bluuebunny/crossref_metadata_2025 huggingface dataset:
Human antibody response to the B oligomer of pertussis toxin
Authors: Lynn F , Burnette W N , Siber G R , Arciniega J L
Published: January 2020

To determine whether antibodies to the B oligomer of pertussis toxin (PT) were present in patients diagnosed with pertussis or vaccinees who had received diphtheria-tetanus-whole-cell pertussis vaccine, we analyzed serum samples from 5 patients and 10 vaccinees by both enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting techniques. Antibodies to the B oligomer were detected by ELISA in all samples containing antibodies to holotoxin. Western immunoblotting procedures were less efficient than ELISA techniques for detecting antibodies to the B oligomer. Antibodies which inhibit the ability of the B oligomer to agglutinate erythrocytes were detected in purified human immunoglobulin preparations. In addition, serum samples containing antibodies to PT inhibited the binding of purified B oligomer and holotoxin to a 165-kDa glycoprotein which has been considered a potential PT receptor in Chinese hamster ovary (CHO) cells. These results suggest that antibodies to the B oligomer contribute to the human serologic response to PT, but their detection and characterization require appropriate methods.
Laboratory evaluation of the inflammatory myopathies
Authors: Rider L G , Miller F W
Published: January 2020

The laboratory plays an important role in the diagnosis, evaluation, and classification of the heterogeneous group of diseases known as the IIM, which are characterized by chronic muscle inflammation. Serial measurements of the levels of muscle-derived enzymes in serum are the traditional laboratory studies used to follow the clinical course of patients with IIM, although other laboratory tests can also be useful in assessing myositis disease activity. Several markers of immune system activation, including cytokines and lymphocyte markers, show promise as possibly more sensitive measures of myositis disease activity. Discovery of a unique group of MSAs over the past decade has provided an immunologic basis for defining relatively homogeneous subsets of patients who share similar clinical features, disease courses, and responses to therapy. Future investigations of novel immunologic activation markers, as well as the cloning and expression of target autoantigens of the MSAs, should allow better diagnostic assays, enhanced prognosis, and a better understanding of the pathogenesis of these disorders.
Human immune response against outer membrane proteins of Moraxella (Branhamella) catarrhalis determined by immunoblotting and enzyme immunoassay
Authors: Helminen M E , Beach R , Maciver I , Jarosik G , Hansen E J , Leinonen M
Published: January 2020

The role of Moraxella (Branhamella) catarrhalis as a respiratory tract pathogen is increasingly recognized. We looked at the human immune response against individual outer membrane proteins of M. catarrhalis and against the 81-kDa CopB protein, which has previously been shown to be a target for protective antibodies. Paired serum samples from six elderly patients with pneumonia were tested by Western blot (immunoblot) analysis by using outer membrane vesicles of M. catarrhalis 035E as antigen. All of the six convalescent-phase serum samples reacted with a protein which migrated at the position of the CopB protein and with a high-molecular-weight protein of M. catarrhalis; three serum samples also reacted with a 34-kDa outer membrane protein. Paired serum samples from 18 patients, 10 of which had M. catarrhalis infection on the basis of previous serology results, were tested by enzyme immunoassay (EIA) with the CopB protein and whole cells of M. catarrhalis 035E as antigens. Nine patients showed a significant rise in EIA titer between acute- and convalescent-phase sera when whole bacterial cells were used as antigens. Six (67%) patient samples that were positive by the EIA with the whole-cell antigen were also positive by the EIA with the CopB antigen, and six of nine patient samples negative by the EIA with the whole-cell antigen were also negative by the EIA with the CopB antigen. These results suggest that both the CopB and a high-molecular-weight protein are major targets of the immune response against M. catarrhalis, and further studies with greater amounts of patient materials are needed to elucidate the usefulness of CopB as an antigen in etiologic studies.
Cross-reactive antigens shared by Pseudomonas aeruginosa, Helicobacter pylori, Campylobacter jejuni, and Haemophilus influenzae may cause false-positive titers of antibody to H. pylori
Authors: Johansen H K , Nørgaard A , Andersen L P , Jensen P , Nielsen H , Høiby N
Published: January 2020

Cystic fibrosis (CF) patients suffer from many of the gastrointestinal conditions which occur in non-CF individuals, e.g., dyspepsia and peptic ulceration. These symptoms may be caused by Helicobacter pylori but could also be due to either pancreatic insufficiency or the intensive antibiotic treatment used in CF patients. Since CF patients chronically infected with Pseudomonas aeruginosa produce antibodies against a wide range of antigens, including antigens common to many other bacteria, e.g., GroEL and lipopolysaccharide, we studied, by the Western blot (immunoblot) technique, the specificity of immunoglobulin G antibodies to H. pylori in Danish CF patients chronically infected with P. aeruginosa, CF patients without P. aeruginosa infection but with Haemophilus influenzae infection, patients with dyspeptic ulcers associated with H. pylori, and patients recovering from acute Campylobacter jejuni or Campylobacter coli infection. Sera from CF patients with chronic P. aeruginosa or H. influenzae infection and patients recovering from acute C. jejuni infection cross-reacted with H. pylori antigens. A strong cross-reacting protein antigen at approximately 14 kDa and minor cross-reactive antigens at approximately 27, 30, and 60 kDa (the heat shock protein GroEL is equivalent to the common antigen of P. aeruginosa) could be demonstrated. The results of this study show that high immunoglobulin G antibody titers against H. pylori in CF patients cannot be regarded as indicating present or past H. pylori infection unless their specificity is proven by absorption studies.
Isolation and preliminary evaluation of a low-molecular-mass antigen preparation for improved detection of Helicobacter pylori immunoglobulin G antibodies
Authors: Andersen L P , Espersen F , Souckova A , Sedlackova M , Soucek A
Published: January 2020

