bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of culture conditions on production of type 5 capsular polysaccharide by human and bovine Staphylococcus aureus strains
Authors: Poutrel B , Gilbert F B , Lebrun M
Published: January 2020

Two Staphylococcus aureus strains, the prototype human Reynolds strain and a bovine isolate, were grown in different complex media and in a synthetic medium (D. Taylor and K. T. Holland, J. Appl. Bacteriol. 66:319-329, 1989) and compared for their ability to produce type 5 capsular polysaccharide. Cell-bound and cell-free type 5 capsular polysaccharide were measured by a new one-step competition enzyme-linked immunosorbent assay. The total production and the proportion of cell-bound type 5 capsular polysaccharide were dependent on the nature of the medium, the duration of the culture, and the strain. Both strains produced more type 5 capsular polysaccharide when cultivated in the synthetic medium than when cultivated in complex media. The best yield of type 5 capsular polysaccharide, about 300 micrograms/ml of medium, was obtained with strain Reynolds grown for 48 h with shaking in the synthetic broth containing glucose as a carbon source.
Immune response to sulfamethoxazole in patients with AIDS
Authors: Daftarian M P , Filion L G , Cameron W , Conway B , Roy R , Tropper F , Diaz-Mitoma F
Published: January 2020

Antibody- and cell-mediated responses to sulfamethoxazole (SMX) were analyzed in AIDS patients with or without a history of hypersensitivity and in negative controls. In 20 of 20 (P < 0.01) human immunodeficiency virus (HIV)-seropositive patients with skin reactions to cotrimoxazole, we found SMX-specific antibodies, while only 9 of 20 and 17 of 20 HIV-seropositive patients without a history of hypersensitivity to cotrimoxazole had SMX-specific immunoglobulin M (IgM) and IgG, respectively. The levels of specific IgM and IgG were higher in patients with skin reactions than in patients without reactions (IgM, 1.0 +/- 0.19 versus 0.47 +/- 0.23 [P < 0.001]; IgG, 0.68 +/- 0.15 versus 0.47 +/- 0.14 [P < 0.001] [mean optical density values +/- standard deviations]). Seronegative controls with no history of exposure to sulfa compounds did not have SMX-specific IgG or IgM antibodies, and controls with a history of intake of SMX with or without reactions had low levels of IgG and IgM. The SMX-specific IgG subclasses were exclusively IgG1 and IgG3. None of the patients had detectable SMX-specific IgE or IgA antibodies nor did they exhibit a cell-mediated response as measured by a lymphocyte proliferation assay. Antibodies to SMX recognized N-acetyl-sulfonamide, N-(2-thiazolyl)-sulfanilamide, sulfadiazine, and sulfisoxazole but did not recognize sulfanilamide or 3-amino-5-methyl isoxazole in an inhibition assay. It is not known whether the SMX-specific antibodies associated with hypersensitivity reactions to SMX in HIV-seropositive patients have a pathogenic role in these reactions. Sulfanilamide or 3-amino-5-methyl isoxazole, on the other hand, could be potential alternative therapies in HIV-seropositive patients with a history of skin reactions to SMX.
Humoral immune response against human cytomegalovirus (HCMV)-specific proteins after HCMV infection in lung transplantation as detected with recombinant and naturally occurring proteins
Authors: van Zanten J , Harmsen M C , van der Giessen M , van der Bij W , Prop J , de Leij L , The T H
Published: January 2020

The humoral immune response to four intracellularly located cytomegalovirus (CMV) proteins was studied in 15 lung transplant recipients experiencing active CMV infections. Five patients had primary infections, and 10 had secondary infections. Antibodies of the immunoglobulin M (IgM) and IgG classes were measured in an enzyme-linked immunosorbent assay (ELISA) system in which procaryotically expressed recombinant proteins were used as a substrate and also in a monoclonal antibody-based capture ELISA which uses naturally occurring proteins as a substrate. The proteins investigated were the lower matrix protein pp65 (ppUL83), the major DNA-binding protein p52 (ppUL44), and the two immediate early proteins IE1 and IE2 (different splicing products of UL123). Higher levels of antibodies were found to pp65 and especially to p52 than to the immediate early antigens. Antibody levels detected in the recombinant protein-based ELISAs were generally lower than antibody responses detected with the matching antigen capture ELISA. Moreover, some patients appeared to have antibodies mainly to epitopes present on naturally occurring proteins. The antibody responses detected in both assays were related to the viral load during infection as assessed by the CMV antigenemia test, which is a quantitative marker for CMV load. It was found that although epitopes on naturally occurring proteins induce higher antibody responses and responses in more patients, antibodies directed to epitopes present on the recombinant proteins were inversely related to the viral load during a CMV infection. Therefore, antibodies to epitopes on the recombinant proteins might be more clinically relevant in this group of lung transplant recipients.
Are soluble factors relevant for polymorphonuclear leukocyte dysregulation in septicemia?
Authors: Wenisch C , Graninger W
Published: January 2020

Polymorphonuclear leukocytes (PMNs) of twelve patients with gram-negative septicemia exhibited a decreased capacity to phagocytize Escherichia coli and generate reactive oxygen products which normalized within 7 days of treatment. Ex vivo exchange of plasma from age-, sex-, and blood-group-identical normal controls resulted in an increase of both phagocytic capacity and reactive oxygen intermediate generation in PMNs of septicemic patients and transiently reduced phagocytosis and reactive oxygen intermediate production in PMNs of normal controls. These results suggest that extrinsic factors are crucial for PMN function.
Development of interleukin 6 and tumor necrosis factor alpha activity in nasopharyngeal secretions of infants and children during infection with respiratory syncytial virus
Authors: Matsuda K , Tsutsumi H , Okamoto Y , Chiba C
Published: January 2020

