bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of absolute CD4+ lymphocyte counts determined by enzyme immunoassay (TRAx CD4 test kit) and flow cytometry
Authors: Moss M W , Carella A V , Provost V , Quinn T C
Published: January 2020

Currently, CD4+ lymphocyte counts are one of the most widely used surrogate markers for monitoring disease progression in and initiating therapy for human immunodeficiency virus-infected individuals. However, the process of obtaining lymphocyte subset counts can be complex and expensive, often rendering the test inaccessible to many patients. In contrast to standard laser-based flow cytometry, the TRAx CD4 Test Kit utilizes an enzyme-linked immunoassay format to provide CD4+ lymphocyte counts by a simple and more cost-effective means. In order to evaluate the utility of the TRAx CD4+ assay in comparison with flow cytometry, heparinized blood samples were drawn from 188 infected and uninfected adult patients and 24 infected pediatric patients and evaluated by both assays. The correlation coefficient for all adult individuals tested was 0.94, and the mean absolute counts (in cells per milliliter, +/- standard deviation) were 510 +/- 358 for TRAx and 480 +/- 361 for flow cytometry. The correlation for the pediatric group was 0.93, with mean absolute counts of 956 +/- 767 for TRAx and 1,521 +/-a 1,438 for flow cytometry. Overall the TRAx CD4 Test Kit performed well in comparison to flow cytometry, and its lower cost and ease of use make it an encouraging alternative for the routine determination of CD4+ lymphocyte counts.
Antibody to native human immunodeficiency virus type 1 envelope glycoproteins induced by IIIB and MN recombinant gp120 vaccines. The NIAID AIDS Vaccine Evaluation Group
Authors: Gorse G J , Patel G B , Newman F K , Belshe R B , Berman P W , Gregory T J , Matthews T J
Published: January 2020

The ability of antibody induced by MN and IIIB recombinant gp120 (rgp120) human immunodeficiency virus type 1 (HIV-1) vaccines the bind to oligomeric native and monomeric recombinant HIV-1 envelope glycoproteins (rgp 120) was measured in 25 uninfected, healthy adult volunteers. A major focus was to evaluate the effect of simultaneous and sequential immunization with vaccines representing different strains of HIV-1 on the ability to broaden cross-reactivity of antibodies against these and other HIV-1 strains. A flow cytometric indirect immunofluorescence assay (FIFA) to detect vaccine-induced antibody to envelope glycoprotein expressed by infected and rgp120-coated target cells was used, MN rgp120 HIV-1 vaccine given alone and coadministered with IIIB rgp120 HIV-1 vaccine elicited antibody which bound to cells infected with HIV-1MN, HIV-IIIB, HIV-1RF, and HIV-1-SF2. The presence of envelope glycoprotein-binding antibody detected by FIFA correlated to a moderate degree with functional antibody against HIV-1MN and HIV-IIIB. Priming immunization with IIIB rgp120 HIV-1 vaccine followed by booster injections of MN rgp120 HIV-1 vaccine resulted in increased cross-reactive antibody binding to these and heterologous clade B HIV-1 strains infecting cells. MN rgp120 HIV-1 vaccine given alone was better able to induce cross-reactive antibody to cells infected with heterologous HIV-1 laboratory strains than was IIIB rgp120 HIV-1 vaccine given alone. The vaccines induced binding antibody to rgp120 possessing the amino acid sequence of a clade E HIV-1 strain as measured by enzyme-linked immunosorbent assay. Levels of antibody binding to cells infected with clade B HIV-1 and cells coated with monomeric rgp120 were greater than that induced by HIV-1IIIB-based gp160 vaccines in previous studies.
Humoral immune response against lipopolysaccharide and cytoplasmic proteins of Brucella abortus in cattle vaccinated with B. abortus S19 or experimentally infected with Yersinia enterocolitica serotype 0:9
Authors: Baldi P C , Giambartolomei G H , Goldbaum F A , Abdón L F , Velikovsky C A , Kittelberger R , Fossati C A
Published: January 2020

The humoral immune responses against three different antigens of Brucella abortus were monitored by enzyme-linked immunosorbent assay in cattle vaccinated with B. abortus S19 or experimentally infected with Yersinia enterocolitica serotype 0:9. Immunoglobulin G (IgG) and IgM responses against (i) B. abortus lipopolysaccharide (LPS), (ii) total cytoplasmic proteins depleted of LPS (LPS-free CYT), and (iii) B. abortus 18-kDa cytoplasmic protein were measured. Vaccinated animals and Yersinia-infected animals developed high anti-LPS IgM and IgG titers, which overlapped with those obtained with sera from B. abortus 544-infected animals used as positive controls. In contrast, only a slight or negative IgG and IgM response against LPS-free CYT and the 18-kDa protein was detected in vaccinated or Yersinia-infected cattle, although its levels were always significantly lower than those of B. abortus 544-infected animals. These data indicate that cytoplasmic proteins of B. abortus could be useful for the differential diagnosis of bovine brucellosis.
A field test for the detection of antibodies to human immunodeficiency virus types 1 and 2 in serum or plasma
Authors: Burgess-Cassler A , Barriga Angulo G , Wade S E , Castillo Torres P , Schramm W
Published: January 2020

