bluuebunny/crossref_metadata_2025 huggingface dataset:
CD69 expression reliably predicts the anti-CD3-induced proliferative response of lymphocytes from human immunodeficiency virus type 1-infected patients
Authors: Prince H E , Lapé-Nixon M
Published: January 2020

Published studies suggest that mitogenic responses of lymphocytes can be reliably assessed by monitoring the expression of lymphocyte surface CD69 after 24 h of culture with the stimulant. We tested this hypothesis by determining the ability of lymphocyte CD69 expression to predict the outcome (normal or abnormal) of lymphocyte proliferative responses to anti-CD3 in a group of human immunodeficiency virus type 1 (HIV-1)-infected patients (n = 47). Cutoff values for defining normal and abnormal CD69 expression and proliferative ([3H]thymidine incorporation) responses were established with lymphocytes from healthy uninfected controls (n = 20). Lymphocytes from 29 HIV-infected patients exhibited an abnormal proliferative response, and those from 25 of the 29 also exhibited abnormal CD69 expression (sensitivity, 86.2%). Similarly, lymphocytes from 18 HIV-infected patients exhibited a normal proliferative response, and those from 16 of the 18 also exhibited normal CD69 expression (specificity, 88.9%). The predictive value of a normal CD69 result was 80%, and the predictive value of an abnormal CD69 result was 92.6%. These findings demonstrate that HIV-1-associated impairments in lymphocyte activation can be reliably detected by the rapid and nonradioactive CD69 expression assay.
Diagnosis of human immunodeficiency virus infection using citrated whole blood
Authors: Sharma U K , Song H F , Willingham F F , Hannig J , Flexner C , Farzadegan H , Nicolau C , Schwartz D H
Published: January 2020

Standard isolation of human immunodeficiency virus type 1 (HIV-1) from peripheral blood mononuclear cells (PBMC) requires 5 to 20 ml of blood, and the centrifugal separation of PBMC is expensive and time-consuming. Whole-blood coculture techniques use small sample volumes, do not require centrifugation, and allow measurement of the total viral burden in peripheral circulation. We compared the results of citrated whole-blood coculture with those obtained by the standard AIDS Clinical Trials Group PBMC semiquantitative culture method and reverse transcription-PCR quantitation of plasma HIV-1 RNA levels. PBMC cocultures were also set up with added erythrocytes (RBCs) to determine if the presence of RBCs affects the replication of HIV-1 in vitro. The mean number of cells required for a p24-positive PBMC coculture was approximately seven times greater than that required for a positive citrated whole-blood coculture (P < 0.01). At volumes of 100, 50, and 25 microl, the sensitivities of the whole-blood coculture were 94.5, 93.6, and 87.3%, respectively. The PBMC culture in the presence of added RBCs was more sensitive than PBMC coculture alone. The citrated whole-blood coculture was simple to perform, produced a reliable diagnosis of HIV infection in adult volunteers, was more sensitive than previously reported techniques even in half the culture time, and showed less variability than the PBMC coculture. Citrated whole-blood coculture may be a useful and efficient tool for diagnosing infection with HIV-1.
Serological response over time to recombinant Neospora caninum antigens in cattle after a neosporosis-induced abortion
Authors: Jenkins M C , Wouda W , Dubey J P
Published: January 2020

Recombinant Neospora caninum tachyzoite antigens were evaluated in an enzyme-linked immunosorbent assay (ELISA) for recognition by serum antibodies (Ab) from Neospora-infected cattle. Serum samples were obtained every 2 to 3 weeks for 8 to 15 months from 10 cows with histories of Neospora-associated abortion. Serum samples were also obtained from offspring of these animals and from a large number of cows that had aborted a fetus, due to infection by Neospora or other organisms, at various times during gestation. All 10 cows had positive ELISA Ab titers to both recombinant N. caninum tachyzoite antigens after abortion, during subsequent gestation, and after parturition. In three cows, there was a noticeable peak in Ab titers early in gestation. Calves born to Neospora-infected cows also had positive titers of Ab to the recombinant tachyzoite antigens, and these titers remained elevated for at least 4 months after birth. A portion of the serum immunoglobulin in calves may have been derived from colostrum of infected cows. A calf born from a seronegative mother had a positive ELISA titer only after being fed colostrum from a seropositive cow. However, precolostral titers in calves born from Neospora-infected cows were high at birth, suggesting that the parasite was transmitted to the fetus via the placenta and induced a humoral immune response therein. The recombinant tachyzoite antigens were also useful for corroborating clinical diagnoses of Neospora-induced abortion. A significant difference (P < 0.05) between anti-recombinant antigen Ab titers in cows that aborted due to Neospora and those in cows that aborted from other causes was found.
Heterogeneity of cell-associated CP5 expression on Staphylococcus aureus strains demonstrated by flow cytometry
Authors: Poutrel B , Rainard P , Sarradin P
Published: January 2020

