Further evaluation and validation of a commercially available competitive ELISA (cELISA) for the detection of antibodies specific to equine arteritis virus (EAV).

The purpose of this study was to further evaluate and validate two commercially available equine arteritis virus (EAV) competitive ELISAs (original and enhanced cELISAs) using archived equine sera from experimentally inoculated animals and field sera submitted for laboratory diagnosis. First, the original and subsequently enhanced cELISAs were compared with the virus neutralisation test (VNT) using a panel of archived serum samples from experimentally inoculated animals. Then, the enhanced cELISA was compared with the VNT using a large panel of archived serum samples. The total number of equine sera tested was 3255, which included sera against 25 different EAV strains. The study confirmed that the enhanced cELISA was more sensitive than the original cELISA. Based on testing sera from experimentally inoculated animals and field sera, the enhanced cELISA had an estimated sensitivity (98.9 percent and 99.6 percent, respectively) and specificity (98.3 percent and 98.7 percent, respectively). The currently marketed enhanced VMRD EAV antibody cELISA test kit (VMRD Inc., Pullman, Washington, USA) has high sensitivity and specificity relative to the VNT. Based on the findings of this study, the authors would propose that the enhanced cELISA should be considered as an alternative approved method to the VNT for the detection of antibodies to EAV.

IP(3) receptor function and localization in myotubes: an unexplored Ca(2+) signaling pathway in skeletal muscle.

We present evidence for an unexplored inositol 1,4,5-trisphosphate-mediated Ca(2+) signaling pathway in skeletal muscle. RT-PCR methods confirm expression of all three known isotypes of the inositol trisphosphate receptor in cultured rodent muscle. Confocal microscopy of cultured mouse muscle, doubly labeled for inositol receptor type 1 and proteins of known distribution, reveals that the receptors are localized to the I band of the sarcoplasmic reticulum, and this staining is continuous with staining of the nuclear envelope region. These results suggest that the receptors are positioned to mediate a slowly propagating Ca(2+) wave that follows the fast Ca(2+) transient upon K(+) depolarization. This slow wave, imaged using fluo-3, resulted in an increase in nucleoplasmic Ca(2+) lasting tens of seconds, but not contraction; the slow wave was blocked by both the inositol trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and the phospholipase C inhibitor U-73122. To test the hypothesis that these slow Ca(2+) signals are involved in signal cascades leading to regulation of gene expression, we assayed for early effects of K(+) depolarization on mitogen-activated protein kinases, specifically extracellular-signal related kinases 1 and 2 and the transcription factor cAMP response element-binding protein (CREB). Within 30-60 seconds following depolarization, phosphorylation of both the kinases and CREB was evident and could be inhibited by 2-aminoethoxydiphenyl borate. These results suggest a signaling system mediated by Ca(2+) and inositol trisphosphate that could regulate gene expression in muscle cells.

The ability of the enzyme-linked immunosorbent assay to detect antibody against Staphylococcus aureus in milk following experimental intramammary infection.

Changes in the milk antibody levels against Staphylococcus aureus were measured at the start of an experimental intramammary instillation of either S. aureus (Study I) or Staphylococcus hyicus (Study II). A commercial enzyme-linked immunosorbent assay system was used. Twenty-one Holstein cows were enrolled in Study I and 15 Holstein cows were used in Study II. Pathogen instillation began 21 days before the start of the non-lactating period. Cows received intramammary antibiotic treatment in all quarters immediately after the last milking, the start of the non-lactating period. Lacteal secretions were collected before the start of the non-lactating period, and during the immediate postpartum period in both studies, and during the non-lactating period in Study I. Milk was cultured for mastitis pathogens and S. aureus antibody levels and somatic cell counts were determined from all samples. There was an approximate 2-week delay in the elevation in antibody levels in response to the instillation of S. aureus. Antibody levels remained elevated in cows with S. aureus intramammary infections postpartum, but were below threshold in cows where intramammary infections were cured during the non-lactating period. Antibody levels were elevated by S. hyicus intramammary infections, remained elevated for the first 12 days postpartum, but were below threshold by day 21 postpartum. Cows with incipient intramammary S. aureus infections might be misclassified as false negatives by the antibody test. However, results suggest that cows with S. hyicus intramammary infections that were not cured would not be misclassified if milk is withheld from test for the first 30 days postpartum, as recommended by the manufacturer of the test.