In response to the need for simple and rapid tests for infectious diseases, we have devised a test for antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 which resembles many contemporary strip-style pregnancy tests in format and ease of use. The test was evaluated with 2,928 serum specimens (1,541 reactive and 1,387 nonreactive) collected and tested at a Mexico City hospital clinic and was compared with a laboratory assay (Abbott) performed simultaneously. The sensitivity and specificity of the test using these serum specimens were 99.68 and 99.71%, respectively (before the code of the blinded study was broken). This compares with 100% sensitivity and 97.55% specificity with the laboratory assay (specificity upon reassay after the code was broken, 99.21%). In a survey of HIV-2 specimens, reactive (positive) specimens were detected in 51 of 51 cases. The test was examined with 21 commercially available (HIV-1) seroconversion panels. The performance of the test was comparable to that of a group of Food and Drug Administration-approved (antibody-based) HIV tests.
Comparison of two 2,3-diacyl trehalose antigens from Mycobacterium tuberculosis and Mycobacterium fortuitum for serology in tuberculosis patients
Authors: Tórtola M T , Lanéelle M A , Martín-Casabona N
Published: January 2020

Immunoglobulin G antibodies against two 2,3-diacyl trehalose (DAT) antigens from Mycobacterium tuberculosis (DATT) and Mycobacterium fortuitum (DATF) were studied by enzyme-linked immunosorbent assay of 356 serum samples. The sera were obtained from non-tuberculosis-infected individuals (282 serum samples) and tuberculosis patients (74 serum samples). Non-tuberculosis-infected individuals were healthy people (120 serum samples; positive purified-protein-derivative skin test, 60 patients; negative purified-protein-derivative skin test, 60 patients) patients with nontuberculosis lung disease (59 serum samples), contacts of sputum-smear-positive tuberculosis patients (57 serum samples), and human immunodeficiency virus-infected patients with nontuberculosis lung disease (46 serum samples). Of the 74 patients with tuberculosis, 14 were human immunodeficiency virus infected. The sensitivity of the method using DATT was 44.5%, and that with DATF was 48.6%. The specificities with both antigens were 99.1%. There were no significant differences between the mean values for both antigens (P = 0.2815). We therefore concluded that both antigens were interchangeable. As M. fortuitum, a fast-growing mycobacterium, could be a good source of antigen DAT, these results deserve consideration in the serology of tuberculosis.
Specificity of monoclonal antibodies elicited by mucosal infection of BALB/c mice with virulent Shigella flexneri 2a
Authors: Hartman A B , Van de Verg L L , Mainhart C R , Tall B D , Smith-Gill S J
Published: January 2020

Protective immunity against shigellosis is thought to be determined by the O-antigen side chains of the lipopolysaccharide (LPS) molecule. To study possible common protective epitopes, monoclonal antibodies reacting with Shigella flexneri 2a LPS were generated from BALB/c mice infected ocularly with the virulent serotype 2a strain S. flexneri 2457T and tested against a panel of S. flexneri LPSs by enzyme-linked immunosorbent and immunoblot assays. Four monoclonal antibodies were identified, all of which showed restricted specificity patterns. Three different patterns of reactivity to LPS possessing the 3,4 group antigen were seen: (i) 2a only, (ii) 2a and 5a, and (iii) 2a, 4a, 5a, and Y. These results have implications for designing a Shigella vaccine that will be protective against related serotypes. Electron microscopy studies showed that the monoclonal antibodies bind to the bacterial surface in a patchy pattern, suggesting their potential use for examining the LPS distribution on the surface of the bacteria.
Detection of serum immunoglobulin M to human cytomegalovirus by western blotting correlates better with virological data than detection by conventional enzyme immunoassay
Authors: Lazzarotto T , Maine G T , Del Monte P , Frush H , Shi K , Landini M P
Published: January 2020

Western blotting (immunoblotting) with proteins separated from purified human cytomegalovirus (HCMV) particles (viral WB) has repeatedly been shown to be a reliable and sensitive method for detecting HCMV-specific immunoglobulin M (IgM). The aim of the present work was to determine whether IgM detected by viral WB correlates with virological diagnosis better than conventional enzyme immunoassay (conv-EIA). The presence of an active HCMV infection was documented on the basis of isolation of virus from urine and/or saliva and on the basis of antigenemia and/or PCR with polymorphonuclear leukocytes for immunocompetent and immunocompromised subjects, respectively. The agreement observed between IgM detected by viral WB and the results obtained by virological detection of HCMV was significantly higher (88.7%) than the agreement of IgM detected by conv-EIA and virological results (67.5%).
Activity of antibodies against Salmonella dublin, Toxoplasma gondii, or Actinobacillus pleuropneumoniae in sera after treatment with electron beam irradiation or binary ethylenimine
Authors: Kyvsgaard N C , Lind P , Preuss T , Kamstrup S , Lei J C , Bøgh H O , Nansen P
Published: January 2020

Viral contamination of biological material may constitute a risk when samples are exchanged between countries, and it may be necessary to subject the material to an inactivation treatment. The present study investigated possible adverse effects on antibody activity subsequent to either electron beam irradiation or binary ethylenimine (BEI) treatment. The treatments were performed with sera obtained from pigs or cattle. For each treatment level, the posttreatment activity was plotted against the pretreatment activity, and regression analyses were carried out. The slope of the regression line was used as an estimate for the relative posttreatment activity. For a Toxoplasma gondii indirect enzyme-linked immunosorbent assay (ELISA) and agglutination assay as well as for a Salmonella dublin indirect ELISA, the posttreatment activity was more than 89% of the pretreatment activity when the samples were irradiated in the frozen state (on dry ice) with up to 46. kGy or when they were treated with 5 or 10 mM BEI for up to 48 h. The samples were more sensitive to irradiation in the liquid state. Thus, samples irradiated with 22.6 kGy retained 98% of their activity in the indirect ELISA when they were irradiated in the frozen state on dry ice but only 35% of their activity when they were irradiated in the liquid state at 0 degrees C. The patterns seen in an S. dublin blocking ELISA and an Actinobacillus pleuropneumoniae complement fixation assay differed in that samples with a low level of pretreatment activity were subject to a relatively greater decrease in activity than samples with a high level of pretreatment activity. The complement fixation assay was particularly sensitive to irradiation of serum. It is concluded that serum samples retain sufficient activity by both methods of virus inactivation, especially when used in indirect ELISA or in the T. gondii agglutination assay.
Comparative study of neutrophil activation by chemiluminescence and flow cytometry
Authors: Lieberman M M , Sachanandani D M , Pinney C A
Published: January 2020

