bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential effects of interleukin-2 and interleukin-4 on immunomodulatory role of platelet-activating factor in human B cells
Authors: Patke C L , Green C G , Shearer W T
Published: January 2020

Platelet-activating factor (PAF), a naturally occurring phospholipid cytokine, is a potent mediator of allergic and inflammatory reactions, as well as a modulator of immune responses. In the present study we showed that PAF is involved in early B-cell activation, as demonstrated by the increased cyclic AMP (cAMP) generation by PAF in a time- and dose-dependent manner in anti-mu antibody- plus B-cell growth factor-activated normal human peripheral blood B lymphocytes. PAF also regulated differentiation by causing a biphasic response on immunoglobulin M (IgM) production with an inhibitory signal generated at 10(-6) M and a stimulatory signal generated at 10(-8) to 10(-10) M. PAF enhanced IgA secretion. The regulation exerted by PAF was shown to be specific because the addition of the PAR antagonist CV-3988 abrogated these effects and the inactive form of PAF, lyso-PAF, induced neither cAMP generation nor immunoglobulin secretion in normal human B cells. Other cytokines, interleukin-2 (IL-2) and IL-4, potent mediators of the immune response, were unable to elicit a cAMP response in B cells. However, the addition of PAF (10(-6) M) with wither IL-2 or IL-4 enhanced cAMP production above the levels enhanced by the addition of PAF alone. IL-2 or IL-4, individually, stimulated IgM production, yet costimulation with PAF resulted in a differential effect between IL-2 and IL-4. PAF down-regulated the IL-4-induced IgM secretion, whereas the IL-2-induced IgM secretion was enhanced. The presence of CV-3988 returned all valued to those obtained with IL-2 or IL-4 alone, demonstrating the specificity of PAF. These data suggest that PAF is an important B-cell immunomodulator which can interact with other leukocyte cell mediators.
Endotoxin levels in sera of elderly individuals
Authors: Goto T , Edén S , Nordenstam G , Sundh V , Svanborg-Edén C , Mattsby-Baltzer I
Published: January 2020

The endotoxin levels in serum of 377 72-year-old individuals were quantitated. The study population was a representative sample of this age group and was participating in a general study of health and disease among the elderly in Göteborg, Sweden. The endotoxin levels in serum were quantified by the chromogenic Limulus amebocyte lysate assay and were correlated with the health status and laboratory findings for each individual. The mean endotoxin levels (+/- 1 standard deviation) in men and women, when excluding four outliers, were 6.6 +/- 3.8 and 6.9 +/- 3.8 pg/ml, respectively. All included, 21.5% of individuals had endotoxin levels equal to or above the sensitivity limit of 10 pg/ml. Strong positive correlations were found between endotoxin levels and plasma triglycerides (P > 0.995) and between endotoxin levels and serum protein (P > 0.9875). The endotoxin activity also correlated with mean corpuscular hemoglobin concentration (P < 0.005, negative correlation), body mass index (P > 0.9875), and decreased appetite (P > 0.9875). A high alcohol consumption was associated with increased endotoxin levels (P = 0.995). There are no previous studies which examine endotoxin levels in serum samples from individuals representative of the population. This study showed that elderly individuals had the same mean level of endotoxin as has been found in other age groups. The increased endotoxin levels seen in heavy drinkers may be explained by a decreased ability of the liver to remove endotoxin. The correlations found between endotoxin and triglycerides, protein, mean corpuscular hemoglobin concentration, decreased appetite, and body mass index are discussed.
Prevalence in Cryptococcus neoformans strains of a polysaccharide epitope which can elicit protective antibodies
Authors: Cleare W , Mukherjee S , Spitzer E D , Casadevall A
Published: January 2020

Monoclonal antibody (MAb) 2H1 binds to an epitope in the capsule of Cryptococcus neoformans that can elicit protective antibodies. The binding of MAb 2H1 to C. neoformans strains was studied by agglutination, immunofluorescence, and phagocytosis assays. The MAb 2H1 epitope was present in all 21 isolates studied, including those recovered from patients with recurrent infections.
A simple method for the propagation of cervical lymphocytes
Authors: Moscicki A B , Hunter S D , Garland S , Quinn M , Crowe S M , Shortman K , Stites D
Published: January 2020

Local immune function is most likely a key influence in the establishment of human papillomavirus infections and its subsequent disease. Unfortunately, little information is known about local cervical immunity, and even less is known about human papillomavirus immunoreactivity. In addition, studies of local immunoreactivity have been hampered by the technical difficulty in obtaining cervical lymphocytes. The objective of the present study was to develop a simple method for the propagation of cervical lymphocytes from biopsy-size specimens. Cervical tissue was obtained from women undergoing a hysterectomy. Cervical samples measuring approximately 3 by 5 by 2 mm were minced and divided into two portions. One portion was digested by standard digestion methods and density gradient lymphocyte separation. The sample was then immunocharacterized for CD4 and CD8 cells by flow cytometry. The other portion was minced into 1-mm3 sections, and each section was placed into a separate well with tissue culture medium and interleukin-2. Lymphocyte counts and immunophenotypic analysis were performed after 18 to 20 days in culture. After 18 to 20 days in culture, the analysis demonstrated that this method of direct lymphocyte culture from a biopsy specimen yielded approximately 1 x 10(6) to 5 x 10(6) lymphocytes. Immunophenotypic studies of the digested sample at day 0 revealed CD4-to-CD8 ratios of between 0.7:1 and 3.5:1, and at days 18 to 20 they revealed ratios of between 2.3:1 and 98:1. In summary, we developed a simple technique for propagating cervical lymphocytes from small tissue samples for the study of the local immune response. Studies are under way to optimize lymphocyte growth and to preserve CD8 populations.
Immunoglobulin gene sequence analysis to further assess B-cell origin of multiple myeloma
Authors: Biggs D D , Kraj P , Goldman J , Jefferies L , Carchidi C , Anderson K , Silberstein L E
Published: January 2020