It was reported previously that two capsular polysaccharides, types 5 and 8 (CP5 and CP8), account for 70 to 80% of Staphylococcus aureus strains isolated from human and animal sources. The capsular material has been shown to play a part in virulence and in resistance to phagocytosis. With a view to investigating the role that CP plays in pathogenicity or protection, relative measurement of cell-associated CP is desirable. Flow cytometry, which permits the analysis of individual bacteria, was used to that end. Thirty isolates expressing CP5, of human (n = 7) and animal (cow, n = 11; goat, n = 3; swine, n = 3; hen, n = 3; and rabbit, n = 3) origin, were cultivated on either brain heart infusion agar (BHI) or modified medium 110 (mod 110) agar. Staphylococci were incubated with a mouse anti-CP5 monoclonal antibody (an immunoglobulin M, which does not react with staphylococcal protein A) and then stained with a fluorescein-labeled anti-murine antibody. The bacteria were washed, sonicated, and analyzed by flow cytometry. Except for three isolates, the expression of cell-bound CP5 was higher when bacteria were cultivated on mod 110 than when they were cultivated on BHI. We found a wide intraisolate phenotypic heterogeneity in surface-exposed CP5 in many strains, which appeared as mixtures of stained and unstained bacteria. Four main patterns could be distinguished on the basis of the distribution of the fluorescence of individual bacteria within the strain population as a function of growth medium. Great variations in both percentages of stained bacteria and fluorescence intensity were recorded among strains regardless of their origin. Flow cytometry analysis provided information on both the relative amounts and the distribution patterns of the surface expression of CP. This information is potentially useful for the evaluation of the part played by the capsule in the interaction of bacteria with host cells or for the study of the activities of antibodies to this target antigen, such as opsonization or prevention of adherence.
Comparison of the nucleic acids of helical and coccoid forms of Helicobacter pylori
Authors: Narikawa S , Kawai S , Aoshima H , Kawamata O , Kawaguchi R , Hikiji K , Kato M , Iino S , Mizushima Y
Published: January 2020

The nucleic acids of the helical and coccoid forms of Helicobacter pylori were studied to determine if the coccoid forms are "viable (capable of growing) but nonculturable." Using a reference strain (NCTC 11638) and five clinical strains, the nucleic acid contents, DNA integrity, and results of PCR and reverse transcription-PCR (RT-PCR) were compared for helical H. pylori and coccoid forms induced using glycochenodeoxycholic acid or bismuth citrate. The DNA and RNA contents of the coccoid forms were respectively 6.8- and 8.1-fold lower than those of helical H. pylori after 3 days of induction and 11.5- and 14.7-fold lower after 7 days. Agarose gel electrophoresis of DNA extracted from the coccoid forms after 3 days of induction showed a smear pattern indicating DNA cleavage, whereas DNA from helical H. pylori showed a single band with a high molecular mass. After 12 days of induction, all RNA samples from 100% coccoid cultures were negative for the mRNA of urease A or the 26-kDa species-specific protein by RT-PCR. However, most RNA samples obtained after 3 or 7 days of induction were positive at low levels despite the lack of recovery from these cultures. These results suggest that the coccoid form of H. pylori has impaired genomic DNA and is in the process of cellular degeneration, thus being still alive but nonincreasable.
Modulatory effect of mycobacterium cell wall extract (Regressin) on lymphocyte blastogenic activity and macrophage cytokine gene transcription in swine
Authors: Vézina S A , Archambault D
Published: January 2020

Mycobacterium cell wall extract (MCWE) (Regressin) contains trehalose dimycolate and muramyl dipeptide, both of which have immunomodulatory properties. The goal of this study was to evaluate the effect of MCWE on the in vitro peripheral blood lymphocyte blastogenic activities to mitogens phytohemagglutinin (PHA) and concanavalin A (ConA) in 6- to 8-week-old piglets. The effect of MCWE on alveolar macrophage tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) gene transcription, as determined by a reverse transcription-PCR assay standardized with the endogenous glyceraldehyde-3-phosphate dehydrogenase gene, was also investigated. The results show enhanced blastogenic lymphocyte activities to mitogens PHA and ConA in MCWE-exposed cell cultures compared to those of control cell cultures. The enhanced blastogenic activity effect of MCWE was dose dependent. The cell background activity (spontaneous [3H]thymidine incorporation) of lymphocyte cultures was also significantly increased in the presence of MCWE, thereby demonstrating a lymphocyte mitogenic effect of MCWE. Cytokine gene transcription analysis showed that the TNF-alpha transcript levels in alveolar macrophage cell cultures stimulated with MCWE for 6 or 16 h were enhanced compared with those in control cell cultures. An enhancement of IL-1beta mRNA levels in cell cultures stimulated for 16 h with MCWE, compared with those in control cell cultures, was also observed. The overall results demonstrate that MCWE can stimulate lymphocyte functional activity and cytokine mRNA expression in swine, thereby indicating its potential use as a clinical immunotherapeutic agent.
Immunoblot analysis for serodiagnosis of tuberculosis using a 45/47-kilodalton antigen complex of Mycobacterium tuberculosis
Authors: Diagbouga S , Fumoux F , Zoubga A , Sanou P T , Marchal G
Published: January 2020

We evaluated the immunoglobulin G (IgG) antibody response to the 45/47-kDa secreted protein of Mycobacterium tuberculosis by immunoblot assay, to assess its potential value for serological diagnosis. Control subjects consisted of healthy volunteers with negative or positive tuberculin skin tests. Most (>98%) scored negative in an immunoblot test when the sera were analyzed at a 1:400 dilution. Approximately 40% of sera (diluted 1 in 400) from tuberculous patients (positive smears) recognized the antigen complex. The sensitivity of the test for patients suffering from extrapulmonary tuberculosis was similar to that for patients suffering from pulmonary tuberculosis but who had negative smears. The frequency of positive reactions among the patients suffering from other pulmonary diseases was similar to that among the control subjects. In tuberculous patients infected with human immunodeficiency virus, the sensitivity of the immunoblot test was significantly lower. Thus, this test based on an antigen complex used in an immunoblot assay to detect the presence of IgG antibody has a specificity of 98% and a sensitivity of 40%. The simultaneous use of different purified antigens, selected at the same high specificity level, may improve the sensitivity of such an assay.
High prevalence of Borna disease virus infection in healthy sheep in Japan
Authors: Hagiwara K , Kawamoto S , Takahashi H , Nakamura Y , Nakaya T , Hiramune T , Ishihara C , Ikuta K
Published: January 2020