Neutrophil activation by phorbol 12-myristate 13-acetate (PMA) and zymosan was assessed by luminol-dependent chemiluminescence (CL) in a novel microtiter plate format and by flow cytometry (FC) based on the oxidation of dihydrorhodamine 123. The results of this comparison demonstrated striking differences in kinetic parameters between these two techniques for neutrophil activation by PMA and zymosan. PMA activation, as determined by FC, was found to be an all-or-none phenomenon in that below a critical concentration of PMA, few cells were positive. Above this concentration, almost all cells were positive; however, the fluorescence intensity of positive cells increased with an increasing PMA concentration until a plateau (maximal) level was reached. In contrast, increasing zymosan concentrations resulted in proportionate increases in the percentage of positive cells until close to 100% of cells were positive. However, the fluorescence intensity of positive cells remained about the same. CL activity increased proportionately with either PMA or zymosan concentration until a maximal level was achieved. The concentration of PMA required for half-maximal activity was about 10-fold higher for FC than for CL, whereas the analogous concentration of zymosan was about 30-fold higher for CL than for FC. In addition, opsonization had only a small negative effect on the ability of zymosan to activate neutrophils, as determined by FC, whereas it had a very large enhancing effect when determined by CL. The differences in kinetic parameters of activation suggest differential sensitivity to particulate (zymosan) versus soluble (PMA) stimulants for FC and CL.
Modification of Escherichia coli B glutathione synthetase with polyethylene glycol for clinical application to enzyme replacement therapy for glutathione deficiency
Authors: Inoue Y , Sugiyama K , Ueminami H , Izawa S , Kimura A
Published: January 2020

Glutathione synthetase of Escherichia coli B was modified with polyethylene glycol, and the properties of the resultant modified enzyme were investigated. The thermal stability of the modified enzyme and its resistance against several proteases increased compared with those of the native enzyme. The modified enzyme was injected intravenously via the rat tail vein, and the circulating life of the enzyme in plasma was monitored. The half-life of the native enzyme was 50 min, whereas that of the modified enzyme was approximately 24 h. The systemic anaphylaxis reaction was tested by using rats intravenously injected with the native and modified enzymes. For the native enzyme, strong reactions such as dyspnea and tumble were observed; however, no symptom or only a very weak reaction, such as scratching, was observed with the modified enzyme.
Characterization of monoclonal antibody B7, which neutralizes the cytotoxicity of Pseudomonas aeruginosa exotoxin A
Authors: Shang H F , Yeh M L , Lin C P , Hwang J
Published: January 2020

A nontoxic Pseudomonas aeruginosa exotoxin A (PE), which has the carboxyl-terminal 38 amino acid residues of native PE deleted, was used as an antigen to immunize BALB/c mice, which were then challenged with native PE in order to raise monoclonal antibodies (MAbs) that can neutralize PE cytotoxicity. A murine MAb against PE, designated MAb B7, was established. MAb B7 was characterized in terms of its ability to neutralize PE cytotoxicity, epitope mapping, inhibition of PE receptor binding, and influence on cellular processing of PE and ADP-ribosylation activities. We found that MAb B7 could neutralize PE cytotoxicity in cell culture and in BALB/c mice. The epitope recognized by MAb B7 was mapped to the carboxyl-terminal amino acid residues 575 to 595 of PE. Consistent with the results of epitope mapping, MAb B7 did not block PE receptor-binding activity or the cellular processing of PE but strongly inhibited the ADP-ribosylating activity of PE. In addition, MAb B7 retained strong binding to PE even at pH 4.0, indicating that the complex of MAb B7 and PE is stable in the phagolysosome. On the basis of these observations, the neutralization of PE cytotoxicity by MAb B7 could be due to its binding to the carboxyl terminus of PE. As a result, MAb B7 may interfere with the interaction of the carboxyl-end amino acid residues REDLK of PE with cellular factors. However, we could not rule out the possibility that MAb B7 directly blocks the ADP-ribosylation activity of PE in the cytosol.
Parameters of disease progression in long-term experimental feline retrovirus (feline immunodeficiency virus and feline leukemia virus) infections: hematology, clinical chemistry, and lymphocyte subsets
Authors: Hofmann-Lehmann R , Holznagel E , Ossent P , Lutz H
Published: January 2020

