Plasmid DNA encoding caprine interferon gamma inhibits antibody response to caprine arthritis-encephalitis virus (CAEV) surface protein encoded by a co-administered plasmid expressing CAEV env and tat genes.

This study characterized immune responses of Saanen goats co-immunized with pUC18 based plasmids expressing caprine arthritis-encephalitis virus (CAEV) rev-env (pENV) or tat-rev-env (pTAT-ENV) and a second plasmid encoding caprine interferon gamma (IFNgamma). A previous study reported that immunization with pENV or pTAT-ENV induces IgG2 biased antibody responses to plasmid encoded CAEV surface envelope protein (SU). We show here that regional lymph nodes (LN) of pENV and pTAT-ENV immunized goats contain a dominant subset of SU activated IFNgamma+ Th1 lymphocytes. Co-immunization with pENV or pTAT-ENV and a caprine IFNgamma cDNA expression plasmid (pcIFNgamma) did not potentiate activation of SU responsive Th1 lymphocytes. However, the antibody response to SU encoded by pTAT-ENV was inhibited by co-immunization with pcIFNgamma. Results indicate that synergistic effects of CAEV Tat and IFNgamma suppress the primary adaptive B cell response to plasmid encoded SU.

Characterization of caprine interleukin-4.

Caprine interleukin-4 (IL-4) cDNA was cloned from RNA of mitogen stimulated goat peripheral blood mononuclear cells utilizing reverse transcriptase-polymerase chain reaction. The sequence of caprine IL-4 cDNA corresponds to a 535 nucleotide mRNA with 5'- and 3'-untranslated regions and a 405 nucleotide open reading frame, the first 66 nucleotides of which encode a putative signal peptide. Mature IL-4 is a 12.8kDa protein containing six cysteine residues and two potential N-linked glycosylation sites and is highly homologous with other ruminant IL-4. The predicted molecular mass of mature unglycosylated IL-4 was confirmed by western blot of recombinant caprine IL-4 expressed in bacteria with a monoclonal antibody against a carboxyterminal peptide derived from the predicted amino acid sequence of bovine IL-4. Eukaryotic expression plasmids containing caprine IL-4 cDNA were used to characterize recombinant IL-4. Transcription of IL-4 mRNA was confirmed by transfection of COS-7 and goat synovial membrane cells, and recombinant IL-4 produced by stably transfected L929 cells inhibited inducible nitric oxide synthase in macrophages. Genetic immunization of mice with a caprine IL-4 cDNA expression plasmid induced antibodies against recombinant caprine IL-4 produced in bacteria.

Immunization with plasmid DNA expressing the caprine arthritis-encephalitis virus envelope gene: quantitative and qualitative aspects of antibody response to viral surface glycoprotein.

Saanen goats were vaccinated intradermally with plasmid DNA expressing caprine arthritis-encephalitis virus (CAEV) rev-env (pENV) or tat-rev-env (pTAT-ENV) or vaccinia virus expressing CAEV env (rWR-63). Sera from all vaccinated goats immunoprecipitated CAEV surface (SU) and transmembrane (TM) glycoproteins with a dominant response to SU. Antibody response to CAEV SU induced by plasmid DNA was relatively biased toward IgG2, whereas vaccinia rWR-63 induced predominantly IgG1 antibodies to SU. Differential IgG isotype bias established by immunization with plasmid or vaccinia vectors was maintained following subcutaneous boost with purified CAEV SU in Freund's incomplete adjuvant (FIA). Goats injected with pUC18 control plasmid followed by immunization with SU-FIA also had IgG2 biased responses, whereas SU-FIA immunization of a goat primed with vaccinia rWR-SC11 without the CAEV env gene induced a predominant IgG1 response. We conclude that pUC based plasmids expressing the CAEV env gene promote stable type 1 biased immune responses to plasmid encoded SU. IgG2 biased response may be due to innate type 1 priming capacity of immunostimulatory CpG motifs in the pUC ampicillin resistance gene.