Previously, immunoglobulin G (IgG) antibodies to five antigens with a relative molecular mass of between 15 and 30 kDa from Helicobacter pylori were found to be significantly more frequent in H. pylori-infected patients than in noninfected patients. In this study, these specific low-molecular-mass (LMW) antigens were separated by ultrafiltration of whole-cell sonicates. The LMW antigen preparation was evaluated by enzyme-linked immunosorbent assay with serum samples from 76 children with abdominal symptoms and 151 adults with dyspeptic symptoms. H. pylori was cultured or seen in 40 (53%) children and 83 (55%) adults. Increased antibody levels to H. pylori were found in serum from 35 (46%) children and 88 (58%) adults. Values for sensitivity, specificity, and predictive value of positive and negative results of the test were higher with LMW antigens than with the heat-stable antigen previously described. The low specificity and predictive value of a positive result were due to seropositive results for 21 persons with a negative culture for H. pylori and negative microscopy results for Helicobacter-like organisms in biopsies from gastric mucosa. Histologically, chronic gastritis was demonstrated in 43% of these persons, and 19% had peptic ulcer, indicating that they have or have had H. pylori infection. Specific antibodies to H. pylori were confirmed in all 21 patients by the Western immunoblot technique. Use of the LMW antigen improved the IgG antibody detection in patients with H. pylori infection, even though the results reflect the difficulties in establishing a true gold standard for diagnosis of H. pylori infection.
Immunoglobulin G subclass-specific antileishmanial antibody responses in Indian kala-azar and post-kala-azar dermal leishmaniasis
Authors: Ghosh A K , Dasgupta S , Ghose A C
Published: January 2020

Antileishmanial antibody responses in the sera of Indian kala-azar (KA) and post-KA dermal leishmaniasis (PKADL) patients were analyzed by enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments using immunoglobulin G (IgG) class- and subclass-specific reagents. All sera showed antileishmanial reactivities in IgG ELISA which followed the order IgG1 > IgG2 > IgG3, with very little IgG4. Immunoblot analysis with IgG class-specific reagents revealed variable patterns of reactivity by KA and PKADL sera, although certain common bands around the 60- to 63-kDa regions were discernible. Sera from antimony-unresponsive KA cases, on the other hand, strongly recognized two bands at around 20 to 22 kDa, in addition to other bands in the high-molecular-mass region. Further analysis showed that the 28-kDa band was preferentially recognized by the IgG2 isotype, while 20- to 22-kDa and 60- to 63-kDa bands were recognized by the IgG1 isotype. Antibodies belonging to the IgG3 isotype reacted to antigens primarily in the region of 14 to 34 kDa and persisted in patients even several months after cure. Immunoblot studies also revealed the presence of a nonspecific band which arose as a result of binding between a 66-kDa leishmanial antigen and streptavidin. Finally, the results presented in this study suggest that certain leishmanial antigens preferentially stimulate the synthesis of a particular IgG subclass(es), depending on the nature of such antigens or their epitopes.
Determination of CD4 and CD8 lymphocyte subsets by a new alternative fluorescence immunoassay
Authors: Denny T N , Jensen B D , Gavin E I , Louzao A G , Vella F A , Oleske J M , Wong W
Published: January 2020

The purpose of this study was to evaluate a new alternative fluorescence immunoassay method (Zymmune CD4/CD8 Cell Monitoring Kit; Zynaxis, Inc., Malvern, Pa.) for determining the absolute CD4+ and CD8+ T-lymphocyte concentrations in whole blood. The investigation was performed as a two-site comparison of the reference whole blood flow cytometric method with the Zymmune method. In this investigation, a total of 166 patient samples were evaluated of which approximately 20% were from human immunodeficiency virus-positive individuals. The mean value for samples performed by the Zymmune CD4 assay was 1,094 (range, 74 to 2,586) cells per microliters, while the reference method yielded a mean of 890 (range, 35 to 2,033) cells per microliter. The correlation coefficient for regression analysis was 0.940. The mean value for samples performed by the Zymmune CD8 assay was 700 (range, 212 to 1,813) cells per microliter, while the reference method yielded a mean of 546 (range, 82 to 2,158) cells per microliter. The correlation coefficient for regression analysis was 0.921. No site-specific differences or trends in CD4 or CD8 values were seen when the data were analyzed by site of collection. The average precision of the CD4 assay varied from 6 to 14%, corresponding to the high and low concentration ranges. For CD8, the average precision varied from 8.3 to 16% over the respective high to low concentration ranges. We conclude that the Zymmune CD4/CD8 Cell Monitoring Kit method provides absolute CD4+ and CD8+ T-lymphocyte concentrations which are equivalent to those given by the reference flow cytometric method.
Presence of neutralizing antibodies to heterologous human immunodeficiency virus type 1 isolates in sera of infected individuals is not predictive of rate of disease progression
Authors: Warren R Q , Wong M T , Melcher G P , Blatt S P , Zapiola I , Bouzas M B , Muchinik G , Anderson S A , Kennedy R C
Published: January 2020