Previous seroepidemiological and molecular epidemiological studies of Borna disease virus (BDV) showed considerably high rates of infection in horses, cattle, cats, and humans in Hokkaido, Japan. Here, we further demonstrate high rates of specific antibodies to BDV and BDV RNA in peripheral blood mononuclear cells (PBMCs) from healthy sheep bred on the same island. The BDV prevalences by immunoblotting and/or reverse transcriptase PCR were 0% (0 of 19) in newborns (<1 month old), 51.7% (15 of 29) in lambs (1 to 6 months old), and 36.7% (11 of 30) in adults (>2 years old). Among animals positive for BDV, 60% of lambs and 45.5% of adults contained BDV RNA in PBMCs while 46.7% of lambs and 90.9% of adults contained specific antibodies to BDV. Thus, it is suggested that virus replication in the blood, as observed in lambs, is usually reduced in adulthood by raising immune responses to BDV.
Measurement of antibodies against meningococcal capsular polysaccharides B and C in enzyme-linked immunosorbent assays: towards an improved surveillance of meningococcal disease
Authors: Andersen J , Berthelsen L , Lind I
Published: January 2020

In order to improve the surveillance of serogroup B and C meningococcal diseases, enzyme-linked immunosorbent assays (ELISAs) specific for anti-B immunoglobulin M (IgM) and anti-C IgM and IgG antibodies were developed. The tests were evaluated by using paired sera from 122 patients with and 101 patients without laboratory evidence of meningococcal disease. Fifty-three of 67 patients (79%) with culture-confirmed serogroup B disease had an anti-B IgM antibody response; anti-B IgM levels waned rapidly in children < or = 4 years of age. Twenty-four of 25 patients (96%) with culture-confirmed serogroup C disease had an anti-C IgM and/or IgG antibody response (IgM, 92%; IgG, 68%). In patients without evidence of meningococcal disease, 19% of children < or = 4 years of age and 69% of those > 4 years of age had intermediate anti-B IgM titers. In contrast, only 1 and 5% of these patients had intermediate titers of anti-C IgM and anti-C IgG, respectively. The ELISAs were shown to be powerful tools for discriminating between serogroup B and C diseases in 96 to 100% of culture-confirmed cases. For 90% of patients with culture-negative meningococcal disease, a serogroup-specific diagnosis could be established by examination of paired sera in the ELISAs. As serogroup B and C meningococci account for practically all cases of meningococcal disease in industrialized countries, the availability of these tests may improve surveillance and prevention.
Microplate hybridization for Borna disease virus RNA in human peripheral blood mononuclear cells
Authors: Fujiwara S , Takahashi H , Nakaya T , Nakamura Y , Nakamura K , Iwahashi K , Kazamatsuri H , Iritani S , Kuroki N , Ikeda K , Ikuta K
Published: January 2020

We developed a simple and sensitive microplate hybridization procedure with which to identify Borna disease virus cDNA in amplified products from human peripheral blood mononuclear cells. The mean values for the positive PCR products were significant compared with those for any of the negative products, indicating that this method can be applied to rapidly diagnose a large number of clinical specimens.
Presence of a neutralizing domain in isolates of rubella virus in Cordoba, Argentina
Authors: Cordoba P , Grutadauria S L , Cuffini C , Zapata M T
Published: January 2020

We studied the presence of a neutralizing epitope of rubella virus (RV) in locally circulating strains in Cordoba, Argentina, using binding by the monoclonal antibody (MAb) H3. This epitope is contained in a sequence of the E1 glycoprotein (E1208-239) represented by the synthetic peptide SP15. H3 MAb showed specific binding to SP15 by enzyme-linked immunosorbent assay (ELISA). One wild-type postnatal isolate, four clones derived from this isolate, and one congenital isolate were reactive with H3 by ELISA. These results suggest that the region of RV represented by SP15 is a domain present in locally circulating strains.
Synthetic-peptide-based enzyme-linked immunosorbent assay for screening human serum or plasma for antibodies to human immunodeficiency virus type 1 and type 2
Authors: Gonzalez L , Boyle R W , Zhang M , Castillo J , Whittier S , Della-Latta P , Clarke L M , George J R , Fang X , Wang J G , Hosein B , Wang C Y
Published: January 2020