Activation of a rel-A/CEBP-beta-related transcription factor heteromer by PGG-glucan in a murine monocytic cell line.

PGG-Glucan is a soluble beta-glucan immunomodulator that enhances a variety of leukocyte microbicidal activities without activating inflammatory cytokines. Although several different cell surface receptors for soluble (and particulate) beta-glucans have been described, the signal transduction pathway(s) used by these soluble ligands have not been elucidated. Previously we reported that PGG-Glucan treatment of mouse BMC2.3 macrophage cells activates a nuclear factor kappa-B-like (NF-kappaB) transcription factor complex containing subunit p65 (rel-A) attached to an unidentified cohort. In this study, we identify the cohort to be a non-rel family member: a CCAAT enhancer-binding protein-beta (C/EBP-beta)-related molecule with an apparent size of 48 kDa, which is a different protein than the previously identified C/EBP-beta p34 also present in these cells. C/EBP-beta is a member of the bZIP family whose members have previously been shown to interact with rel family members. This rel/bZIP heteromer complex activated by PGG-Glucan is different from the p65/p50 rel/rel complex induced in these cells by lipopolysaccharide (LPS). Thus, our data demonstrate that PGG-Glucan uses signal transduction pathways different from those used by LPS, which activates leukocyte microbicidal activities and inflammatory cytokines. We further show that heteromer activation appears to use protein kinase C (PKC) and protein tyrosine kinase (PTK) pathways, but not mitogen-activated protein kinase p38. Inhibitor kappa-B-alpha (IkappaB-alpha) is associated with the heteromer; this association decreases after PGG-Glucan treatment. These data are consistent with a model whereby treatment of BMC2.3 cells with PGG-Glucan activates IkappaB-alpha via PKC and/or PTK pathways, permitting translocation of the rel-A/CEBP-beta heteromer complex to the nucleus and increases its DNA-binding affinity.

PGG-glucan, a soluble beta-(1,3)-glucan, enhances the oxidative burst response, microbicidal activity, and activates an NF-kappa B-like factor in human PMN: evidence for a glycosphingolipid beta-(1,3)-glucan receptor.

PGG-Glucan, a soluble beta-(1,6)-branched beta-(1,3)-linked glucose homopolymer derived from the cell wall of the yeast Saccharomyces cerevisiae, is an immunomodulator which enhances leukocyte anti-infective activity and enhances myeloid and megakaryocyte progenitor proliferation. Incubation of human whole blood with PGG-Glucan significantly enhanced the oxidative burst response of subsequently isolated blood leukocytes to both soluble and particulate activators in a dose-dependent manner, and increased leukocyte microbicidal activity. No evidence for inflammatory cytokine production was obtained under these conditions. Electrophoretic mobility shift assays demonstrated that PGG-Glucan induced the activation of an NF-kappaB-like nuclear transcription factor in purified human neutrophils. The binding of 3H-PGG-Glucan to human leukocyte membranes was specific, concentration-dependent, saturable, and high affinity (Kd approximately 6 nM). A monoclonal antibody specific to the glycosphingolipid lactosylceramide was able to inhibit activation of the NF-kappaB-like factor by PGG-Glucan, and ligand binding data, including polysaccharide specificity, suggested that the PGG-Glucan binding moiety was lactosylceramide. These results indicate that PGG-Glucan enhances neutrophil anti-microbial functions and that interaction between this beta-glucan and human neutrophils is mediated by the glycosphingolipid lactosylceramide present at the cell surface.

