Showing papers in "Clinical and Vaccine Immunology in 1998"
TL;DR: In newly borne colostrum-deprived calves given the E. coli orally, intramuscular inoculation of phage delayed appearance of the bacterium in the blood and lengthened life span and there is considerable potential for this approach to bacterial-disease therapy.
Abstract: A lytic bacteriophage, which was previously isolated from sewage and which attaches to the K1 capsular antigen, has been used to prevent septicemia and a meningitis-like infection in chickens caused by a K1 1 bacteremic strain of Escherichia coli. Protection was obtained even when administration of the phage was delayed until signs of disease appeared. The phage was able to multiply in the blood. In newly borne colostrum-deprived calves given the E. coli orally, intramuscular inoculation of phage delayed appearance of the bacterium in the blood and lengthened life span. With some provisos there is considerable potential for this approach to bacterial-disease therapy.
302 citations
TL;DR: The complete assignment of the proton chemical shifts obtained by nuclear magnetic resonance (NMR) spectroscopy of de-O-acetylated glucuronoxylomannans (GXMs) from Cryptococcus neoformans permitted the high-resolution determination of the total structure of any GXM.
Abstract: The complete assignment of the proton chemical shifts obtained by nuclear magnetic resonance (NMR) spectroscopy of de-O-acetylated glucuronoxylomannans (GXMs) from Cryptococcus neoformans permitted the high-resolution determination of the total structure of any GXM. Six structural motifs based on an alpha-(1-->3)-mannotriose substituted with variable quantities of 2-O-beta- and 4-O-beta-xylopyranosyl and 2-O-beta-glucopyranosyluronic acid were identified. The chemical shifts of only the anomeric protons of the mannosyl residues served as structure reporter groups (SRG) for the identification and quantitation of the six triads present in any GXM. The assigned protons for the mannosyl residues resonated at clearly distinguishable positions in the spectrum and supplied all the information essential for the assignment of the complete GXM structure. This technique for assigning structure is referred to as the SRG concept. The SRG concept was used to analyze the distribution of the six mannosyl triads of GXMs obtained from 106 isolates of C. neoformans. The six mannosyl triads occurred singularly or in combination with one or more of the other triads. The identification and quantitation of the SRG were simplified by using a computer-simulated artificial neural network (ANN) to automatically analyze the SRG region of the one-dimensional proton NMR spectra. The occurrence and relative distribution of the six mannosyl triads were used to chemotype C. neoformans on the basis of subtle variations in GXM structure determined by analysis of the SRG region of the proton NMR spectrum by the ANN. The data for the distribution of the six SRGs from GXMs of 106 isolates of C. neoformans yielded eight chemotypes, Chem1 through Chem8.
173 citations
TL;DR: A commercially available indirect immunofluorescence assay (IFA) based on B. henselae-infected human larynx carcinoma cells (test A) was evaluated, showing that the seroprevalence of antibodies toB.
Abstract: Serologic testing for the presence of antibodies to Bartonella henselae is a widely accepted diagnostic procedure for laboratory confirmation of the diagnosis of cat scratch disease (CSD). In this study a commercially available indirect immunofluorescence assay (IFA) based on B. henselae-infected human larynx carcinoma cells (test A) was evaluated. Sera from 42 patients with CSD (20 confirmed by PCR) and 270 sera from healthy controls (consisting of 63 cat owners, 65 individuals whose last close contact with cats was >6 months previously, and 142 persons who had never been exposed to cats) were investigated for antibodies to B. henselae. All patients with CSD had titers of immunoglobulin G (IgG) to B. henselae of 128 or higher (test A; sensitivity, 100%). Of the 270 controls 189 (70%) were seronegative (titer, <64), 38 (14.1%) had titers of 64, 30 (11.1%) had titers of 128, 9 (3.3%) had titers of 256, and 4 (1.5%) had high titers, 512 (test A; specificity, 70%). Of the cat owners and individuals who had never had close contact with cats, 71.4 and 71.12%, respectively, were seronegative, and titers of 64, 128, 256, and 512 were found in 14.3 and 16.2%, 1.6 and 10.5%, 9.5 and 0.7%, and 3.2 and 1.4%, respectively. The sera from the patients and from the first 100 healthy adults without a history of close contact with cats were additionally tested with a second commercially available IFA, based on Vero cells infected with B. henselae and Bartonella quintana (test B). The sensitivity and specificity of test B were 93 and 73%, respectively. For patients with CSD the cross-reactivity between B. henselae and B. quintana in this test was 95%. Both systems are highly sensitive but less specific for detection of IgG antibodies to B. henselae in samples from patients with clinically apparent CSD. For detection of IgM antibodies, test A seems to be more sensitive (88%) and more specific (95%) than test B (sensitivity and specificity of 64 and 86%, respectively). The data show that the seroprevalence of antibodies to B. henselae in German individuals is high (30%). Low antibody levels are not sufficient evidence of active or prior infection.
155 citations
TL;DR: The results suggest that NO and nitrogen-derived oxidants may play a role in the initiation of demyelination in acute-MS lesions but not in the later phase of the disease.
Abstract: We have examined the localization of inducible nitric oxide synthase (iNOS) and nitrotyrosine (the product of nitration of tyrosine by peroxynitrite, a highly reactive derivative of nitric oxide [NO]) in demyelinating lesions from (i) two young adult patients with acute multiple sclerosis (MS), (ii) a child with MS (consistent with diffuse sclerosis), and (iii) five adult patients with chronic MS. Previous reports have suggested a possible correlation between iNOS, peroxynitrite, related nitrogen-derived oxidants, and the demyelinating processes in MS. We have demonstrated iNOS-immunoreactive cells in both acute-MS and diffuse-sclerosis-type lesions. In acute-MS lesions, iNOS was localized in both monocytes/macrophages and reactive astrocytes. However, foamy (myelin-laden) macrophages and the majority of reactive astrocytes were iNOS negative. In specimens from the childhood MS patient, iNOS protein was present only in a subpopulation of reactive or hypertrophic astrocytes. In contrast, no iNOS staining was detected in chronic-MS lesions. Immunohistochemical staining of acute-MS lesions with an antibody to nitrotyrosine revealed codistribution of iNOS- and nitrotyrosine-positive cells, although nitrotyrosine staining was more widespread in cells of the monocyte/macrophage lineage. In diffuse-sclerosis-type lesions, nitrotyrosine staining was present in hypertrophic astrocytes, whereas it was absent in chronic-MS lesions. These results suggest that NO and nitrogen-derived oxidants may play a role in the initiation of demyelination in acute-MS lesions but not in the later phase of the disease.