To further characterize the B-cell origin of multiple myeloma, our laboratory performed immunoglobulin gene sequence analyses of four cases of myeloma (three immunoglobulin A and one immunoglobulin G). Three tumors expressed VH3 genes and one expressed a VH1 gene, while the light chains included two V lambda and one V kappa III; one light chain was not isolated. The closest homology to published germ line genes ranged from 91 to 97%. In two cases, the expressed VH genes were compared with the putative germ line precursor VH genes isolated from autologous granulocyte DNA and appeared to have mutated randomly from the germ line gene. By sequencing multiple clonal isolates from each tumor sample, we found no evidence for ongoing mutation in three cases; in one case, however, clonotypic heterogeneity was evident. The analysis of DH- and JH-region genes revealed (i) limited or absent N nucleotide insertions (two of four cases), (ii) the presence of a DH-JH junction resulting from sequence overlap between the DH and JH genes (one of four cases), (iii) the absence of somatic mutations (two of four cases), and (iv) restricted JH gene usage of a JH6 polymorphism (three of four cases). These analyses of DH and JH genes suggest that multiple myeloma, similar to what has been proposed for chronic lymphocytic leukemia, may derive from B cells which have rearranged during fetal development rather than during adult life.
Enhancement of natural killer cell activity in human immunodeficiency virus-infected subjects by in vitro treatment with biologic response modifier OK-432
Authors: Huang X L , Fan Z , Murayama T , Rinaldo C
Published: January 2020

A decrease in natural killer (NK) cell function has been related to the progression of human immunodeficiency virus (HIV) infection. In the present study, we assessed the ability of a streptococcus-derived biologic response modifier, OK-432, to augment NK lysis of uninfected K562 and U937 cells and HIV-infected U937 cells by peripheral blood mononuclear cells (PBMC) from HIV-seropositive homosexual men. Optimal two- to fourfold increases in lysis of the three targets were observed after pretreatment of PBMC from HIV-negative subjects for 4 h with 2 micrograms of OK-432 per ml. This effect was related primarily to gamma interferon (IFN-gamma) production induced by OK-432 and was not linked to production of tumor necrosis factors alpha and beta or to monocytes in the cultures. The enhancing effect of OK-432 on NK cell function was diminished but still evident in PBMC from subjects with relatively early-phase (< 3-year) HIV infection and high CD4+ cell counts and was lower in subjects with longer-term HIV infection (> 3 years), in association with reduced production of IFN-gamma. Augmentation of NK cell activity in HIV-infected men by OK-432 was comparable to that induced by treatment of cells with 1,000 U of IFN-alpha or interleukin 2 per ml. The data suggest that the NK cell-enhancing effects of OK-432 are at least in part mediated by IFN-gamma and that OK-432 may be effective in treatment of patients with early-phase HIV infection.
Assignment of Neisseria meningitidis serogroup A and C class-specific anticapsular antibody concentrations to the new standard reference serum CDC1992
Authors: Holder P K , Maslanka S E , Pais L B , Dykes J , Plikaytis B D , Carlone G M
Published: January 2020

A new standard meningococcal reference serum designated CDC1992 was prepared to replace meningococcal reference sera ECG and PB-2, which are not available in sufficient quantities for continued use as primary reference sera. CDC1992 was prepared from 14 healthy adult volunteers who underwent plasmapheresis 4 to 12 weeks postvaccination with a single dose of a Neisseria meningitidis quadrivalent polysaccharide vaccine. Total and/or class-specific meningococcal serogroup A and C anticapsular antibody concentrations (in micrograms per milliliter) were assigned to CDC1992 by using homologous and heterologous enzyme-linked immunosorbent assay (ELISA) formats. The reference serum ECG was used as a reference standard to assign total anticapsular antibody concentrations to CDC1992 by a homologous ELISA format. A heterologous ELISA format, with the Haemophilus influenzae type b standard reference serum FDA 1983, was used to assign total and class-specific antibody concentrations to CDC1992. Alkaline phosphatase-labeled mouse anti-human monoclonal antibody conjugates were used as secondary antibodies in both ELISA formats. The total, immunoglobulin G (IgG), IgA, and IgM antibody concentrations, assigned to CDC1992 for serogroup A were 135.8, 91.8, 20.1, and 23.9 micrograms/ml, respectively, and those for serogroup C were 32.0, 24.1, 5.9, and 2.0 micrograms/ml, respectively. Meningococcal serogroup A and C antibody concentrations were in good agreement when homologous and heterologous ELISA format results were compared. Total and class-specific serogroup A and C antibody concentrations were determined in six adult quality control serum samples from the Centers for Disease Control and Prevention by using the homologous ELISA and our assigned antibody concentrations for CDC1992. Antibody concentrations in reference sera ECG and PB-2 were measured in order to provide a historical link to previous studies. The general acceptance of CDC1992 as the standard reference serum and the assigned antibody concentrations will allow investigators to compare antibody levels in serum to those in a single reference preparation.
Monoclonal antibodies to Legionella Mip proteins recognize genus- and species-specific epitopes
Authors: Helbig J H , Ludwig B , Lück P C , Groh A , Witzleb W , Hacker J
Published: January 2020