These studies were undertaken to examine whether the presence of human immunodeficiency virus type 1 (HIV-1)-neutralizing antibodies in sera of infected individuals would alter the rate of disease progression. HIV-1-infected individuals (n = 87) were initially examined for neutralizing activity in vitro against both laboratory and tissue culture-adapted clinical heterologous HIV-1 isolates. The neutralizing activities of sera were determined by a 90% or greater reduction in HIV-1 p24 levels in vitro. In a cross-sectional analysis of all infected individuals, we observed that sera from asymptomatic individuals neutralized a significantly greater number of heterologous HIV-1 isolates than sera from symptomatic patients. Patients who could be followed up longitudinally (n = 24) were then studied to determine the impact of neutralizing antibodies on the rate of disease progression. We observed no significant difference between the numbers of HIV-1 isolates neutralized in vitro by sera from patients who remained clinically stable and by those from patients who progressed rapidly. Our data indicated that the presence or absence of neutralizing antibodies to heterologous HIV-1 isolates was not associated with the rate of disease progression.
Monoclonal antibodies to Coxiella burnetii that cross-react with strain Nine Mile
Authors: Sekeyová Z , Kovácová E , Kazár J , Toman R , Olvecká S
Published: January 2020

Results are presented to show the binding properties of five monoclonal antibodies directed to Coxiella burnetii Priscilla with cross-reactions to the Nine Mile strain. The monoclonal antibodies preferentially recognize phase I epitopes by ELISA and recognize phase II epitopes by immunoblotting but do not allow differentiation between so-called chronic and acute strains of C. burnetii. The only difference in reactivity was in the staining pattern revealed after reactions with lipopolysaccharide I antigens.
Immune complex transfer enzyme immunoassay that is more sensitive and specific than western blotting for detection of antibody immunoglobulin G to human immunodeficiency virus type 1 in serum with recombinant pol and gag proteins as antigens
Authors: Hashida S , Hashinaka K , Nishikata I , Oka S , Shimada K , Saito A , Takamizawa A , Shinagawa H , Yano S , Kojima H
Published: January 2020

Antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) in serum was detected by ultrasensitive enzyme immunoassays (immune complex transfer enzyme immunoassays) with recombinant reverse transcriptase (rRT), p17 (rp17) and p24 (rp24) of HIV-1 as antigens and beta-D-galactosidase from Escherichia coli as the label. The immune complex, comprising 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate, antibody IgG to HIV-1, and recombinant protein-beta-D-galactosidase conjugate, was trapped on polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with epsilon N-2,4-dinitrophenyl-L-lysine, and transferred to polystyrene beads coated with affinity-purified (anti-human IgG gamma-chain) IgG. Bound beta-D-galactosidase activity was assayed by fluorometry. The assays were highly reproducible with no serious serum interference, and they were much more sensitive than Western immunoblotting for the corresponding antigens. Signals with rRT, rp17, and rp24 for asymptomatic carriers were at least 56,000-, 680-, and 22-fold higher, respectively, than those for seronegative individuals, and neither indeterminate nor false-positive results were observed, whereas some serum samples were false negative or false positive by Western blotting for p17 and/or p24 antigen. In some cases, seroconversion was detected earlier than by conventional methods. Therefore, these assays are suggested to be more useful than conventional methods not only for the confirmation of antibody IgGs to RT, p17, and p24 of HIV-1 in serum but also for the detection of seroconversion.
Characteristics of viral protein expression by Epstein-Barr virus-infected B cells in peripheral blood of patients with infectious mononucleosis
Authors: Wagner H J , Hornef M , Middeldorp J , Kirchner H
Published: January 2020

The frequency of Epstein-Barr virus (EBV) antigen-positive B cells in the peripheral blood of patients with infectious mononucleosis compared with that for latently EBV-infected individuals was examined by immunocytochemistry. B cells positive for Epstein-Barr nuclear antigen (EBNA) 1, EBNA2, and latent membrane protein were frequently found in all peripheral B lymphocyte preparations from 25 patients suffering for 3 to 28 days from infectious mononucleosis by using monoclonal antibodies and the alkaline phosphatase anti-alkaline technique. There was a significant decrease in the number of positive B cells during the course of disease. EBNA1-positive B cells were detected in 0.01 to 2.5% of total B cells (median, 0.8%), EBNA2-positive B cells were detected in 0.01 to 4.5% of total B cells (median, 0.9%), and latent membrane protein-positive B cells were detected in 0.01 to 1.8% of total B cells (median, 0.5%), depending on the duration of clinical signs. In contrast, we did not find any EBNA1- or EBNA2-positive B cells in 2 x 10(6) peripheral blood B lymphocytes of 10 latently EBV-infected individuals, whereas aliquots of the same cell preparations were EBV DNA positive by a PCR assay. Therefore, it appears to be possible to detect infectious mononucleosis by immunocytochemical determination of latent EBV products, which might be of relevance for the diagnosis of EBV reactivations in immunosuppressed patients.
Molecular analysis of highly enriched populations of T-cell-depleted monocytes
Authors: Aleixo L F , Goodenow M M , Sleasman J W
Published: January 2020

CD4+ T lymphocytes and monocytes/macrophages are important components of the immune system. Blood monocytes are usually targeted for studies of the human macrophage lineage cells because of their accessibility through blood sampling. Most separation techniques currently available to obtain human monocytes either require large volumes of blood or do not yield a monocyte fraction sufficiently depleted of other cell types. We have developed a simple strategy to isolate a highly enriched population of monocytes from small volumes (< 6 ml) of peripheral blood by using an anti-CD14 monoclonal antibody and magnetic microspheres. Yields of monocytes ranged from 75 to 80% of CD14+ cells in peripheral blood. CD4+ T cells were subsequently selected from the monocyte-depleted peripheral blood by using an anti-CD4 monoclonal antibody and immunomagnetic beads. The effectiveness of immunomagnetic selection to yield a monocyte population highly depleted of T cells was analyzed by using a sensitive molecular strategy based on PCR amplification and detection of T-cell receptor (TCR) gene rearrangements. The relative frequency of rearranged TCRs within the monocyte population was compared with the frequency of rearranged TCRs within the CD4+ T-cell fraction from the same individual. Molecular analysis indicated that a viable monocyte population which contains fewer than 2% residual T lymphocytes can be consistently selected from small aliquots of blood.
A family cluster of an immune escape variant of hepatitis B virus infecting a mother and her two fully immunized children
Authors: Ho M S , Lu C F , Kuo J , Mau Y C , Chao W H
Published: January 2020

