Identification of vaccine candidate peptides in the NcSRS2 surface protein of Neospora caninum by using CD4+ cytotoxic T lymphocytes and gamma interferon-secreting T lymphocytes of infected holstein cattle

Previously, our laboratory showed that Holstein cattle experimentally infected with Neospora caninum develop parasite-specific CD4+ cytotoxic T lymphocytes (CTL) that lyse infected, autologous target cells through a perforin-granzyme pathway. To identify specific parasite antigens inducing bovine CTL and helper T-lymphocyte responses for vaccine development against bovine neosporosis, the tachyzoite major surface proteins NcSAG1 and NcSRS2 were targeted. In whole tachyzoite antigen-expanded bovine T-lymphocyte lines, recombinant NcSRS2 induced potent memory CD4+- and CD8+-T-lymphocyte activation, as indicated by proliferation and gamma interferon (IFN-gamma) secretion, while recombinant NcSAG1 induced a minimal memory response. Subsequently, T-lymphocyte epitope-bearing peptides of NcSRS2 were mapped by using overlapping peptides covering the entire NcSRS2 sequence. Four experimentally infected cattle with six different major histocompatibility complex (MHC) class II haplotypes were the source of immune cells used to identify NcSRS2 peptides presented by Holstein MHC haplotypes. NcSRS2 peptides were mapped by using IFN-gamma secretion by rNcSRS2-stimulated, short-term T-lymphocyte cell lines, IFN-gamma enzyme-linked immunospot (ELISPOT) assay with peripheral blood mononuclear cells, and 51Cr release cytotoxicity assay of rNcSRS2-stimulated effector cells. Four N. caninum-infected Holstein cattle developed NcSRS2 peptide-specific T lymphocytes detected ex vivo in peripheral blood by IFN-gamma ELISPOT and in vitro by measuring T-lymphocyte IFN-gamma production and cytotoxicity. An immunodominant region of NcSRS2 spanning amino acids 133 to 155 was recognized by CD4+ T lymphocytes from the four cattle. These findings support investigation of subunit N. caninum vaccines incorporating NcSRS2 gene sequences or peptides for induction of NcSRS2 peptide-specific CTL and IFN-gamma-secreting T lymphocytes in cattle with varied MHC genotypes.     

Safety and immunogenicity of a prototype enterotoxigenic Escherichia coli vaccine administered transcutaneously

Transcutaneous immunization (TCI) is a new method for vaccine delivery that has been shown to induce immunity relevant to enteric disease vaccines. We evaluated the clinical safety and immunogenicity of a recombinant subunit vaccine against enterotoxigenic Escherichia coli (ETEC) delivered by TCI. Adult volunteers received patches containing the recombinant ETEC colonization factor CS6, either with heat-labile enterotoxin (LT) or patches containing CS6 alone. The vaccine was administered at 0, 1, and 3 months, and serum antibodies and antibody-secreting cells (ASCs) were assessed. Among the 26 volunteers that completed the trial, there were no responses to CS6 in the absence of LT. In the groups receiving both CS6 and LT, 68 and 53% were found to have serum anti-CS6 immunoglobulin G (IgG) and IgA, respectively; 37 and 42% had IgG and IgA anti-CS6 ASCs. All of the volunteers receiving LT had anti-LT IgG, and 90% had serum anti-LT IgA; 79 and 37% had anti-LT IgG and IgA ASCs. Delayed-type hypersensitivity (DTH), suggesting T-cell responses, was seen in 14 of 19 volunteers receiving LT and CS6; no DTH was seen in subjects receiving CS6 alone. This study demonstrated that protein antigens delivered by a simple patch could induce significant systemic immune responses but only in the presence of an adjuvant such as LT. The data suggest that an ETEC vaccine for travelers delivered by a patch may be a viable approach worthy of further evaluation.     

Biochemical identification of citrobacteria in the clinical laboratory.

We biochemically identified 235 Citrobacter strains to the species level on the basis of the recently proposed taxonomic changes of Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993). Citrobacter isolates were initially identified as C. koseri or as members of the C. freundii complex or C. amalonaticus group on the basis of indole production, formation of H2S, malonate utilization, and acid production from D-arabitol and adonitol. On the basis of the results of these tests, 68% of the Citrobacter strains were identified as members of the C. freundii complex, 25% were C. koseri, and 8% were members of the C. amalonaticus group. By using a 15-test system recently proposed by Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993) to help identify new species in the C. freundii complex and C. amalonaticus group, 81% of the C. freundii complex strains and 100% of the C. amalonaticus strains could be definitively assigned to one of the previously established or recently designated species or hybridization groups of the genus Citrobacter. Within the C. freundii complex, C. freundii predominated overall (37%), followed by C. youngae (24%), C. braakii (13%), and C. werkmanii (6%). Only one strain each of C. sedlakii and Citrobacter DNA group 11 was identified in this study. Among C. amalonaticus complex members, all were identified as C. amalonaticus with the singular exception of one fecal isolate of C. farmeri. C. freundii and C. koseri were the two Citrobacter species most commonly (80 of 93 [86%]) isolated from extraintestinal sources (genitourinary tract, wounds, blood).     