133 citations
TL;DR: The presence of activated T cells and TH1 cytokine transcripts in chronic joint lesions of patients with OA suggests that T cells contribute to chronic inflammation in a large proportion of these patients.
Abstract: The synovial membrane in osteoarthritis (OA) often exhibits inflammatory infiltrates, but the role of T cells in these infiltrates is not known. T-cell activation antigens were analyzed by immunohistochemistry, and T-cell cytokine transcripts were measured by competitive PCR in synovial membranes from patients with OA and rheumatoid arthritis (RA). Lymphoid cell aggregates, containing primarily CD3 1 T lymphocytes, were found in 65% of patients with OA. Mononuclear cells expressing the activation antigens CD69, CD25, CD38, CD43, CD45RO, and HLA class II were present in both patient groups, although in higher numbers in patients with RA. Interleukin 2 (IL-2) transcripts were found in 10 of 18 patients with OA versus 12 of 13 patients with RA (P 5 0.03). Gamma interferon (IFN-g) transcripts were detected in 9 of 18 patients with OA versus 10 of 13 patients with RA (not significant), whereas IL-10 transcripts were found in nearly all patients. IL-4 and IL-5 were not detected in any patients. The levels of IFN-g and IL-2 transcripts, normalized for T-cell number equivalents, were not statistically different between OA and RA, but the levels of IFN-g, normalized for total cell number equivalents, were lower in OA than in RA (P 5 0.01). Synovial membranes that expressed IL-2 and IFN-g transcripts were more likely to have heavier infiltrations of T cells and cells bearing activation markers than synovial membranes that did not express these cytokines. The presence of activated T cells and TH1 cytokine transcripts in chronic joint lesions of patients with OA suggests that T cells contribute to chronic inflammation in a large proportion of these patients. Osteoarthritis (OA), although a heterogeneous disease, is generally believed by rheumatologists to be primarily a disease of biomechanical alteration (18). However, apart from the relatively rare type of erosive inflammatory OA which clearly shows a strong inflammatory component, certain patients with OA exhibit inflammatory infiltrates in the synovial membrane (SM) (15, 17, 23, 28). These mononuclear infiltrates have not been fully characterized, and their possible role in the pathogenesis of the disease is not clearly understood. In certain patients with OA, mononuclear cell infiltrates in SM may resemble those found in rheumatoid arthritis (RA). In RA, significant evidence demonstrating that T cells play a significant role in the pathogenesis of the disease has accumulated (reviewed in reference 46). This evidence includes the amelioration of the disease by treatments directed against T cells, the association of the disease with certain HLA-DR4 alleles, and the presence in the SM of patients with RA of infiltrating T cells which express activation antigens, produce cytokines, and contain oligoclonal populations of T cells (reviewed in reference 46). T-cell-derived cytokines are major determinants of the outcome of immune responses. TH1 cytokines (interleukin 2 [IL-2] and gamma interferon [IFN-g]) are associated with macrophae activation, enhancement of cell-mediated cytotoxicity, delayed-type hypersensitivity responses, and effective responses to intracellular pathogens (38, 48, 62). TH2 cytokines (IL-4 and IL-5) are associated with allergic diseases, helminthic infections, and progressive infections by intracellular bacteria (38). A biased cytokine pattern is also found in animal models of autoimmune disease. For example, in experimental allergic encephalomyelitis, IL-2 and IFN-g, but not IL-4, are expressed in the brain of rats at the peak of disease, whereas during recovery, the expression of IL-2 and IFN-g decrease with the concomitant appearance of IL-4 (24). Also, in nonobese diabetic mice, IL-4 production is compromised, while administration of IL-4 to prediabetic mice prevents the development of diabetes (44). Although several studies have examined the TH1/TH2 cytokine pattern in SM of patients with RA and have reported the prevalence of a TH1 pattern (9, 25, 33, 42, 47, 51, 58), the role of T cells and the pattern of TH1/TH2 cytokines in patients with OA are largely unknown. In this study, we employed (i) immunohistochemistry with a panel of monoclonal antibodies (MAbs) to antigens expressed on activated T cells to characterize the mononuclear cell infiltrates, and (ii) reverse transcriptase (RT) PCR and competitive PCR to detect and quantitate T-cell cytokine transcripts in SM from patients with OA.
133 citations
TL;DR: The N-MAT proved to be highly sensitive and specific for both naturally and experimentally infected animals, highly reproducible between and within readers, easy to use on large sample sizes without requiring special equipment, and useful in testing serum from any species without modification.
Abstract: Current serologic tests used to detect antibodies to Neospora caninum require species-specific secondary antibodies, limiting the number of species that can be tested. In order to examine a wide variety of animal species that may be infected with N. caninum, a modified direct agglutination test (N-MAT) similar to the Toxoplasma gondii modified direct agglutination test (T-MAT) was developed. This test measures the direct agglutination of parasites by N. caninum-specific antibodies in serum, thus eliminating the need for secondary host-specific anti-isotype sera. The N-MAT was compared to the indirect fluorescent-antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA) with a “gold standard” serum panel from species for which secondary antibodies were available (n = 547). All positive samples tested were from animals with histologically confirmed infections. Up to 16 different species were tested. The N-MAT gave a higher sensitivity (100%) and specificity (97%) than the ELISA (74 and 94%, respectively) and had a higher sensitivity but a lower specificity than the IFAT (98 and 99%, respectively). The reduced specificity of the N-MAT was due to false-positive reactions in testing fetal fluids with particulate matter or severely hemolyzed serum. Overall, the N-MAT proved to be highly sensitive and specific for both naturally and experimentally infected animals, highly reproducible between and within readers, easy to use on large sample sizes without requiring special equipment, and useful in testing serum from any species without modification.