Cytokine (interleukin 6 [IL-6] and tumor necrosis factor alpha [TNF-alpha]) activity in nasopharyngeal secretions of 21 infants and children (19 days to 16 months old) infected with primary respiratory syncytial virus was determined by an enzyme-linked immunosorbent assay. IL-6 and TNF-alpha were detectable in 100% (21 of 21) and 67% (14 of 21) of cases during the course of infection, respectively. Generally, TNF-alpha activity was high in the acute phase and declined thereafter, sometimes to undetectable levels. IL-6 activity was also highest in the acute phase and declined thereafter in infants younger than 5 months, while in patients older than 5 months, it-increased during the course of the disease to peak in the early convalescent phase. These observations suggest that inflammatory cytokines are produced in vivo in infants and children in response to primary respiratory syncytial virus infection and may be involved in disease pathogenesis. However, the mechanism of induction of cytokines may be different for infants and children in different age groups.
Impaired expression of interleukin 2 receptor and CD45RO antigen on lymphocytes from children with acute lymphoblastic leukemia in response to cytomegalovirus and varicella-zoster virus
Authors: Mizutani K , Ito M , Nakano T , Kamiya H , Sakurai M
Published: January 2020

The expression of CD25 and CD45RO in lymphocytes from patients with acute lymphoblastic leukemia in response to cytomegalovirus and varicella-zoster virus antigens was analyzed by flow cytometry. It is suggested that the defects in the immune response to cytomegalovirus and varicella-zoster virus in patients with acute lymphoblastic leukemia on chemotherapy are in the expression of CD25 and in the switching of CD45RA cells to CD45RO cells.
Characterization of the recognition site and diagnostic potential of an enterovirus group-reactive monoclonal antibody
Authors: Samuelson A , Forsgren M , Sällberg M
Published: January 2020

The epitope for a monoclonal antibody reactive against different enteroviruses was characterized. The epitope could be located to residues 40 to 48 of VP1, and the most-essential residues for monoclonal antibody binding coincided with those conserved among the majority of known enteroviruses, indicating a high diagnostic potential.
Use of hydroethidine and flow cytometry to assess the effects of leukocytes on the malarial parasite Plasmodium falciparum
Authors: van der Heyde H C , Elloso M M , vande Waa J , Schell K , Weidanz W P
Published: January 2020

Flow cytometry was evaluated as a method of assessing in vitro the effects of leukocytes on blood-stage Plasmodium falciparum. Hydroethidine is converted by metabolizing cells to ethidium, a nucleic acid fluorochrome. After incubation with hydroethidine, viable and dead leukocytes and parasitized and uninfected erthrocytes could all be identified on the basis of fluorescence intensity and size. Leukocytes can therefore be eliminated from further analysis; this allows assessment, at any parasite developmental stage, of the level of parasitemia within erythrocytes in the presence of any of several types of leukocytes. Whether leukocytes actually kill intraerythrocytic parasites can therefore be determined and the level of cytotoxicity can be assessed. The ability of leukocytes to prevent merozoites from invading new erythrocytes, i.e., inhibition of parasite invasion, can also be assessed by this method. When erythrocytes containing schizont-stage parasites were cocultured with different leukocyte populations and the level of parasitemia was determined after merozoite release and invasion, only cultures containing gamma delta T cells inhibited parasite invasion. The different blood-stage forms of the parasite vary in nucleic acid content, which allows each of the developmental stages to be distinguished by flow cytometry; this permits assessment of changes in parasite development in the presence of leukocytes. Monocyte-derived macrophages (MDMs) appeared to have an effect on parasite development. In this instance, when erythrocytes containing ring-form parasites were cocultured with MDMs and harvested 24 h later, the parasites in cultures containing MDMs were at the late schizont stage, whereas parasites in control cultures were early trophozoites; this finding suggests that MDMs accelerate parasite development. Together, these results indicate that flow cytometry is potentially useful for measuring the following effects mediated by leukocytes: (i) level of cytotoxicity, (ii) changes in parasite development, and (iii) inhibition of parasite invasion.
Development of monoclonal antibodies against Hantaan virus nucleocapsid protein
Authors: Ha I S , Choi Y , Park Y S , Cheong H I , Koo J W , Kim I S , Kim E C , Kim S , Lee J S
Published: January 2020

Forty-five hybridoma cell lines producing monoclonal antibodies against Hantaan virus, the etiologic agent of hemorrhagic fever with renal syndrome, were generated by fusion of P3-X63-Ag8.V653 myeloma cells with spleen cells of mice immunized with inactivated Hantaan virus vaccine. Among these, 38 antibodies were identified as binding to the 48-kDa nucleocapsid protein by immunoblot assay or radioimmunoprecipitation. Twenty-six of them were of the immunoglobulin G1 (IgG1), nine were of the IgG2a, and three were of the IgA isotype. According to cross-reactivities with other serotypes of the genus Hantavirus, the antibodies were classified into three groups: 6 antibodies specific to the Hantaan serotype (group I), 20 antibodies cross-reacting with Hantaan and Seoul serotypes (SR-11, Tchoupitoulas, and R22) (group II), and 12 antibodies cross-reacting with Hantaan, Seoul, and Prospect Hill serotypes (group III). None of the antibodies cross-reacted with the Puumala serotype. With a panel of antibodies of different cross-reactivities, serotypes of Hantavirus could be differentiated. Thirty-eight monoclonal antibodies against Hantaan virus nucleocapsid protein which have different cross-reactivities between serotypes were developed. These results confirmed the presence of multiple serotype-specific epitopes on the nucleocapsid protein of Hantaan virus, which can be utilized in differentiation of serotypes.
Interlaboratory variability of CD8 subset measurements by flow cytometry and its applications to multicenter clinical trials. NAID/NICHD Women and Infants Transmission Study Group
Authors: Landay A L , Brambilla D , Pitt J , Hillyer G , Golenbock D , Moye J , Landesman S , Kagan J
Published: January 2020

