Immunohistochemistry of the canine vomeronasal organ

The canine's olfactory acuity is legendary, but neither its main olfactory system nor its vomeronasal system has been described in much detail. We used immunohistochemistry on paraffin-embedded sections of male and female adult dog vomeronasal organ (VNO) to characterize the expression of proteins known to be expressed in the VNO of several other mammals. Basal cell bodies were more apparent in each section than in rodent VNO and expressed immunoreactivity to anticytokeratin and antiepidermal growth factor receptor antibodies. The thin layer of neurone cell bodies in the sensory epithelium and axon fascicles in the lamina propria expressed immunoreactivity to neurone cell adhesion molecule, neurone-specific beta tubulin and protein gene product 9.5. Some neurones expressed growth-associated protein 43 (GAP43): and a number of those also expressed neurone-specific beta tubulin-immunoreactivity. Some axon fascicles were double labelled for those two proteins. The G-protein alpha subunits Gi and Go, involved in the signal transduction pathway, showed immunoreactivity in the sensory cell layer. Our results demonstrate that the canine vomeronasal organ contains a population of cells that expresses several neuronal markers. Furthermore, GAP43 immunoreactivity suggests that the sensory epithelium is neurogenic in adult dogs.     

An anti-idiotype antibody which mimics the inner-core region of lipopolysaccharide protects mice against a lethal challenge with endotoxin.

Recently, we described the generation and characterization of an Armenian hamster Ab2 beta anti-idiotype monoclonal antibody (MAb4G2) specific for the binding site of a mouse monoclonal antibody, MAbY1-4A6, directed against the conserved 2-keto-3-deoxyoctulosonate (Kdo)-containing inner-core region of lipopolysaccharide (LPS) (S. K. Field, M. Pollack, and D. C. Morrison, Microb. Pathog. 15:103-120, 1993). In that study, mice and hamster immunized with MAb4G2 generated serum immunoglobulin G and M (IgG and IgM) antibodies which cross-react with Salmonella minnesota R595-chemotype rough mutant LPS (Re-LPS). In this report, we demonstrate that in C3Heb/FeJ mice, MAb4G2 elicits an immune response which is characterized by specific binding of antibody to Re-LPS, as assessed by enzyme-linked immunosorbent assay. The practical use of MAb4G2 as a potentially effective therapeutic agent against gram-negative bacterial sepsis is suggested by the demonstration that immunization of these mice with MAb4G2 results in significant protection of D-galactosamine-sensitized animals against an otherwise lethal dose of Re-LPS. Assessment of the temporal changes in Re-LPS-specific serum antibody titers from mice immunized with MAb4G2 or Re-LPS over a 40-day period indicates that immunization with Re-LPS elicits significantly higher titers of serum IgM antibodies compared with those in animals immunized with MAb4G2. Conversely, two immunizations with MAb4G2 result in an up to 10-fold increase in anti-Re-LPS-specific IgG serum antibody titers relative to those obtained in mice immunized with Re-LPS. Nineteen days after the secondary boost with MAb4G2, anti-Re-LPS-specific IgG serum antibody titers were significantly higher (three- to fourfold) compared with those in Re-LPS-treated animals. Initial immunization with the anti-idiotype antibody primes animals for enhanced secondary responses to Re-LPS, as assessed by the titers of anti-Re-LPS-specific IgG profiles. These data suggest the potential utility of MAb4G2 as a candidate vaccine against the lethal properties of gram-negative bacterial LPS.     

Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction.

A new family of vectors has been produced which facilitates the cloning and expression of immunoglobulin variable regions cloned by polymerase chain reaction (PCR). The vectors are designed to express the cloned variable regions joined to human constant regions and take advantage of priming in the leader sequence so that no amino acid changes will be introduced into the mature antibody molecule. Both the heavy chain and light chain vectors utilize a murine VH promoter provided with an EcoRV restriction site so that the amplified variable regions can be directly cloned into a functional promoter. For the heavy chain an NheI restriction site has been generated at the first two amino acids of CH1 and the cloned leader and variable region are fused directly to the CH1 domain of the constant region. When the leader and variable regions of the light chain were fused directly to C kappa, no expression was observed. Therefore the light chain expression vector was designed with a SalI restriction site for cloning into a splice junction 3' of the variable region; VL then is joined to C kappa by splicing. Both vectors direct the expression of functional, fully assembled immunoglobulin molecules with the expected molecular weight. Use of redundant oligomers to prime the PCR permits the cloning and expression of recombinant antibodies without any prior information as to their sequence and makes it possible to rapidly generate recombinant antibodies from any monoclonal antibody producing cell line.     

Identification of Actinomyces (Corynebacterium) pyogenes with the API 20 Strep system.

A total of 62 strains of Actinomyces pyogenes (previously Corynebacterium pyogenes) were examined by the API 20 Strep system (API System, La Balme Les Grottes, Montalieu-Vercieu, France). The system was shown to be reliable and rapid when the tests were compared with standard identification methods. No confusion occurred with streptococcal profiles in the current API 20 Strep data base.     

