a historically successful vaccination campaign, there is concern in the global community that either Variola virus (VARV), the causative agent of smallpox, or another species of Orthopoxvirus could be used as agents of bioterrorism. Therefore, development of countermeasures to Orthopoxvirus infection is a crucial focus in biodefense research, and these efforts rely on the use of various animal models. Smallpox typically presented as a generalized pustular rash with 30 to 40% mortality, and although smallpox-like syndromes can be induced in cynomolgus macaques with VARV, research with this virus is highly restricted; therefore, animal models with other orthopoxviruses have been investigated. Monkeypox virus causes a generalized vesiculopustular rash in rhesus and cynomolgus macaques and induces fatal systemic disease in several rodent species. Ectromelia virus has been extensively studied in mice as a model of orthopoxviral infection in its natural host. Intranasal inoculation of mice with some strains of vaccinia virus produces fatal bronchopneumonia, as does aerosol or intranasal inoculation of mice with cowpox virus. Rabbitpox virus causes pneumonia and fatal systemic infections in rabbits and can be naturally transmitted between rabbits by an aerosol route similar to that of VARV in humans. No single animal model recapitulates all known aspects of human Orthopoxvirus infections, and each model has its advantages and disadvantages. This article provides a brief review of the Orthopoxvirus diseases of humans and the key pathologic features of animal models of Orthopoxvirus infections.

Is accelerated partner therapy a feasible and acceptable strategy for rapid partner notification in the UK?: a qualitative study of genitourinary medicine clinic attenders.

Partner notification (PN) in the UK is of limited effectiveness. Expedited partner therapy improves PN outcomes but does not comply with existing UK professional guidance. We developed two new strategies, known as accelerated partner therapy (APT), based on elements of PN practice for which there is evidence of efficacy, and which conform to UK prescribing guidance. We explored the acceptability and feasibility of these models qualitatively in genitourinary medicine clinic attenders. Both strategies were viewed favourably. Preference was influenced by age, relationship type, whether participants were delivering or receiving APT and whether the sex partner was aware of the participant's clinic visit. APT provides a new approach to PN, which has strong patient support and complies with existing UK regulations. The complex factors that influence patients' choice of PN method suggest that provision of a range of PN options including APT may be central to improving the effectiveness of PN in the UK.

Members of the Toll-like receptor family of innate immunity pattern-recognition receptors are abundant in the male rat reproductive tract.

Protecting developing and maturing spermatozoa and reproductive tissues from microbial damage is an emerging aspect of research in reproductive physiology. Bacterial, viral, and yeast infections of the testis and epididymis can hinder maturation and movement of spermatozoa, resulting in impaired fertility. Toll-like receptors (TLRs) are a broad family of innate immunity receptors that play critical roles in detecting and responding to invading pathogens. Objectives of this study were to determine if organs of the rat male reproductive tract express mRNAs for members of the TLR family, to characterize expression patterns for TLRs in different regions of the epididymis, and to determine if TLR adaptor and target proteins are present in the male reproductive tract. Messenger RNA for Tlr1-Tlr9 was abundantly expressed in testis, epididymis, and vas deferens, as determined by RT-PCR, while Tlr10 and Tlr11 were less abundantly expressed. Tlr mRNA expression showed no region-specific patterns in the epididymis. Immunoblot analysis revealed relatively equal levels of protein for TLRs 1, 2, 4, and 6 in testis, all regions of the epididymis and vas deferens, and lower levels of TLRs 3, 5, and 9-11. TLR7 was primarily detected in the testis. The TLR adapter proteins, myeloid differentiation primary response gene 88 and TLR adaptor molecule 1, as well as v-rel reticuloendotheliosis viral oncogene homolog and NFKBIA, were prominent in testis, epididymis, and vas deferens. The abundant expression of a majority of TLR family members together with expression of TLR adaptors and activation targets provides strong evidence that TLRs play important roles in innate immunity of the male reproductive tract.

Substrate specificity of isopenicillin N synthase.

Highly purified isopenicillin N synthase (IPNS) from two sources (naturally occurring in Penicillium chrysogenum and that expressed in Escherichia coli via a cloned gene derived from Cephalosporium acremonium) have been isolated and utilized in vitro to test synthetic modifications of the natural substrate, (L-alpha-amino-delta-adipyl)-L-cysteinyl-D-valine (ACV). A very sensitive procedure utilizing the ability of beta-lactams to induce the synthesis of beta-lactamase was employed to determine whether an ACV analogue could serve as a substrate for IPNS. A wide variety of amino and carboxyl terminal tripeptide substitutions were examined and found to elicit positive beta-lactamase induction profiles. However, none of these modifications were found to function as efficiently as a substrate as ACV. One of the beta-lactam products which was formed from the reaction of IPNS and the tripeptide analogue was independently synthesized and evaluated for antibacterial activity. Modification of the L-cysteine residue in the second position of ACV resulted in tripeptides that were unable to serve as substrates. Conversion of the D-valine residue in the third position of ACV to an aromatic amino acid or to a highly electronegative residue such as trifluorovaline resulted in elimination of substrate activity and creation of an inhibitor of the enzyme.

Management of malignant ventricular arrhythmias using antitachycardia devices.