After several years of latency, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) cause fatal disease in the cat. The aim of this study was to determine laboratory parameters characteristic of disease progression which would allow a better description of the asymptomatic phase and a better understanding of the pathogenesis of the two infections. Therefore, experimentally infected cats (FIV and/or FeLV positive) and control animals were observed over a period of 6.5 years under identical conditions. Blood samples were analyzed for the following: complete hematology, clinical chemistry, serum protein electrophoresis, and determination of CD4+ and CD8+ lymphocyte subsets. The following hematological and clinical chemistry parameters were markedly changed in the FIV-infected animals from month 9 onwards: glucose, serum protein, gamma globulins, sodium, urea, phosphorus, lipase, cholesterol, and triglyceride. In FeLV infection, the markedly changed parameters were mean corpuscular volume, mean corpuscular hemoglobin, aspartate aminotransferase, and urea. In contrast to reports of field studies, neither FIV-positive nor FeLV-positive animals developed persistent leukopenia, lymphopenia, or neutropenia. A significant decrease was found in the CD4+/CD8+ ratio in FIV-positive and FIV-FeLV-positive animals mainly due to loss of CD4+ lymphocytes. In FeLV-positive cats, both CD4+ and, to a lesser degree, CD8+ lymphocytes were decreased in long-term infection. The changes in FIV infection may reflect subclinical kidney dysfunction, changes in energy and lipid metabolism, and transient activation of the humoral immune response as described for human immunodeficiency virus (HIV) infections. The changes in FeLV infection may also reflect subclinical kidney dysfunction and, in addition, changes in erythrocyte and immune function of the animals. No severe clinical signs were observed in the FIV-positive cats, while FeLV had a severe influence on the life expectancy of persistently positive cats. In conclusion, several parameters of clinical chemistry and hematology were changed in FIV and FeLV infection. Monitoring of these parameters may prove useful for the evaluation of candidate FIV vaccines and antiretroviral drugs in cats. The many parallels between laboratory parameters in FIV and HIV infection further support the importance of FIV as a model for HIV.
A simplified method for Ca2+ flux measurement on isolated human B cells that uses flow cytometry
Authors: Gergely L , Cook L , Agnello V
Published: January 2020

A method for Ca2+ flux measurement on isolated human peripheral B cells that uses flow cytometry is described. B cells were isolated by anti-CD19 magnetic bead sorting, and Ca2+ flux was measured with the fluo-3 reagent on a standard single-laser flow cytometer. The response of B-cell stimulation by anti-immunoglobulin B (anti-IgM), anti-IgD, protein A, concanavalin A, and ionomycin was determined. Percentage of responder B cells, the level of Ca2+, and the time of peak stimulation were measured. Bound anti-CD19 monoclonal antibody coupled with small paramagnetic particles did not affect Ca2+ flux. All the isolated B cells responded maximally at 10s with stimulation by 8 microg of ionomycin. The average isolated preparation contains 70% IgM+ and 85% IgD+ cells, all of which showed peak stimulation with 10 microg of anti-IgM and anti-IgD per ml, respectively, at 30s. Only at high concentrations of 80 microg/ml, concanavalin A produced a slower response, peaking at 90 s after stimulation. Stimulation with 20 microg of protein A per ml resulted in Ca2+ flux in only 40 to 60% of cells that had a rapid response and maximal stimulation resembling the pattern of activation of ionomycin. B cells from three patients with mixed cryoglobulinemia with high concentrations of monoclonal rheumatoid factors showed stimulation with aggregated IgG, whereas those from healthy control subjects did not, demonstrating the applicability of the methodology to detection of specific antigen stimulation of B cells. This methodology may be useful in testing the functional capacity of B cells in a variety of diseases. The methodology may also prove useful in studying antigen-specific B-cell responses when they involve a significant percentage of B cells.
CD4+ T-cell lymphopenia in Q fever endocarditis
Authors: Sabatier F , Dignat-George F , Mège J L , Brunet C , Raoult D , Sampol J
Published: January 2020

Valvular endocarditis is the most serious complication of chronic Q fever, an infectious disease due to Coxiella burnetii. Although its pathogenesis is poorly understood, the role of the immune system has been evoked. The aim of this study was to investigate lymphocyte subsets in the peripheral blood of infected patients by analyzing the distribution of T- and B-lymphocyte subsets. Since various infectious diseases have been found to be associated with modified antigen expression, we also measured the antigen density of the main lymphocyte markers by quantitative flow cytometry. The absolute values of CD3+ T cells and CD19+ B cells were lower in infected subjects than in controls. The decrease in the CD4+ T-cell count was more pronounced than that in the CD8+ T-cell count, leading to a significantly lower CD4/CD8 ratio in patients. The decreases in CD4+ T cells and CD19+ B cells were correlated with levels of C. burnetii-specific immunoglobulin G, showing that CD4+ lymphopenia is related to the activity of chronic Q fever. Quantitation of antigen expression on lymphocytes showed that CD3, CD4, CD8, and CD19 were expressed similarly in patients and controls. In contrast, CD2 and CD11a expression levels, which are both related to naive and memory phenotypes, were modified in patients. The study of CD45RO and CD45RA expression by CD4+ T cells provided evidence that lymphopenia preferentially affected unprimed lymphocytes.
Vitamin A levels and human immunodeficiency virus load in injection drug users
Authors: Semba R D , Farzadegan H , Vlahov D
Published: January 2020

Although low plasma vitamin A levels are associated with increased mortality and higher vertical transmission during human immunodeficiency virus (HIV) infection, it is unknown whether plasma low vitamin A levels are a marker for circulating HIV load. We conducted a cross-sectional study within a prospective cohort study of injection drug users in order to evaluate the relationship between plasma vitamin A levels and HIV viral load. Plasma vitamin A level was measured by high-performance liquid chromatography. Infectious viral load was measured by quantitative microculture of serial fivefold dilutions of 10(6) peripheral blood mononuclear cells. A total of 284 HIV-infected adults (79 women, 205 men) were studied. Plasma vitamin A levels consistent with deficiency were found in 28.9% of adults. A total of 38.0% of women and 25.3% of men had vitamin A deficiency (P < 0.04). The median infectious viral load for the entire study population was 8 infectious units per million cells. No significant relationship between plasma vitamin A levels and infectious viral load was observed in these injection drug users. This study suggests that there is no correlation between HIV viral load and plasma vitamin A levels in injection drug users, and these variables may represent independent risk factors during HIV infection. HIV-infected adult women appear to be at higher risk of developing vitamin A deficiency.
Lack of association of Borna disease virus and human T-cell leukemia virus type 1 infections with psychiatric disorders among Japanese patients
Authors: Kubo K , Fujiyoshi T , Yokoyama M M , Kamei K , Richt J A , Kitze B , Herzog S , Takigawa M , Sonoda S
Published: January 2020