A hepatitis B virus (HBV) immune escape variant which results from a substitution of glycine by arginine at position 145 (arginine-145) in the immunodominant neutralization epitope of the S protein was found to infect one child in a seroepidemiologic survey of 1,812 vaccinated children. The child's mother and a younger brother were also infected by the same HBV variant, despite a greater than accepted level of protective antibody (anti-HBV surface antigen) in both the index child (30 mIU/ml) and the brother (> 20,000 mIU/ml). Our findings suggest the perinatal or horizontal transmission of the arginine-145 HBV variant from mother to child, but the ability of this variant to propagate among the vaccinated children remains unknown.
Correlation of lactoferrin with neutrophilic inflammation in body fluids
Authors: Martins C A , Fonteles M G , Barrett L J , Guerrant R L
Published: January 2020

We have reported that lactoferrin, a 77-kDa iron-binding glycoprotein found in secondary neutrophil granules, provides a useful marker of fecal leukocytes in fecal specimens from patients with inflammatory diarrhea (R. L. Guerrant, V. Araujo, E. Soares, K. Kotloff, A. A. M. Lima, W. H. Cooper, and A. G. Lee, J. Clin. Microbiol. 30:1238-1242, 1992). In order to determine the usefulness of this marker of neutrophilic inflammation in different body fluids, we examined blood, gingival swabs, sputum, and saliva using antilactoferrin antibodies (lactoferrin latex agglutination [LFLA]). LFLA titers in whole blood samples were < or = 1:4 in all eight samples from patients with neutropenia (absolute neutrophil count [ANC] = < 150 polymorphonuclear cells [PMNs] per microliter), < or = 1:8 in samples from 13 individuals with moderate leukocyte counts (ANC = 150 to 8,000), and 1:8 to 1:32 in samples from six patients with neutrophilia (ANC > 8,000). While the overlap precludes a useful role in the identification of neutropenia, these data confirm that lactoferrin titers of > 1:100 indeed indicate inflammation in fluid specimens. On quantitative elution of lactoferrin from gingival swabs, all 7 patients with dental plaque had titers of 1:200 to 1:400; 9 of 12 patients with clinical gingivitis had LFLA titers of 1:200 to 1:1,600, while all 7 individuals with healthy gums and teeth and 4 edentulous patients had LFLA titers of < or = 1:100. Eight purulent sputum samples had titers of > or = 1:400 (7 were 1:1,600) while 11 normal saliva samples showed titers of < or = 1:100. Lactoferrin titers in sputum, gingival swabs, and whole blood correlate with the presence of neutrophils or inflammation in these specimens and may offer a convenient rapid test for inflammatory processes.
Alterations in peripheral blood mononuclear cell cytokine production in response to phytohemagglutinin in multiple sclerosis patients
Authors: Crucian B , Dunne P , Friedman H , Ragsdale R , Pross S , Widen R
Published: January 2020

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS). The disease is characterized by inflammatory lesions in the white matter of the CNS, consisting of a specific immune response to the myelin sheath. We investigated peripheral blood mononuclear cell (PBMC) cytokine production by enzyme-linked immunosorbent assays of 21 MS patients and 19 age-matched normal controls in response to the T-cell mitogen phytohemagglutinin (PHA). Peripheral blood mononuclear cells were cultured in medium alone or in medium with 5 micrograms of PHA per ml for 48 h, and culture supernatants were collected for analysis. Cytokines selected for study were interleukin-10 (IL-10), gamma interferon (IFN-gamma), IL-2, and IL-4. All cytokine activities described were expressed as concentrations per 500,000 cells. We found that 48% (10 of 21) of the MS patients produced small but detectable levels of IL-10 in medium alone, compared with 26% (5 of 18) of the controls. We found that the MS patients produced significantly higher quantities of IL-10 protein than the controls in response to PHA (mean supernatant concentrations of IL-10 for patients and controls, 421 and 204 pg/ml, respectively [P < 0.05]). No significant differences were detected in the production of IL-2, IFN-gamma, and IL-4 between patients and controls in response to PHA, although patients appeared to display a trend toward decreased production of IFN-gamma.
Immunoglobulin and complement complexes in blood following infection with human immunodeficiency virus type 1
Authors: Peng X X , Wainberg M A , Tao Y , Brenner B G
Published: January 2020

Freely soluble and complexed plasma immunoglobulin A (IgA), IgG, IgM, C1q, C3, and factor B in 36 human immunodeficiency virus type I (HIV-1)-seronegative controls, 69 asymptomatic HIV+ subjects, and 117 individuals with symptomatic HIV-associated disease were characterized. Levels of free and complexed IgG and IgA, and to a lesser extent free C1q and complexed IgM, increased with HIV-1 infection. In stark contrast, both HIV+ groups showed three- to sixfold declines in complexed C3, C1q, and factor B levels. The asymptomatic HIV+ population showed declines in levels of C3-bound IgA, IgG2, and IgG4 complexes. The asymptomatic group showed reductions in C3-complexed IgM, IgA, IgG2, and IgG4 levels. HIV infection is associated with complement-deficient immune complexes.
Production of agglutinating monoclonal antibody against antigen 8 specific for Cryptococcus neoformans serotype D
Authors: Ikeda R , Nishimura S , Nishikawa A , Shinoda T
Published: January 2020