A synthetic-peptide-based enzyme-linked immunosorbent assay (EIA) capable of screening for antibodies to both human immunodeficiency virus type 1 (HIV-1) and HIV-2 has been developed for use in blood banks and diagnostic laboratories. Microtiter wells are coated with two synthetic peptides, one corresponding to the highly conserved envelope region of HIV-1 and another corresponding to the conserved envelope region of HIV-2. Overall, sensitivity was 100% in 303 individuals diagnosed with AIDS and 96 individuals diagnosed with AIDS-related complex, 14.8% in a study of 500 high-risk group members, 99.9% in 600 EIA repeatedly reactive (RR)-HIV-1 Western blot (WB)-positive repository specimens, and 100% for 222 geographically diverse HIV-1 specimens and 216 confirmed HIV-2-positive specimens evaluated. The specificity was determined to be 99.72% for a total of 13,004 serum and plasma samples from random volunteer donors evaluated across five blood banks. Forty donors who were found to be EIA RR-WB indeterminate but nonreactive on the United Biomedical, Inc., test (UBI HIV 1/2 EIA) were prospectively followed as an additional measure of specificity. None of the 40 low-risk cases evolved into a positive WB pattern at follow-up. The sensitivity and specificity of this new assay are comparable to those of other Food and Drug Administration-licensed HIV-1 and HIV-1-HIV-2 assays that are currently available in the United States. The UBI HIV 1/2 EIA affords laboratories another choice in the detection of antibodies for HIV-1 and HIV-2 with a test based on an alternative antigen format.
Hemagglutination is a novel biological function of lipopolysaccharide (LPS), as seen with the Vibrio cholerae O139 LPS
Authors: Alam M , Miyoshi S , Tomochika K , Shinoda S
Published: January 2020

It has been generally thought that the polysaccharide moiety of lipopolysaccharide (LPS) maintains only serological specificity, while the lipid A portion determines various biological functions. However, we found that hemagglutination was a common function of the polysaccharide moiety of LPSs from important human enteropathogenic bacteria. Of the LPSs examined, Vibrio cholerae O139 LPS showed the highest hemagglutinating activity. Glycoproteins, such as mucin and fetuin, showed efficient inhibition of the hemagglutinating ability. Since cell-mediated hemagglutination is known to be correlated with bacterial adherence, hemagglutination induced by the polysaccharide moiety is interpreted to indicate that cell-surface LPS is a potential adhesin.
Antibodies to the hepatitis B e antigen (HBeAg) can be induced in HBeAg-transgenic mice by adoptive transfer of a specific T-helper 2 cell clone
Authors: Hultgren C , Milich D R , Sällberg M
Published: January 2020

Production of antibody to hepatitis B e antigen (HBeAg); i.e., anti-HBe antibody,) in HBeAg-transgenic mice is believed to be mediated by T-helper 2 (Th2) cells. Injection of an HBeAg-specific Th2 clone into HBeAg-transgenic H-2k mice induced anti-HBe antibody production, confirming the function of Th2 cells in this model system.
Laboratory findings in cows after experimental infection with Ehrlichia phagocytophila
Authors: Pusterla N , Huder J , Wolfensberger C , Braun U , Lutz H
Published: January 2020

The goal of this study was to assess various hematological variables in 10 cows after experimental infection with Ehrlichia phagocytophila. Blood samples were collected at regular intervals for examination of leukocytes for Ehrlichia organisms and for determination of hematological and biochemical variables. In addition, PCR amplification was performed throughout the disease period on blood and milk samples for the detection of E. phagocytophila organisms. The time of seroconversion and the duration of serum titers indicating positivity were determined by indirect immunofluorescence. For all cows, E. phagocytophila organisms were first detected microscopically in leukocytes 5 to 8 days postinfection and could be demonstrated for a period of 6 to 14 days. For all cows, the appearance of E. phagocytophila organisms in leukocytes coincided with transient erythropenia, leukopenia, and thrombocytopenia and a decrease in hematocrit and hemoglobin concentration. For five lactating cows, E. phagocytophila organisms were identified in leukocytes of milk samples during the acute phase of the disease, which, we believe, has not previously been reported. E. phagocytophila DNA was detected in blood samples by nested PCR from 1 to 2 days before to 2 to 12 days after the organisms were identified microscopically. In milk samples, E. phagocytophila DNA was detected for an average of 11 days.
Reactions of polyclonal and neutralizing anti-p54 monoclonal antibodies with an isolated, species-specific 54-kilodalton protein of Chlamydia pneumoniae
Authors: Wiedmann-Al-Ahmad M , Schuessler P , Freidank H M
Published: January 2020

A recently described 54-kDa protein has been detected in six type strains and three patient isolates of Chlamydia pneumoniae by immunoblotting with sera from patients positive for antibodies to C. pneumoniae by the microimmunofluorescence test. This protein was not found in either C. trachomatis E or C. psittaci Z 432 as an antigen, confirming its species specificity. The 54-kDa protein was isolated by continuous-elution electrophoresis and immunoglobulin G monoclonal antibodies (MAbs) against the isolated antigen were produced. MAb 8B11E6 reacted only with the 54-kDa band of C. pneumoniae and not with C. trachomatis E or C. psittaci in a Western immunoblot assay. This antibody was purified and tested for neutralizing activity together with three additional anti-p54-active MAbs (8B11E6, 8B11B4, and 10F1C1). In Buffalo green monkey cells, all of the MAbs significantly reduced the infectivity of C. pneumoniae elementary bodies, whereas no neutralizing activity could be observed with C. trachomatis E or C. psittaci Z 432. These results not only confirm the species specificity of the 54-kDa protein but also indicate that this protein might play an important role in the pathogenesis of C. pneumoniae infection. Furthermore, the results suggest a possible protective role of anti-p54 antibodies in an adaptive immune response.
Selective modulation of lipopolysaccharide-induced death and cytokine production by various muramyl peptides
Authors: Parant M A , Pouillart P , Le Contel C , Parant F J , Chedid L A , Bahr G M
Published: January 2020

