bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a Human Gamma Interferon Enzyme Immunoassay and Comparison with Tuberculin Skin Testing for Detection ofMycobacterium tuberculosisInfection
Authors: Desem Nuket , Jones Stephen L. 
Published: December 2019

ABSTRACTA sensitive two-step simultaneous enzyme immunoassay (EIA) for human gamma interferon (IFN-γ) has been developed and used as an in vitro test for human tuberculosis (TB) in comparison with tuberculin skin testing. The EIA was shown to be highly sensitive, detecting less than 0.5 IU of recombinant human IFN-γ per ml within a linear detection range of 0.5 to 150 IU/ml. The assay was highly reproducible and specific for native IFN-γ. In addition, the assay detected chimpanzee, orangutan, gibbon, and squirrel monkey IFN-γs. Cross-reactions with other human cytokines or with IFN-γs derived from mice, cattle, or Old World monkeys were not evident. The assay was used to detect TB infection by incubating whole blood overnight with human, avian, and bovine tuberculin purified protein derivatives (PPDs), as well as positive (mitogen)- and negative-control preparations. The levels of IFN-γ in plasma supernatants were then determined. Blood from 10 tuberculin skin test-positive individuals responded predominantly to the human tuberculin PPD antigen and to a lesser extent to bovine and avian PPD antigens. By contrast, blood from 10 skin test-negative individuals showed minimal responses or no response to any of the tuberculin PPDs. Detectable levels of IFN-γ were present in all blood samples stimulated with mitogen. In vivo tuberculin reactivity was correlated with IFN-γ responsiveness in vitro. These results support the further study of the blood culture–IFN-γ EIA system as an alternative to skin testing for the detection of human TB infection.
Effects of Colloidal Resuscitation Fluids on Reticuloendothelial Function and Resistance to Infection after Hemorrhage
Authors: van Rijen Ed A. M. , Ward Jason J. , Little Roderick A. 
Published: December 2019

ABSTRACT

The effects of three resuscitation fluids, hydroxyethyl starch (HES), Haemaccel, and fresh autologous blood, on reticuloendothelial system phagocytic and catabolic functions and resistance to infection after 40% hemorrhages in BALB/c mice were studied. The mice, anesthetized with isoflurane, were bled over a 10-min period, left hypovolemic for 30 min, and then resuscitated with their shed blood or the same volume of asanguineous fluid. Normothermia was maintained throughout the experiments. The uptake and catabolism of intravenously injected double-labelled sheep erythrocytes (
51
Cr-
125
I-SRBC) in liver and spleen were determined at 1 and 48 h after hemorrhage. No significant changes in the uptake or catabolism of SRBC in liver or spleen were found at 1 h after hemorrhage and resuscitation with any of the fluids. However, at 48 h a significant increase in liver uptake of SRBC was seen in animals resuscitated with either Haemaccel or HES compared to that in animals resuscitated with shed blood or in animals subjected to a sham operation. The increase in liver uptake was accompanied by a small decrease in spleen uptake in animals resuscitated with Haemaccel but not with HES. No great changes in catabolic activity were seen at 48 h, although activity levels tended to be higher in animals resuscitated with Haemaccel. Separate groups of animals were challenged by an intraperitoneal injection with live
Escherichia coli
at 1 or 48 h after hemorrhage and resuscitation. Sixty-four percent of the animals resuscitated with shed blood survived the challenge with
E. coli
at 1 h after hemorrhage, whereas only 10 and 0% survival was seen for animals resuscitated with Haemaccel and HES, respectively. At 48 h survival was 80% for shed-blood-resuscitated animals and 60 and 70% for Haemaccel- and HES-resuscitated animals, respectively.
Measurement of Induced Cytokines in AIDS Clinical Trials Using Whole Blood: A Preliminary Report
Authors: Wallis Robert S. , Lederman Howard M. , Spritzler John , Devers Jennifer L. , Georges Daniel , Weinberg Adriana , Stehn Susan , Lederman Michael M. , Group the Actg Inducible Cytokines Focus 
Published: December 2019