The effect of patients' race on their attitudes toward medical students' participation in ambulatory care visits.

PURPOSE: To ascertain the preconceptions of ambulatory patients seeking care in internal medicine practices toward medical students' participation in their care. METHOD: The authors developed a self-administered, seven-item survey that sought patients' demographic information and their attitudes toward medical students' participation in their ambulatory care. In 1998, this survey was given to patients seen at four distinct internal medicine ambulatory clinic settings. RESULTS: Analysis of 516 completed surveys found neutral responses to the statement: "I would benefit from having a medical student involved in my care." Respondents indicated a lack of comfort in having medical students either answer their questions or examine them in the absence of a doctor. The responses did not differ when analyzed as a function of clinic site, age, gender, education, or annual income. Non-Caucasian respondents rated the benefit of having a student present significantly lower than did Caucasian respondents. They also indicated greater concern about being examined by a student alone, that the presence of a student would make the visit last longer, and that the gender of the student was important to them. CONCLUSIONS: Patients generally have neutral feelings as to whether they would benefit from medical students' participation in their ambulatory care. Caucasian patients are significantly more favorably inclined to medical student involvement than are non-Caucasian patients.

Cloning and characterization of a family of cDNAs from human histiocyte macrophage cells encoding an arginine-rich basic protein related to the 70 kD U1-snRNP splicing factor.

This paper describes the cloning and characterization of five cDNA members of a novel family of mRNAs, termed hm-1, isolated from human U937 macrophage cells. Two family members (clones 46 and 11) show complete mRNA features [including ribosome binding sites (RBS), polyadenylation signals, and poly(A) tails], and encode the same protein (designated HM-1), but differ substantially in their 5' untranslated regions. The three other cDNAs (clones 20, 60, and 38) appear to represent partial cDNAs. The protein sequences deduced from the five hm-1 cDNAs are identical (some truncated), except for one Trp --> Cys substitution. Full-length HM-1 is 246 amino acids long, has a predicted MW of 29431, is rich in arginine residues, has a pI of 10.25, and a mean hydrophobicity index of -1.23. HM-1 contains no obvious hydrophobic N-terminal cleavable signal sequence, and no potential N-glycosylation sites, but does contain three highly conserved motifs present in U1-70K splicing factors, and contains numerous C-terminal Arg/Asp and Arg/Glu dipeptides characteristic of "RD" family members that function as regulators of mRNA splicing. Northern hybridizations indicate that hm-1 is a family of mRNAs differentially expressed in a variety of human tissues.

PGG-Glucan activates NF-kappaB-like and NF-IL-6-like transcription factor complexes in a murine monocytic cell line.

PGG-Glucan (Betafectin) is a novel soluble beta-glucan immunomodulator that enhances leukocyte microbicidal activities without inducing inflammatory cytokines. Although several different receptors for soluble and particulate beta-glucans have been described, the signal transduction pathway(s) used by soluble beta-glucans have not been elucidated. We report that in a murine monocytic cell line (BMC2.3) PGG-Glucan activates nuclear factor-kappaB (NF-kappaB)-like and NF-interleukin-6 (IL-6)-like transcription factors. Electrophoretic mobility shift assays showed that PGG-Glucan activation of the factors is time- and concentration-dependent. The NF-kappaB-like complex includes subunit p65 (rel-A) as one of its components, but apparently not p50 (kappaB1), p52 (kappaB2), p68 (rel-B), or p75 (C-rel) family members. The NF-IL-6-like complex contains subunit C/EBP-beta (NF-IL-6alpha) as one of its components, but apparently not C/EBP-alpha or C/EBP-delta (NF-IL-6beta). As expected, lipopolysaccharide (LPS) activated p65/p50 NF-kappaB and C/EBP-beta NF-IL-6 complexes, increased the nuclear titer of p65 and p50 antigens, and increased cytokine (IL-1beta, tumor necrosis factor alpha) mRNA production. In contrast, PGG-Glucan increased the nuclear titer of p65, but apparently not p50, and did not induce cytokine mRNA production. These data demonstrate that PGG-Glucan utilizes signal transduction pathways different from those used by LPS. The data suggest that activation of the PGG-Glucan-stimulated factors is not sufficient to stimulate cytokine mRNA transcription.