A hybridoma (clone CRND-8) that produced agglutinating monoclonal antibody (MAb) against Cryptococcus neoformans serotype D was established by using a soluble capsular polysaccharide-keyhole limpet hemocyanin conjugate for immunization. The isotype was immunoglobulin M(kappa). Specificity was determined by cell slide agglutination and enzyme-linked immunosorbent assay (ELISA). In both tests, the MAb reacted to serotypes D and A-D but not to serotypes A, B, and C. Furthermore, the specificity of the MAb determined by ELISA was the same as that of polyclonal antibody factor serum (PAb factor) 8, which showed high-level reactivity with serotypes D and A-D. These results supported the deduced specificity of the PAb-based antigenic factor 8. A total of 15 isolates of serotypes D and A-D but no serotype A isolates reacted with the MAb in cell slide agglutination tests. CRND-8 MAb can be used in place of PAb factor 8 for serotyping C. neoformans isolates and for the analysis of the antigen 8 epitope.
Purification and seroreactivity of pneumococcal surface adhesin A (PsaA)
Authors: Tharpe J A , Russell H
Published: January 2020

Pneumococcal surface adhesin A (PsaA) is a 37-kDa common protein antigen of Streptococcus pneumoniae. In the present study, the protein was purified so that its immunoreactivity could be determined. PsaA was released and purified from cells by lysis in the presence of n-laurylsarcosine; this was followed by ammonium sulfate precipitation and subsequent preparative isoelectric focusing. A capture antibody enzyme-linked immunosorbent assay was used to determine the immunoreactivity of purified PsaA. The assay had a 67% sensitivity for sera from patients with bacteremic pneumococcal pneumonia. A specificity of 97% was estimated on the basis of a lack of reactivity with sera from patients with pneumonia caused by other organisms. PsaA is a potential vaccine candidate and may be useful as an antigen in a diagnostic assay for pneumococcal disease.
Utilization of tests for Lyme disease antibody at a university hospital
Authors: Nachamkin I , Riddle D L , Feldman M , Edelstein P H
Published: January 2020

We performed a retrospective study on patients who had a positive screening antibody test result for antibody to Borrelia burgdorferi to determine the clinical indicators used by physicians to order this test. Eighty-two evaluable patients who were screen positive (indirect enzyme-linked immunosorbent assay) between August 1991 and March 1993 were included. Additional tests, isotype-specific capture immunoglobulin enzyme immunoassay and Western blot (immunoblot) analysis (immunoglobulin G), were performed on positive samples. Of 82 patients with a positive screening test result, 54 (66%) had no serologic evidence of Lyme disease on the basis of additional testing (positive predictive value, 34%). Only 28 of 82 patients (34%) had clinical indicators suggestive of Lyme disease. Antibody screening tests may provide misleading information if they are not accompanied by more specific assays. Inappropriate testing of patients without indications of Lyme disease is frequently performed, and the ordering practices of physicians should be reassessed.
Immune responses to the R4 protein antigen of group B streptococci and its relationship to other streptococcal R4 proteins
Authors: Fasola E L , Flores A E , Ferrieri P
Published: January 2020

The R antigen, a trypsin-resistant protein observed in group A, C, F, G, and L streptococci, has also been found in group B streptococci (GBS). Although four species of the R antigen have been described for GBS, the R4 protein is the most prevalent in GBS isolates recovered from humans. This study examined the prevalence of antibodies against the R4 antigen by Western blot (immunoblot) (WB) in sera from 40 mothers colonized with GBS serotype II and III and from 26 noncolonized mothers; 92.5% of the colonized mothers had anti-R4 antibodies, compared with 54% of the noncolonized mothers (P < 0.001). Findings of antibodies in neonatal cord sera (n = 14) were concordant with maternal results by WB analysis for 71% of mother-infant pairs colonized with serotype II and for 57% of pairs colonized with serotype III. Of mothers known to be colonized with type II/R4 or III/R4, 100% (n = 12) had antibody against R4 by WB. This study also evaluated the prevalence of antibody to the GBS R4 antigen in 48 sera from individuals with high and low group A streptococcal anti-DNase B titers. Of those individuals with an anti-DNase B titer of > 640, 64% had a positive WB for anti-R4 antibody, compared with 30% of individuals with low anti-DNase B titers (P < 0.05). The R4 antigen of GBS had immunologic identity to the R4 antigen of group A streptococci. Overall, the findings suggested that antibodies to the streptococcal R4 antigen were commonly present in GBS-colonized mothers and that transplacental passage of these antibodies occurred. The presence of antibody to R4 in non-GBS-colonized individuals may be due to immunologic responses to past exposure to the R antigen present in GBS or other streptococcal groups.
Antigenicity of cell wall mannans of Candida albicans NIH B-792 (serotype B) strain cells cultured at high temperature in yeast extract-containing sabouraud liquid medium
Authors: Okawa Y , Goto K , Nemoto S , Akashi M , Sugawara C , Hanzawa M , Kawamata M , Takahata T , Shibata N , Kobayashi H , Suzuki S
Published: January 2020