Borna disease virus (BDV) infection has been suspected to be a possible etiological factor in human psychiatric disorders and recently in chronic fatigue syndrome. Evidence of the correlation of BDV infection with these disorders remained unclear. Kagoshima is known to be one of the major areas in which human T-cell leukemia virus type 1 (HTLV-1) is endemic; this is the first isolated human retrovirus that causes adult T-cell leukemia with neurological symptoms. The present study aimed to clarify whether BDV and HTLV-1 infections are associated with psychiatric disorders among Japanese patients. Subjects were 346 patients with psychiatric disorders (schizophrenia, 179; mood disorder, 123; and others, 44) and 70 healthy controls. Anti-BDV antibodies from plasma samples were screened by the indirect immunofluorescence (IF) method using BDV-infected MDCK cells. Results revealed that only three samples were found to be weakly positive for BDV in the IF assay and seronegative by Western blot (immunoblot) assay. Furthermore, BDV-p24 related RNA in peripheral blood mononuclear cells from 106 of 346 psychiatric patients and 12 or 70 healthy controls by p24-reverse transcription PCR was examined. Two mood disorder patients were positive for BDV-p24 RNA but seronegative. To detect anti-HTLV-1 antibodies the plasma samples were screened by the particle agglutination method and no significant difference in seropositivity for anti-HTLV-1 antibody was found between the patients and healthy controls. These results also suggested that there is a lack of association between BDV and HTLV-1 infections with psychiatric disorders among Japanese patients.
Antibody to human immunodeficiency virus type 1 (HIV-1) gp160 in mucosal specimens of asymptomatic HIV-1-infected volunteers parenterally immunized with an experimental recombinant HIV-1 IIIB gp160 vaccine. The National Institute of Allergy and Infectious Diseases-sponsored AIDS Vaccine Evaluation Group
Authors: Lambert J S , Viscidi R , Walker M C , Clayman B , Winget M , Wolff M , Schwartz D H
Published: January 2020

Twenty-two human immunodeficiency virus type 1 (HIV-1)-infected, asymptomatic volunteers with CD4 cell counts of >600 cells/mm3 who were enrolled in a phase I immunotherapy trial comparing two schedules of immunization of an HIV-1 IIIB-based recombinant gp160 (rgp160) experimental vaccine were evaluated for rgp160-specific antibodies in parotid saliva, genital secretions, and serum. When the study was unblinded, it was determined that five volunteers had received rgp160 on a month 0, 1, 2, 3, 4, and 5 immunization schedule, seven volunteers had received rgp160 on a month 0, 1, 2, and 5 schedule, five had received alum/deoxycholate placebo, and seven had received a licensed hepatitis B virus vaccine. Five volunteers consented to the donation of parotid saliva but not genital secretions. Prior to immunization, parotid saliva specimens were available for 11 of 22 volunteers, seminal plasma (SP) specimens were available for 7 of 22 volunteers, cervicovaginal lavage (CVL) specimens were available for 5 of 22 volunteers, and serum was available for 22 of 22 volunteers. These baseline specimens and specimens collected at 1 and 7 months after the final immunizations were assessed by enzyme-linked immunosorbent assay for immunoglobulin G (IgG) and IgA antibodies specific for HIV-1 LAI rgp160 or HIV-1 MN rgp160. No augmentation in HIV rgp160-specific IgG or IgA antibody production in either parotid saliva or serum specimens of vaccinees compared to that in controls was observed after immunization. There were insufficient numbers of SP or CVL specimens available for statistical comparisons between vaccinees and controls. Overall, anti-LAI rgp160 IgG antibodies were detected in the parotid saliva specimens of 20 of 22 volunteers, the seminal plasma specimens of 11 of 11 volunteers, and the CVL specimens of 6 of 6 volunteers and in 21 of 22 serum specimens. Fewer volunteers expressed anti-LAI rgp160 IgA antibodies in mucosal or serum specimens: 11 of 22 parotid saliva specimens, 3 of 11 SP specimens, 3 of 5 CVL samples, and 12 of 22 sera.
Evaluation of a method for counting absolute numbers of cells with a flow cytometer
Authors: Nicholson J K , Stein D , Mui T , Mack R , Hubbard M , Denny T
Published: January 2020

We evaluated a method for performing absolute cell counts of lymphocyte populations with a flow cytometer. In this method, TruCount, test tubes that contain a known number of brightly fluorescent polystyrene beads are provided by the manufacturer. Whole anticoagulated blood is accurately pipetted into the tubes and mixed with fluorochrome-labeled monoclonal antibodies, the erythrocytes are lysed, and this mixture is analyzed on the flow cytometer. Absolute counts of lymphocyte subsets are calculated by determining the ratio of beads to the cell population of interest and then multiplying this ratio by the number of beads in the tube. We found this method to be reproducible. The values we obtained by the TruCount method were 5 to 10% higher than those obtained by conventional methods (flow cytometry and automated hematology) used to determine absolute numbers of cells. We believe that these differences are due to the methods of determining absolute cell counts and not to faulty identification of lymphocyte subsets.
Differential humoral response against alpha- and beta-linked mannose residues associated with tissue invasion by Candida albicans
Authors: Jouault T , Delaunoy C , Sendid B , Ajana F , Poulain D
Published: January 2020