Immune response to caprine arthritis-encephalitis virus surface protein induced by coimmunization with recombinant vaccinia viruses expressing the caprine arthritis-encephalitis virus envelope gene and caprine interleukin-12.

The objective of this study was to determine if interleukin (IL)-12 can focus an antigen specific type 1 immune response characterized by activation of Th1 lymphocytes and production of IgG2 antibodies in vivo. Saanen goats co-immunized with recombinant vaccinia viruses expressing caprine IL-12 (rRB-IL12) and the caprine arthritis-encephalitis virus (CAEV) envelope (env) gene (rWR-63) were evaluated for development of immune responses to the CAEV env encoded surface glycoprotein (SU). Immune responses were defined by: (i) SU antibody titers; (ii) the ratio of SU IgG1 and IgG2 antibodies; (iii) interferon gamma (IFNgamma) and IL-4 gene expression and proliferative response of SU stimulated lymph node mononuclear cells (LNMC). Apart from enhancement of IFNgamma and IL-4 gene expression in SU stimulated LNMC, rRB-IL12 did not affect the immune response to rWR-63 encoded SU. Thus, localized production of exogenous species specific IL-12 at the site of immunization did not focus initial priming of antigen reactive Th lymphocytes. These results are in contrast to previous studies using inbred mice and raise questions regarding the use of cytokine adjuvants to focus immune responses in outbred animals.

Serologic and molecular characterization of an abortigenic strain of equine arteritis virus isolated from infective frozen semen and an aborted equine fetus.

A virus isolated from an aborted equine fetus was determined to be antigenically distinct from several other strains of equine arteritis virus (EAV) by use of a neutralization assay with a large panel of neutralizing monoclonal antibodies. The virus was readily neutralized by polyclonal equine anti-EAV serum. Comparative nucleotide and amino acid sequence analyses indicated that the virus (WA97) isolated from the aborted fetus was virtually identical to a virus (S1971) isolated from imported semen used to inseminate another mare on the farm. Phylogenetic analysis indicated that the WA97/S1971 virus was more related to European than to North American strains of EAV. These sensitive molecular procedures may be useful for epidemiologic investigations of EAV infections. Screening and certification of stallions and frozen equine semen would prevent dissemination of pathogenic strains of EAV.

Non-thermoregulatory shivering in patients recovering from isoflurane or desflurane anesthesia.

BACKGROUND: Although cold-induced shivering is an obvious source of postanesthetic tremor, other causes may contribute. Consistent with this theory, the authors had previously identified an abnormal clonic component of postoperative shivering and proposed that it might be nonthermoregulatory. A subsequent study, however, failed to identify spontaneous muscular activity in normothermic volunteers. These data suggested that the initial theory was erroneous or that a yet-to-be identified factor associated with surgery might facilitate shivering in patients after operation. Therefore, the authors tested the hypothesis that some postoperative tremor is nonthermoregulatory. METHODS: One hundred twenty patients undergoing major orthopedic operation were observed. They were grouped randomly to receive maintenance anesthesia with nitrous oxide and isoflurane (0.8 +/- 0.4%) or desflurane (3.4 +/- 1.1%). Twenty patients in each group were allowed to become hypothermic, whereas normal body temperatures were maintained in the others (tympanic membrane temperature exceeding preinduction values). Arteriovenous shunt vasoconstriction was evaluated using forearm-minus-fingertip skin-temperature gradients; gradients less than 0 degrees C identified vasodilation. Postanesthetic shivering was graded by a blinded investigator. Tremor in patients who were normothermic and vasodilated was considered nonthermoregulatory. RESULTS: Thermoregulatory responses were similar after isoflurane or desflurane anesthesia. Approximately 50% of the unwarmed patients shivered. Shivering was observed in 27% of the patients who were normothermic; 55% of this spontaneous muscular activity occurred in vasodilated patients. Among the normothermic patients, 15% fulfilled the authors' criteria for nonthermoregulatory tremor. CONCLUSIONS: The incidence of postoperative shivering is inversely related to core temperature. Therefore, it was not surprising that shivering was most common among the hypothermic patients. The major findings, however, were that shivering remained common even among patients who were kept scrupulously normothermic and that many shivered while they were vasodilated. Thus, postoperative patients differ from nonsurgical volunteers in demonstrating a substantial incidence of nonthermoregulatory tremor.