Monoclonal antibodies (MAbs) against the virulence-associated Mip protein of Legionella spp. were raised by immunizing BALB/c mice with (i) Legionella pneumophila, (ii) Legionella micdadei, and (iii) purified recombinant native Mip protein cloned from L. pneumophila Philadelphia 1. Following screening of seeded wells by immunoblot analysis with homologous antigens, eight Mip-specific MAbs were found. These MAbs were chosen to investigate the antigenic diversity of Mip proteins in the genus Legionella. Mip was detected in 82 Legionella strains representing all 34 species tested. One of these MAbs, obtained from immunization with L. micdadei, recognized an epitope common to all Legionella species tested by immunoblot analysis. Another MAb was discovered to be specific for the Mip protein of L. pneumophila. The remaining six MAbs recognized 18 to 79% of Legionella species included in this study. By making use of the MAbs introduced in this study, it could be shown that, based on Mip protein epitope expression, Legionella species can be divided into at least six antigenetically distinct groups. As demonstrated by 43 L. pneumophila strains representing all serogroups, no antigenic diversity of Mip proteins was found for this species. In addition, 18 non-Legionella species, including Chlamydia trachomatis, Neisseria meningitidis, Pseudomonas aeruginosa, and Saccharomyces cerevisiae, all of which are known to carry genes homologous to the Legionella mip genes, were reacted against all eight MAbs. No cross-reactivity was detectable in any of those strains.
A novel method to chemically immobilize antibody on nylon and its application to the rapid and differential detection of two Vibrio parahaemolyticus toxins in a modified enzyme-linked immunosorbent assay
Authors: Honda T , Miwatani T , Yabushita Y , Koike N , Okada K
Published: January 2020

A new method of chemically immobilizing antibody on nylon was developed. The method consists of serial treatments with HCl, polyethylene imine, and maleic anhydride methylvinyl ether copolymer, which resulted in the stable immobilization of sufficient amounts of antibodies on nylon. This principle was used to differentially detect two immunologically related but nonidentical hemolysins (thermostable direct hemolysin [TDH] and TDH-related hemolysin [TRH]) of Vibrio parahaemolyticus in a modified enzyme-linked immunosorbent assay with antibodies immobilized on nylon slips (NSIT). The results (dark purple color on nylon slips) were easily evaluated by the naked eye. The results with NSIT were compatible with those obtained by using DNA probes or a conventional bacterial culture test, not only with cultured specimens but also with clinical specimens (diarrheal stool samples). Furthermore, the NSIT differentially detected TDH and TRH in a single test. The antibody immobilization method developed here is applicable to various immunological detection methods and may improve their sensitivity and specificity.
Studies of the specificity and cross-reactions of antibodies to lipid A found in juvenile arthritis
Authors: Miller J J , Olds L C
Published: January 2020

This work was started to determine whether the immunoglobulin G (IgG) reactions with monophosphoryl lipid A (MPL) found in children with arthritis were due to contaminants, a specific site on lipid A, or polyspecific binding. Different lots of MPL were examined by electrophoresis and immunoblot. Competitive inhibition of enzyme-linked immunosorbent assays (ELISAs) by analogs of MPL and biologic materials of clinical interest was used to determine the specificity of the binding site and potential cross-reactions. IgG in all patient sera tested reacted with a single band just < 6.5 kDa on immunoblots of all lots of MPL tested. The ELISAs were inhibited best by analogs of lipid A with an exposed diglucosamine core and intact polar domains. The anti-MPL was also inhibited by fetal bovine collagen types I and II and in some instances by cardiolipin, but not by keratan sulfate, proteoglycan, or DnaK heat shock protein. Lot variation was a persistent technical problem, but no protein contaminant could be demonstrated in any lot. The ELISA and immunoblot results confirmed each other. Immunoblots detected a single band of MPL reactive with IgG. This antibody remains of interest because of its disease association and correlations and because it cross-reacts with collagen and cardiolipin.
Detection and quantification of live, apoptotic, and necrotic human peripheral lymphocytes by single-laser flow cytometry
Authors: Liegler T J , Hyun W , Yen T S , Stites D P
Published: January 2020

Regulation of peripheral lymphocyte number involves a poorly understood balance between cell renewal and loss. Disrupting this balance leads to a large number of disease states. Methods which allow qualitative and quantitative measurements of cell viability are increasingly valuable to studies directed at revealing the mechanisms underlying apoptotic and necrotic cell death. Here, we have characterized a method using single-laser flow cytometry that differentiates and quantifies the relative number of live, apoptotic, and late-stage apoptotic and necrotic peripheral lymphocytes. Following in vitro gamma irradiation and staining with acridine orange in combination with ethidium bromide, three distinct populations were seen by bivariate analysis of green versus red fluorescence. The identity of each distinct fluorescent population (whether live, apoptotic, or necrotic) was determined by sorting and examination of cellular morphology by electron microscopy. This flow cytometric method is directly compared with the techniques of trypan blue exclusion and DNA fragmentation to quantify cell death following exposure to various doses of in vitro gamma irradiation and postirradiation incubation times. We extend our findings to illustrate the utility of this method beyond analyzing radiation-induced apoptotic peripheral blood mononuclear cells (PBMC); similar fluorescent patterns are shown for radiation- and corticosteroid-treated murine thymocytes, activated human PBMC, and PBMC from human immunodeficiency virus-infected individuals. Our results demonstrate that dual-parameter flow cytometric analysis of acridine orange-ethidium bromide-stained lymphocytes is overall a superior method with increased sensitivity, greater accuracy, and decreased subjectivity in comparison with the other methods tested. By using standard laser and filter settings commonly available to flow cytometric laboratories, this method allows rapid measurement of a large number of cells from a heterogeneous sample.
Cell response to a salt-extractable and sonicated Brucella melitensis 16M antigen in human brucellosis
Authors: Moreno-Lafont M C , López-Merino A , López-Santiago R
Published: January 2020