Cultivation of Candida albicans NIH B-792 (serotype B) at high temperature (37 degrees C) for 48 h in yeast extract-containing Sabouraud liquid medium (YSLM) provided the following findings in comparison with the findings obtained after incubation at 27 degrees C. Growth of the blastoconidia of this strain was decreased, with a dry weight of 9%, and the cells were deficient in cytokinesis. The cells did not undergo agglutination with serum factor 5 from a commercially available serum factor kit (Candida Check). Mannan (B-37-M) obtained from the cells cultured at 37 degrees C had partially lost its reactivity against serum factor 4 and lost most of its reactivity against serum factor 5 in an enzyme-linked immunosorbent assay (ELISA) in contrast to that (B-27-M) at 27 degrees C. Both cells and mannan prepared by cultivation first at 37 degrees C and then at 27 degrees C entirely recovered their reactivities with serum factors 4 and 5. 1H-nuclear magnetic resonance analysis also revealed that B-37-M had lost a beta-1,2-linked mannopyranose unit and retained a phosphate group. Similar changes were observed in the three other serotype B strains used in the study. The beta-1,2-linked mannooligosaccharides longer than mannotetraose were not included among the products released from B-37-M by mild acid treatment. The results of the inhibition ELISA with a series of beta-1,2-linked mannooligosaccharides from biose to octaose (M2 to M8, respectively) showed that the reactivity against serum factor 4 was inhibited most strongly by the oligosaccharides M4 to M8 and that the reactivity against serum factor 5 was inhibited completely by relatively longer oligosaccharides, M5 to M8, indicating their participation as the antigenic factor 5 epitopes.
Specific serodiagnosis of human leishmaniasis with recombinant Leishmania P2 acidic ribosomal proteins
Authors: Soto M , Requena J M , Quijada L , Alonso C
Published: January 2020

The Leishmania P2 proteins have been analyzed as potential tools for the immunodiagnosis of human mucocutaneous and visceral leishmaniasis. Two recombinant Leishmania infantum proteins, rLIP2a and rLip2b, were used. The analysis indicated that the rLiP2a and rLiP2b proteins are recognized by 76% (16 of 21) and 42% (9 of 21), respectively, of sera from patients with mucocutaneous leishmaniasis and by 50% (5 of 10) and 40% (4 of 10), respectively, of sera from patients with visceral leishmaniasis. The Leishmania P2 proteins were engineered to have deletions of particular amino acids from the carboxyl-terminal region in order to avoid cross-reactivity with sera from patients with systemic lupus erythematosus and Chagas' disease, since it is known that this region is the main target of the autoantibodies present in sera from these patients. The results show that while the modified recombinant proteins rLiP2a-Q and rLiP2b-Q, in which the five carboxyl-terminal amino acids had been deleted, maintain the leishmaniasis-specific epitopes, they do not react with sera from patients with autoimmune disease and Chagas' disease. For this reason, and also because the sera from patients with tuberculosis and leprosy, diseases that have to be considered in a differential clinical diagnosis of infectious diseases, do not react with the rLiP2a-Q or rLiP2b-Q protein, we think that the engineered proteins may be considered specific tools for the immunodiagnosis of mucocutaneous and visceral leishmaniasis.
Detection of altered T helper 1 and T helper 2 cytokine production by peripheral blood mononuclear cells in patients with multiple sclerosis utilizing intracellular cytokine detection by flow cytometry and surface marker analysis
Authors: Crucian B , Dunne P , Friedman H , Ragsdale R , Pross S , Widen R
Published: January 2020

Production of T helper 1 and T helper 2 cytokines was investigated in peripheral blood mononuclear cells (PBMCs) from multiple sclerosis (MS) patients by a newly described technique, detection of intracellular cytokines by flow cytometry in conjunction with immunophenotype analysis. T-cell gamma interferon (IFN-gamma) production and interleukin 10 (IL-10) production were examined after PBMC activation with T-cell mitogens at 5 and 24 h, and monocyte spontaneous production of IL-10 and production after PBMC activation with lipopolysaccharide (LPS) for 24 h were also examined. The data indicate that MS patients have decreased percentages of T cells capable of secreting IFN-gama compared with healthy controls, and this change is detectable at 5 and 24 h. the patients displaying decreased T-cell production of IFN-gamma were essentially confined to a group being treated with the newly approved drug Betaseron (berlex Labs, Cedar Knolls, N.J.), a recombinant form of IFN-beta (rIFN-beta 1b). By gating of the entire lymphocyte population, analysis of IFN-gama production in T cells (CD3+ versus that in non-T cells (CD3+) was possible. The percentage of IFN-gamma-producing lymphocytes that was made up of T cells was essentially unchanged between the Betaseron-treated patients, non-Betaseron-treated patients, and controls, indicating that the suppression of IFN-gamma production displayed by betaseron-treated MS patients was a nonspecific suppression of all IFN-gamma-producing lymphocytes as opposed to a suppression of T-cell production only. The data seem to indicate that treatment of MS with Betaseron corresponds to an inhibition of the lymphocyte's ability to produce IFN-gamma. No changes were detected in T-cell production of IL-10 at either time point. We also observed that MS patients in general appear to have small percentages of peripheral blood monocytes spontaneously producing slight but detectable levels of IL-10. No difference was seen regarding monocyte production of IL-10 after PBMC activation with LPS between MS patients and controls. Both populations responded with high percentages of monocytes producing IL-10. The data seem to indicate that treatment of MS with Betaseron, known to decrease the exacerbation rate of relapsing-remitting MS, corresponds to a suppression of peripheral blood lymphocyte production of IFN-gamma. Monocyte production of IL-10 may also play a role in regulating the disease process.
Enzyme-linked immunosorbent assay-based detection of antibodies to antigenic subtypes of infectious bursal disease viruses of chickens
Authors: Jackwood D J , Henderson K S , Jackwood R J
Published: January 2020