127 citations
TL;DR: During the past decade, the use of oral fluids, including saliva, gingival crevicular fluid, and mucosal transudates have been widely used for monitoring drugs, hormones, and a variety of other molecules and chemical substances for over 50 years.
Abstract: Oral fluids, including saliva, gingival crevicular fluid, and mucosal transudates have been widely used for monitoring drugs, hormones, and a variety of other molecules and chemical substances for over 50 years ([34][1], [35][2], [53][3], [54][4],[68][5]). During the past decade, the use of oral
123 citations
TL;DR: The results indicate that behavioral interventions may have salutary immunological and clinical health effects following bereavement among HIV-1-infected individuals and that this bereavement support group intervention might have similarSalutary effects in the general population.
Abstract: A randomized, controlled, clinical trial was conducted to examine the impact of a semistructured, 10-week, once weekly, 90-min/session bereavement support group intervention on immunological, neuroendocrine, and clinical health status in human immunodeficiency virus type 1-seropositive (HIV-1+) and HIV-1-seronegative (HIV-1−) homosexual men, compared to a standard of care control condition. A total of 119 homosexual men (74 HIV-1+and 45 HIV-1−) were assessed at baseline, 10 weeks, and 6 months follow-up. At the 6-month follow-up assessment, the intervention groups exhibited significant beneficial effects compared to controls on changes in CD4 cell, total T-lymphocyte, and total lymphocyte counts, when baseline levels, antiretroviral medication use, CDC stage of disease, and other potentially confounding factors were accounted for. There was no statistically significant effect on the CD4/CD8 ratio or on the CD8 cell count. The effect on CD4 cell count was associated with group attendance and with changes in plasma cortisol level. Plasma cortisol levels decreased significantly among intervention subjects, compared to controls. A significantly reduced number of health care visits over the 6-month follow-up period among the intervention subjects supported the clinical relevance of the immunological changes observed for both HIV-1+ and HIV-1−individuals. These results indicate that behavioral interventions may have salutary immunological and clinical health effects following bereavement among HIV-1-infected individuals. The effect in HIV-1− individuals suggests that this bereavement support group intervention might have similar salutary effects in the general population. Potential effects of such interventions on clinical HIV disease progression are of interest and should be studied.
123 citations
TL;DR: The rK39 enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for Leishmania donovani infection in the Sudan as mentioned in this paper.
Abstract: The rK39 enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for Leishmania donovani infection in the Sudan. rK39 ELISA proved more sensitive than DAT in diagnosis of kala-azar (93 and 80%, respectively); both tests may remain positive up to 24 months after treatment. For patients with post-kala-azar dermal leishmaniasis and individuals with subclinical infection, rK39 ELISA performed as well as DAT but could detect infection 6 months earlier in approximately 40% of patients. Conversion in DAT and rK39 ELISA also occurred in leishmanin skin test (LST)-positive individuals, suggesting active parasite replication (rK39 is an amastigote antigen) in these presumably immune individuals. In contrast to DAT, rK39 ELISA also detected infection in randomly selected LST-positive individuals (in four of six) and endemicity (LST-negative) controls (in one of five). rK39 ELISA appears more sensitive than DAT and may prove an important tool in epidemiological studies.
121 citations
TL;DR: By employing conditions that favor detection of higher-avidity IgG antibody, the modified ELISA provides results that correlate closely with measurements of antibody functional activity that are thought to be important in protection against meningococcal disease.
Abstract: The standardized enzyme-linked immunosorbent assay (ELISA) for measurement of serum immunoglobulin G (IgG) antibody responses to meningococcal C polysaccharide has been modified to employ assay conditions that ensure specificity and favor detection primarily of high-avidity antibodies. The modified and standard assays were used to measure IgG antibody concentrations in sera of toddlers vaccinated with meningococcal polysaccharide vaccine or a meningococcal C conjugate vaccine. The results were compared to the respective complement-mediated bactericidal antibody titers. In sera obtained after one or two doses of vaccine, the correlation coefficients, r, for the results of the standard assay and bactericidal antibody titers were 0.45 and 0.29, compared to 0.85 and 0.87, respectively, for the modified assay. With the standard assay, there were no significant differences between the geometric mean antibody responses of the two vaccine groups. In contrast, with the modified assay, 5- to 20-fold higher postvaccination antibody concentrations were measured in the conjugate than in the polysaccharide group. Importantly, the results of the modified assay, but not the standard ELISA, paralleled the respective geometric mean bactericidal antibody titers. Thus, by employing conditions that favor detection of higher-avidity IgG antibody, the modified ELISA provides results that correlate closely with measurements of antibody functional activity that are thought to be important in protection against meningococcal disease.
119 citations
TL;DR: OMT levels correlated better with levels in serum than did saliva and appear to reflect systemic immune activation in HIV infection, which could be useful in the assessment of disease activity in patients with HIV infection or chronic inflammatory diseases.
Abstract: Oral fluids are convenient alternatives to blood sampling for evaluating significant metabolic components. Two forms of oral fluids, oral mucosal transudates (OMT) and saliva, were collected and compared for content of soluble products of immune activation. The data confirm that OMT and saliva represent distinct body fluids. The concentrations, outputs, and analyte/protein ratios of β-2-microglobulin (β2M), soluble tumor necrosis factor alpha receptor II (sTNFαRII), and neopterin were measured. Both the OMT and the saliva of most of the individuals in the control healthy populations had measurable levels of all three activation markers. When the immune system is activated, as in human immunodeficiency virus (HIV) infection, the levels of β2M and sTNFαRII are increased in both OMT and saliva compared to those in a healthy control population. OMT levels correlated better with levels in serum than did saliva and appear to reflect systemic immune activation in HIV infection. Because acquisition of oral fluids is noninvasive and easily repeatable, measurement of β2M and/or sTNFαRII content in OMT could be useful in the assessment of disease activity in patients with HIV infection or chronic inflammatory diseases.