Candida albicans mannan is the major cell wall antigen that elicits antibodies considered to be of little diagnostic value. It comprises epitopes corresponding to sequences of alpha- and beta-1,2-linked mannose residues. Both types of oligomannosidic epitopes may also be present on the glycosidic portions of other C. albicans molecules, i.e., mannoproteins (MP) (either structural or enzymatic) and glycolipids. The human humoral responses against beta-1,2- and alpha-linked oligomannosides were investigated by C. albicans Western blotting by considering the elective distribution of beta-1,2-oligomannosidic epitopes over a 14- to 18-kDa phospholipomannan (PLM) and the presence of alpha-mannosidic epitopes over heavily glycosylated MP. Western blotting of 51 control sera confirmed the presence of antibodies against C. albicans as a commensal member of the indigenous microflora; an immunoglobulin G (IgG) reactivity linked to enzyme-linked immunosorbent assay mannan signals was found for both PLM (beta-1,2-Man residues) and MP (alpha-Man residues). Despite strong reactivities against mannan and MP, IgG from 21 hospitalized patients with mycological evidence of deep-tissue invasion by C. albicans very significantly failed to react or reacted only faintly with PLM. This downregulation of anti-beta-1,2-oligomannosidic epitopes, associated with tissue invasion by C. albicans, was confirmed in 3 of 4 AIDS patients with extended oroesophageal candidosis. The application of a dissociation procedure proved that the absence of PLM reactivity was not due to the presence of immune complexes. These data provide the first evidence for a qualitative modification of the human antimannan antibody response associated with the C. albicans commensal-pathogenic transition.
Phagocytosis and killing of Staphylococcus aureus: effects of stress and depression in children
Authors: Bartlett J A , Demetrikopoulos M K , Schleifer S J , Keller S E
Published: January 2020

While a large body of literature depicting relationships between depression or stress and immunity exists, few such studies have dealt with children, and none investigated myeloid cell-derived immunity. We investigated both phagocytosis and bactericidal activity against Staphylococcus aureus in children with major depressive disorder (MDD). We found that both MDD and stress influence the bactericidal but not the phagocytic activity of polymorphonuclear leukocytes. The data support the existence of psychobiologic effects in children and suggest possible mechanisms by which depression and stress may affect health.
Perioperative variation in phagocytic activity against Candida albicans measured by a flow-cytometric assay in cardiovascular-surgery patients
Authors: Tran T L , Auger P , Marchand A R , Carrier M , Pelletier C
Published: January 2020

Candidiasis is an opportunistic fungal infection that frequently occurs following modifications of host defenses. Major surgery can be responsible for such alterations, and therefore it increases the risk of fungal infection. The purpose of this study was to evaluate the perioperative impairment of leukocyte function in patients after cardiovascular surgery by measuring the phagocytic activity against Candida albicans by a flow-cytometric method. The average postsurgical decrease in phagocytosis in our patients was 11.4%. By univariate analysis, three factors, all related to antibiotic therapy, were significantly associated with an important decrease in phagocytosis; the use of antimicrobial therapy before surgery, the number of different antibiotics taken, and the length of antibiotic treatment. The results of our study showed that the use of antibiotics in patients undergoing cardiovascular surgery alters the normal phagocytic activity of the host immune system against C. albicans and that flow cytometry is a rapid and simple technique that helps in early identification of patients at high risk for Candida infections. The mechanisms by which surgery and antibiotics decrease phagocytosis remain to be elucidated.
Production of reassortant viruses containing human rotavirus VP4 and SA11 VP7 for measuring neutralizing antibody following natural infection
Authors: Gorrell R J , Bishop R F
Published: January 2020

The outer capsid proteins VP4 and VP7 of group A rotaviruses are both targets of neutralizing antibody produced following natural infection in humans. Of interest is the relative importance and immunodominance of each protein in the generation of a protective immune response. In order to measure neutralizing antibody responses to VP4 and VP7 separately, reassortants bearing VP4 of each of the major human rotavirus P types with VP7 of SA11 origin were successfully produced by neutralizing monoclonal antibody selection. The resulting reassortants, together with reassortants representing each of the major VP7 types, were antigenically characterized with serotype-specific neutralizing monoclonal antibodies and hyperimmune sera. The neutralization proteins of human rotavirus origin were found to be unaffected antigenically by reassortment. The abilities of these reassortants to discriminate between VP4 and VP7 immune responses were evaluated with postinfection sera collected from three patients infected with either a P1A[8],G1, a P1B[4],G2, or a P1A[8],G4 rotavirus strain. The reassortants were shown to be capable of separating the neutralizing antibody responses to VP4 and VP7, with each patient showing a different immune response with respect to VP4 or VP7 immunodominance. These reassortants can now be applied to analyses of individual immune responses to VP4 and VP7 proteins after primary rotavirus infections and reinfections in humans.
Distinct genotypic distributions of cytomegalovirus (CMV) envelope glycoprotein in bone marrow and renal transplant recipients with CMV disease
Authors: Woo P C , Lo C Y , Lo S K , Siau H , Peiris J S , Wong S S , Luk W K , Chan T M , Lim W W , Yuen K Y
Published: January 2020

