bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence heterogeneity of the major antigenic protein 1 genes from Cowdria ruminantium isolates from different geographical areas
Authors: Reddy G R , Sulsona C R , Harrison R H , Mahan S M , Burridge M J , Barbet A F
Published: January 2020

The genes for the immunodominant major antigenic protein 1 (MAP1) of Cowdria ruminantium from four African and two Caribbean isolates were cloned, restriction mapped, and sequenced to identify conserved epitopes for development of serodiagnostic tools for heartwater. Restriction length polymorphisms were observed among the respective MAP1 genes analyzed and were confirmed by sequencing. The sequence data generated for these isolates were compared with data for the previously reported Senegal isolate MAP1 gene. These sequences were found to differ from each other by 0.6 to 14.0%. These differences translate into a 0.8 to 10.0% variation in the predicted protein sequence. In the entire coding sequence, several amino acid substitutions were identified in addition to deletions or insertions at three regions of the gene. These variable regions are referred to as variable regions I, II, and III. From the sequence data, an evolutionary distance tree was constructed; this tree suggested that at least two genetically distinct C. ruminantium strains exist in the Caribbean: the isolate from Antigua is similar to that from Senegal, while the isolate from Guadeloupe is closely related to that from Sudan.
Lack of agreement among two commercial enzyme-linked immunosorbent antibody assays and a conventional immunofluorescence-based method for detecting islet cell autoantibodies
Authors: Baron E J , Weber D E , Weide L G
Published: January 2020

Two commercial enzyme-linked immunosorbent assays (ELISAs) for antibodies associated with development of insulin-dependent (type 1) diabetes mellitus (IDDM) were evaluated in conjunction with a conventional indirect immunofluorescent-antibody-islet cell antibody (ICA) test and a radioimmunoprecipitation method for detection of insulin autoantibodies in sera from a selected group of patients. The anti-ICA ELISA was positive for only 1 to 17 serum samples from newly diagnosed IDDM patients but yielded false-positive results with 2 of 6 serum samples containing non-diabetes-related autoantibodies. Although the anti-glutamic acid decarboxylase ELISA did not show positive results for sera with other autoantibodies, it was positive for only 4 of 29 serum samples from recently diagnosed IDDM patients and for 49% of 37 indirect immunofluorescent-antibody-ICA test-positive sera. Until the antibodies associated with the development of diabetes are better characterized, allowing better standards for comparison, it will be difficult to evaluate commercial assays in this field.
An unusual presentation of Sjögren's syndrome
Authors: Crooks G W , Zweiman B
Published: January 2020

A 44-year-old woman presented with a long history of nausea and vomiting and was found to have hypokalemic metabolic acidosis with elevation of the urine bicarbonate level consistent with the new onset of renal tubular acidosis, type I. Her laboratory findings and histologic picture of a buccal biopsy sample of two minor salivary glands were consistent with Sjögren's syndrome. Treatment with steroids markedly improved her symptoms.
Neuraminidase-specific antibody responses to inactivated influenza virus vaccine in young and elderly adults
Authors: Powers D C , Kilbourne E D , Johansson B E
Published: January 2020