TL;DR: The results obtained provide a normal range for the production of these cytokines under specified conditions in vitro by peripheral blood mononuclear cells cultured from healthy immunocompetent subjects after mitogen stimulation was determined.
Abstract: The production of interleukin 2 (IL-2) gamma interferon, IL-4, tumor necrosis factor alpha (TNF-alpha), TNF-beta, IL-5, and IL-10 in vitro by peripheral blood mononuclear cells cultured from healthy immunocompetent subjects after mitogen stimulation was determined. The mitogens used were concanavalin A, phytohemagglutinin, pokeweed mitogen, and Staphylococcus aureus Cowen. The results obtained provide a normal range for the production of these cytokines under specified conditions in vitro.
TL;DR: The profiles of some cytokines, i.e., IL-1RA, IL-6, and TNF-α, showed age-related increases that overlapped with known patterns of physical growth, and sIL-2R and sE-selectin instead declined with time.
Abstract: Serum cytokine levels were measured in 275 healthy children of different ages (3 to 17 years). Interleukin-1 receptor antagonist (IL-1RA), soluble IL-2R (sIL-2R) (sCD25), IL-6, IL-8, tumor necrosis factor alpha (TNF-α), soluble TNF receptor type II (sTNF-RII) (sCD120b), gamma interferon (IFN-γ), soluble intercellular adhesion molecule 1 (sICAM-1) (sCD54), soluble E selectin (sE-selectin) (ELAM-1; sCD62E), sCD14, and neopterin were measured with commercial test kits. The mean levels of IL-1RA, sIL-2R, TNF-α, sICAM-1, sE-selectin, and sCD14 were higher than in healthy adults. In contrast, IFN-γ and IL-8 were hardly detectable in children and thereby significantly lower than in adults. In the case of TNF-α, sICAM-1, sE selectin, and sCD14, there was a high interindividual variability, apparently unrelated to disease. The profiles of some cytokines, i.e., IL-1RA, IL-6, and TNF-α, showed age-related increases that overlapped with known patterns of physical growth. Of note, sIL-2R and sE-selectin instead declined with time. Because of the remarkable age-dependent variability in healthy pediatric subjects, disease-related changes, as well as therapy-dependent alterations, should be considered with caution.
TL;DR: Results indicate that IL-8 plays a major role in neutrophil recruitment during mastitis, and an understanding of its role will be of help in designing strategies for immunomodulatory therapies for mastitis.
Abstract: Due to its association with low-quality milk and a decrease in milk production in bovines, mastitis is a major cause of economic loss. Additionally, mastitis can be harmful to suckling newborns and can cause damage to the mammary gland. In mastitic mammary secretions there is a substantial increase in somatic cells, specifically neutrophils. In this study we examined the ability of mastitic and nonmastitic mammary secretions to cause in vitro neutrophil chemotaxis using a microchemotaxis assay. Also, the role of the inflammatory chemokine interleukin-8 (IL-8) in neutrophil recruitment during mastitis was addressed in these in vitro experiments. We found that both nonmastitic and mastitic mammary secretions were chemotactic, not chemokinetic, for neutrophils. The neutrophil chemotactic activity in mastitic, but not nonmastitic, mammary secretions was blocked by anti-IL-8 antibodies. Molecular mass separation of the active components showed that the chemotactic activity of the mastitic secretions was present in the 10-kDa-or-less fraction and was blocked by anti-IL-8 antibodies. These results indicate that IL-8 plays a major role in neutrophil recruitment during mastitis. An understanding of its role will be of help in designing strategies for immunomodulatory therapies for mastitis.
TL;DR: A collection of 12 monoclonal antibodies raised against porcine reproductive and respiratory syndrome (PRRS) virus was used to study the antigenic structure of the virus nucleocapsid protein, suggesting that the carboxy-terminal region plays an important role in maintaining local protein conformation.
Abstract: A collection of 12 monoclonal antibodies (MAbs) raised against porcine reproductive and respiratory syndrome (PRRS) virus was used to study the antigenic structure of the virus nucleocapsid protein (N). The full-length N gene, encoded by open reading frame 7, was cloned from the Canadian PRRS virus, PA-8. Deletions were introduced into the N gene to produce a series of nine overlapping protein fragments ranging in length from 25 to 112 amino acids. The individual truncated genes were cloned as glutathione S-transferase fusions into a eukaryotic expression vector downstream of the T7 RNA polymerase promoter. HeLa cells infected with recombinant vaccinia virus expressing T7 RNA polymerase were transfected with plasmid DNA encoding the N protein fragments, and the antigenicity of the synthesized proteins was analyzed by immunoprecipitation. Based on the immunoreactivities of the N protein deletion mutants with the panel of N-specific MAbs, five domains of antigenic importance were identified. MAbs SDOW17, SR30, and 5H2.3B12.1C9 each identified independent domains defined by amino acids 30 to 52, 69 to 123, and 37 to 52, respectively. Seven of the MAbs tested specifically recognized the local protein conformation formed in part by the amino acid residues 52 to 69. Furthermore, deletion of 11 amino acids from the carboxy terminus of the nucleocapsid protein disrupted the epitope configuration recognized by all of the conformation-dependent MAbs, suggesting that the carboxy-terminal region plays an important role in maintaining local protein conformation.
TL;DR: To test human sera for the presence of neutralizing antibodies and to type field strains, neutralization tests using CaCo2 tissue-culture-adapted astrovirus strains 1 to 7 and the corresponding rabbit reference sera were developed and Typing by NT was concordant with typing by ELISA and genotyping, with one exception.