Cloning and expression of caprine interferon-gamma.

Caprine interferon-gamma (IFN-gamma) cDNA was cloned from mitogen stimulated peripheral blood mononuclear cell (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The cDNA open reading frame (ORF) is 498bp, encoding a putative 166 amino acid (aa) protein (19327Da). The predicted aa sequence homology of caprine IFN-gamma and the corresponding ovine, bovine and cervine cytokine is 98.8%, 95.2% and 92.8%, respectively. IFN-gamma cDNA was subcloned and expressed in two different plasmids under the control of either the human cytomegalovirus (CMV) immediate early promoter or the caprine arthritis-encephalitis virus long terminal repeat (CAEV LTR). Recombinant caprine IFN-gamma (rCaIFN-gamma) secreted by transfected COS-7 cells shared at least two antigenic epitopes with recombinant bovine IFN-gamma (rBoIFN-gamma) and exhibited biological activity in the vesicular stomatitis virus (VSV) cytopathic effect reduction assay. In-vivo expression of IFN-gamma cDNA promoted by the CAEV LTR was confirmed by the intramuscular (IM) injection of Balb/C mice with plasmid followed by Western blot analysis of mouse serum against purified rCaIFN-gamma produced in E. coli.

Type 1 and type 2 cytokine gene expression by viral gp135 surface protein-activated T lymphocytes in caprine arthritis-encephalitis lentivirus infection.

Peripheral blood mononuclear cells (PBMC) from Saanen goats experimentally infected with the lentivirus caprine arthritis-encephalitis virus (CAEV) were evaluated by semiquantitative reverse transcriptase PCR for gamma interferon (IFN-gamma), interleukin-4 (IL-4), and IL-2 gene expression following in vitro stimulation with purified CAEV gp135 surface protein (SU). Studies examined three goats with chronic arthritis and four clinically asymptomatic goats at 5 years postinfection. SU-responsive IFN-gamma mRNA-positive cells and IL-4 mRNA-positive cells in PBMC from infected goats reflected differences in lymphokine balance associated with disease status. IFN-gamma mRNA-positive cells were dominant in PBMC from asymptomatic goats, whereas SU-responsive IL-4 mRNA-positive cells were dominant in PBMC from goats with arthritis. IL-2 gene expression was not responsive to SU stimulation of PBMC from either asymptomatic or arthritic goats. Lymphokine mRNA profiles in SU-stimulated PBMC were dependent on the presence of CD4+ T lymphocytes. The results indicate that asymptomatic goats have a dominant population of CAEV SU-reactive T-helper 1 (Th1)-like lymphocytes in PBMC whereas goats with clinical arthritis have a dominant population of SU-reactive Th2-like lymphocytes.

Prouterina wescotti n. gen., n. sp. (Trematoda: Prouterinidae N. Fam.) from the brain, lungs, and nasal sinuses of a black bear (Ursus americanus) from Idaho.

Prouterina wescotti gen. n. and sp. n. (Trematoda: Prouterinidae N. Fam.) is described from a free-ranging black bear (Ursus americanus) which died in May 1995 in northern Idaho (USA). Adult digenetic trematodes were detected in brain, lungs, and nasal sinuses, and were likely responsible for the emaciated condition, copious nasal discharge, neurological signs, and death of the bear. Mature trematodes recovered from the bear were conical with small spines on the tegument. The anterior end was broad and tapered gradually toward the posterior. Mean (+/-SE) size of the mature trematodes was 3.67 (+/-0.08) by 2.14 (+/-0.04) mm (n = 80). Eggs are operculated, gold, and 68.2 (+/-0.42) by 41.4 (+/-0.41) microns (n = 75). Suckers are well developed and located in the anterior half of the body, with the genital pore just posterior to the ventral sucker. Testes are tandem and the ovary is lateral and slightly anterior to the anterior testes just posterior to the ventral sucker. The uterus is predominantly anterior to the ventral sucker and is the most distinctive feature of the trematode.