Pretreatment of animals with the adjuvant muramyl dipeptide enhances both the production of circulating tumor necrosis factor and the sensitivity to the lethal effect of a lipopolysaccharide (LPS) challenge. The present study examined the capacity of various adjuvant muramyl dipeptide derivatives to potentiate responsiveness to LPS administration. Cytokine levels in serum were determined at various time intervals after LPS administration by bioassays and immunoassays; the cytokines examined were tumor necrosis factor, interleukin-1, interleukin-6, and gamma interferon. The time course of cytokine response was not modified by the pretreatment, but most of the levels were strongly enhanced. However, of the four compounds which were found to be potent priming agents, only two caused an increased sensitivity to LPS lethality, showing that elevated titers of cytokines in serum were not correlated with host sensitization. Interestingly, previous studies have shown that these two compounds also display neurobiological properties, implying a possible role of the central nervous system in LPS lethality. However, two hydrophilic derivatives with low activity as priming agents were capable of decreasing the toxicity of LPS when given after the challenge in galactosamine-sensitized mice. These results illustrate the diversity of responses elicited by immunological priming. They raise unanswered questions on the importance of endogenous mediators in the pathophysiological alterations during toxic shock.
Immunomodulatory spectrum of lipids associated with Mycobacterium avium serovar 8
Authors: Barrow W W , Davis T L , Wright E L , Labrousse V , Bachelet M , Rastogi N
Published: January 2020

Lipid fractions obtained from Mycobacterium avium serovar 8 were assessed for the ability to affect various immune functions of human peripheral blood mononuclear cells (PBM). Lipids included a total lipid fraction and fractions eluted from silicic acid column separation of that total lipid fraction, using chloroform and chloroform-methanol combinations. Lipid fractions were assayed for total carbohydrate and total 6-deoxyhexose content and were assessed for the ability to influence human macrophage function and the capacity to induce secretion of prostaglandin E2 (PGE2) and tumor necrosis factor alpha in PBM. The total lipid and serovar-specific glycopeptidolipid (GPL) fractions both induced significant levels of tumor necrosis factor alpha, as well as PGE2, in PBM exposed to a sublethal concentration of 100 micrograms lipid per 2 x 10(6) cells. In addition, the same concentrations of the 5 to 7% and GPL fractions induced significant levels of leukotriene B4 in PBM. Comparison of carbohydrate and 6-deoxyhexose contents of each fraction suggested a relationship to carbohydrate content and ability of fractions to induce immune modulator secretion. Analysis of GPL fractions from M. avium serovars 4 and 20 revealed that those GPL lacked the ability to induce PGE2. These results are explained by considering the difference in the carbohydrate residues of the oligosaccharide moieties.
Heat stress alters the virulence of a rifampin-resistant mutant of Francisella tularensis LVS
Authors: Bhatnagar N B , Elkins K L , Fortier A H
Published: January 2020

We have studied the stress response of a rifampin-resistant mutant of Francisella tularensis LVS. This mutant, Rif 7, was avirulent with an intraperitoneally administered 50% lethal dose greater than 10(7) CFU in a murine model of infection. Exposure of Rif 7 to heat stress for 5 h in vitro resulted in a 2-log decrease in its 50% lethal dose (P < 0.02). The increase in virulence was dependent on the time of exposure to high temperature and was maximal at 5 h. Envelope preparations from heat-stressed cells showed increased levels of several proteins. Notable among these were polypeptides with approximate molecular masses of 16, 60, and 75 kDa. Increases in both virulence and envelope protein levels were reversed when heat-treated cells were subsequently grown at 37 degrees C. Inhibition of protein synthesis by actinomycin D during heat stress blocked the increase in virulence of Rif 7. Cell-free media from the heat-stressed Rif 7 reacted with the whole spectrum of bacterial proteins were not toxic to mice. Hyperimmune serum against Rif 7 reacted with the whole spectrum of bacterial proteins in Western blots (immunoblots), although its reaction with 34- and 45-kDa proteins and two 60- and 75-kDa proteins upregulated during heat stress was weak. Other stress conditions, low iron and low pH, caused similar increases in the virulence of Rif 7. However, examination of the protein profile did not reveal any major common polypeptides induced by different stresses. Heat-treated Rif 7 bacteria were fully able to replicate in macrophages in vitro and in the host tissues, even though heat treatment only partially restored virulence.
Human T-cell activation by 14- and 18-kilodalton nuclear proteins of Leishmania infantum
Authors: Suffia I , Quaranta J F , Eulalio M C , Ferrua B , Marty P , Le Fichoux Y , Kubar J
Published: January 2020

Leishmanial antigens which stimulate T lymphocytes from primed individuals may be candidates for a vaccine. We recently found a significant concordance between the humoral response specific for two proteins from Leishmania infantum promastigotes, p14 and p18, and a positive leishmanin delayed-type hypersensitivity reaction, testifying to the occurrence of cell-mediated immunity. In this communication, we describe a partial characterization of these antigens and an in vitro analysis of their capacity to activate primed human T cells. We showed, by immunofluorescent staining and through analysis of subcellular fractions by Western immunoblotting, that in stationary-phase promastigotes, p14 and p18 were located only in the parasite nuclei; in the middle of the log phase, a transitory and only weak expression outside the nucleus was detected. We then showed that p14 and p18 antigens shared a common epitope(s). Finally, we analyzed the in vitro proliferation and interleukin-2 production induced by leishmanial proteins in human peripheral blood mononuclear cells from sensitized subjects. We showed that in some individuals who have been exposed to L. infantum the specific response to the whole lysate was mostly due to the nuclear antigens. We demonstrated directly the capacity of nitrocellulose-bound p14 and p18 to activate in vitro all of the tested primed peripheral blood mononuclear cells, which contrasted with a lack of stimulatory activity of other membrane-bound leishmanial proteins. Taken together, our results suggest that an antigenic determinant(s) dominant for some individuals might exist on both antigens.
Adoptive transfer of T lymphocytes to T-cell-depleted mice inhibits Escherichia coli translocation from the gastrointestinal tract
Authors: Gautreaux M D , Gelder F B , Deitch E A , Berg R D
Published: January 2020

Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal (GI) tract to extraintestinal sites, such as the mesenteric lymph node (MLN), spleen, liver, kidneys, and blood. Previously, we reported that depletion of CD4+ and/or CD8+ T cells promotes bacterial translocation from the GI tract to the MLN. In the present study, CD4+ and/or CD8+ T cells, harvested from donor mice, were adoptively transferred to mice previously depleted of T cells by thymectomy plus intraperitoneal injection of rat anti-mouse T-cell monoclonal antibodies. The adoptively transferred CD4+ and/or CD8+ T cells inhibited the translocation of Escherichia coli from the GI tract. Migration of the adoptively transferred T cells to the spleens and MLNs of the recipient mice was determined by utilizing Thy 1.1+ donor cells adoptively transferred into mice whose cells express the Thy 1.2 marker. These results provide further evidence of the importance of T cells in the host immune defense against bacterial translocation from the GI tract.
Generation of interleukin-8 from human monocytes in response to Trichomonas vaginalis stimulation
Authors: Shaio M F , Lin P R , Liu J Y , Yang K D
Published: January 2020

Neutrophils are the predominant inflammatory cells found in the vaginal discharges of patients with Trichomonas vaginalis infection. We have investigated the possible role of interleukin-8 (IL-8) in the inflammatory response elicited by T. vaginalis infection. This study has shown that T. vaginalis induces blood monocytes to produce large amounts of bioactive IL-8, mainly by membrane components of T. vaginalis (MTV). Monocyte-derived IL-8 induced by MTV was dose and time dependent. The peak level of IL-8 was 102 +/- 11 ng/ml of conditioned media (mean +/- standard error; n = 5) obtained from MTV-stimulated monocytes (MTVCM) at 36 h of cultivation. With a multichamber chemotactic assay, we found an optimal neutrophil chemotaxis (177 +/- 14 migrated cells) induced by MTVCM collected at 16 h of cultivation when the level of IL-8 was 42 +/- 8 ng/ml. A neutralizing monoclonal antibody directed against IL-8, but not the irrelevant antibodies, significantly blocked the neutrophil chemotactic activity (decreased from 153 +/- 6 to 23 +/- 3 migrated cells; n = 3 [P < 0.001]) induced by MTVCM. Moreover, the maximum increase of the IL-8 mRNA level from MTV-treated monocytes was observed after a 5-h cultivation and decreased thereafter. Monocytes cocultured with MTV in the presence of a neutralizing monoclonal antibody directed against tumor necrosis factor alpha, but not against IL-1 beta, decreased IL-8 production by 25% (P < 0.05), indicating that the release of IL-8 in MTV-stimulated monocytes is partially dependent on tumor necrosis factor alpha. The capacity of MTV-induced monocytes to synthesize IL-8 suggests that these cells can contribute to the induction of the acute inflammatory response seen in T. vaginalis infection.
Escherichia coli cytotoxic necrotizing factor 1: evidence for induction of actin assembly by constitutive activation of the p21 Rho GTPase
Authors: Fiorentini C , Donelli G , Matarrese P , Fabbri A , Paradisi S , Boquet P
Published: January 2020

Cytotoxic necrotizing factor type 1 (CNF1) induces in HEp-2 cells an increase in F-actin structures, which was detectable by fluorescence-activated cell sorter analysis 24 h after addition of this factor to the culture medium. Increase in F-actin was correlated with the augmentation of both the cell volume and the total cell actin content. Actin assembly-disassembly is controlled by small GTP-binding proteins of the Rho family, which have been reported recently to be modified by CNF1 treatment. Clostridium difficile toxin B and Clostridium botulinum exoenzyme C3, both known to act on the Rho GTPase, were used as biological tools to study the effect of CNF1 on this protein. CNF1 incubated before, during, or after exposure to the chimeric toxin C3B (which is the product of a genetic fusion between the DNA coding for C3 and the one coding for the B fragment of diphtheria toxin) protected HEp-2 cells from the disruption of F-actin structures caused by inactivation of the Rho GTPase through its ADP-ribosylation. On the other hand, C. difficile toxin B cytopathic effect was not observed upon preincubation of cells with CNF1. Toxins acting through a Rho-independent mechanism, such as cytochalasin D and Clostridium spiroforme iota-like toxin, could not be modified in their cellular activities by CNF1 treatment. All of our results suggest that CNF1 modifies the Rho molecule, thus probably protecting this GTPase from further bacterial toxin modification.
Purification and characterization of an exopolysaccharide of Burkholderia (Pseudomonas) pseudomallei
Authors: Steinmetz I , Rohde M , Brenneke B
Published: January 2020