ABSTRACTMeasures of immune function have become increasingly important as endpoints in AIDS clinical trials, with respect to both modulation and reconstitution of immunity by experimental therapies. Measurement of immune function in this setting requires the development of robust analytic approaches suitable for the clinical laboratory. Experiments were performed to evaluate the suitability of using cultured heparinized (“whole”) blood for induction of tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), two cytokines critical in AIDS pathogenesis. TNF-α expression ranged from 229 to 769 pg/ml in lipopolysaccharide (LPS)-stimulated cultures and was not detected in unstimulated cultures. IFN-γ expression ranged from 0 to 112,000 pg/ml in phytohemagglutinin A (PHA)-stimulated cultures and from 0 to 789 pg/ml in antigen-stimulated cultures. The mean coefficient of variation observed in three weekly determinations was 0.47 for TNF-α and ranged from 0.12 to 1.73 for IFN-γ. These values indicate that sample sizes of 8, 24, and 29 subjects would be sufficient to detect twofold changes in LPS-induced TNF-α and in PHA- and antigen-induced IFN-γ, respectively, if two baseline and two treatment determinations were obtained, and if the interpatient variability of changes in true levels from baseline to follow-up is negligible compared to the variability in the three weekly measurements. Measurement of LPS-induced TNF-α and mitogen- or antigen-induced IFN-γ can be performed simply and reproducibly in human immunodeficiency virus-infected persons by the whole-blood culture method. Further studies are warranted to determine the effect of overnight shipping on assay reproducibility and to determine the extent to which responses can be reliably detected in subjects with low CD4 cell numbers.
Increased Interleukin-6 Levels in Nasal Lavage Samples following Experimental Influenza A Virus Infection
Authors: Gentile Deborah , Doyle William , Whiteside Theresa , Fireman Philip , Hayden Frederick G. , Skoner David 
Published: December 2019

ABSTRACT
Interleukin-6 (IL-6) is a pleotropic cytokine implicated in the pathogenesis of local inflammation during viral upper respiratory infections. This study determined if experimental influenza A virus infection causes local IL-6 production. Seventeen healthy, adult subjects were intranasally inoculated, by course drops, with a safety-tested strain of influenza A/Kawasaki/86 (H1N1) virus. Nasal lavage samples were collected, symptoms were recorded, and expelled nasal secretions were weighed once before and then daily for 8 days after the virus inoculation. Lavage samples were submitted for virus culture and were examined for IL-6 and IL-4 by enzyme-linked immunosorbent assay. The IL-6, but not IL-4, levels were significantly increased in the nasal lavage samples of the 12 subjects who shed virus but not in those of the 5 subjects who did not shed virus. Moreover, the elevations in IL-6 levels were related temporally to the development of nasal symptoms and secretions but not to systemic symptoms. These results suggest a role for locally produced IL-6 in the pathogenesis and expressed symptomatology of influenza A virus infection.
Selection of Recombinant, Library-Derived Antibody Fragments against p24 for Human Immunodeficiency Virus Type 1 Diagnostics
Authors: de Haard Hans J. W. , Kazemier Bert , Koolen Marck J. M. , Nijholt Liekle J. , Meloen Rob H. , van Gemen Bob , Hoogenboom Hennie R. , Arends Jan-Willem 
Published: December 2019

ABSTRACTBy application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of VHand Vκregions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.
A Competitive Enzyme-Linked Immunosorbent Assay for Measuring the Levels of Serum Antibody toHaemophilus influenzaeType b
Authors: Mariani Massimo , Luzzi Enrico , Proietti Daniela , Mancianti Silvia , Casini Daniele , Costantino Paolo , van Gageldonk Pieter , Berbers Guy 
Published: December 2019