Recent studies have demonstrated the utility of measuring subsets of CD8+ T cells as prognostic markers in epidemiology cohort studies of human immunodeficiency virus (HIV)-infected patients. Most of these studies evaluating the value of CD8+ T-cell subsets have been performed at single centers, and few data are available on variability in the measurement of the CD8+ cell populations in multicenter trials. In the current study, we addressed this question by evaluating interlaboratory variability from the five laboratories enrolled in the Women and Infants Transmission Study sponsored by the National Institutes of Health. This study evaluated 35 HIV-positive and 28 HIV-negative proficiency testing samples sent to the laboratories for evaluation. The study focused on the robust coefficient of variation (RCV) for CD38 (11%), HLA-DR (21%), and CD57 (15%) expression on the CD8+ population. Data from the current study indicated that the variability in these measurements is greater than that for CD3+ CD4+ (RCV, 5%) and CD3+ CD8+ (RCV, 5%) cells. Knowledge of the variability of the CD8+ subset measurements should guide investigators in the design and analysis of clinical trials and epidemiology studies. Ability to obtain improved interlaboratory agreement on CD8+ subset measurements will facilitate further evaluation of these markers in HIV studies.
Cytokines in the stools of children with complicated shigellosis
Authors: Azim T , Halder R C , Sarker M S , Ahmed S , Hamadani J , Chowdhury A , Qadri F , Salam M A , Sack R B , Albert M J
Published: January 2020

The pathogenesis of the systemic complications, leukemoid reaction and hemolytic uremic syndrome, associated with Shigella dysenteriae type 1 infection is not well understood. The excessive production of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6), has been suggested as a possible factor. We measured IL-6 and TNF-alpha in stools of 56 children with S. dysenteriae 1 infection and 29 children without any apparent infection, all age 12 to 60 months. Sixteen children with S. dysenteriae 1 infection had leukemoid reaction or hemolytic uremic syndrome (complicated shigellosis), while the others did not (uncomplicated shigellosis). Stool IL-6 and TNF-alpha concentrations were higher in children with uncomplicated shigellosis than in children with complicated shigellosis (P = 0.009 and < 0.001, respectively) or in uninfected children (P < 0.001). It is concluded that complicated infection is not associated with higher concentrations of the proinflammatory cytokines IL-6 and TNF-alpha in stool.
Flow cytometry, a new approach to detect anti-live trypomastigote antibodies and monitor the efficacy of specific treatment in human Chagas' disease
Authors: Martins-Filho O A , Pereira M E , Carvalho J F , Cançado J R , Brener Z
Published: January 2020

Sera from patients chronically infected with Trypanosoma cruzi display antibodies that bind to epitopes of living trypomastigotes, known as lytic antibodies (LA), and are detected by a complement-mediated lysis test. Conventional serology antibodies (CSA) are also present in sera from patients with chronic infections but, in contrast to LA, are unable to recognize viable trypomastigotes. The presence of LA has been used as an important element in the criterion of cure in human Chagas' disease. Using flow cytometry technology, we introduced a new and sensitive immunomethod for the detection of anti-live trypomastigote membrane-bound antibodies. On the basis of serological tests (LA and CSA detection) and parasitological assays such as hemoculture (HE), patients were classified into the following groups: chronically infected untreated patients (NT) and treated not-cured patients (TNC), with positive HE and both LA and CSA in their sera; "dissociated" HE-negative patients (DIS), in whom LA was not detected whereas CSA were present; a group of cured HE-negative patients (CUR), who were both LA and CSA negative; and, as control, a group of non-chagasic individuals (NC). Sera from these patients were assayed by incubation with live bloodstream trypomastigotes, which were subsequently exposed to fluorescein isothiocyanate-conjugated anti-human immunoglobulin G. The parasites were then fixed, run in the cytometer, and identified on basis of their size and granularity gain adjustments. On the basis of experience with the complement-mediated lysis test, a level of 20% of parasites being fluorescein isothiocyanate fluorescence positive was used as a cutoff between effective and ineffective treatments. With this criterion, our results indicated that sera from NT and TNC patients were antibody positive whereas all sera from DIS, CUR, and NC patients did not contain membrane-bound antibodies. This new approach is a tool to easily identify anti-live T. cruzi membrane-bound antibodies that can be used to monitor the efficacy of Chagas' disease treatment.
Nucleocapsid protein N of Lelystad virus: expression by recombinant baculovirus, immunological properties, and suitability for detection of serum antibodies
Authors: Meulenberg J J , Bende R J , Pol J M , Wensvoort G , Moormann R J
Published: January 2020