We compared the immunological responses of leukocytes taken from healthy negative controls, laboratory workers immunized with the phenol-insoluble French vaccine against brucellosis, patients acutely ill with brucellosis, and patients chronically infected with Brucella melitensis. A salt-extractable antigen (protein-rich but with traces of lipopolysaccharide) and a sonicated suspension from B. melitensis 16M were used as antigens for in vitro lymphocyte proliferation test. Quantitation of T cells showed that the ratio of CD4+/CD8+ cells decreased as the condition of the patient deteriorated. An assay to quantitate the cell-mediated immunity showed that the activities of mononuclear cells stimulated with concanavalin A increased as the disease progressed as well.
Oral fluid as a specimen for detection and confirmation of antibodies to human immunodeficiency virus type 1
Authors: Granade T C , Phillips S K , Parekh B , Pau C P , George J R
Published: January 2020

Paired serum and oral fluid specimens (n = 287) were collected with the Omni-Sal device and were assayed for the presence of antibodies to human immunodeficiency virus type 1 (HIV-1). Enzyme immunoassays (EIAs)--Abbott 3A11, an Organon Teknika Corporation research-use-only test, and the Murex GACELISA--were used per the manufacturers' inserts or were modified slightly to accommodate the oral fluid specimens. Compared with serum Western blot (immunoblot) results, each EIA had a sensitivity of 100% and the specificities were 89.6% for the Abbott 3A11 EIA, 96.5% for the GACELISA, and 97.8% for the Organon Teknika Corporation EIA. Specificities based on specimens that were repeatedly reactive were 99.3% for all EIAs. A miniaturized Western blot technique used for confirmatory testing of both the serum and oral fluid specimens found 149 of the 287 samples to be HIV-1 antibody positive in both sample types. The Western blot banding patterns observed for the serum and oral fluid specimens were essentially identical. Immunoglobulin G concentrations were determined for all oral fluid specimens and ranged from < 0.5 to > 40.0 micrograms/ml. Immunoglobulin G concentrations did not correlate with the ability of any of the EIAs to detect HIV-1-specific antibody or with the ability of the modified Western blot to detect HIV-1 protein-specific antibodies.
Impairment of monocytic function after influenza virus infection
Authors: Louie M , Yoo J , Moran T , Mayer L , Sperber K
Published: January 2020

In order to analyze the immunosuppression associated with influenza virus infection, we investigated monocytic function in macrophage hybridoma cell lines 5 weeks after infection with two strains of influenza virus. Clones 30 and 63, chosen for stability in long-term culture, were infected with two strains of influenza virus, X-31 and PR-8. Uniform infection of both cell lines was confirmed by intracytoplasmic staining with the antihemagglutinin strain-specific monoclonal antibodies PY 102 and PY 206. One week after infection, clones 30 and 63 lost their ability to stimulate tetanus toxoid-specific major histocompatibility complex (MHC)-matched responder T cells. Coincident with the inability to stimulate MHC-matched T cells, there was diminished surface expression of class II MHC antigens and LFA-1-alpha and LFA-3 compared with that in uninfected cells: DR, 2.5 versus 10.6% (mean channel 0.3 versus 1.5); DQ, 1.6 versus 15.6% (mean channel 0.3 versus 3.0); DP, 5.0 versus 30.9% (mean channel 0.3 versus 2.0). LFA-1-alpha expression was reduced (13.1 versus 20.0%; mean channel 1.5 versus 2.0) while LFA-3 expression remained the same (22.2 versus 324%; mean channel 3.0 versus 3.3). Class I MHC surface antigen expression was unaltered. Cytokine secretion was also perturbed, as interleukin 1-alpha (IL-1-alpha) and IL-1-beta production was lost 1 week after infection. Production of IL-12 and IL-10 was unchanged, while IL-6 production was increased. The viability of the T cells cocultured with 63Flu was unaltered, demonstrating that the inability of the MHC-restricted T cells to proliferate in response to tetanus toxoid was not due to a toxic effect of 63Flu. Interestingly, other accessory functions, including the ability to support mitogen- and anti-CD3-mediated T-cell proliferation, remained intact. These data suggest that alteration of macrophage function relating to viral infection occurs at multiple levels and may contribute to the immunosuppression observed following influenza virus infection.
Age-dependent humoral responses of children to mycobacterial antigens
Authors: Fairchok M P , Rouse J H , Morris S L
Published: January 2020