Antibody responses of cows during an outbreak of neosporosis evaluated by indirect fluorescent antibody test and different enzyme-linked immunosorbent assays.

Serum samples from 70 (33 aborting and 37 non-aborting) dairy cows from a herd in California were analyzed for Neospora caninum antibodies in different laboratories by various serologic assays including enzyme-linked immunosorbent assay (ELISA) with recombinant antigens (Nc4.1 and Nc14.1), kinetic ELISA, whole tachyzoite lysate ELISA, immunostimulating complex (iscom) ELISA, antigen capture competitive inhibition ELISA, and by the indirect fluorescent antibody test. Eighteen percent of pregnant cows in this herd had aborted within 2 mo of the index case. All 70 cows had antibodies to N. caninum by at least 1 of the tests. Antibody levels to N. caninum in aborting cows as a group were higher than in nonaborting cows. However, it was concluded that no serological test could be used to establish definitively that N. caninum caused the abortion in an individual cow.

Stroke rehabilitation: indications, outcomes, recent developments.

Following the development of fixed neurological deficit due to hemorrhagic or ischemic stroke, neurorehabilitation is important in the acute, subacute, and chronic stages to develop strategies to prevent complications and return patients to their maximal functional and vocational independence. This article reviews the potential stroke complications and strategies for effective management.

Serologic responses of cattle and other animals infected with Neospora caninum.

OBJECTIVE: To examine cross-reactivity among Neospora caninum and closely-related apicomplexans. DESIGN: Sera from animals were examined for antibody production to N caninum and cross-reactivity to Toxoplasma gondii. ANIMALS: Cattle were experimentally infected with 3 tissue cyst-forming protozoan parasites N caninum, T gondii, and Sarcocystis sp, and calves were monospecifically inoculated with the intestinal coccidia, Eimeria bovis and Cryptosporidium parvum. Similar studies were done in laboratory rabbits inoculated with N caninum, T gondii, Hammondia hammondi, and Sarcocystis sp. Additionally, sera were obtained from ewes, lambs, goats, sows, cats, rats, and mice inoculated with N caninum tachyzoites. PROCEDURE: The indirect fluorescent antibody (IFA) and ELISA antibody tests (cattle only) were used to examine reactivity to N caninum; the modified direct agglutination, Sabin-Feldman dye, and IFA tests were used to evaluate reactivity to T gondii. RESULTS: Serologic cross-reactivity among N caninum, T gondii, and Sarcocystis sp was none or minimal by the IFA test. There was some reactivity to N caninum by the use of ELISA in cattle inoculated with Sarcocystis sp. CONCLUSIONS: The IFA test for N caninum was specific for the diagnosis of neosporosis in animals.

Genes encoding giant danio and golden shiner ependymin.

Ependymin (EPN) is a brain glycoprotein that functions as a neurotrophic factor in optic nerve regeneration and long-term memory consolidation in goldfish. To date, true epn genes have been characterized in one order of teleost fish, Cypriniformes. In the study presented here, polymerase chain reactions were used to analyze the complete epn genes, gd (1480 bp), and sh (2071 bp), from Cypriniformes giant danio and shiner, respectively. Southern hybridizations demonstrated the existence of one copy of each gene per corresponding haploid genome. Each gene was found to contain six exons and five introns. Gene gd encodes a predicted 218-amino acid (aa) protein GD 93 percent conserved to goldfish EPN, while sh encodes a predicted 214-aa protein SH 91 percent homologous to goldfish. Evidence is presented classifying proteins previously termed "EPNs" into two major categories: true EPNs and non-EPN cerebrospinal fluid glycoproteins. Proteins GD and SH contain all the hallmark, features of true EPNs.