The ORF7 gene, encoding the nucleocapsid protein N of Lelystad virus (LV), was inserted downstream of the P10 promoter into Autographa californica nuclear polyhedrosis virus (baculovirus). The resulting recombinant baculovirus, designated bac-ORF7, expressed a 15-kDa protein in insect cells. This protein was similar in size to the N protein expressed by LV in CL2621 cells when it was analyzed on sodium dodecyl sulfate-polyacrylamide gels. The N protein expressed by bac-ORF7 was immunoprecipitated with anti-ORF7 was immunoprecipitated with anti-ORF7 peptide serum, porcine convalescent-phase anti-LV serum, and N protein-specific monoclonal antibodies, indicating that this N protein had retained its native antigenic structure. The recombinant N protein was immunogenic in pigs, and the porcine antibodies raised against this protein recognized LV in an immunoperoxidase monolayer assay. However, pigs vaccinated twice with approximately 20 micrograms of N protein were not protected against a challenge with 10(5) 50% tissue culture infective doses of LV. Experimental and field sera directed against various European and North American isolates reacted with the N protein expressed by bac-ORF7 in a blocking enzyme-linked immunosorbent assay. Therefore, the recombinant N protein may be useful for developing diagnostic assays for the detection of serum antibodies directed against different isolates of LV.
Nasopharyngeal antibodies to pneumococcal pneumolysin in children with acute otitis media
Authors: Virolainen A , Jero J , Käyhty H , Karma P , Eskola J , Leinonen M
Published: January 2020

Pneumolysin, an intracellular protein toxin of all clinically relevant pneumococcal serotypes, is released in vivo during the autolysis of pneumococci and is believed to pave the way for intact pneumococci to invade and cause disease. Therefore, antibodies to pneumolysin should prevent its destructive function. We measured antibodies to pneumococcal pneumolysin in acute- and convalescent-phase nasopharyngeal aspirate samples of 120 children (median age, 2.5 years) with acute otitis media by enzyme immunoassay. Nasopharyngeal immunoglobulin M (IgM) and IgG class antibodies to pneumolysin were rarely detectable, whereas IgA class antibody was detected often, occurred independently of serum IgA antibody in serum, and correlated with the presence of the secretory component in pneumococcal antibody, indicating local production of IgA antibodies. Nasopharyngeal IgA antibody to pneumolysin was detected in 93% of the children already in the acute phase of otitis. Twenty percent of the children developed at least a threefold rise in the pneumolysin-specific IgA antibody concentration by the convalescent phase of otitis, with the youngest at 6 months of age, regardless of the pneumococcal findings in the nasopharynx or middle ear fluid. We suggest that nasopharyngeal IgA antibody to pneumolysin can be produced early in life by pneumococcal colonization.
Selective increases in antibody isotypes and immunoglobulin G subclass responses to secreted antigens in tuberculosis patients and healthy household contacts of the patients
Authors: Hussain R , Dawood G , Abrar N , Toossi Z , Minai A , Dojki M , Ellner J J
Published: January 2020

Mycobacterium tuberculosis-specific antibodies (immunoglobulin M [IgM], IgE, IgG, and IgG subclasses) were determined in 164 tuberculosis patients (pulmonary involvement, n = 135; lymph node involvement, n = 29), 59 healthy household contacts (HC), and 51 healthy endemic donors (EC) by a quantitative enzyme-linked immunosorbent assay for reactivity with culture filtrate. Among the isotypes, significant differences between tuberculosis patient groups with either pulmonary or lymph node involvement and healthy control groups (HC and EC) were detected only for IgG (P < 0.001) and IgG1 (P < 0.001) antibodies. Pulmonary patients also showed a significant difference with IgM (P < 0.01) and IgE (P < 0.05) antibodies. HC showed elevation of only IgM antibodies compared with EC, indicating that IgM antibodies may be an indicator of recent infection with M. tuberculosis. These results suggest that the switching of IgM antibody response to IgG1 is a critical event in disease progression. Polyclonal IgG1, IgG3, and IgE antibodies also showed significant elevation (P < 0.05) in patients compared with EC. A strong correlation (rho = 0.254; P < 0.003) was observed between M. tuberculosis-specific IgG1 and polyclonal IgG1 in patients, suggesting that activations of antigen-specific and polyclonal antibodies are related events. No correlation was found between IgG1 antibodies and purified protein derivative skin test results. Since IgG1 antibody responses to culture filtrate are present only after disease establishment, IgG1 responses could provide a useful diagnostic marker of disease.
Modulation of immune cell proliferation by glycerol monolaurate
Authors: Witcher K J , Novick R P , Schlievert P M
Published: January 2020

Previous studies have shown that glycerol monolaurate (GML), a surfactant commonly used in a wide variety of food and cosmetic products, inhibits the production of a variety of exotoxins by group A streptococci and staphylococci. Given the highly lipophilic nature of the structure of GML, it is suspected that the surfactant exerts its toxin inhibition effects via interaction with the cell membrane. The present study attempted to characterize some of the potential targets of GML action using the model system of lymphocyte activation. Results from murine splenocytes show that GML stimulates proliferation at concentrations between 10(-5) and 5 micrograms/ml/5 x 10(5) splenocytes. At concentrations greater than 5 micrograms/ml, GML inhibited lymphocyte proliferation and blocked the proliferative effects of the lymphocyte mitogens phorbol myristate acetate and concanavalin A and the potent T-cell mitogen toxic shock syndrome toxin-1. Studies using purified immune cell subsets indicated that GML at a concentration of 0.1 microgram/ml optimally induced proliferation of T cells but did not affect B cells. At higher concentrations, GML inhibited the toxic shock syndrome toxin-1 mitogenic effects on T cells, but did not inhibit the lipopolysaccharide-induced stimulation of B cells, suggesting that GML preferentially affects the T-cell population. GML-induced proliferation was blocked by the immunosuppressive drug cyclosporin A, suggesting that GML may be exerting its T-cell-proliferative effects along the calcium-dependent inositol phospholipid signal transduction pathway.
Detection of anti-neutrophil cytoplasmic antibodies after acute Plasmodium falciparum malaria
Authors: Wenisch C , Wenisch H , Bankl H C , Exner M , Graninger W , Looareesuwan S , Rumpold H
Published: January 2020