In the United States, disseminated infection with environmental mycobacteria, including the Mycobacterium avium complex, is the most common opportunistic bacterial infection seen in AIDS patients. However, the source and relative degree of exposure to environmental mycobacteria during childhood are unknown. To examine the age-related exposure to mycobacteria, we obtained serum samples from 150 children ranging in age from 6 months to 18 years. Each sample was tested against both M. avium (serovar 1) sonic extracts and mycobacterial lipoarabinomannan, using an enzyme-linked immunosorbent assay (ELISA). All serum samples were also subjected to immunoblot analysis with the sonic extract antigen. These studies established that elevated ELISA values (P < 0.0001) and increased immunoblot reactivity (P < 0.0001) against mycobacterial antigens were both associated with increasing age. The seroreactivity differences were most striking when comparing the age groups of children below the age of 6 with the older age groups. Our results suggest that the development of humoral immune responses to mycobacterial antigens in children correlates with increasing age and that there may be an environmental factor predisposing to mycobacterial exposure which is related to advancing age.
Early appearance of antibodies to simian immunodeficiency virus in saliva and serum of infected macaques
Authors: Meiller T F , Narayan O , Joag S V , Overholser C D
Published: January 2020

Simian immunodeficiency virus (SIV) infection in macaques is an important animal model for human immunodeficiency virus infection in humans. This study evaluated the temporal development of antibodies to SIV in the parotid saliva of macaques inoculated with the virus and compared these findings with the development of antibodies to SIV in the animals' sera. Three animals (ages, 14, 18, and 18 years) were inoculated with the macrophagetropic strain SIVmac239. Prior to inoculation and at consecutive weekly intervals during a four-week period following the initial virus inoculations, parotid saliva and serum were collected from each animal. A fourth animal (age, 9 years) served as a negative control, and the fifth and sixth animals (ages, 2 and 22 years) served as positive controls (6 and 18 months postinoculation, respectively) with SIVmac239. Saliva and serum samples were reacted against SIV antigen in Western blots (immunoblots) prepared in the standard fashion to determine the presence of antibodies. The reactions of these antigen-antibody complexes with biotinylated anti-human immunoglobulin A (IgA), IgM, and IgG and biotinylated anti-human secretory component (SC) determined the class of antibody present or the presence of SC in the original parotid saliva or serum samples. In infected animals, the IgM to SIV was detectable in serum and saliva at 13 days, and antiviral IgA and IgG in serum and saliva were detectable at 20 to 27 days postinoculation. The antibody to SC reacted to saliva from only two animals at 20 and 27 days, and long-term positive controls were positive for SC in saliva, indicating that either secretory IgA or secretory IgM was present in these samples. Antibodies to SIVmac239 antigens have therefore been detected in saliva as early as 13 days postinfection. Saliva may be as useful as serum as a diagnostic specimen and/or disease-monitoring method in this important animal model.
Evidence obtained with monoclonal antibodies that O antigen is the major antigen responsible for the cross-reactivities between serotypes 4 and 7 of Actinobacillus (Haemophilus) pleuropneumoniae
Authors: Rodríguez Barbosa J I , Gutiérrez Martín C B , Tascón R I , Suárez J , Rodríguez Ferri E F
Published: January 2020

Monoclonal antibodies (MAbs) against Actinobacillus (Haemophilus) pleuropneumoniae serotype 4 (reference strain M62 and field isolate F6) were produced and characterized. Three hybridoma clones were raised against strain M62, and 13 were raised against strain F6. The predominant antibody class was immunoglobulin M (IgM), although IgG2a and IgG2b were also obtained. Three of the MAbs produced to field isolate F6 (5C5, 1E10, and 5H7) did not recognize the reference strain of serotype 4, another (6F7) was reactive with both reference strains of serotypes 4 and 7, and the remaining 12 MAbs reacted only with the reference strain of the homologous serotype. All epitopes recognized by MAbs, except for one (6F7), were sensitive to periodic acid oxidation, and all of them were resistant to boiling in the presence of sodium dodecyl sulfate and reducing conditions, as evidenced by immunodot. Enhanced chemioluminiscence-immunoblot assays revealed that 10 MAbs (3E12, 5B8, 7C3, 6F7, 7F5, 7E6, 5G4, 4F1, 7E10, and 4B8) recognized a ladder-like banding pattern, which is in accordance with the O side chain antigen of lipopolysaccharide (LPS), while the remaining 6 MAbs (5C5, 5H7, 1E10, 6D11, 6B4, and 5E4) blotted with high-molecular-weight regions composed of a single banding pattern. The suitability of MAbs for serotyping of 78 field isolates was also examined. A high correlation was found between the results previously established by indirect hemagglutination with polyclonal sera and those obtained by enzyme-linked immunosorbent assay with MAbs. According to the different immunoreactivity of MAbs, three groups were established: group I (MAbs 3E12, 5B8, 7C3, 6F7, and 7F5), group II (MAbs 7E6, 5G4, 4F1, 7E10, and 4B8), and group III (MAbs 5C5, 5H7, and 1E10). MAbs 6D11, 6B4, and 5E4 could not be included in any of the described above. At least six different immunodominant epitopes on the O antigen of the A. pleuropneumoniae serotype 4 LPS were identified. Finally, the implications of the effect of the O antigen of LPS in cross-reactions between serotypes 4 and 7 are clearly evidenced.
Tumor necrosis factor alpha upregulates human microglial cell production of interleukin-10 in vitro
Authors: Sheng W S , Hu S , Kravitz F H , Peterson P K , Chao C C
Published: January 2020