Asymptomatic carotid disease: changing perspectives.

Information acquired in the last decade has clarified the risk of carotid stenosis and defined multiple causes of stroke. Bruits are associated with very low risk of stroke. Severe stenosis (> 70%) is rare in the general population and only implies a 3%/year stroke risk. Strokes have many causes with as few as 15% due to surgically accessible carotid disease. Studies to date have shown no benefit to prophylactic endarterectomy. Patients with carotid stenosis undergoing surgery have also been shown to be at no significant increased risk. The acceptable risk for surgery on asymptomatic disease is no more than 3%. Whether this can be achieved in the community is unclear. The ideal management of carotid stenosis is unclear. Surgery for asymptomatic disease must be very low risk to be beneficial and, to date, has not been shown to improve the natural history of the problem.

Cloning and sequencing the genes encoding goldfish and carp ependymin.

Ependymins (EPNs) are brain glycoproteins thought to function in optic nerve regeneration and long-term memory consolidation. To date, epn genes have been characterized in two orders of teleost fish. In this study, polymerase chain reactions (PCR) were used to amplify the complete 1.6-kb epn genes, gf-I and cc-I, from genomic DNA of Cypriniformes, goldfish and carp, respectively. Amplified bands were cloned and sequenced. Each gene consists of six exons and five introns. The exon portion of gf-I encodes a predicted 215-amino-acid (aa) protein previously characterized as GF-I, while cc-I encodes a predicted 215-aa protein 95% homologous to GF-I.

Frequency of reisolation of Staphylococcus aureus from multiple sequential milk samples.

Bacterial cultures were performed on multiple sequential composite samples of milk from 1,172 cows in 9 dairy herds. If the initial diagnosis of Staphylococcus aureus infection was based on the first positive culture, an average of 37.8% of subsequent cultures on the same cows were negative for S aureus. However, if the initial diagnosis of S aureus infection was confirmed by 2 or 2 of 3 sequential positive cultures and any conversions from S aureus positive to negative were confirmed by 2 or 2 of 3 sequential negative cultures, then only 17.0% converted to a negative diagnosis. Conversion of cows from S aureus culture-positive to -negative varied between herds; 8.1 to 69% for single cultures and 0.0 to greater than 40% for confirmed cultures.

Mechanisms of cell shape change: the cytomechanics of cellular response to chemical environment and mechanical loading.

Processes such as cell locomotion and morphogenesis depend on both the generation of force by cytoskeletal elements and the response of the cell to the resulting mechanical loads. Many widely accepted theoretical models of processes involving cell shape change are based on untested hypotheses about the interaction of these two components of cell shape change. I have quantified the mechanical responses of cytoplasm to various chemical environments and mechanical loading regimes to understand better the mechanisms of cell shape change and to address the validity of these models. Measurements of cell mechanical properties were made with strands of cytoplasm submerged in media containing detergent to permeabilize the plasma membrane, thus allowing control over intracellular milieu. Experiments were performed with equipment that generated sinusoidally varying length changes of isolated strands of cytoplasm from Physarum polycephalum. Results indicate that stiffness, elasticity, and viscosity of cytoplasm all increase with increasing concentration of Ca2+, Mg2+, and ATP, and decrease with increasing magnitude and rate of deformation. These results specifically challenge assumptions underlying mathematical models of morphogenetic events such as epithelial folding and cell division, and further suggest that gelation may depend on both actin cross-linking and actin polymerization.

A synthetic DNA encoding a modified human urokinase resistant to inhibition by serum plasminogen activator inhibitor.