ABSTRACTA competitive ELISA method is described for the measurement of total antibodies to the capsular polysaccharide ofHaemophilus influenzaetype b (HibCPS) in human sera. The competitive method showed an excellent correlation to the radioantigen binding assay (RABA, or Farr assay) and improved correlation of sera with low titers with respect to the more conventional noncompetitive method. Overestimation of samples in the low concentration range was no longer observed with the competitive ELISA method. The free HibCPS competition allowed us to eliminate the day-to-day background variation typical of some sera; thus, only values representing the true anti-HibCPS response were determined. The use of precoated microplates, which could be stored up to 8 months, greatly improved the speed of the procedure. An overall correlation coefficient of 0.9660 was found when 407 serum samples with a wide variety of anti-HibCPS antibody levels were tested with the competitive ELISA and RABA. The regression line was very close to the ideal line, with a slope of 1.0045 and an intercept of −0.1996. A subset of 96 serum samples representative of all pre- and postimmunization samples was used to compare the competitive ELISA with a previously described ELISA method. The competitive method performed in two laboratories in different countries showed a better correlation with the RABA. The correlation factors were 0.9770 and 0.9816, respectively, while a factor of 0.9547 was found with the previously described noncompetitive procedure, which was better for this method than previously reported (r= 0.917). Therefore, the competitive ELISA is proposed for the assay of anti-HibCPS titers in sera from vaccinated subjects.
A Rapid Flow Cytometric Method to Explore Cellular Immunity againstToxoplasma gondiiin Humans
Authors: Kahi Sandrine , Cozon Grégoire J. N. , Greenland Timothy , Wallon Martine , Gay-Andrieu Françoise , Peyron François 
Published: December 2019

ABSTRACTTo assess cell-mediated immunity toToxoplasma gondii, we evaluated the expression of the activation antigens CD69, CD71, and CD25 on T lymphocytes by flow cytometry after specific in vitro stimulation of whole blood from 127T. gondii-positive and 63T. gondii-negative patients. T lymphocytes from many seropositive individuals did not express CD69 at 24 h afterT. gondiiantigen stimulation, but CD71 and CD25 were easily detectable on T cells from seropositive individuals 7 days after specific activation. CD25 was mainly expressed by stimulated CD4+T cells, and its detection on total T cells was both a sensitive (98%) and a specific (97%) indicator of priorT. gondiiinfection. These results make flow cytometric detection of CD25 an excellent candidate for screening cell-mediated immunity toT. gondiiin vitro and an interesting tool for the diagnosis of congenital infection.
Evaluation of a Novel Mononuclear Cell Isolation Procedure for Serological HLA Typing
Authors: Schlenke Peter , Klüter Harald , Müller-Steinhardt Michael , Hammers Hans-Jörg , Borchert Kerstin , Bein Gregor 
Published: December 2019

ABSTRACT

Despite recent advances in DNA-based genotyping, the microcytotoxicity test is still broadly used for the determination of human leukocyte class I antigens in patients as well as organ donors and also for the detection of HLA antibodies. Excellent purity and viability of peripheral blood mononuclear cells (PBMC) are essential for reliable HLA typing results. Background staining and cell loss can contribute to impaired typing results or even cause misinterpretations. A novel isolation procedure using cell preparation tubes (CPT) with prefilled Ficoll was compared with the standard Ficoll gradient. We determined the recovery, purity, and viability of the PBMC after several periods of storage. Finally, the isolated cells were used for HLA class I typing, and background reactivities were scored. By using the CPT method, the recovery of PBMC was significantly higher than recovery with the standard technique (
P
≤ 0.001). Contamination by granulocytes increased considerably during the storage time for the standard protocol, whereas purity remained stable when CPT were used (
P
≤ 0.001). With both methods, lymphocyte viability declined markedly over time. We found significantly more dead cells by using the CPT methods. Due to high background scores, HLA typing was impossible after 48 h. The isolation of PBMC by the CPT method resulted in a higher yield and improved purity compared to those obtained with the standard gradient technique. The decreasing viability after 48 h limits the use of both methods for HLA typing and HLA antibody screening.
Preparation of a Human Standard for Determination of the Levels of Antibodies to Oxidatively Modified Low-Density Lipoproteins
Authors: Koskinen Sinikka , Enockson Candace , Lopes-Virella Maria F. , Virella Gabriel 
Published: December 2019