Interleukin (IL)-10 appears to play an important regulatory role in the systemic inflammatory response; however, production of IL-10 within the human central nervous system has not been described. Using cultures of human fetal microglial cells, the resident macrophages of the brain, we investigated the production and regulation of bioactive IL-10. Lipopolysaccharide stimulated acute release of tumor necrosis factor (TNF)-alpha (peak by 8 h) and delayed production of IL-10 (over a 48-h period) in microglial cell cultures. Treatment of microglial cell cultures with TNF-alpha and IL-6 resulted in a dose-dependent release of IL-10. These cytokines also induced expression of IL-10 mRNA. Treatment of microglial cell cultures with IL-10 markedly inhibited TNF-alpha and IL-6 production. These findings suggest that during inflammation within the brain, acute release of TNF-alpha and IL-6 by activated microglia could promote subsequent release of IL-10, which functions to minimize the potential neurotoxic effects of proinflammatory cytokines.
Differences between Taylorella equigenitalis strains in their invasion of and replication in cultured cells
Authors: Bleumink-Pluym N M , ter Laak E A , Houwers D J , van der Zeijst B A
Published: January 2020

The ability of Taylorella equigenitalis, the causative agent of contagious equine metritis, to invade and replicate in equine derm cells was studied. The kinetics of invasion and replication were determined for four T. equigenitalis strains. On the basis of these experiments, a simpler assay in which the invasive as well as the replicative properties of a particular strain could be determined was developed. This assay was used to characterize 32 strains, which had previously been typed by field inversion gel electrophoresis of genomic restriction fragments. The invasiveness of T. equigenitalis strains ranged from 3 to 0.015 bacteria per cell and seemed to be associated with the contagiousness of the infection. The replication index (number of intracellular bacteria per cell at 24 h after inoculation divided by the number of intracellular bacteria per cell at 4 h after inoculation) varied from 1 to 857 and seemed to be associated with the severity of the symptoms of contagious equine metritis. There was no association between the invasiveness and the replication index of the strains, nor was there an association of invasion and replication with field inversion gel electrophoresis grouping.
Evaluation of serological assays for identification of parvovirus B19 immunoglobulin M
Authors: Tolfvenstam T , Rudén U , Broliden K
Published: January 2020

Three different enzyme immunoassays (EIAs) (Parvoscan-B19, IBL parvovirus B19, and IDEIA parvovirus B19) and one immunofluorescence assay (Biotrin Parvo B19 IFA) were evaluated for detection of parvovirus B19 immunoglobulin M (IgM) antibodies in 203 clinical serum samples. An IgM antibody capture radioimmunoassay was used as a reference test. Serum specimens obtained from patients with clinical symptoms suggestive of parvovirus B19 infections were used to evaluate the sensitivities of the assays, which were shown to be comparable for the Biotrin IFA and IDEIA (97%) and lower for the other two EIAs (90%). In order to test the specificity of the assays, clinical serum samples with IgM antibodies against other viruses were examined, as well as sera with rheumatoid factor activity and sera from healthy pregnant women. The specificities of B19 IgM antibody detection were 96% for the Biotrin IFA, 96% for IDEIA, 90% for Parvoscan, and 88% for the IBL assay. These results show that all four assays can be recommended for diagnostic purposes, although false-positive results may be seen with other acute viral infections, healthy pregnant women, and rheumatoid factor-positive samples.
T-cell proliferative response to human papillomavirus type 16 peptides: relationship to cervical intraepithelial neoplasia
Authors: Nakagawa M , Stites D P , Farhat S , Judd A , Moscicki A B , Canchola A J , Hilton J F , Palefsky J M
Published: January 2020

The incidence of human papillomavirus (HPV)-related cervical intraepithelial neoplasia (CIN) and cervical cancer is increased with immunodeficiency, but the role of immune response, including cell-mediated immunity, in disease prevention is not well understood. In this study, T-cell proliferative responses to six synthetic peptides with predicted immunogenic determinants from the HPV-16 E4, E6, E7, and L1 open reading frames were analyzed in 22 sexually active women with new-onset CIN and 65 sexually active women without cervical disease, characterized by cytology, colposcopy, and HPV testing. T-cell proliferative responses were demonstrated to all six HPV-16 peptides. Although not statistically significant, rates of reactivity to E6 (24-45) were higher among sexually active women without disease (26%) than among women with current CIN (7%), as was the overall number of peptides stimulating a response. Women with CIN may not respond to selected HPV antigens as well as women without disease do.
Novel enzyme-linked immunosorbent assays for the detection of anti-Fc gamma receptor autoantibodies
Authors: Lamour A , LeCorre R , Jamin C , Soubrane C , Khayat D , Youinou P
Published: January 2020

There is a substantial interest in the role of antineutrophil antibodies since Fc gamma receptors (Fc gamma Rs) have been identified as the target for the majority of such autoantibodies. Antineutrophil antibodies have long been detected by an indirect immunofluorescence technique. Following optimization of the flow cytometric method of detection, we developed three enzyme-linked immunosorbent assays (ELISAs), each specific for autoantibodies against one of the three classes of human Fc gamma R. Fc gamma RI and Fc gamma RII were purified from cultured cells, and Fc gamma RIII was produced as a recombinant molecule. These were then used as capture agents in the respective ELISAs. When applied in parallel to a sizeable group of patients with primary Sjögren's syndrome, both methods established that anti-Fc gamma R autoantibodies were heterogeneous. This finding indicates that different populations of partly cross-reactive antibodies are detectable by these two methods.
Prevalence of neutralizing antibody to respiratory syncytial virus in sera from mothers and newborns residing in the Gambia and in The United States
Authors: Suara R O , Piedra P A , Glezen W P , Adegbola R A , Weber M , Mulholland E K , Greenwood B M , Whittle H
Published: January 2020