Four of 30 patients with Plasmodium falciparum infection in Bangkok, Thailand, were positive for anti-neutrophil cytoplasmic antibodies by indirect immunofluorescence 1 month after antimalarial therapy. No myeloperoxidase, proteinase 3, lactoferrin, or elastase reactivity was found. Since no evidence of vasculitis was seen in these patients, anti-neutrophil cytoplasmic antibody production in malaria-infected susceptible patients probably represents a secondary response, indicating neutrophil activation.
Long-term human serum antibody responses after immunization with whole-cell pertussis vaccine in France
Authors: Grimprel E , Bégué P , Anjak I , Njamkepo E , François P , Guiso N
Published: January 2020

Three hundred sixty children were tested for pertussis serology 0.5 to 1.58 months after complete whole-cell pertussis vaccination. An immunoblot assay was used to detect serum antibodies to pertussis toxin, filamentous hemagglutinin, adenylate cyclase-hemolysin, and pertactin, and agglutination was used for detection of anti-agglutinogen antibodies. Antibodies against pertussis toxin, pertactin, and agglutinogens decreased rapidly after vaccination but increased secondarily, suggesting exposure to infected persons. In contrast, anti-filamentous hemagglutinin antibodies persisted and anti-adenylate cyclase-hemolysin antibodies increased continuously, suggesting either cross-reaction with non-Bordetella antigens or exposure to Bordetella isolates expressing these two antigens, including Bordetella pertussis. These data suggest that unrecognized pertussis is common in France despite massive and sustained immunization in infants and that vaccinated children become susceptible to infection more than 6 years after their last vaccination.
Phenotype of lymphocytes associated with the inflammatory reaction to silicone gel breast implants
Authors: Katzin W E , Feng L J , Abbuhl M , Klein M A
Published: January 2020

The tissue response to silicone gel breast implants typically includes an inflammatory infiltrate that consists of macrophages, foreign body-type giant cells, and a variable number of lymphocytes and plasma cells. The phenotype of the lymphocytic component was investigated with three-color flow cytometry. Lymphocytes were obtained by collecting fluid from the space between the implant and the fibrous capsule or by washing cells from the fibrous capsule at the time of implant removal with total capsulectomy. Eighty-nine percent of the implant-associated lymphocytes were T cells. Twenty-five percent of the CD3+ T cells coexpressed HLA-DR compared with only 7.9% of matched peripheral blood lymphocytes. Sixty-eight percent of the implant-associated T cells coexpressed CD4 and CD29, while only 3% of the T cells coexpressed CD4 and CD45RO. The expression of HLA-DR and the predominance of CD29+ CD4+ T cells indicate that there is immune activation with the potential for stimulating antigen-specific antibody production. The role of silicone gel breast implants in immune activation and its clinical significance require further investigation.
Silicate antibodies in women with silicone breast implants: development of an assay for detection of humoral immunity
Authors: Shen G Q , Ojo-Amaize E A , Agopian M S , Peter J B
Published: January 2020

Silicon, in the form of sodium silicate (Na2SiO3), adsorbed onto bovine serum albumin (BSA)-precoated plates served as the solid-phase antigen in an enzyme immunoassay to detect silicate-reactive antibodies in the plasma of 40 symptomatic women with silicone breast implants, 91 asymptomatic women with silicone breast implants, 50 healthy control women, and 52 women with rheumatic diseases and without silicone breast implants, Silicate-reactive antibodies of immunoglobulin G (IgG) or IgM isotypes were detected in the plasma of 30% (12 of 40) of the symptomatic women with silicone breast implants; 9% (8 of 91) of the asymptomatic women with silicone breast implants; 5% (1 of 20) of the women without implants who had systemic lupus erythematosus; and 0% (0 of 32) of the women without implants who had either Sjögren syndrome, scleroderma, or rheumatoid arthritis. Only 2% (1 of 50) of the sera from the healthy control women contained silicate-reactive antibodies. Preincubation of sera with silicate and eight other metal compounds (including SiO2) demonstrated that the IgG and IgM antibodies bound specifically to silicate, because preincubation with Na2SiO3 inhibited more than 90% of the activity, whereas CrO3, Li2SO4, MgSO4, NiSO4, HgCl2, ZrOCl2, BeSO4, and SiO2 failed to inhibit the IgG or IgM antibody binding to the silicate-BSA plates. Furthermore, the F(ab')2 portion and not the Fc portion of the silicate-reactive IgG was reactive with BSA-bound silicate in the enzyme immunoassay. The assay for silicate-reactive antibodies was quantified by assigning arbitrary units to a standard curve composed of serial twofold dilutions of high-positive (ten times higher than the cutoff) silicate antibody sera. This novel assay is a useful method for detecting and quantifying humoral immune response to silicate.
Utility of flow cytometric detection of CD69 expression as a rapid method for determining poly- and oligoclonal lymphocyte activation
Authors: Simms P E , Ellis T M
Published: January 2020