An enzyme-linked immunosorbent assay (ELISA) using a fragment of the infectious bursal disease virus (IBDV) VP2 gene expressed in baculovirus was developed. A 944-bp portion of the VP2 gene from the Del-A strain of IBDV was ligated into the pAc360 transfer vector and transfected into baculovirus, Recombinant baculoviruses were identified by dot blot hybridization. The recombinant baculovirus 9A5 expressed a 57-kDa VP2 fusion protein, which was immunoprecipitated. This baculovirus-expressed VP2 was used as an antigen in an ELISA (Ohio State University [OSU]-ELISA). Titers of sera from specific-pathogen-free chickens infected with different strains of IBDV in our laboratory were determined by the OSU-ELISA, commercial ELISAs, and a virus neutralization assay. The results indicate that all sera from the specific-pathogen-free chickens infected with IBDV strains contained high titers of neutralizing antibodies. Each of these antisera also tested positive with the commercial ELISA kits. The OSU-ELISA did not detect antibodies to all strains of IBDV tested. This ELISA detected antibodies to the antigenically similar Delaware variant strains Del-A, Del-E, and GLS but did not detect or detected very poorly antibodies to antigenically heterologous classic IBDV strains STC, D78, and BVM. Although the antiserum to the IN strain of IBDV contained a virus-neutralizing antibody titer, the OSU-ELISA did not detect antibodies in this serum, suggesting IN is antigenically heterologous to Del-A. Titers to the IN strain were detected with the commercial ELISA kits. The OSU-ELISA detected antibodies to the SAL strain, suggesting this virus is antigenically similar to Del-A, which is supported by previously reported vaccination and challenge studies. In conclusion, the OSU-ELISA could be used to detect antibodies to a subgroup of IBDV strains, while commercially available ELISA kits detected antibodies to all the antigenic subtypes of IBDV tested.
Immunoreactivity of specific epitopes of PrPSc is enhanced by pretreatment in a hydrated autoclave
Authors: Yokoyama T , Momotani E , Kimura K , Yuasa N
Published: January 2020

An abnormal protein (PrPSc) accumulates in animals affected with scrapie. Immunoblotting procedures have been used widely to detect PrPSc. Blotted membranes were subjected to pretreatment in a hydrated autoclave, and the subsequent immunoreactivity of PrPSc was examined. The immunoreactivity of PrPSc to antisera against the synthetic peptides of the mouse PrP amino acid sequences 199 to 208 and 213 to 226 was enhanced by the pretreatment. However, the reactivity to antisera of peptide sequences 100 to 115 and 165 to 174 was not affected. The antibody-binding ability of the specific epitopes which are located close to the C-terminal end of PrP27-30 the proteinase-resistant portion of PrPSc, was enhanced by pretreatment in a hydrated autoclave. This pretreatment increased the sensitivity of PrPSc, and it would be useful for diagnosis of scrapie.
Mapping of the H7-serospecific domain of Escherichia coli flagellin
Authors: Kwang J , Wilson R , Yang S , He Y
Published: January 2020

The amino acid sequences responsible for H7 and H23 flagellum serology have been identified by using a genetic approach. The H7-specific domain was located between amino acids 352 and 374 of the H7 flagellin. The sequencing data also demonstrated that the difference between the H7 and H23 flagellins in this region results from a single substitution at amino acid 366 (Ser-->Thr). The common epitopes for H7 and H23 were located between amino acids 284 and 366.
Chemiluminescent and flow cytometric analysis of gamma interferon preincubation on neonatal and adult rat polymorphonuclear leukocytes
Authors: Wittler R R , Lieberman M M , Paine D D , Muehlbauer S L , Lima J E , Sachanandani D M , Pinney C A
Published: January 2020

Gamma interferon (IFN-gamma) has multiple immunomodulating effects and has been postulated as a possible immunopotentiating agent for the prevention or treatment of neonatal infections. This report describes the effect of rat recombinant IFN-gamma on the oxidative burst activity and CD11b expression of neonatal and adult rat polymorphonuclear leukocytes (PMNL). Oxidative burst activity was assessed by chemiluminescence and dihydrorhodamine flow cytometry. Neonatal PMNL exhibited significantly less oxidative burst activity than did adult PMNL. IFN-gamma mildly enhanced the chemiluminescence response of PMNL from both the rat pups and adults, but this effect was not statistically significant when analyzed by a multivariate model of repeated-measures analysis of variance for both chemiluminescence and dihydrorhodamine flow cytometry. CD11b expression was also not significantly enhanced by IFN-gamma.
Specific antibody reactivity against a 110-kilodalton Actinobacillus actinomycetemcomitans protein in subjects with periodontitis
Authors: Fleming T F , Selmair I , Schmidt H , Karch H
Published: January 2020

The purpose of the present study was to determine the serum immunoglobulin A (IgA), IgM, and IgG reactivities against proteins of Actinobacillus actinomycetemcomitans in patients with periodontitis. Serum samples from 20 patients with early-onset periodontitis, 20 patients with adult periodontitis, and 20 age- and sex-matched healthy controls were assessed by immunoblot analysis. IgG antibody reactivity against a sarcosyl-insoluble 110-kDa protein of A. actinomycetemcomitans was detected in 65 and 45% of patients with early-onset periodontitis and adult peritonitis, respectively, and IgA antibodies against this protein were found in 70 and 55% of these patients, respectively. However, control subjects showed no IgG reactivity, and IgA antibodies against the sarcosyl-insoluble 110-kDa protein were detected in only 5% of the patients (P < 0.05). There was no IgM antibody reactivity against this protein in any of the diseased or healthy subjects. The sensitivity and specificity of serum IgA antibody reactivity against the 110-kDa protein in detecting subgingival A. actinomycetemcomitans infection, as determined by PCR, were 77 and 66%, respectively. The results of the study indicated that the sarcosyl-insoluble 110-kDa protein is a potential candidate for use in the serodiagnosis of periodontal disease.
Self-assembly of in vitro-translated human papillomavirus type 16 L1 capsid protein into virus-like particles and antigenic reactivity of the protein
Authors: Iyengar S , Shah K V , Kotloff K L , Ghim S J , Viscidi R P
Published: January 2020