Cardioverter defibrillators were implanted in 26 patients at Charleston Area Medical Center for management of cardiac arrest (7 patients), and drug refractory sustained ventricular tachycardia (19 patients). A variety of operative approaches and concomitant surgical procedures were utilized in the implantation of these devices. No operative deaths occurred. A superficial wound infection was the only operative complication. During the follow-up period (9.3 +/- 5 months), 11 of 26 patients (46 percent) had a defibrillator discharge and one death occurred (3 percent), which was due to heart failure. Patients with malignant ventricular arrhythmias may present with sustained monomorphic ventricular tachycardia with associated syncope, pre-syncope or without any associated symptoms. Unfortunately, cardiac arrest may be the initial presentation. The use of antitachycardia devices such as implantable cardioverter defibrillators and antitachycardia pacemakers has allowed physicians to more successfully treat patients with malignant ventricular arrhythmias. In a significant number of patients with these arrhythmias, such devices are now used as first-line therapy.

Climate change and the evolution of high-latitude terrestrial vegetation and floras.

Our understanding of polar vegetation and climate through time has expanded enormously in the past five years as a consequence of improved logistics, detailed studies of plant fossils in their proper sedimentological context, and the development of sophisticated physiognomic methods for extracting the climate signal present in plant fossil assemblages. These revelations are particularly timely in that climate change is most strongly expressed at the poles, and polar conditions play a critical role in determining global climate. By studying the evolution and change in polar vegetation, valuable insights on possible future biotic responses to global warming can be obtained.

Analysis of the role of cysteine residues in isopenicillin N synthetase activity by site-directed mutagenesis.

The predicted amino acid sequences of isopenicillin N synthetase from both Cephalosporium acremonium and Penicillium chrysogenum have two cysteine residues in analogous positions (Cys-106 and Cys-255 in the C. acremonium numbering). To examine the role of these cysteine residues in the activity of the C. acremonium enzyme, we used site-directed in vitro mutagenesis to change these cysteine residues to serine residues. Mutation of Cys-255 reduces specific activity approximately equal to 50%, whereas mutation of Cys-106 or mutation of both Cys-106 and Cys-255 reduces specific activity about 97%. This suggests that the cysteines are important but not essential for IPNS activity. Alkylation of IPNS also almost completely inactivated the enzyme, but residual activity could have been due to incomplete alkylation. Atomic substitution via genetic manipulation in this case is a more accurate means of assessing the role of sulfhydryl moieties in enzyme activity.

Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium.

The enzyme isopenicillin N synthetase (IPS) catalyses the oxidative condensation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV) to isopenicillin N, which is a central reaction in the pathway to clinically important penicillins and cephalosporins. Here we report the cloning, characterization and expression in Escherichia coli of the gene encoding the IPS protein in Cephalosporium acremonium. The IPS gene was identified by purifying IPS protein, determining the first 23 amino-terminal amino acids, preparing a set of synthetic oligonucleotides encoding a portion of the determined amino-acid sequence, and probing a cosmid genome library with the mixed oligonucleotides. A cosmid hybridizing with the probe was isolated and the IPS gene was localized and sequenced. The IPS gene encodes a polypeptide of relative molecular mass (Mr) 38,416. When this open reading frame was cloned into an E. coli expression vector and inserted into E. coli, the recombinant E. coli produced a new protein co-migrating with authentic IPS as the major protein of the cell (approximately 20% of cell protein). Crude cell extracts condensed LLD-ACV to a penicillinase-sensitive molecule whose antibacterial activity indicated that it was isopenicillin N.

The efficacy of 5-fluorouracil, mitomycin C, and methyl CCNU in advanced gastrointestinal malignancy.

50 patients with advanced gastrointestinal malignancies were treated with 5-fluorouracil (5FU), 1-(2-chloroethyl)3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU) and mitomycin C (Mito C). 43 patients, with diagnoses of colorectal, pancreatic or gastric cancer, were evaluable. 13 patients (30%) achieved complete remissions, and 4 achieved partial remissions. The median durations of responses in colorectal, pancreatic and gastric disease were 11.0, 11.0, and 11.5 months, respectively. Survival was definitely prolonged in responding patients with pancreatic and gastric carcinomas. The combination was well tolerated. Mucositis, leukopenia, and thrombocytopenia were the major toxicities that occurred. The quality of life was improved in all responding patients.

Depletion of total hemolytic complement in sera from hamsters bearing herpes simplex type 2-induced tumors.

Total hemolytic complement (CH50) levels were compared in sera from normal hamsters and hamsters bearing tumors derived from herpes simplex virus type 2-transformed cells CH50 in normal sera ranged from 160 to 212 while CH50 in tumor bearer sera ranged from 82 to 146. Preincubation of tumor bearer sera with cell surface proteins (CSP) from homologous herpes simplex virus type 2-derived tumor cells resulted in a 66% depletion of CH50 whereas preincubation with heterologous herpes simplex virus type 1-derived tumor CSP resulted in a decrease of 26%. The depletion of CH50 appeared to occur via the classic complement pathway. Similar results were seen using CSP from herpes simplex virus-infected cells although overall depletion of CH50 was considerably less than that seen using tumor cell material. Using the complement subcomponent 1q(C1q)-binding test, tumor bearer sera and tumor bearer sera preincubated with homologous CSP were shown to contain increased levels of immune complexes not present in normal serum. These results indicate that the observed depletion of complement activity in sera from tumor-bearing hamsters could be the result of complement pathway activation by antigen-antibody complexes.

Evaluation of psychological stress as an investigative tool.

Involuntary physiologic changes are related to psychological stress, and their analysis has broad applications in psychophysiologic and pharmacologic research as well as other fields. Analyzing patterns of significant stress by means of Psychological Stress Evaluation testing provides an accurate assessment of a person's psychological set in a controlled situation.