Effect of naltrexone on food intake, hunger, and satiety in obese men

Increasing doses of naltrexone (25 to 200 mg) given over 4 consecutive days reduced intake of laboratory luncheon meals by 30% in 17 obese men. Meal size remained suppressed in the laboratory during the week following naltrexone administration. Water intake in the laboratory and body weight were not affected. Rates of ingestion and subjective ratings suggested that naltrexone reduced appetite rather than promoted early satiation. Nausea and other side effects occurred on 1 or 2 days during the naltrexone week in seven subjects whose food intake was reduced. Food intake was also reduced in seven of the remaining 10 subjects who reported no adverse reactions. These results suggest that a conditioned taste aversion or a conditioned anorexia may have developed in some subjects.     

Ganglioside modulation of lipopolysaccharide-initiated complement activation

Highly purified preparations of mouse gangliosides have been demonstrated to bind to purified preparations of lipopolysaccharide (LPS). In some instances, the binding has been demonstrated to be dependent upon the presence of sialic acid in the ganglioside preparation. The binding of gangliosides to LPS from the deep rough Salmonella minnesota Re mutant has suggested that the interaction involves the lipid A-2-keto-3-deoxyoctulosanate region of the LPS macromolecule. The interaction between gangliosides and LPS has been demonstrated to result in an abrogation of lipid A dependent activation of the classical pathway of serum complement by Re LPS. Surprisingly, however, the presence of sialic acid containing glycolipids has been shown to enhance significantly the capacity of LPS to initiate activation of the alternative pathway of complement. These data suggest that sialic acid can enhance as well as inhibit the formation of a stable alternative-pathway C3 convertase.     

Rabbit antisera to the variable region domains of an anti-alpha(1----6) dextran using E. coli-produced VL and VH fusion proteins as immunogens.

Bacteria were engineered for the expression of mouse immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) fusion proteins. cDNAs encoding the VL and VH of anti-alpha(1----6)dextran hybridoma protein 19.22.1 were inserted into the pATH 10 prokaryotic expression vector downstream of trp operon sequences. V domains joined to approximately 330 amino acids of the trp E gene product encoded by the expression plasmids accumulated at high levels in E. coli. In addition, the VL domain was expressed with a 15 amino acid extension at low levels in lon mutant bacteria. The trp E-VL and trp E-VH proteins were used to raise antisera in rabbits and the V specificity of the sera demonstrated.     

Distribution of TT virus (TTV), TTV-like minivirus,and related viruses in humans and nonhuman primates

TT virus (TTV) and TTV-like minivirus (TLMV) are small DNA viruses with single-stranded, closed circular, antisense genomes infecting man. Despite their extreme sequence heterogeneity (>50%), a highly conserved region in the untranslated region (UTR) allows both viruses to be amplified by polymerase chain reaction (PCR). TTV/TLMV infection was detected in 88 of 100 human plasma samples; amplified sequences were differentiated into TTV and TLMV by analysis of melting profiles, showing that both viruses were similarly prevalent. PCR with UTR primers also detected frequent infection with TTV/TLMV-related viruses in a wide range of apes (chimpanzees, gorillas, orangutans, gibbons) and African monkey species (mangabeys, drills, mandrills). These findings support the hypothesis for the co-evolution of TTV-like viruses with their hosts over the period of primate speciation, potentially analogous to the evolution of primate herpesviruses.     

Immunological and biophysical properties of hepatitis B antigen labeled by the chloramine-T and by the lactoperoxidase methods.

Optimal conditions were sought for the radiolabeling of microgram quantities of hepatitis B surface antigen (HBs Ag) employing the chloramine-T or lactoperoxidase iodination procedures. Preparations of HBsAg labeled by these procedures are referred to as chloramine-T preparations and lactoperoxidase preparations, respectively. Labeled HBsAg having specific activities between 10-20 muCi/mug were found to display the greatest degree of sensitivity for unlabeled HBsAg and for anti-HBs using a double-antibody radioimmunoassay (RIA-DA). Increasing the specific activity above this level redulted in a decreased affinity of labeled 1251-HBs Ag for anti-HBs, indicating that soluble antigenic alterations had developed. At equivalent specific activities, chloramine-T preparations competed less effectively for unlabeled HBs Ag than lactoperoxidase preparations, and anti-HBs endpoint titers were slightly reduced, especially among preparations of high specific activity (greater than or equal to 65 muCi/mug). Chloramine-T preparations of HBs Ag (sp. act. 15--30 muCi/mug) showed essentially no antigenic deterioration over a 2-month period at minus 196 degrees C or minus 70 degrees C. Utilization of optimally labeled 1251-HBs Ag has increased the sensitivity of the RIA-DA for unlabeled HBs Ag 30-fold to a level below 1 ng/ml and enhanced antiamine-T method revealed that only the most acidic population was labeled (pH 3.75+/-0.5). In contrast, six antigenic components with distinct pI values ranging from 3.7 to 5.2 were detected by RIA-DA in both unlabeled HBs ag and in the chloramine-T preparation. This indicated that the chloramine-T method did not radically change the relative number or charge of each of the pI populations present in purified preparations of HBs Ag. Analysis of HBs Ag iodinated by the lactoperoxidase procedure revealed the presence of three of four populations of particles with pI values ranging from 3.9 to 4.5, suggesting that this procedure labels HBs Ag more uniformly.