Abstract: Astroviruses are a new family of positive-stranded RNA viruses that cause gastroenteritis in a wide range of animals and in humans. Seven types of astrovirus, tentatively considered serotypes, have been distinguished by enzyme-linked immunosorbent assays (ELISA) or immunoelectron microscopy, but it is unclear whether the serotype designation is used properly. To test human sera for the presence of neutralizing antibodies and to type field strains, neutralization tests (NT) using CaCo2 tissue-culture-adapted astrovirus strains 1 to 7 and the corresponding rabbit reference sera were developed. In rabbits, neutralizing antibodies were predominantly serotype specific, with the exception of low-level cross-reactivity in astrovirus serotype 4 reference serum with astrovirus serotype 1 virus. Similarly, in humans, no evidence of cross-reactivity was found for the serotype combinations tested (all except the combination 1 and 7 and the combination 6 and 7). Typing by NT was concordant with typing by ELISA and genotyping, with one exception. The seroprevalence rates of neutralizing antibodies in an age-stratified sample of the population in Utrecht Province (n = 242) were 91% for astrovirus serotype 1, 69% for astrovirus serotype 3, 56% for astrovirus serotype 4, 36% for astrovirus serotype 5, 31% for astrovirus serotype 2, 16% for astrovirus serotype 6, and 10% for astrovirus serotype 7. Acquisition of antibodies was slower among persons seropositive for astrovirus serotype 5 than among those seropositive for astrovirus serotypes 1 to 4, suggesting that the epidemiology of serotype 5 astrovirus is different from that of astrovirus serotypes 1 to 4.
TL;DR: No differences in antibody titers against any of the tested antigens could be detected between the duodenal ulcer patients and the asymptomatic H. pyloricarriers, either in sera or in gastric aspirates.
Abstract: In this study we have determined systemic and local antibody responses against different Helicobacter pylori antigens in H. pylori-infected and noninfected subjects. In addition, we studied whether differences in antibody responses between patients with duodenal ulcers and asymptomatic H. pylori carriers might explain the different outcomes of infection. Sera and in most instances gastric aspirates were collected from 19 duodenal ulcer patients, 15 asymptomatic H. pylori carriers, and 20 noninfected subjects and assayed for specific antibodies against different H. pylori antigens, i.e., whole membrane proteins (MP), lipopolysaccharides, flagellin, urease, the neuraminyllactose binding hemagglutinin HpaA, and a 26-kDa protein, by enzyme-linked immunosorbent assay. The H. pylori-infected subjects had significantly higher antibody titers against MP, flagellin, and urease in both sera and gastric aspirates compared with the noninfected subjects. Furthermore, the antibody titers against HpaA were significantly elevated in sera but not in gastric aspirates from the infected subjects. However, no differences in antibody titers against any of the tested antigens could be detected between the duodenal ulcer patients and the asymptomatic H. pylori carriers, either in sera or in gastric aspirates.
TL;DR: A standardization of the PorA VR typing nomenclature is proposed that incorporates serologic information from traditional PorA serosubtyping with molecular data from predicted VR sequences, which will be useful in future classification and applied epidemiologic studies of N. meningitidis.
Abstract: Neisseria meningitidis isolates are conventionally classified by serosubtyping, which characterizes the reactivities of the PorA outer membrane protein variable-region (VR) epitopes with monoclonal antibodies (MAbs). A newer method (PorA VR typing) uses predicted amino acid sequences derived from DNA sequence analysis. The resulting classification schemes are not standardized, offering conflicting and sometimes irreconcilable data from the two methods. In this paper, we propose a standardization of the PorA VR typing nomenclature that incorporates serologic information from traditional PorA serosubtyping with molecular data from predicted VR sequences. We performed a comprehensive literature and database search, generating a collection of strains and DNA sequences that reflects the diversity within PorA that exists to date. We have arranged this information in a comprehensive logical model that includes both serosubtype and PorA VR type assignments. Our data demonstrate that the current panel of serosubtype-defining MAbs underestimates PorA VR variability by at least 50%. Our proposal for VR typing is informative because amino acid sequence and serologic information, when serosubtype-defining MAbs are available, can be deduced simultaneously from the PorA VR designation. This scheme will be useful in future classification and applied epidemiologic studies of N. meningitidis, being a systematic way of selecting PorA vaccine candidates and analyzing vaccine coverage and failure.
TL;DR: Although HIV-infected patients had a significantly lower number of positive antigen-driven LPA responses compared with uninfected controls, most of the specimen processing variables had similar effects on HIV-positive and -negative samples.
Abstract: Lymphocyte proliferation assays (LPA), which can provide important information regarding the immune reconstitution of human immunodeficiency virus (HIV)-infected patients on highly active antiretroviral therapy, frequently involve shipment of specimens to central laboratories. In this study, we examine the effect of stimulant, anticoagulant, cell separation, storage, and transportation on LPA results. LPA responses of whole blood and separated peripheral blood mononuclear cells (PBMC) to different stimulants (cytomegalovirus, varicella-zoster virus, candida and tetanus toxoid antigens, and phytohemagglutinin) were measured using fresh specimens shipped overnight and frozen specimens collected in heparin, acid citrate dextrose (ACD), and citrate cell preparation tubes (CPT) from 12 HIV-infected patients and uninfected controls. Odds ratios for positive LPA responses were significantly higher in separated PBMC than in whole blood from ACD- and heparin-anticoagulated samples obtained from HIV-infected patients and from ACD-anticoagulated samples from uninfected controls. On separated PBMC, positive responses were significantly more frequent in fresh samples compared with overnight transportation for all antigens and compared with cryopreservation for the candida and tetanus antigens. In addition, viral antigen LPA responses were better preserved in frozen PBMC compared with specimens shipped overnight. CPT tubes yielded significantly more positive LPA results for all antigens, irrespective of the HIV patient status compared with ACD, but only for the candida and tetanus antigens and only in HIV-negative controls compared with heparin. Although HIV-infected patients had a significantly lower number of positive antigen-driven LPA responses compared with uninfected controls, most of the specimen processing variables had similar effects on HIV-positive and -negative samples. We conclude that LPA should be performed on site, whenever feasible, by using separated PBMC from fresh blood samples collected in either heparin or ACD. However, if on-site testing is not available, optimal transportation conditions should be established for specific antigens.