P gene mutations associated with oculocutaneous albinism type II (OCA2)

Oculocutaneous albinism type II (OCA2) is the most common form of albinism in humans. OCA2 has been previously associated with mutations of the P gene, the human homologue to the murine pink-eyed dilution gene. The P gene encodes a 110 kDa protein containing 12 potential membrane spanning domains and is associated with melanosomal membranes. The specific function of the P protein is currently unknown but is thought to be involved in tyrosinase processing and transport. We report nine novel mutations in the P gene associated with OCA2. These include two missense mutations, c.1938A>C (p.Ile646Val) and c.1556T>C (p.Val519Ala); one nonsense mutation c.612G>A (p.Trp204X); five frameshift mutations: c.2372_2373delTC, c.1555delG, c.1938_1939insC, c.2050delT, and c.1045_1046delAT; and a splice site mutation c.1951+1G>A. We also report 12 novel polymorphisms including one amino acid substitution, c.2365_2366GC>CA (p.Ala789Glu). At present, there is no functional assay to determine if a mutation is truly pathogenic. The presence of numerous polymorphisms of the P gene in the coding region, several of which result in amino acid substitutions, makes molecular diagnosis problematic. To ensure accurate molecular diagnosis, further mutational analysis will be necessary to produce a comprehensive list of mutations associated with OCA2. This information will also help define the critical functional domains of the P protein. Mutations associated with OCA2 can be found in the Albinism Database (http://albinismdb.med.umn.edu).     

Psychiatric complications of anabolic steroid abuse

The authors review the literature from human and animal studies on the neurochemical and pathological psychiatric effects of supraphysiological doses of anabolic-androgenic steroids (AAS) and discuss the AAS use and abuse patterns, additional drug use patterns, and personality and behavioral characteristics of AAS abusers.     

Identification of Aeromonas strains to the genospecies level in the clinical laboratory.

One hundred thirty-three strains of Aeromonas (human, n = 102; animal, n = 16; environmental, n = 15) previously identified to the DNA group level by molecular methods were biochemically analyzed for 58 properties. On the basis of the use of between 9 and 16 selected tests, 132 of the 133 strains (99%) could be assigned to their correct hybridization group using this biochemical scheme. The results suggest a feasible approach for identifying aeromonads to genospecies level under appropriate conditions.     

Repertoire of transcribed peripheral blood T-cell receptor beta chain variable-region genes in acute rheumatic fever.

Patients with severe group A streptococcal infections have abnormalities in the Vbeta repertoire of peripheral blood T cells that are consistent with superantigen stimulation by cytoplasmic membrane proteins. The purpose of this study was to determine whether similar changes in Vbeta repertoire could be found for patients with acute rheumatic fever (ARF). The mean Vbeta repertoire of peripheral blood T cells in nine hospitalized ARF patients was similar to that of 34 controls and did not change during 6 months of follow-up in 6 of the ARF subjects. We were unable to detect changes in the Vbeta repertoire of peripheral blood T cells from patients with ARF that could be attributed to the influence of a superantigen.     

Glial Fibrillary Acidic Protein Expression in Pleomorphic Adenoma of Salivary Gland: An Immunoelectron Microscopic Study

Previous immunocytochemical studies of pleomorphic adenomas have demonstrated consistent labeling with glial fibrillary acidic protein (GFAP). Cross-reactivity with other intermediate filaments of similar structure and chemical composition has been suggested to account for this seemingly inappropriate pattern of immunoreactivity. To investigate further this phenomenon, we examined five pleomorphic adenomas by immunoelectron microscopy. Ultrastructural features were similar to those described by other investigators, with ductal epithelium being surrounded by myoepithelial cells and modified cells becoming detached to form the isolated stellate and spindle cells of the stroma. As part of this process, many neoplastic myoepithelial cells appeared to lose their specialized ultrastructural features, assuming a rather undifferentiated appearance. Single and double immunoelectron microscopic labeling showed vimentin filaments in all these neoplastic myoepithelial cells. In contrast, GFAP filaments were identified only in the most undifferentiated cells. Such restriction of GFAP filaments to an ultrastructurally definable subset of neoplastic cells provides strong evidence against nonspecific staining due to cross-reactivity. Given the previously described coexpression of vimentin and GFAP by neoplastic cartilage, it appears likely that this immunophenotype in neoplastic myoepithelial cells reflects early chondroid differentiation.     

Development of automated immunoassays for immune status screening and serodiagnosis of rubella virus infection

The fully automated IMx immunoassay analyzer was used to develop a system for the detection of IgG and IgM antibodies to rubella virus for immune status screening and diagnosis of primary infections. Reagents and assay protocol software were developed using rubella virus sensitized microparticles as the solid phase to capture specific antibodies from serum samples. Anti-human IgG or IgM antibody coupled to alkaline phosphatase enzyme followed by methylumbelliferyl phosphate substrate was used to detect the presence or absence of antibodies specific to the antigens on the solid phase. To evaluate the efficacy of the IMx rubella IgG assay, immune status screening was performed with a clinical patient population of 501 sera. When compared to an IgG specific enzyme immunoassay and passive hemagglutination assay the agreement was greater than 99%. The IMx rubella IgM assay was utilized to determine the presence of rubella specific IgM antibodies in 462 sera. These results were compared to IgM specific enzyme immunoassay results and also demonstrated greater than 99% agreement. Seroconversion following rubella vaccination of susceptible individuals was demonstrated by IgG and IgM antibody responses as early as two weeks postvaccination. In addition to automation, the IMx system offers rapid assay times and calibration curve storage without sacrificing clinical efficacy.