Using DNA synthesis technology we constructed two synthetic DNAs, designated syn-uPA-DNA and mut-uPA-DNA. Syn-uPA-DNA contains the complete coding sequence of human presecretion form of single-chain u-PA. Mut-uPA-DNA was derived from syn-uPA-DNA by deleting 18 base pairs coding for amino acids Arg179-Ser184. Each synthetic DNA was inserted into a bovine papilloma viral genome-based expression vector to obtain expression in mouse cells. The results indicate that both syn-uPA and mut-uPA proteins are secreted predominantly in single-chain form. The single-chain form of both enzymes can be completely converted to two-chain form by treatment with plasmin. Both enzymes are as active as natural urokinase (std-uPA) isolated from urine. Analysis of enzymatic activity indicates that under conditions where syn-uPA and std-uPA are completely inhibited by endothelial-type plasminogen activator inhibitor (PAI-1), mut-uPA retains 90% activity. In identical experiments with placental-type PAI (PAI-2), mut-uPA retains 80% activity. Syn-uPA is capable of forming a approximately 100-kDa complex with PAI, whereas mut-uPA can not. PAI-treated mut-uPA has kinetic properties similar to untreated syn-uPA or std-uPA. Overall, the data indicate that amino acids Arg179-Ser184 function at least in part as a binding site for PAI. Resistance to PAI inhibition may increase the potency of mut-uPA as a thrombolytic agent.

Analysis of binding of a technetium-99m-labeled monoclonal antibody to lentivirus-infected cells.

Caprine arthritis-encephalitis (CAE) is a model for the study of lentiviral infections. The authors' hypothesis is that radioimmunodetection has the potential to detect lentiviral proteins at the surface of infected cells. A monoclonal antibody (CAEV92A1) specific for a CAE virus (CAEV)-associated glycoprotein and a control antibody were radiolabeled with technetium-99m (99mTc) using the pretinning method. Cell binding assays were used to evaluate immunoreactivity and binding properties of 99mTc-labeled antibodies to CAEV-infected cells. 99mTc-CAEV92A1 bound preferentially to paraformaldehyde-fixed and live CAEV-infected cells. 99mTc-CAEV92A1 did not appear to be shed rapidly from its binding site.

The mechanics of notochord elongation, straightening and stiffening in the embryo of Xenopus laevis.

We have examined the biomechanical development of the notochord of Xenopus early tail-bud embryos by: (1) quantifying morphological and mechanical changes in the embryo during stages 20-28, and (2) conducting manipulative experiments to elucidate mechanical roles of various components of the notochord. The notochord, which is composed of a stack of flat cells surrounded by a connective tissue sheath, elongates dramatically and begins straightening between stages 21 and 25. At this time the fiber density in the notochord sheath goes up, the osmotic activity of the notochord cells increases, vacuoles within these cells swell, the internal pressure of the notochord increases 2- to 3-fold, and the flexural stiffness of the notochord rises by an order of magnitude. We suggest that the tendency of the notochord cells to osmotically swell is resisted by the sheath, thereby permitting the internal pressure to rise. This pressure increase results in the greater stiffness that permits the notochord to elongate and straighten without being buckled by the surrounding tissues.

Performance studies of an enzyme-linked immunosorbent assay for detecting Staphylococcus aureus antibody in bovine milk.

An enzyme-linked immunosorbent assay (ELISA) for detecting Staphylococcus aureus antibody in bovine milk samples was examined for repeatability. A set of 51 bovine milk samples from 4 universities with confirmed culture results was assembled, and a panel of 30 milk samples was randomly selected. When the selected panel was tested at the collection laboratory, there was 97% agreement between the ELISA and the culture test. The panel was tested with the ELISA by the 4 university laboratories. Results were scored by both visual and optical density reader methods. When compared to reference ELISA results, the university laboratory ELISA results showed an agreement of 99.8% for negative samples, 98% for positive samples, and 99% for all samples. Additional studies on 19 milk samples that cultured positive for bacteria other than S. aureus showed 100% specificity. Overall comparison of ELISA and culture results showed high agreement between the 2 techniques. Disagreement appeared to result from explainable differences in antibody and bacterial levels and not from errors in either of the 2 techniques.