The human papillomavirus type 16 (HPV-16) L1 capsid protein is the major component of the HPV virion. We prepared L1 protein of HPV-16 in a cell-free system. The L1 gene was cloned in an expression plasmid and transcribed and translated in vitro in a rabbit reticulocyte lysate. The expressed protein had the molecular mass (55 kDa) expected for the L1 protein, and it assembled into virus-like particles that closely resembled papillomavirus virions. The protein retained conformational epitopes, as evidenced by its reactivity with monoclonal antibodies which recognize only intact viral particles. In radioimmunoprecipitation assays with sera from college women grouped by their genital tract HPV DNA status, high reactivity was found in 68% of HPV-16 DNA-positive women, in 23% of women with other HPVs, and in 19% of HPV-negative women. In comparison, none of the sera of children were reactive. The results of the radioimmunoprecipitation assays showed a significant correlation with results obtained with the same sera in an enzyme-linked immunosorbent assay with virus-like particles produced in baculovirus (chi-square test for linear trend, P = 0.0023). Although the amounts of L1 protein obtained are small, the ability to produce virus-like particles by in vitro translation may be useful in the study of virus assembly, virus binding, and the immunological response to HPV infection.
Salivary binding antibodies induced by human immunodeficiency virus type 1 recombinant gp120 vaccine. The NIAID AIDS Vaccine Evaluation Group
Authors: Gorse G J , Yang E Y , Belshe R B , Berman P W
Published: January 2020

Salivary binding antibodies induced by candidate human immunodeficiency virus type 1 (HIV-1) vaccines in healthy, HIV-1 uninfected volunteers were assessed in a clinical trial evaluating intramuscularly injected HIV-1MN recombinant gp120 (rgp120) vaccine alone or with HIV-1IIIB rgp120 vaccine. The two rgp120 vaccines induced envelope glycoprotein-specific immunoglobulin G (IgG) and IgA antibodies in whole saliva and serum.
Mycobacterium tuberculosis induces apoptosis in gamma/delta T lymphocytes from patients with advanced clinical forms of active tuberculosis
Authors: Duarte R , Kindlelán J M , Carracedo J , Sánchez-Guijo P , Ramírez R
Published: January 2020

Antigens from inactivated Mycobacterium tuberculosis H37Ra induce activation in a subpopulation of gamma/delta (gamma/delta) T lymphocytes in a manner that resembles that of superantigens from alpha/beta T cells. After culture in vitro with H37Ra proteins, gamma/delta T lymphocytes from patients with advanced clinical forms of active tuberculosis (ACF-TBC) display cytotoxic activity against homotypic target cells exposed to H37Ra. Cytotoxicity by gamma/delta T lymphocytes from ACF-TBC patients occurs in a range similar to that observed in healthy subjects. Following activation, H37Ra-stimulated gamma/delta T lymphocytes from healthy subjects did proliferate in the presence of exogenous recombinant human interleukin 2. However, under the same conditions, gamma/delta T lymphocytes from ACF-TBC patients not only did not proliferate but died by apoptosis. These results suggest that in gamma/delta T lymphocytes from patients with ACF-TBC, antigens from M. tuberculosis may induce cell activation that leads to apoptotic cell death.
Monoclonal antibodies to P24 and P61 immunodominant antigens from Nocardia brasiliensis
Authors: Salinas-Carmona M C , Castro-Corona M A , Sepúlveda-Saavedra J , Perez L I
Published: January 2020

We prepared a Nocardia brasiliensis cell extract and purified two immunodominant antigens with molecular weights of 61,000 and 24,000. The isolated proteins were shown to be reasonably pure when analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8 to 18% polyacrylamide gradient) and stained with Coomassie blue and silver nitrate. By using an immunoelectrotransfer blot method (Western blotting), we demonstrated that these two purified proteins reacted strongly with serum from N. brasiliensis-infected mycetoma patients. To obtain anti-P61 and anti-P24 monoclonal antibodies (MAbs), we used an N. brasiliensis cell extract as the antigen for the first immunization; 2 weeks later female mice were reimmunized with a semipurified antigen containing the P24 or P61 fraction. A booster injection was given 3 days before the fusion was carried out. Two hybrids that reacted strongly with P24 were cloned by limiting dilution, the generated MAbs were analyzed for isotyping, and their specificity was tested in a Western blot assay with cell extracts from Nocardia asteroides and Mycobacterium tuberculosis cultures. Anti-P24 MAbs were shown to be specific for N. brasiliensis HUJEG-1 and did not cross-react with either the N. asteroides or M. tuberculosis strains used. However, additional studies with several N. asteroides and N. brasiliensis strains are needed to investigate whether there are cross-reactions between strains or species when these MAbs are used. The anti-P61 and anti-24 MAbs were used to locate the antigen in N. brasiliensis cells by immunofluorescence. The lack of reaction with intact cells suggests that the P24 and P61 antigens are not exposed in the complete bacterial cell surface or that the recognized epitopes are different. Only one anti-P61 MAb that reacted specifically with the N. brasiliensis cell extract was obtained.