A prospective study of the spectrum of glycoprotein B (gB) and glycoprotein H (gH) genotypes of cytomegalovirus (CMV) was conducted with five categories of patients: viremic bone marrow-transplant (BMT) recipients who developed CMV disease after BMT (n = 22), viremic BMT recipients without CMV disease (n = 11), viremic renal-transplant recipients who developed CMV disease after transplantation (n = 14), viremic renal-transplant recipients without CMV disease (n = 13), and premature babies with asymptomatic congenital CMV infections (n = 13). Genotypic stability was observed because the gB and gH genotypes of multiple isolates obtained from a single patient were identical. The distribution of gH genotypes in patients of all groups studied were similar. However, there was a unique distribution of the gB genotype in the first category of patients, i.e., BMT recipients with CMV disease, which was distinct from those of all other categories (P < 0.05). CMV isolates from 54% of BMT recipients with CMV disease exhibited gB type 2, while isolates from 46, 50, 69, and 77% of the BMT recipients without CMV disease, renal-transplant recipients with and those without CMV disease, and premature babies with congenital CMV infection, respectively, were of gB type 1. An analysis of the clinical characteristics of BMT recipients with CMV disease indicated that all underwent either an allogeneic or matched, unrelated donor transplant, and half had severe acute graft-versus-host disease (grades 2 to 4). The statistically significant genotypic difference between CMV isolates from BMT recipients with and without CMV disease was not observed between isolates from renal-transplant recipients with and without CMV disease. We speculate that differences in pathogenesis in different patient groups might account for these observations. These findings would also facilitate decision making about the choice of recombinant CMV glycoprotein vaccine required to immunize transplant donors and the subsequent adoptive transfer of immunity to BMT recipients. When the source of CMV DNA required for genotyping was investigated among renal-transplant recipients, direct use of peripheral blood leukocytes was 95% effective compared to the effectiveness of cells obtained from conventional culture of peripheral blood specimens.
O-antigen seroepidemiology of Klebsiella clinical isolates and implications for immunoprophylaxis of Klebsiella infections
Authors: Trautmann M , Ruhnke M , Rukavina T , Held T K , Cross A S , Marre R , Whitfield C
Published: January 2020

To provide a database for the development of an O-antigen-polysaccharide-containing vaccine against Klebsiella spp., we examined the O-antigen seroepidemiology of 378 Klebsiella clinical isolates collected prospectively in two university centers. Strains were typed by competitive enzyme-linked immunosorbent assay with rabbit antisera specific for serogroups O1 to O12 and monoclonal antibodies (MAbs) specific for serogroups O1, O2ab, O2ac, and the genus-specific core antigen. The numbers of isolates (percentages) of individual O serogroups were as follows: 148 (39.2) for serogroup O1, 40 (10.6) for serogroup O2ab, 4 (1.1) for serogroup O2ac, 89 (23.6) for serogroup O3, 2 (0.5) for serogroup O4, 32 (8.5) for serogroup O5, none for serogroups O7, O9, and O12, and 21 (5.6) for serogroup O11. Forty-two (11.1) of the strains were non-O-typeable. O-serogroup distributions were virtually identical between isolates from invasive infections and those from noninvasive infections or colonizations. A vaccine containing the O-specific polysaccharides of serogroups O1, O2ab, O3, and O5 would cover 82% of clinically occurring O-antigen specificities. Three hundred thirty-eight of 378 isolates (89.4%) reacted with the genus-specific MAb V/9-5, which recognizes an epitope of the outer core region of Klebsiella lipopolysaccharide. Antibodies directed against this epitope may represent a further alternative for O-antigen-targeted immunoprophylaxis of Klebsiella infections. These data support further experimental investigations on the protective potential of O-antigen-based vaccines and/or hyperimmune globulins in Klebsiella infection.
Soluble interleukin-2 receptor is a thyroid hormone-dependent early-response marker in the treatment of thyrotoxicosis
Authors: Smallridge R C , Tsokos G C , Burman K D , Porter L , Cranston T , Sfikakis P P , Solomon B L
Published: January 2020

Thyrotoxic patients exhibit increased levels of immune activation molecules (soluble interleukin-2 receptor [sIL-2R], intercellular adhesion molecule-1 [ICAM-1], and endothelial-leukocyte adhesion molecule-1 [ELAM-1]) in serum, although the clinical significance of these measurements remains unclear. In a randomized 4-week study, we have recently shown that in the treatment of hyperthyroidism, the combination of cholestyramine and methimazole (MMI) resulted in faster lowering of serum thyroid-hormone levels than did MMI alone. Stored serial serum samples from patients participating in this randomized treatment trial were analyzed for sIL-2R, soluble ICAM-1 (sICAM-1), and soluble ELAM-1 (sELAM-1). The levels of all three molecules were elevated in patients with hyperthyroidism. Although the levels of sICAM-1 and sELAM-1 remained elevated through the 4-week follow-up period in both groups of patients, the sIL-2R levels (normal levels, 1.0 to 4.2 ng/ml) decreased significantly in the 10 patients who received cholestyramine in addition to MMI (week 0, 14.2 +/- 1.5 ng/ml; week 2, 10.8 +/- 1.2 ng/ml; week 4, 8.9 +/- 1.5 ng/ml). In eight patients who received MMI alone, sIL-2R decreased less rapidly (week 0, 12.3 +/- 1.4 ng/ml; week 2, 12.3 +/- 1.3 ng/ml; week 4, 10.9 +/- 1.3 ng/ml). sICAM-1 and sELAM-1 were elevated at baseline but did not decrease during therapy. In the former group, free thyroxine and free triiodothyronine decreased faster. These data show that levels of sIL-2R in serum, but not those of sICAM-1 and sELAM-1, may be of clinical use in the early follow-up evaluation of medically treated patients.
Field evaluation of an immunoglobulin G anti-F1 enzyme-linked immunosorbent assay for serodiagnosis of human plague in Madagascar
Authors: Rasoamanana B , Leroy F , Boisier P , Rasolomaharo M , Buchy P , Carniel E , Chanteau S
Published: January 2020