ABSTRACT
As part of our ongoing effort to develop a standardized competitive enzyme immunoassay for human autoantibodies to oxidized low-density lipoprotein (oxLDL), we have generated a reference human antibody standard and revised some of the conditions of the assay. The preparation of the standard involved purification of human anti-oxLDL antibodies by affinity chromatography using immobilized oxLDL. The total concentration of antibody in this purified human oxLDL antibody was established by adding the concentrations of immunoglobulin G (IgG), IgA, and IgM determined in the standard by radial immunodiffusion. The isolated antibody was used to calibrate a whole human serum standard, which was used to calibrate the assays to detect antibody in serum samples. We also revisited the general conditions for performance of our competitive assay. We determined that 1/20 was the ideal dilution for performing the absorption step, and that 1/20 and 1/40 were optimal dilutions to assay oxLDL antibody in unknown serum samples. We also established that the optimal concentration of oxLDL for absorption of free antibody in serum samples was 200 μg of oxLDL/ml; no significant decrease in the reactivity of samples with immobilized oxLDL was observed when higher concentrations of oxLDL were used for absorption. The minimum detection level of the assay is 0.65 mg/liter. Because serum samples are diluted 1/20 and 1/40 for the assay, the minimal concentration of antibody detectable in serum is 20-fold higher, i.e., 13 mg/liter. The intraassay coefficient of variation calculated from seven determinations of three samples containing antibody concentrations of 240, 340, and 920 mg/liter ranged from 8 to 6.1%. The interassay coefficients of variation for sera with antibody levels of 100 to 594 mg/liter varied from 9.2 to 7.0%, and for isolated antibodies with concentrations of 52 to 111 mg/liter, the coefficients varied from 5.8 to 3.9%.
Proposed Standardization of
Neisseria meningitidis
PorA Variable-Region Typing Nomenclature
Authors: Sacchi Claudio T. , Lemos Ana P. S. , Brandt Mary E. , Whitney Anne M. , Melles Carmo E. A. , Solari Claude A. , Frasch Carl E. , Mayer Leonard W. 
Published: December 2019

ABSTRACT

Neisseria meningitidis
isolates are conventionally classified by serosubtyping, which characterizes the reactivities of the PorA outer membrane protein variable-region (VR) epitopes with monoclonal antibodies (MAbs). A newer method (PorA VR typing) uses predicted amino acid sequences derived from DNA sequence analysis. The resulting classification schemes are not standardized, offering conflicting and sometimes irreconcilable data from the two methods. In this paper, we propose a standardization of the PorA VR typing nomenclature that incorporates serologic information from traditional PorA serosubtyping with molecular data from predicted VR sequences. We performed a comprehensive literature and database search, generating a collection of strains and DNA sequences that reflects the diversity within PorA that exists to date. We have arranged this information in a comprehensive logical model that includes both serosubtype and PorA VR type assignments. Our data demonstrate that the current panel of serosubtype-defining MAbs underestimates PorA VR variability by at least 50%. Our proposal for VR typing is informative because amino acid sequence and serologic information, when serosubtype-defining MAbs are available, can be deduced simultaneously from the PorA VR designation. This scheme will be useful in future classification and applied epidemiologic studies of
N. meningitidis
, being a systematic way of selecting PorA vaccine candidates and analyzing vaccine coverage and failure.
Differentiation betweenMycobacterium bovisBCG-Vaccinated andM. bovis-Infected Cattle by Using Recombinant Mycobacterial Antigens
Authors: Buddle Bryce M. , Parlane Natalie A. , Keen Denise L. , Aldwell Frank E. , Pollock John M. , Lightbody Ken , Andersen Peter 
Published: December 2019

ABSTRACTTuberculosis continues to be a worldwide problem for both humans and animals. The development of tests to differentiate between infection withMycobacterium tuberculosisorMycobacterium bovisand vaccination withM. bovisBCG could greatly assist in the diagnosis of early infection as well as enhance the use of tuberculosis vaccines on a wider scale. Recombinant forms of four major secreted proteins ofM. bovis—MPB59, MPB64, MPB70, and ESAT-6—were tested in a whole-blood gamma interferon (IFN-γ) assay for differentiation between cattle vaccinated with BCG and those experimentally infected withM. bovis. BCG vaccination induced minimal protection in the present study, with similar numbers of animals infected withM. bovisin BCG-vaccinated and nonvaccinated groups. Following vaccination with BCG, the animals produced moderate IFN-γ responses to bovine purified protein derivative (PPDB) but very weak responses to the recombinant antigens. Cattle from both the BCG-vaccinated and nonvaccinated groups which wereM. bovisculture positive following challenge produced IFN-γ responses to PPDB and ESAT-6 which were significantly stronger than those observed in the correspondingM. bovisculture-negative animals. IFN-γ responses to MPB59, MPB64, and MPB70 were significantly weaker, and these antigens could not discriminate between vaccinated animals which develop disease and the culture-negative animals. The results of the study indicate that of the four antigens tested in the IFN-γ assay, only ESAT-6 would be suitable for differentiating BCG-vaccinated animals from those infected with bovine tuberculosis.
Giant Syncytia and Virus-Like Particles in Ovarian Carcinoma Cells Isolated from Ascites Fluid
Authors: Rakowicz-Szulczynska Eva M. , McIntosh David G. , Smith McClure L. 
Published: December 2019