CD69 is a lymphoid activation antigen whose rapid expression (< or = 2 h postactivation) makes it amenable for the early detection of T-cell activation and for subset activation analyses. In the present study we evaluated the utility of flow cytometric detection of CD69 expression by T cells activated with polyclonal stimuli (anti-CD3 and staphylococcal enterotoxin B [SEB]) and oligoclonal stimuli (tetanus toxoid and allogeneic cells) using flow cytometry. Following activation of T cells with anti-CD3 or SEB, CD69 is detectable at < or = 4 h following activation, with anti-CD3 peaks at 18 to 48 h. Dose titration experiments indicated that CD69 expression largely paralleled that in [3H]thymidine incorporation assays, although the former offered a more sensitive measure of T-cell activation at limiting doses of activator than [3H]thymidine incorporation when cells were activated with either anti-CD3 or SEB. However, activation of T cells with either tetanus toxoid or allogeneic stimulator cells failed to induce detectable CD69 expression at up to 7 days of culture. Subset analyses of anti-CD3- and SEB-activated T cells indicated that populations other than T cells can express CD69 following stimulation with T-cell-specific stimuli, indicating that CD69 can be induced indirectly in non-T cells present in the population. These findings indicate that CD69 is a useful marker for quantifying T-cell and T-cell subset activation in mixed populations but that its utility might be restricted to potent stimuli that are characterized by their ability to activate large numbers of cells with rapid kinetics.
A monoclonal antibody reactive with a common epitope of Moraxella (Branhamella) catarrhalis lipopolysaccharides
Authors: Oishi K , Tanaka H , Sonoda F , Borann S , Ahmed K , Utsunomiya Y , Watanabe K , Nagatake T , Vaneechoutte M , Verschraegen G , Matsumoto K
Published: January 2020

A hybrid cell line producing a monoclonal antibody (MAb) against Moraxella (Branhamella) catarrhalis lipopolysaccharide (LPS) was established. The specificity of the MAb 1B12 to purified rough LPSs from six strains of M. catarrhalis was ascertained by enzyme-linked immunosorbent assay (ELISA), competitive-inhibition ELISA, and immunoblotting. MAb 1B12 bound to live bacterial cells and culture supernatants from a total of 34 strains of M. catarrhalis, including 12 strains with different LPS serotypes. No cross-reactions with smooth and rough LPSs from selected enterobacterial and nonenterobacterial strains, with other respiratory pathogens, or with Neisseria species were observed. These data suggest that MAb 1B12 recognizes a common epitope of M. catarrhalis LPS which differs from serotype determinants.
Antibody responses to defined epitopes in the Epstein-Barr virus BZLF1-encoded transactivator protein among human immunodeficiency virus-infected patients
Authors: Tedeschi R , Dillner J , De Paoli P
Published: January 2020

The Epstein-Barr virus BZLF1-encoded replication activator (ZEBRA) is a key mediator of reactivation from latency to the viral productive cycle. In the present study, the serum antibody responses against three defined ZEBRA epitopes (designated ZEBRA-1, -19, and -22) were determined for 50 human immunodeficiency virus (HIV)-seropositive patients and 100 matched healthy control subjects. The anti-ZEBRA responses were more commonly found among HIV-seropositive patients than among healthy controls for all the three ZEBRA epitopes tested (P < 0.0003, P < 0.003, and P < 0.001, respectively). Comparison of ZEBRA antibody levels with the degree of immunodeficiency (CD4 cell counts), CDC grouping, and HIV p24 antigen positivity showed little association, suggesting that induction of ZEBRA antibodies is an early event after HIV infection.
High-dose catecholamine treatment decreases polymorphonuclear leukocyte phagocytic capacity and reactive oxygen production
Authors: Wenisch C , Parschalk B , Weiss A , Zedwitz-Liebenstein K , Hahsler B , Wenisch H , Georgopoulos A , Graninger W
Published: January 2020

Flow cytometry was used to study phagocytic function (uptake of fluorescein isothiocyanate-labeled bacteria) and release of reactive oxygen products (dihydrorhodamine 123 converted to rhodamine 123) following phagocytosis by neutrophil granulocytes of heparinized whole blood treated with adrenaline, noradrenaline, dopamine, dobutamine, or orciprenaline. Reduced neutrophil phagocytosis and reactive oxygen production were seen at 12 micrograms of adrenaline per liter (72% each compared with control values); at 120 micrograms of noradrenaline (72% each), dobutamine (83 and 80%, respectively), and orciprenaline (81 and 80%, respectively) per liter; and at 100 micrograms of dopamine per liter (66 and 70%) (P < 0.05 for all). At these dosages, neutrophil chemotaxis was reduced to < 50% of control values for all catecholamines. Treatment with catecholamines at lower dosages had no significant effect on phagocytosis or generation of reactive oxygen products or chemotaxis. The phagocytic capacity of granulocytes was related to the generation of reactive oxygen products (r = 0.789; P < 0.05). The results demonstrate that catecholamines have a suppressive effect on the response of phagocytic cells to bacterial pathogens at high therapeutic levels in blood.
Physical mapping of the Borrelia miyamotoi HT31 chromosome in comparison with that of Borrelia turicatae, an etiological agent of tick-borne relapsing fever
Authors: Takahashi Y , Fukunaga M
Published: January 2020

We report the construction of physical maps of chromosomes for Borrelia miyamotoi HT31 (a new species of Borrelia) and Borrelia turicatae (relapsing fever agent) by pulsed-field gel electrophoresis of DNA fragments generated by digestion of chromosomal DNA with rare-cutting restriction endonucleases and reciprocal hybridization. The size of the B. miyamotoi HT31 chromosome was calculated to be approximately 925 kilobase pairs, and the chromosome for B. turicatae was estimated to be 951 kilobase pairs. The chromosomes of B. miyamotoi HT31 and B. turicatae consisted of single linear molecules. The locations of several genes have been assigned to the chromosome maps by Southern hybridization by using specific gene probes. Comparison of the genetic maps of the two species of Borrelia provided evidence that the gene order on the chromosomes is quite similar to that of Borrelia burgdorferi sensu lato strains and is highly conserved in the genus Borrelia.
Detection of Toxoplasma gondii tachyzoites and bradyzoites in blood, urine, and brains of infected mice
Authors: Nguyen T D , de Kesel M , Bigaignon G , Hoet P , Pazzaglia G , Lammens M , Delmee M
Published: January 2020