TL;DR: The results showed that secondary mumps vaccine failure occurs not infrequently, even among school age children under condition in which the vaccine coverage is low (i.e., 33% in the study population), and therefore, vaccinees are prone to be exposed to wild-type viruses.
Abstract: To characterize patients with mumps vaccine failure, avidity testing was performed with the Enzygnost Anti-Parotitis Virus/IgG kit using a single-dilution–6 M urea denaturation method. Five groups of patients were tested. Group 1 consisted of 29 patients with primary mumps infections; group 2 was 20 children and adults with a definite history of natural infection; group 3 was 7 patients with a recent mumps vaccination, 1 of whom developed parotid gland swelling and aseptic meningitis; group 4 was 14 patients with mumps vaccine failure; and group 5 was 6 patients with recurrent episodes of parotitis in addition to a history of vaccination. On the basis of the results of groups 1 and 2, an avidity of ≦31% was determined to be low, and ≧32% was determined to be high. Avidity maturation from low to high appears to occur around 180 days after the acute illness. The results of group 3 showed that the vaccine-induced immunoglobulin G (IgG) had very low avidity. Among the 14 patients in group 4, 12 patients, including 7 with a positive IgM response, were diagnosed as having secondary vaccine failures. The results of group 5 suggested the possibility that the avidity of the mumps vaccine-induced IgG remains low or borderline. These results showed that secondary mumps vaccine failure occurs not infrequently, even among school age children under condition in which the vaccine coverage is low (i.e., 33% in our study population), and therefore, vaccinees are prone to be exposed to wild-type viruses. Avidity testing should provide information useful for the analysis of mumps virus infections.
TL;DR: A role for locally produced IL-6 in the pathogenesis and expressed symptomatology of influenza A virus infection is suggested.
Abstract: Interleukin-6 (IL-6) is a pleotropic cytokine implicated in the pathogenesis of local inflammation during viral upper respiratory infections. This study determined if experimental influenza A virus infection causes local IL-6 production. Seventeen healthy, adult subjects were intranasally inoculated, by course drops, with a safety-tested strain of influenza A/Kawasaki/86 (H1N1) virus. Nasal lavage samples were collected, symptoms were recorded, and expelled nasal secretions were weighed once before and then daily for 8 days after the virus inoculation. Lavage samples were submitted for virus culture and were examined for IL-6 and IL-4 by enzyme-linked immunosorbent assay. The IL-6, but not IL-4, levels were significantly increased in the nasal lavage samples of the 12 subjects who shed virus but not in those of the 5 subjects who did not shed virus. Moreover, the elevations in IL-6 levels were related temporally to the development of nasal symptoms and secretions but not to systemic symptoms. These results suggest a role for locally produced IL-6 in the pathogenesis and expressed symptomatology of influenza A virus infection.
TL;DR: A 13% prevalence of IgA deficiency in patients with the chromosome 22q11.2 deletion syndrome is reported, which confirms the occurrence of significant humoral deficits in this predominantly cellular immunodeficiency.
Abstract: We wished to determine the prevalence of immunoglobulin A (IgA) deficiency in patients with the chromosome 22q11.2 deletion syndrome. A total of 32 patients with the chromosome 22q11.2 deletion were examined for IgA deficiency. We report a 13% (n = 4) prevalence of IgA deficiency in patients with this syndrome. The odds ratio of IgA deficiency in this population is 14.20 (P
TL;DR: It is proposed that collection of new antigens such as those recognized here will be useful in serologic tests for detecting and monitoring H. pylori infection and may also serve as potential targets for antimicrobial agent or vaccine development.
Abstract: There is great interest in characterizing the proteins of the gastric pathogen Helicobacter pylori, especially those to which humans respond immunologically, because of the potential importance of such proteins in diagnosis and vaccine development. Two-dimensional gel electrophoresis was used to separate and identify potential antigens of H. pylori ATCC 43504. Over 30 proteins were reactive in Western blots with pooled sera from 14 infected patients. These proteins were analyzed by N-terminal sequence analysis. Fourteen proteins were determined to be distinct from any proteins previously described from H. pylori; the others were previously isolated and characterized proteins. Analysis of eight distinct H. pylori strains showed that most of these antigens were produced by all of the strains. We propose that collection of new antigens such as those recognized here will be useful in serologic tests for detecting and monitoring H. pylori infection and may also serve as potential targets for antimicrobial agent or vaccine development.
TL;DR: Paired serum and oral-fluid specimens collected from blood donors and patients attending local sexually transmitted disease clinics in Trinidad and Tobago and the Bahamas and tested for the presence of human immunodeficiency virus type 1 (HIV-1) antibodies confirm the suitability of OF specimens for detection of HIV-1 antibodies.
Abstract: Paired serum and oral-fluid (OF) specimens (n = 4,448) were collected from blood donors and patients attending local sexually transmitted disease clinics in Trinidad and Tobago and the Bahamas and were tested for the presence of human immunodeficiency virus type 1 (HIV-1) antibodies. Sera were tested by Abbott AB HIV-1/HIV-2 (rDNA) enzyme immunoassay (EIA), and positive specimens were confirmed by Cambridge HIV-1 and HIV-2 Western blotting (WB). OF specimens were collected with the OraSure collection device and were tested by Murex GACELISA and by two EIAs from Organon Teknika (the Oral Fluid Vironostika HIV-1 Microelisa System [OTC-L] and the Vironostika HIV-1 Microelisa System [OTC-M]). EIA-reactive OF specimens were confirmed by miniaturized WB (OFWB). GACELISA detected all 474 HIV-1 seropositive specimens (sensitivity, 100%). OTC-L detected 470 positive specimens (sensitivity, 99.2%), while OTC-M detected 468 positive specimens (sensitivity, 98.8%). Specificities ranged from 99.2 to 100% for the three assays. Concordance of OFWB with serum WB was 99.4%, and banding patterns determined by the two methods were similar. The immunoglobulin G (IgG) concentration of OF specimens ranged from 0.21 to 100 microg/ml, with a mean of 17.1 microg/ml. Significant differences in OF IgG concentrations were observed between HIV antibody-positive and HIV antibody-negative persons (31.94 versus 15.28 microg/ml, respectively [P < 0.0001]). These data further confirm the suitability of OF specimens for detection of HIV-1 antibodies. Currently available HIV-1 antibody assays provide sensitivities and specificities with OF specimens comparable to those achieved with serum specimens.