ABSTRACT
Ovarian cancer cells were isolated from ascites fluid of 30 different patients diagnosed with cystadenocarcinoma of ovaries. Large colonies of malignant ASC cells were observed during the first week of cell growth in vitro. Colony formation was followed by fusion of cells and formation of large multinucleated and highly vacuolated syncytia. In contrast, cells isolated from the ascites fluid produced by patients with benign mucinous cystadenoma of ovaries did not form syncytia. Nonmalignant Brenner tumor cells, isolated from the ascites fluid, also did not form syncytia. Syncytia, but not the nonmalignant tumor cells, were immunofluorescence stained with an anti-human immunodeficiency virus type 1 (HIV-1) gp120 monoclonal antibody (MAb) and MAb RAK-BrI. Both MAbs recognized cancer-associated antigens RAK (for Rakowicz markers) p120, p42, and p25. Exposure of ASC cells to either the anti-HIV-1 gp120 MAb or MAb RAK-BrI inhibited syncytium formation. PCR with HIV-1 Env-derived primers revealed DNA sequences with over 90% homology to HIV-1 gp41 in syncytia and in ovarian cancer cells but not in normal ovary cells. Electron microscopic analysis revealed viral particles, hexagonal in shape (90 nm in diameter), with a dense central core surrounded by an inner translucent capsid and dense outer shell with projections. Negative staining detected membrane-covered particles (100 to 110 nm in diameter) in the cell culture medium. Incubation of normal breast cells with viral particles resulted in drastic morphological changes and syncytium formation by the transformed breast cells. The cytopathic effects of the identified virus resembled those of spumaviruses, which, in addition to their epitopic and genetic homology to HIV-1, might suggest a common phylogeny.
Viability and Recovery of Peripheral Blood Mononuclear Cells Cryopreserved for up to 12 Years in a Multicenter Study
Authors: Kleeberger Cynthia A. , Lyles Robert H. , Margolick Joseph B. , Rinaldo Charles R. , Phair John P. , Giorgi Janis V. 
Published: December 2019

ABSTRACT

The Multicenter AIDS Cohort Study (MACS), an ongoing prospective study of the natural history of human immunodeficiency virus (HIV), has stored biologic specimens, including peripheral blood mononuclear cells (PBMC), from 5,622 participants for up to 12 years. The purpose of the present analysis was to evaluate the quality of the PBMC in the MACS repository in order to test the validity and feasibility of nested retrospective studies and to guide the planning of future repositories. PBMC were collected from MACS participants at four centers at 6-month intervals from 1984 to 1995, cryopreserved, and transported to a central repository for storage. A total of 596 of these specimens were subsequently tested for viability and used to evaluate cell function, to conduct immunophenotype analysis, or to isolate HIV. Simple linear regression models were applied to evaluate trends in recovery and viability over time and by center. Results indicated that from a nominal 10
7
cells cryopreserved per vial at all four centers, the median number of viable cells recovered was at least 5 × 10
6
(50% of the number stored) and the median viability was at least 90%. Results suggested that cryopreserved cells can be stored for at least 12 years with no general tendency toward cell loss over time. Furthermore, there were no statistically significant changes in the percent cell viability according to the length of time frozen, regardless of HIV serostatus or the level of CD4
+
lymphocytes. Storing 10
7
PBMC per vial yields sufficient viable cells for phenotypic and/or functional analysis. Results from the MACS provide the basis for the planning of future repositories for use by investigators with similar research goals.