The prevalence of maternal respiratory syncytial virus (RSV)-neutralizing antibodies has been documented in developed countries, but there is little information from developing countries. We assessed the prevalence of RSV-neutralizing antibody in sera from Gambian women and their newborns and compared them with their American counterparts during a similar period. The geometric mean titers of maternal antibodies to RSV subgroup A in the two populations were similar, while titers of antibodies to RSV subgroup B in Gambian mothers were significantly higher (8.7 +/- 1.4 versus 7.9 +/- 1.3 [mean +/- standard deviation], P < 0.001). The titers of neutralizing antibody in newborns in both populations correlated with the neutralizing-antibody titers of their mothers. Thus, the status of neutralizing antibody to both major RSV subgroups was comparable among infants and mothers in a developing country, The Gambia, and those in a developed country, the United States.
Humoral antibody to Mobiluncus curtisii, a potential serological marker for bacterial vaginosis
Authors: Schwebke J R , Morgan S C , Hillier S L
Published: January 2020

While bacterial vaginosis (BV) is a polymicrobial syndrome, Mobiluncus spp. are the organisms most highly associated with this condition. It is possible that serum antibody to Mobiluncus spp. could be used as a serological marker for BV. Using immunofluorescence techniques, we studied the prevalence of antibody to M. curtisii among three cohorts-pregnant women, pediatric patients, and sexually inexperienced women. The prevalence of antibody in each of these three groups was 75, 6, and 0%, respectively. Of the three pediatric patients with antibody to Mobiluncus curtisii, two were neonates, and the only class of antibody detected was immunoglobulin G. Among the cohort of pregnant women, the presence of antibody could not be correlated with a clinical history of BV. Serum antibody to M. curtisii could be a useful serological marker for BV. The lack of correlation of antibody positivity to historical information regarding BV suggests that unrecognized or undiagnosed episodes of BV may be common.
Investigation of immunofluorescence cross-reactions against Trichinella spiralis by western blot (immunoblot) analysis
Authors: Robert F , Weil B , Kassis N , Dupouy-Camet J
Published: January 2020

Immunofluorescence cross-reactions in Trichinella spiralis serodiagnosis are sometimes difficult to identify. We compared the results of an indirect immunofluorescence assay and the profiles obtained by Western blot (immunoblot) analysis for three groups of patients: 10 T. spiralis-infected patients, 10 patients with autoimmune diseases, and 7 patients with parasitic diseases other than trichinellosis. The degree of immunofluorescence cross-reaction was variable. Western blotting allowed us to differentiate Trichinella infection from other parasitic diseases. In 3 of 10 serum samples from patients with autoimmune diseases, bands which had the same sizes as Trichinella bands were observed, and they could correspond to shared epitopes such as heat shock proteins.
Identification of serum and urine proteins responsible for enhanced pigment production by group B streptococci as amylases
Authors: Rosa-Fraile M , Sampedro A , Ruiz-Bravo A , Sanbonmatsu S , Gimenez-Gallego G
Published: January 2020

The serum and urine proteins responsible for enhanced pigment production in Streptococcus agalactiae in culture media were purified by chromatography and were identified as amylases by comparison of their amino acid composition with that calculated for proteins with known sequences. Similar pigment-enhancing activity was displayed by other amylases of nonanimal origin and by maltooligosaccharides.
Monoclonal antibodies to the enterotoxin of Bacteroides fragilis: production, characterization, and immunodiagnostic application
Authors: Qadri F , Mohi M G , Chowdhury A , Alam K , Azim T , Sears C , Sack R B , Albert M J
Published: January 2020

A monoclonal antibody, ICT11, specific for the toxin of enterotoxigenic Bacteroides fragilis (ETBF) neutralized the cytotoxic effect of the toxin on human colonic cell line HT-29/C1. In an evaluation using 115 diarrheal stool specimens and culture as the "gold standard," the assay showed a sensitivity of 85% and a specificity of 100%. An ICT11-based sandwich enzyme-linked immunosorbent assay showed a sensitivity of 100% and a specificity of 98% for direct detection of toxin from stool samples compared with those of culture. Thus, ICT11-based assays will be useful for screening for ETBF.
Serologic reactivity of a synthetic peptide from human immunodeficiency virus type 1 gp41 with sera from a Mexican population
Authors: Gevorkian G , Soler C , Viveros M , Padilla A , Govezensky T , Larralde C
Published: January 2020

The reactivities of 1,172 serum samples obtained from asymptomatic human immunodeficiency virus type 1 (HIV-1)-positive and HIV-1-negative individuals residing in Mexico to a synthetic disulfide-looped peptide from the HIV-1 gp41 (amino acids 602 to 616 [IWGCSGKLICTTAVP] were examined by an enzyme-linked immunoadsorbent assay (ELISA) procedure. Antibodies to the synthetic peptide were detected in 261 of 268 serum samples from HIV-positive individuals (sensitivity, 97.4%). The peptide also reacted with 12 of 904 serum samples from control HIV-negative individuals (specificity, 98.7%). Western blots (immunoblots) of four of the seven serum samples that produced false-negative results in the ELISA showed that three of them reacted weakly with gp41 and strongly with gp120, p55, and/or p24. Potential diagnostic difficulties raised by the reported C1q binding capacity of this peptide were also evaluated: few and weak false-positive results were found among sera from patients with rheumatoid arthritis (1 of 31) and neurocysticercosis (2 of 111). In fact, strong reactivity with the peptide spotted an undetected HIV infection underlying clinical neurocysticercosis.
Development of a rapid and specific colony-lift immunoassay for detection and enumeration of Campylobacter jejuni, C. coli, and C. lari
Authors: Rice B E , Lamichhane C , Joseph S W , Rollins D M
Published: January 2020