Bacteriological isolation of Yersinia pestis is the reference test for confirming plague infection, but recovery of the pathogen from human samples is usually very poor. When the etiology of the disease cannot be bacteriologically confirmed, it may be useful to possess alternative tests such as detection of specific circulating antibodies to help guide the diagnosis. In the present study, the immunoglobulin G (IgG) anti-F1 enzyme-linked immunosorbent assay (ELISA) has been applied to various human sera to evaluate its large-scale applicability in the high-endemicity plague foci of Madagascar. The sensitivity of the test was found to be 91.4%, and its specificity was 98.5%. The positive and negative predictive values were 96 and 96.6%, respectively. Seroconversion was observed on day 7 after onset of the disease. Patients with a positive ELISA result could be separated into high (82%) and low (18%) IgG anti-F1 responders. Cross-reactions with eight other infectious diseases prevalent in Madagascar were scarce and were found in 1 of 27 Mycobacterium tuberculosis-, 3 of 34 Schistosoma haematobium-, and 1 of 41 Salmonella-infected patients. Finally, the efficiency of the IgG anti-F1 ELISA was evaluated during the Mahajanga, Madagascar, plague outbreak of 1995. When the number of ELISA-positive patients was added to the number of bacteriologically confirmed and probable cases, the number of positive patients was increased by 35%. In conclusion, although it does not replace bacteriology, IgG anti-F1 ELISA is a useful and powerful tool for retrospective diagnosis and epidemiological surveillance of plague outbreaks.
Differences in humoral responses to the p24 antigen between Ethiopian and Swedish human immunodeficiency virus type 1-infected patients may suggest influences from a T-helper 2-like phenotype
Authors: Kebede T , Britton S , Fehniger T , Vahlne A , Sällberg M
Published: January 2020

The levels of human immunodeficiency virus type 1 p24 antibodies and p24 antigen among 256 Ethiopians and Africans and 86 Swedes were compared. The elevated levels of total anti-p24 immunoglobulin G (IgG) and anti-p24 IgG4 among the Ethiopian and African patients suggest influences from T-helper-cell type 2-like responses.
Reduced antibody reactivity to hepatitis C virus antigens in hemodialysis patients coinfected with hepatitis B virus
Authors: Devesa M , Khudyakov Y E , Capriles F , Blitz L , Fields H A , Liprandi F , Pujol F H
Published: January 2020

Antibody reactivities to hepatitis C virus (HCV) antigens and to synthetic peptides derived from different parts of the HCV genome (core, NS4, and NS5) were evaluated in HCV-infected hemodialysis patients. In the RIBA 3 assay, NS5 was significantly less recognizable by sera of hemodialysis patients compared to other HCV-infected subjects. Among hemodialysis patients, those coinfected with hepatitis B virus (HBV) (positive for hepatitis B surface antigen [HBsAg+]) showed a reduction in reactivity to C33 and C100. Sera of only 23% of the hemodialysis patients (37 of 161) reacted with more than three of eight peptides tested, significantly fewer than the 60% (12 of 20) of the sera of other HCV-infected patients tested (P = 0.001). This immunosuppression was also manifested by a reduced frequency of recognition of additional peptides on follow-up. An even more reduced reactivity was observed among the HBV-coinfected patients (HBsAg+). The low-responder hemodialysis patients were not infected with any particular genotype of HCV, and the same HCV genotypes observed in the whole group of hemodialysis patients (1a, 1b, 2a, and 3a) were found circulating in the low-responder group. Even in this low-responder population, the good performance of two peptides (peptide 716, corresponding to a portion of the core, and peptide 59, corresponding to a portion of NS4) corroborates the immunodominance of the conserved epitopes within these peptides.
Immunomagnetic selection of purified monocyte and lymphocyte populations from peripheral blood mononuclear cells following cryopreservation
Authors: Sleasman J W , Leon B H , Aleixo L F , Rojas M , Goodenow M M
Published: January 2020

Cryopreservation is a method commonly used to store human blood samples. We sought to determine if cryopreserved peripheral blood mononuclear cells (PBMC) could be separated effectively into distinct populations by using monoclonal antibodies and immunomagnetic microspheres. PBMC obtained from healthy blood donors and from human immunodeficiency virus-infected subjects were cryopreserved for as long as 18 months. Recovered cells were separated into CD14+ monocytes and CD4+ T-cell subsets by immunomagnetic selection. Flow cytometry analysis indicated >95% depletion of monocytes from PBMC following immunomagnetic selection with anti-CD14. A highly enriched population of CD4+ T cells was obtained from the CD14-depleted cell fraction by using an anti-CD4 monoclonal antibody and detachable immunomagnetic beads. The CD4+ T cells were subsequently separated into CD4+ CD45RO and CD4+ CD45RA fractions. Each fraction contained >90% enrichment for the respective subpopulation and <5% of the reciprocal subpopulation. No significant differences in cell surface expression of leukocyte markers, in efficiency of selection of PBMC subpopulations, or in mitogen-induced proliferation were detected in freshly isolated or cryopreserved cells. Efficient recovery of cryopreserved specimens means that targeted assays can be performed on selected, prospectively stored samples once clinical endpoints have been achieved.