Burkholderia pseudomallei (basonym Pseudomonas pseudomallei) is the causative organism of melioidosis, a disease which is recognized as a major public health problem primarily in Southeast Asia and Northern Australia. In this paper, we report on the identification, purification, and characterization of a species-specific exopolysaccharide of B. pseudomallei. After immunization of mice with a B. pseudomallei strain exhibiting mucoid growth characteristics, we isolated an immunoglobulin G1 monoclonal antibody (MAb) (3015) with specificity for a carbohydrate structure as determined by immunoblotting following sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Electron microscopy studies with MAb 3015 revealed reactivity with an exopolysaccharide with a capsule-like appearance in the immunizing strain. All of the mucoid and nonmucoid B. pseudomallei strains tested from geographically different tropical regions were recognized by MAb 3015 in an enzyme-linked immunosorbent assay or immunoblot, indicating that the exopolysaccharide is constitutively expressed among this species. Intensive testing for cross-reactivity including members of all the Pseudomonas rRNA groups showed no cross-reactivity except in the case of the closely related species Burkholderia mallei. A protocol for purification of the exopolysaccharide which is based principally on mechanical separation from the cell surface followed by repetitive ethanol precipitation steps and finally affinity chromatography using MAb 3015 was established. The exopolysaccharide yielded was of high purity. Gel permeation chromatography was performed, and the molecular mass was estimated to be > 150 kDa. Sera from patients with melioidosis were strongly reactive with the purified exopolysaccharide, indicating its in vivo expression and immunogenicity in natural infection. The diagnostic value of the exopolysaccharide and its role in the pathogenesis of disease must still be determined.
Evidence of recombination and an antigenically diverse immunoglobulin A1 protease among strains of Streptococcus pneumoniae
Authors: Lomholt H
Published: January 2020

The genetic relationships among 114 isolates of Streptococcus pneumoniae representing mainly nine serotypes that frequently cause severe childhood disease in Northern Europe were examined by use of multilocus enzyme electrophoresis. A comparison was made of the corresponding antigenic variations of excreted immunoglobulin A1 (IgA1) proteases detected by enzyme neutralization assays. Allelic variation at 13 gene loci among 70 electrophoretic types disclosed a comparatively low mean genetic diversity per locus (H = 0.319). In contrast, IgA1 proteases showed extensive antigenic diversity as 17 different inhibition types were distinguished. A lack of overall clonality was apparent from the linkage equilibrium of alleles harbored by 28 isolates chosen to represent the genetic diversity of the study population. However, certain clones, such as those marked by identical electrophoretic type, serotype, and IgA1 protease type, persisted for a sufficiently long time to enable clonal spread between distant geographic areas. Among clonally related isolates, examples illustrating a shift of capsular serotype or IgA1 protease type supported the view that recombination occurs in vivo in corresponding genes. In conclusion, over time, horizontal genetic exchange appears to be sufficiently frequent to disrupt the clonal structure otherwise generated by binary fission in natural populations of S. pneumoniae. The clonal instability combined with considerable antigenic heterogeneity renders the pneumococcal IgA1 protease less attractive as a potential component of future vaccines.
Growth hormone activation of human monocytes for superoxide production but not tumor necrosis factor production, cell adherence, or action against Mycobacterium tuberculosis
Authors: Warwick-Davies J , Lowrie D B , Cole P J
Published: January 2020

We have previously demonstrated that growth hormone (GH) is a human macrophage-activating factor which primes monocytes for enhanced production of H2O2 in vitro. This report extends our observations to other monocyte functions relevant to infection. We find that GH also primes monocytes for O2- production, to a degree similar to the effect of gamma interferon. Neither macrophage-activating factor alone stimulates monocytes to release bioactive tumor necrosis factor. However, GH, unlike gamma interferon, does not synergize with endotoxin for enhanced tumor necrosis factor production. In further contrast, GH does not alter monocyte adherence or morphology, while phagocytosis and killing of Mycobacterium tuberculosis by GH-treated monocytes are also unaffected. Therefore, despite the multiplicity of the effects of GH on the immune system in vivo, its effects on human monocytes in vitro appear to be limited to priming for the release of reactive oxygen intermediates.
A conserved peptide sequence of the Plasmodium falciparum circumsporozoite protein and antipeptide antibodies inhibit Plasmodium berghei sporozoite invasion of Hep-G2 cells and protect immunized mice against P. berghei sporozoite challenge
Authors: Chatterjee S , Wery M , Sharma P , Chauhan V S
Published: January 2020

Minutes after injection into the circulation, malaria sporozoites enter hepatocytes. The speed and specificity of the invasion process suggest that it is receptor mediated. The region II sequence of Plasmodium falciparum circumsporozoite (CS) protein includes a nonapeptide (WSPCSVTCG) which is highly conserved in all of the CS proteins sequenced to data, including the one from Plasmodium berghei. We have found that two peptides based on the P. falciparum region II sequence, P18 (EWSPCSVTCGNGIQVRIK) and P32 (IEQYLKKIKNS ISTEWSPCSVTCGNGIQVRIK), significantly inhibited P. berghei sporozoite invasion into Hep-G2 cells in vitro. This inhibition was enhanced if either peptide was preincubated with Hep-G2 cells prior to sporozoite invasion. We confirm that region II is a sporozoite ligand for the hepatocyte receptor; moreover, despite the few differences between P. falciparum and P. berghei region II sequences around the nonapeptide sequence (66% homology), the functional characteristics of the motif sequences are not affected. Since the conserved motifs represent a crucial sequence involved in Plasmodium sporozoite invasion of hepatocytes, antibodies to region II should inhibit sporozite invasion into hepatocytes. Indeed, we found that polyclonal antibodies generated to the P. falciparum-based peptide P32 inhibited P. berghei sporozoite invasion of Hep-G2 cells. Furthermore, inbred mice (C57BL/6) immunized with P32 were protected against a lethal challenge of P. berghei sporozoites. Our results suggest that the conserved region II of the CS protein contains crucial B- and T-cell epitopes, that such peptide sequences from the human malaria parasite P. falciparum can be screened in the P. berghei rodent model, and, finally, that region II can be considered useful as one of the components of a malaria vaccine.
Mapping of a surface-exposed, conformational epitope of the P6 protein of Haemophilus influenzae
Authors: Bogdan J A , Apicella M A
Published: January 2020