Different techniques for identifying Toxoplasma gondii were compared. PCR was used to amplify part of the major surface antigen P30 gene of T. gondii. Amplified-DNA detection with the DNA enzyme immunoassay (PCR-DEIA) was more sensitive than ethidium bromide staining after agarose gel electrophoresis and as sensitive as nested PCR. PCR-DEIA, using common enzyme-linked immunosorbent assay (ELISA) methods, avoids agarose gel electrophoresis for the identification of amplified products. T. gondii can also be detected with equal sensitivity in infected fibroblasts, but only after at least 8 days of cell culture. PCR-DEIA is thus recommended because of its sensitivity and convenience for detecting early parasitemia in the surveillance of toxoplasmosis among pregnant women and immunocompromised hosts. The courses of infection in mice infected with two strains of T. gondii were compared. Tachyzoites of the virulent strain T. gondii RH, killing the host in 4 days, were identified in urine specimens and blood samples of mice 24 to 94 h after inoculation but not in brains, but no antibodies were detected. After intraperitoneal inoculation with cysts of the low-level virulence Beverley strain of T. gondii, parasites were identified in blood samples 4 days later and up to 17 days (but not in urine specimens) and in the brain from day 6 through day 525. By ELISA, high antibody titers were found from day 11 to day 525, with parasitemia preceding the appearance of antibodies. The usefulness of PCR-DEIA tests in conjunction with the search for circulating antibodies for the early diagnosis of toxoplasmosis in humans is discussed.
Hydrogen peroxide generation by polymorphonuclear leukocytes exposed to peritoneal dialysis effluent
Authors: Daniels I , Bhatia K S , Porter C J , Lindsay M A , Morgan A G , Burden R P , Fletcher J
Published: January 2020

In the presence of peritoneal dialysis effluent (PDE), human polymorphonuclear leukocytes (PMN) showed reduced production of hydrogen peroxide and hypochlorous acid (H2O2 and HOCl, respectively) when at rest and when stimulated with both soluble (formylmethionyl-leucyl-phenylalanine and phorbol myristate acetate) and particulate (Staphylococcus epidermidis) agonists. This effect occurred in a concentration-dependent manner between 0 and 70%. (vol/vol) dialysis effluent. The inhibition of H2O2 and HOCl observed in resting, formy-methionylleucyphenyalanine-stimulated, and S. epidermidis-stimulated PMN was confined to a low-molecular-mass (< 10,000-Da) fraction of PDE, whereas the inhibition of the PMA response was equally dispersed throughout both low (< 10,000-Da)- and high-molecular-mass (> 10,000-Da) fractions. Human serum albumin, a major component of PDE, also inhibited H2O2 and HOCl production by PMN; however, results from cell-free systems suggested that human serum albumin was not wholly responsible for the inhibition of PMN function seen with PDE. The solute(s) responsible did not affect myloperoxidase but very rapidly scavenged H2O2 and HOCl. These data suggest that the factors capable of affecting H2O2 and HOCl production by PMN accumulate in uremia and are removed from the circulation into dialysis effluent.
Molecular pathology of the X-linked hyper-immunoglobulin M syndrome: detection of wild-type transcripts in a patient with a complex splicing defect of the CD40 ligand
Authors: Ameratunga R , McKee J , French J , Prestidge R , Fanslow W , Marbrook J
Published: January 2020

The X-linked hyper-immunoglobulin M syndrome (XHIM) is a primary immune deficiency disorder characterized by an inability to produce immunoglobulin isotypes other than immunoglobulin M (IgM) and IgD. Recently, a B-cell surface antigen (CD40) and its conjugate T-cell counterstructure (CD40 ligand) were shown to mediate immunoglobulin isotype switching in the presence of cytokines such as interleukin 4. Most patients with XHIM have been shown to have mutations of the extracellular domain of the CD40 ligand. Here we describe a novel point mutation of an intronic splice acceptor site which results in a complex splicing defect of the CD40 ligand in a patient with XHIM. In addition to two species of deleted transcripts, wild-type transcripts were also detected in this individual. The demonstration of wild-type CD40 ligand transcripts may be an explanation for previous observations suggesting that some XHIM patients are able to undergo immunoglobulin isotype switching in vivo.
A novel method for monitoring Mycobacterium bovis BCG trafficking with recombinant BCG expressing green fluorescent protein
Authors: Luo Y , Szilvasi A , Chen X , DeWolf W C , O'Donnell M A
Published: January 2020

To better understand intracellular and extracellular trafficking of Mycobacterium bovis bacillus Calmette-Guérin (BCG) when used as an intravesical agent in the treatment of transitional cell carcinoma (TCC) of the bladder, recombinant BCG (rBCG) expressing the jellyfish green fluorescent protein (GFP) was created. When the MB49.1 murine TCC cell line was incubated with GFP-expressing rBCG, internalization of the pathogen could be directly visualized by UV microscopy and quantitated by flow cytometry. The in vitro internalization of the GFP rBCG by the bladder tumor cells was temperature dependent, occurring most readily at 37 degrees C and being severely inhibited at 4 degrees C. Optimum internalization was achieved in vitro at a 10:1 BCG-to-tumor cell ratio over 24 h during which approximately 16% of the tumor cells became infected. Cytochalasin B, a phagocytosis inhibitor, abrogated the ingestion by almost 100% at a concentration of 200 micrograms/ml, indicating that contractile microfilaments likely played an important role in this process. By using mitomycin, a DNA cross-linking reagent, to inhibit proliferation of MB49.1 cells, clearance of about 40% of the green rBCG was achieved by 3 days postinfection. No significant difference between the GFP rBCG and wild-type BCG was observed in the ability to induce the expression of cell membrane proteins of major histocompatibility classes I and II, ICAM-I and -II, B7-1 and -2, of Fas from MB49.1 cells or cytokine production from mouse spleen cells. These results indicate that GFP rBCG may serve as a useful substitute for wild-type BCG in future studies of in vivo trafficking experimental and clinical immunotherapy.
Human humoral responses to antigens of Mycobacterium tuberculosis: immunodominance of high-molecular-mass antigens
Authors: Laal S , Samanich K M , Sonnenberg M G , Zolla-Pazner S , Phadtare J M , Belisle J T
Published: January 2020