TL;DR: The EIA was used to detect TB infection by incubating whole blood overnight with human, avian, and bovine tuberculin purified protein derivatives (PPDs), as well as positive (mitogen)- and negative-control preparations and was highly reproducible and specific for native IFN-γ.
Abstract: A sensitive two-step simultaneous enzyme immunoassay (EIA) for human gamma interferon (IFN-γ) has been developed and used as an in vitro test for human tuberculosis (TB) in comparison with tuberculin skin testing. The EIA was shown to be highly sensitive, detecting less than 0.5 IU of recombinant human IFN-γ per ml within a linear detection range of 0.5 to 150 IU/ml. The assay was highly reproducible and specific for native IFN-γ. In addition, the assay detected chimpanzee, orangutan, gibbon, and squirrel monkey IFN-γs. Cross-reactions with other human cytokines or with IFN-γs derived from mice, cattle, or Old World monkeys were not evident. The assay was used to detect TB infection by incubating whole blood overnight with human, avian, and bovine tuberculin purified protein derivatives (PPDs), as well as positive (mitogen)- and negative-control preparations. The levels of IFN-γ in plasma supernatants were then determined. Blood from 10 tuberculin skin test-positive individuals responded predominantly to the human tuberculin PPD antigen and to a lesser extent to bovine and avian PPD antigens. By contrast, blood from 10 skin test-negative individuals showed minimal responses or no response to any of the tuberculin PPDs. Detectable levels of IFN-γ were present in all blood samples stimulated with mitogen. In vivo tuberculin reactivity was correlated with IFN-γ responsiveness in vitro. These results support the further study of the blood culture–IFN-γ EIA system as an alternative to skin testing for the detection of human TB infection.
TL;DR: This work represents the most complete and integrated data set of PorB VR sequences and MAb reactivities of serogroup B and C meningococci produced to date and enabled the expansion of the panel of MAbs currently available for serotyping, by including MAbs of previously undetermined specificities.
Abstract: The current serological typing scheme for Neisseria meningitidis is not comprehensive; a proportion of isolates are not serotypeable. DNA sequence analysis and predicted amino acid sequences were used to characterize the structures of variable-region (VR) epitopes on N. meningitidis PorB proteins (PorB VR typing). Twenty-six porB gene sequences were obtained from GenBank and aligned with 41 new sequences. Primary amino acid structures predicted from those genes were grouped into 30 VR families of related variants that displayed at least 60% similarity. We correlated VR families with monoclonal antibody (MAb) reactivities, establishing a relationship between VR families and epitope locations for 15 serotype-defining MAbs. The current panel of serotype-defining MAbs underestimates by at least 50% the PorB VR variability because reagents for several major VR families are lacking or because a number of VR variants within some families are not recognized by serotype-defining MAbs. These difficulties, also reported for serosubtyping based on the PorA protein, are shown as inconsistent results between serological and sequence analyses, leading to inaccurate strain identification and incomplete epidemiological data. The information from this study enabled the expansion of the panel of MAbs currently available for serotyping, by including MAbs of previously undetermined specificities. Use of the expanded serotype panel enabled us to improve the sensitivity of serotyping by resolving a number of formerly nonserotypeable strains. In most cases, this information can be used to predict the VR family placement of unknown PorB proteins without sequencing the entire porB gene. PorB VR typing complements serotyping, and a combination of both techniques may be used for full characterization of meningococcal strains. The present work represents the most complete and integrated data set of PorB VR sequences and MAb reactivities of serogroup B and C meningococci produced to date.
TL;DR: The assigned IgG antibody values for most types were confirmed by a different method and extended antibody assignments to several additional types and large differences were found between the assigned values and those obtained by the mHSA ELISA for types 3 and 19F.
Abstract: An enzyme-linked immunosorbent assay (ELISA) and the antibody concentrations assigned to different pneumococcal capsular polysaccharide types were used to estimate concentrations of antibody to additional pneumococcal types in reference serum 89SF and to confirm assigned antibody values. This was possible because the slopes of curves of antibody binding to all polysaccharide types evaluated (1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) were similar. The point estimates for total anti-pneumococcal antibody and immunoglobulin G (IgG) antibody determined by cross-standardization by an ELISA based on use of methylated human serum albumin (mHSA) to improve the efficiency of polysaccharide binding to the ELISA plate differed by less than 40% from those reported by Quataert et al. (Clin. Diagn. Lab. Immunol. 2:590–597, 1995) for types 1, 4, 6B, 7F, 9V, 14, 18C, and 23F. However, large differences were found between the assigned values and those obtained by our mHSA ELISA for types 3 and 19F. The mHSA ELISA and the direct polysaccharide coat ELISA may not measure antibodies to the same epitopes on polysaccharides of types 3 and 19F. The functional importance of these different antibody specificities is being investigated. We have thus confirmed the assigned IgG antibody values for most types by a different method and have extended antibody assignments to several additional types.
TL;DR: All ELISA tests are applicable as an additional diagnostic tool in cases of abortion in cattle and for monitoring of congenitally infected calves and for herd screening, the lysate-based ELISAs appear to be more adequate because of their higher sensitivities.