Acute lymphoblastic leukemia, hypercalcemia, and pseudohyperkalemia in a dog.

A 5-month-old sexually intact male Chesapeake Bay Retriever was evaluated for lameness of 2 weeks' duration and lymphocytosis. Acute lymphoblastic leukemia was diagnosed on the basis of results of cytologic and cytochemical evaluation of a bone marrow aspirate. Serum biochemical abnormalities included hypercalcemia and hyperkalemia. Hypercalcemia was likely paraneoplastic; hyperkalemia was believed to be a result of release of potassium from large numbers of lymphocytes in vitro (pseudohyperkalemia). The dog was euthanatized, and necropsy revealed infiltration of the hepatic vasculature and sinusoids, renal parenchyma, mesenteric and peripheral lymph nodes, bone marrow, and iridial tissue with neoplastic cells. Unique features of this case include the young age of the dog and the hypercalcemia and hyperkalemia associated with acute lymphoblastic anemia.

Coronary artery atherosclerosis revisited in Korean war combat casualties.

To confirm earlier studies of a high prevalence of coronary atherosclerosis in combat casualties of the Korean and Vietnam wars, we examined previously uncut coronary arteries from the hearts of 94 American male combat casualties (mean age, 20.5 years) from the Korean War using computerized planimetry and microscopic evaluation. Six (6.4%) men had severe atherosclerosis (75% to 90% cross-sectional area luminal narrowing) in one or more coronary arteries; five of the six had fibrous plaques, and one had a complicated plaque. Our prevalence is similar to that reported previously (5%). Therefore, to determine whether the decrease in the incidence of severe coronary atherosclerosis today is the result of decreased plaque formation in the young, we will need to examine at least 772 men to detect a 50% decline with 85% to 90% confidence.

Fatal metoprolol overdose.

Metoprolol, a beta 1-adrenergic blocking agent, has been found effective in the treatment of hypertension. A death due to deliberate ingestion of metoprolol is described, including the case history, postmortem toxicologic findings, and identification and quantitation of the drug by high pressure liquid chromatography and gas chromatography/mass spectrometry. Metoprolol levels were found to be 4.7 mg/L in blood, 194 mg/L in urine, 3.3 mg/L in vitreous humor, 3.9 mg/L in pleural fluid, 254 mg/L in bile, 7.1 mg/kg in kidney, and 6.3 mg/kg in liver.

Prostatic acid phosphatase, aspermia, and alcoholism in rape cases.

In a few alleged rape cases, examination of vaginal secretions will be negative for spermatozoa but positive for significant levels of prostatic acid phosphatase. These laboratory results can occur in cases in which the accused is known to have sired children. The most common etiologic factors for the aspermia are vasectomies and chronic alcoholism with or without cirrhosis.

Drug identification through analysis of maggots.

The examination of insect larvae from decomposed bodies not only can serve in the estimation of the postmortem interval but also can be used in qualitative identification of drug substances.

Unilateral lobar pneumonia with empyema caused by Neisseria gonorrhoeae.

A 62-year-old woman died of a combination of pneumonia, empyema, emphysema, and severe heart disease. The pneumonia was due to Neisseria gonorrhoeae, a rarely if ever reported causative agent in pulmonary infection. Autopsy ruled out other sites from which the organism could have originated.

Management of the rape victim.

If a rape crisis center and specialized personnel are not available, the family physician can adequately examine and treat the rape victim as well as collect and properly handle all the required medical evidence. The detailed history has several facets. Laboratory confirmation can be obtained for prior consensual intercourse, sodomy and other special aspects. Treatment is indicated for the prevention of venereal disease and pregnancy. Psychologic support is essential. The physician's examination and testimony can play an important legal role.