The selection of antigens of Mycobacterium tuberculosis for most studies of humoral responses in tuberculosis patients has been restricted to molecules that were either immunodominant in immunized animals or amenable to biochemical purification rather than those that were reactive with the human immune system. Delineation of antigens that elicit humoral responses during the natural course of disease progression in humans has been hindered by the presence of cross-reactive antibodies to conserved regions on ubiquitous prokaryotic antigens in sera from healthy individuals and tuberculosis patients. The levels of cross-reactive antibodies in the sera were reduced by preadsorption with Escherichia coli lysates, prior to studying their reactivity against a large panel of M. tuberculosis antigens to which the human immune system may be exposed during natural infection and disease. Thus, reactivity against pools of secreted, cellular, and cell wall-associated antigens of M. tuberculosis was assessed by an enzyme-linked immunosorbent assay (ELISA). Initial results suggested that the secreted protein preparation contained antigens most frequently recognized by the humoral responses of pulmonary tuberculosis patients. The culture filtrate proteins were subsequently size fractionated by preparative polyacrylamide gel electrophoresis, characterized by reaction with murine monoclonal antibodies to known antigens of M. tuberculosis by an ELISA, and assessed for reactivity with tuberculous and nontuberculous sera. Results show that a secreted antigen of 88 kDa elicits a strong antibody response in a high percentage of patients with pulmonary tuberculosis. This and other antigens identified on the basis of their reactivity with patient sera may prove useful for developing serodiagnosis for tuberculosis.
Prevalence of antibodies to human papillomavirus (HPV) type 16 virus-like particles in relation to cervical HPV infection among college women
Authors: Viscidi R P , Kotloff K L , Clayman B , Russ K , Shapiro S , Shah K V
Published: January 2020

A human papillomavirus type 16 (HPV-16) virus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA) was used to measure serum antibody to capsid proteins in 376 sexually active college women who were also screened for the presence of genital HPVs by PCR and interviewed for demographic and behavioral risk factors for HPV infection. The seroprevalence was 46% in women with HPV-16 DNA in the genital tract. The corresponding values for women who harbored other HPV types or no HPV in the genital tract were 30 and 19%, respectively (HPV-16 group versus no-HPV group; odds ratio [OR], 3.7; 95% confidence interval [CI], 1.5 to 8.9). The antibody response was significantly higher among women with a high viral load than among those with a low viral load (median optical density value, 0.838 versus 0.137, P = 0.009). Comparable levels of seroreactivity were observed among women infected with HPV types distantly or closely related genetically to HPV-16. Seroreactivity was significantly associated with an age of 25 to 30 years (OR, 2.3; 95% CI, 1.2 to 4.4), three or more lifetime sexual partners (OR, 2.9; 95% CI, 1.1 to 10), and history of a sexually transmitted disease other than HPV (OR, 3.1; 95% CI, 1.5 to 6.3). The percent seropositivity increased linearly with number of lifetime sexual partners until reaching a plateau at 35% for women with more than six partners (chi for linear trend, P < 0.001). The low sensitivity of HPV-16 VLP-based ELISA may limit the usefulness of the assay as a diagnostic test for HPV-16 infection. However, the assay appears to have adequate specificity and should be useful as an epidemiological marker of HPV-16 infection and sexual behavior.
Inter- and intrainstitutional evaluation of automated volumetric capillary cytometry for the quantitation of CD4- and CD8-positive T lymphocytes in the peripheral blood of persons infected with human immunodeficiency virus. Site Investigators and the NIAID New CD4 Technologies Focus Group
Authors: O'Gorman M R , Gelman R
Published: January 2020

Volumetric capillary cytometry (VCC) is a new technology that involves the detection and enumeration of dually fluorochrome-labeled cells in a precise volume. We compared the accuracy and precision of VCC with the accuracy and precision of flow cytometry and hematology (F&H) for the measurement of the absolute numbers of CD4 and CD8 T cells in the whole blood of patients infected with human immunodeficiency virus. Five laboratories, each with a different F&H system and certified by the National institute of Allergy and Infectious Diseases flow cytometry proficiency testing program, were shipped aliquots of the same samples from a central site in addition to procuring samples locally. In general, the VCC technology generated CD4 and CD8 T-cell counts which were lower than those obtained with F&H. Intralaboratory variability of replicate CD4 T-cell determinations was similar for both technologies except in the local samples with CD4 counts less than 200/microliters, where the VCC variability was higher than the F&H variability. Interlaboratory variability on replicate CD4 T-cell counts made by VCC was significantly less than that when counts were made by F&H. The VCC instrument has automated CD4 and CD8 T-cell enumeration in whole blood and has consolidated the process to a single platform. Its performance in this evaluation indicates that it may represent a viable alternative to F&H for obtaining absolute T-cell subset counts.