Abstract: The performance of three enzyme-linked immunosorbent assays (ELISA) for detection of antibodies to Neospora caninum in bovine sera was evaluated by using various categories of sera. Two commercial ELISA methods, one based on chemically fixed intact tachyzoites and one based on a sonicate lysate of whole tachyzoites, were compared with an in-house ELISA based on a detergent lysate of whole tachyzoites. A brief description of the development of the latter ELISA is also given. There was good agreement among all three tests with regard to postabortion sera. By using acute-phase abortion sera from cows with confirmed N. caninum-induced and non-N. caninum-induced abortions, satisfactory levels of sensitivity and specificity were calculated for all tests. In addition, similar test results were obtained with postpartum samples from dams and calves. However, considerable differences were found between test results of sequential samples and cross-sectional and total-herd samples. Apparently, these discrepancies were due to different sensitivities of the tests for detection of low antibody levels in chronically infected animals. It is suggested that these differences were primarily due to the use of different antigens and different test sample dilutions. It is concluded that all tests are applicable as an additional diagnostic tool in cases of abortion in cattle and for monitoring of congenitally infected calves. For herd screening, the lysate-based ELISAs appear to be more adequate because of their higher sensitivities. Neoporosis is a newly recognized protozoan disease. The etiological agent was first isolated from a paralyzed dog and was named Neospora caninum (11). Later it was discovered that Neospora-like protozoa may cause abortion in cattle (27). The bovine and the canine parasite were shown to be identical
TL;DR: With the first serum sample of the pair of serum samples, the ELISA was able to diagnose significantly more cases of dengue than the HAI assay, and the IgG capture ELISA could be used to distinguish between primary and secondary infection.
Abstract: A commercially available capture enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulin M (IgM) and IgG antibodies produced during dengue infection (PanBio Dengue Duo) was evaluated with paired serum specimens from 176 patients. Diagnosis was based on a hemagglutination inhibition (HAI) assay, with patients having either primary dengue (n 5 90), secondary dengue (n 5 58), or no dengue (n 5 28) infection. The combined use of IgM and IgG (sensitivity, 99%; specificity, 96%) was superior to the use of IgM alone (sensitivity, 88%; specificity, 96%) or IgG alone (sensitivity, 85%; specificity, 96%). Furthermore, with the first serum sample of the pair of serum samples, the ELISA was able to diagnose significantly more cases of dengue than the HAI assay (55% versus 14%). The results of the IgG capture ELISA gave a significant correlation with those of the HAI assay (r 5 0.91; P < 0.0001), and the IgG capture ELISA could be used to distinguish between primary and secondary infection. The best distinction was observed when an IgG cutoff ratio of 3.0 was used, with 88% of primary infections and 98% of secondary infections being correctly classified. This ELISA should prove to be useful in the clinical diagnosis of dengue infection. Dengue is the most important mosquito-borne disease in the world in terms of morbidity, mortality, and economic costs, with an estimated 100 million cases per year (9). Serology is useful in the diagnosis of dengue infections and in differentiating between primary and secondary infections (3, 4, 7). Patients with a primary infection produce an immunoglobulin M (IgM) response to dengue virus 3 to 5 days after the onset of fever, and the IgM titer continues to rise for 1 to 3 weeks and is detectable for up to 6 months. Anti-dengue virus IgG antibodies are produced approximately 2 weeks after infection and are maintained for life, although at a hemagglutination inhibition (HAI) assay titer of #1:640 (3, 5). In contrast, during secondary infection IgM may take a long time to be detected or may be undetectable, while the IgG titer rises rapidly from 1 to 2 days after the onset of symptoms (3, 4). The HAI assay titer rises to $1:2,560, and these levels persist for 30 to 40 days before returning to levels of #1:640 (3). Traditionally, HAI assays have been used for the diagnosis of dengue. The HAI assay requires paired serum specimens collected at least 7 days apart and is considered positive if a fourfold or greater increase in antibody titer is demonstrated (2). Furthermore, a single serum sample demonstrating a titer of $1:2,560 is diagnostic of a secondary dengue infection (11). Doubts concerning the general applicability of the HAI assay have been raised due to variations in the potencies of the hemagglutinins made in different laboratories. Commercially available enzyme-linked immunosorbent assays (ELISAs) offer improvements over the HAI assay for the serological diagnosis of dengue infections. ELISAs reduce interlaboratory variation in dengue serology through the use of a standard calibrator serum sample. Unlike for HAI assays, pretreatment of sera (i.e., acetone extraction) is not required, and a diagnosis can be made from the results for a single serum sample. Differentiation between primary and secondary infections may also be made with a single dilution of serum rather than with a series of dilutions. A commercially available capture ELISA for the detection of IgM and IgG antibodies during dengue infection has recently become available (PanBio Dengue Duo). In this study, the Dengue Duo ELISA has been compared to the HAI assay by using paired serum specimens from patients with or without dengue infection.
TL;DR: The general profiles of expression of the five cytokine genes were similar but not identical, suggesting some shared regulatory mechanisms, and the various stimuli caused induction of all cytokines with quantitative, not qualitative differences.
Abstract: Sequential gene expression of two type 1 cytokines (interleukin 2 [IL-2] and gamma interferon), one type 2 cytokine (IL-10), two monokines (IL-6 and tumor necrosis factor alpha), and one cytokine receptor (IL-2 receptor [IL-2R]) in normal human peripheral blood mononuclear cells (PBMC) following in vitro stimulation was investigated by reverse transcription-PCR methods. Two stimuli were utilized: phytohemagglutinin (PHA), which acts on the CD2 molecule and T-cell receptors, and anti-CD3 monoclonal antibody, which acts on the CD3 molecule and on T-cell receptors. Increased expression of all studied genes occurred between 1 and 4 hours after stimulation, except for that of the gene encoding IL-10, which was delayed. Expression of all but one of the genes was transient, with a maximal mRNA accumulation at about 8 h on average. IL-2R mRNA expression was an exception, showing a prolonged increase (72 h). The general profiles of expression of the five cytokine genes were similar but not identical, suggesting some shared regulatory mechanisms. When responses to four additional stimuli (pokeweed mitogen, Candida albicans, and IL-2 at high and low doses) were compared, similar profiles of cytokine gene expression were found. Thus, the various stimuli caused induction of all cytokines with quantitative, not qualitative, differences. Altogether, the present data are useful for defining the kinetics of gene expression for key cytokines in response to standard immune-cell stimuli.