Contamination of retail poultry by Campylobacter spp. is a significant source of human diarrheal disease. We have developed a colony-lift immunoassay (CLI) for the detection of Campylobacter jejuni, C. coli, and C. lari isolated from such sources and grown on selective agar medium or on filter membranes. This technique has been successfully utilized to quantify Campylobacter colonies within 18 to 28 h after sampling. Hydrophobic, high-protein-binding membranes were prewet with methanol and used to imprint bacterial cells from the agar or filter membrane, while leaving colonies intact and viable. The membranes were air dried, peroxidase neutralized, blocked with bovine serum albumin in phosphate-buffered saline, and hybridized for 5 min with an affinity-purified, horseradish peroxidase-labeled goat anti-Campylobacter antibody preparation (Kirkegaard and Perry Laboratories). The membranes were washed briefly, exposed to a 3,'5,5'-tetramethylbenzidine membrane substrate, rinsed in deionized water, and allowed to dry. Lifted colonies of Campylobacter were identified by a blue color reaction on the membrane. Replicas of the membranes were made by marking the location of the Campylobacter colonies on clear transparencies, which were subsequently utilized to locate the original colony on the filter membrane or agar plate. The specificity of this antibody preparation has been evaluated against a wide range of Campylobacter spp., including American Type Culture Collection type and references strains, retail poultry isolates, and isolates obtained from cloacal swabs of live commercial broiler chickens. Specificity against numerous non-Campylobacter spp. obtained from the same sources was also evaluated. The CLI provided a rapid and simple means for detection and enumeration of enteropathogenic Campylobacter organisms. We have successfully combined this CLI procedure with methods recently developed in our laboratories for retail meat and poultry sampling. Potentially, broader applications for use of this technique include detection and enumeration of campylobacters from clinical, veterinary, and environmental samples.
Limits of the microimmunofluorescence test and advantages of immunoblotting in the diagnosis of chlamydiosis
Authors: Biendo M , Eb F , Lefebvre J F , Orfila J
Published: January 2020

The Western blot (immunoblot) patterns of 56 serum specimens, all examined previously by the microimmunofluorescence (MIF) test for species-specific Chlamydia antibodies, were analyzed. Predominant specific-antibody activity was directed to the 170-, 155-, 145-, 120-, 115-, 100-, 57-, and 38-kDa proteins of Chlamydia trachomatis and to the 175-, 130-, 110-, 98-, and 30-kDa proteins of Chlamydia pneumoniae. All of these antigens appeared to be species specific. The reactivity with 90-, 80-, 75-, 62- or 60-, and 55-kDa proteins and the major outer membrane protein appeared to be genus specific. Fourteen serum samples which had identical titers of immunoglobulin G as determined by the MIF test were investigated by Western blotting. We found that nine serum samples had anti-C. trachomatis protein profiles and two had anti-C. pneumoniae protein profiles. The double seropositivity observed by MIF corresponded with cross-reactivity to genus-specific antibodies. As for the three remaining serum specimens, we observed identical protein profiles for C. trachomatis and C. pneumoniae, confirming the double seropositivity experienced with the MIF test. Western blotting can differentiate between specific reactions and interfering noise from other, partly cross-reacting chlamydial species.
Production of T-helper cell subsets and cytokines by lymphocytes from patients with chronic mucocutaneous candidiasis
Authors: Kobrynski L J , Tanimune L , Kilpatrick L , Campbell D E , Douglas S D
Published: January 2020

Chronic mucocutaneous candidiasis (CMC) is a heterogeneous group of disorders characterized by recurrent and persistent superficial candidal infections. Cytokine-induced dysregulation of T-helper cell function has been described in other immune-deficient states but has not been studied in CMC patients. We studied T-helper cell subsets by flow cytometry and cytokine production by stimulated lymphocytes in six CMC patients, two healthy pediatric controls, and five healthy adult controls. Peripheral blood lymphocytes were stimulated in vitro with phytohemagglutinin or Candida albicans extract, and the production of interleukin-2R (IL-2R), IL-4, IL-10, and gamma interferon in the supernatants was measured by enzyme-linked immunosorbent assay. CMC patients had a decrease in the CD29+/CD29+ cell population compared with the numbers in controls (P < 0.02). The percentage of CD4+/CD45RA+ cells was greater in patients than in controls, but the difference was not significant. There was no difference in the production of IL-10 or gamma interferon by the patient lymphocytes. CMC patients produced more IL-4 than the controls (P < 0.001), whereas the controls tended to produce more IL-2R than the patients (P = 0.19). These findings support the concept that a decrease in CD4+/CD29+ T-helper inducer cells along with T-helper cell dysregulation may lead to defective memory responses to antigens in CMC patients and a decrease in cell-mediated immunity due to inhibition of TH1 cells by increased levels of IL-4.
Macrofragment localization of the toxin A and toxin B genes of Clostridium difficile
Authors: Norwood D A , Sands J A
Published: January 2020

We report the physical mapping of the toxin A and B genes to the bacterial chromosome of Clostridium difficile ATCC 43594 by pulsed-field gel electrophoresis. Single and double digestions with restriction endonucleases NruI and SacII allowed localization of the toxin genes to a specific 577-kb fragment and estimation of genome size to be approximately 3.8 megabases. This effort represents the initial step in the construction of a physical map of the whole genome.