P6 is an outer membrane protein of Haemophilus influenzae that is antigenically conserved and considered a candidate component of future H. influenzae vaccines. P6 contains a surface-exposed epitope recognized by monoclonal antibody (MAb) 3B9. This epitope has been shown to be distinct from that recognized by the P6-specific MAbs 7F3 and 4G4 in a competitive inhibition enzyme-linked immunosorbent assay (ELISA). MAb 3B9 did not bind to synthetic P6-specific sequential and overlapping hexameric peptides. Five peptides made to correspond to P6 sequences with high probabilities of surface exposure did not inhibit binding of MAb 3B9 to P6. An antiserum to one of the peptides, designated SP66, inhibited binding of MAb 3B9 to P6. A rabbit antiserum to P6 bound to sequential hexameric peptides, Gly-87AsnThrAspGluArgGlyThr-94, which were in the SP66 region of P6. This antiserum inhibited the binding of P6 to MAb 3B9 in a competitive inhibition ELISA. P6 mutations with His and Ala substitutions at residues Thr-88 and Asn-89 still bound MAb 3B9. MAb 3B9 reacted with Escherichia coli OmpA and Salmonella typhimurium OmpA. Sequence comparisons of P6 with these proteins indicated that the residue in the SP66 region responsible for binding is either Gly-87, Asp-90, or Gly-93. Mercaptoethanol reduction abolished MAb 3B9 binding to E. coli OmpA and S. typhimurium OmpA. In these proteins, immediately downstream of the second cysteine, there is an ArgArg dipeptide which is identical to and aligns with Arg-147Arg-148 in P6. This dipeptide has a high probability of surface exposure in P6. Mutagenesis of the Arg-147Arg-148 to an AlaAla dipeptide in P6 abolished binding of MAb 3B9, demonstrating that it was either a portion of the epitope or important in the protein folding necessary for expression of this epitope. This study demonstrates that MAb 3B9 recognizes a conserved conformational determinant on the surface of H. influenzae that is composed of two discontinuous regions of P6.
Reduced virulence of Candida albicans PHR1 mutants
Authors: Ghannoum M A , Spellberg B , Saporito-Irwin S M , Fonzi W A
Published: January 2020

Candida albicans mutants lacking PHR1 exhibit a pH-dependent morphogenic defect which is expressed at pH 7.5, a pH comparable to that of mammalian blood (S. M. Saporito-Irwin, C. E. Birse, P. S. Sypherd, and W. A. Fonzi, Mol. Cell. Biol. 15:601-613, 1995). The in vivo relevance of this expression pattern was tested in a mouse model of systemic candidiasis. A phr1/phr1 mutant was found to be less virulent than an isogenic Phr1+ strain and exhibited altered morphological development in vivo. These results indicate that PHR1 contributes to the virulence of C. albicans.
Trypanosoma cruzi immunosuppressive factor decreases the interleukin-2 mRNA level in cultured normal activated human lymphocytes
Authors: Majumder S , Kierszenbaum F
Published: January 2020

We show here that the marked inhibition of interleukin-2 production seen in hosts acutely infected with Trypanosoma cruzi can be reproduced in vitro by adding a T. cruzi suspension filtrate to cultures of activated human peripheral blood mononuclear cells. This effect was not due to an accelerated decay of interleukin-2 mRNA and appeared to be selective since the levels of other mRNA molecules relevant to lymphocyte activation were not noticeably decreased. The use of normal lymphoid cells in this in vitro system makes it possible to examine the mechanisms used by T. cruzi to induce abnormalities in lymphocyte functions that also occur in infected hosts.
Mapping of the delayed-type hypersensitivity-inducing epitope of secreted protein MPT64 from Mycobacterium tuberculosis
Authors: Oettinger T , Holm A , Mtoni I M , Andersen A B , Hasløov K
Published: January 2020

The gene encoding the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was expressed in Escherichia coli K-12 and purified as a recombinant protein. The purified recombinant MPT64 elicited delayed-type hypersensitivity (DTH) in outbred guinea pigs sensitized with Mycobacterium bovis BCG Tokyo. The skin reactions were comparable to those obtained with native MPT64. No skin reactions were observed when either recombinant MPT64 or native MPT64 was used in guinea pigs sensitized with M. bovis BCG Danish 1331. Amino- and carboxy-terminal deletion mutants of MPT64 were purified as fusion proteins for the mapping of DTH-inducing epitopes on recombinant MPT64 by use of the guinea pig skin test model. The part of the molecule responsible for the biological activity was located at the carboxy-terminal end. Further studies with overlapping synthetic peptides have pinpointed the biological activity at a single DTH-inducing epitope consisting of 15 residues between amino acids Gly-173 and Ala-187. Screening by PCR of 56 clinical isolates of M. tuberculosis from Danish and Tanzanian patients demonstrated the presence of mpt64 in all of the strains. These results point to MPT64 as a possible candidate for a skin test reagent specific for diagnosis of human tuberculosis.