Little information is available on the potential role of antibody to influenza virus neuraminidase (NA) in vaccine-induced immunity. In the present study, serologic responses to the N1Texas/91 and N2Beijing/92 NA components of trivalent inactivated influenza virus vaccine were measured by NA inhibition (NI) and enzyme-linked immunosorbent assay (ELISA), and the results for adults aged 18 to 45 (young) or > or = 65 (elderly) years were compared. The two age groups had comparable rates (32 to 50%) of NI response. In contrast, ELISA immunoglobulin G (IgG) antibody responses to N1 and N2 NAs occurred in 70 to 71 and 67 to 83%, respectively, of young subjects but in only 3 to 18 and 18 to 35%, respectively, of elderly subjects. prevaccination mean ELISA IgG and IgA NA antibody titers were generally lower for the young adults than they were for the elderly, whereas the corresponding NI titers were comparable. In young adults, plaque size-reducing NA antibody increases were positively associated with ELISA but not with NI antibody increases. There were no apparent age-related differences in the immunoglobulin isotype distribution of the anti-NA response, with IgG being the dominant class and IgG1 the dominant subclass of serum antibody. Anti-hemagglutinin antibody responses to H1Texas/91 and H3Beijing/92 were greater in magnitude and frequency than the corresponding NA-specific responses to N1Texas/91 and N2Beijing/92 when measured by hemagglutination inhibition and NI, respectively, but not when measured by ELISA. The discordance between NI and ELISA for measurement of NA-specific vaccine responses may reflect the relative insensitivity of NI in discriminating differences when initial antibody titers are low.
Clonal diversity of Streptococcus mitis biovar 1 isolates from the oral cavity of human neonates
Authors: Fitzsimmons S , Evans M , Pearce C , Sheridan M J , Wientzen R , Bowden G , Cole M F
Published: January 2020

The clonal diversity of 101 isolates of the pioneer bacterium Streptococcus mitis biovar 1 obtained from the oral cavities of 40 human neonates 1 to 3 days, 2 weeks, and 1 month postpartum was examined by using rRNA gene restriction patterns. There was a high degree of genetic diversity, with the 101 isolates comprising 93 unique PvuII ribotypes. There were eight identical pairs of ribotype patterns, and seven of the eight pairs were obtained from individual neonates. Only one identical pair comprised isolates obtained from different neonates. In all but two cases, isolates with matching ribotypes were obtained at one visit. Two pairs of isolates with matching ribotype patterns were obtained from neonates on successive visits. The ribotype patterns of the isolates were examined by cluster analysis. The isolates forming each cluster were very similar, yet each cluster was well separated from its neighbors. When several isolates were obtained from individual neonates at a particular visit, in some instances they were contained in a single cluster, whereas in other cases each isolate was contained in a separate cluster. Isolates obtained from individual neonates on successive visits tended to be contained in different clusters. This high degree of diversity, which has been observed in other mucosal commensal bacteria, may serve as a mechanism for avoiding immune elimination of these bacteria.
Prevalence of antibodies to feline parvovirus, calicivirus, herpesvirus, coronavirus, and immunodeficiency virus and of feline leukemia virus antigen and the interrelationship of these viral infections in free-ranging lions in east Africa
Authors: Hofmann-Lehmann R , Fehr D , Grob M , Elgizoli M , Packer C , Martenson J S , O'Brien S J , Lutz H
Published: January 2020

While viral infections and their impact are well studied in domestic cats, only limited information is available on their occurrence in free-ranging lions. The goals of the present study were (i) to investigate the prevalence of antibodies to feline calicivirus (FCV), herpesvirus (FHV), coronavirus (FCoV), parvovirus (FPV), and immunodeficiency virus (FIV) and of feline leukemia virus (FeLV) antigen in 311 serum samples collected between 1984 and 1991 from lions inhabiting Tanzania's national parks and (ii) to evaluate the possible biological importance and the interrelationship of these viral infections. Antibodies to FCV, never reported previously in free-ranging lions, were detected in 70% of the sera. In addition, a much higher prevalence of antibodies to FCoV (57%) was found than was previously reported in Etosha National Park and Kruger National Park. Titers ranged from 25 to 400. FeLV antigen was not detectable in any of the serum samples. FCoV, FCV, FHV, and FIV were endemic in the Serengeti, while a transient elevation of FPV titers pointed to an outbreak of FPV infection between 1985 and 1987. Antibody titers to FPV and FCV were highly prevalent in the Serengeti (FPV, 75%; FCV, 67%) but not in Ngorongoro Crater (FPV, 27%; FCV, 2%). These differences could be explained by the different habitats and biological histories of the two populations and by the well-documented absence of immigration of lions from the Serengeti plains into Ngorongoro Crater after 1965. These observations indicate that, although the pathological potential of these viral infections seemed not to be very high in free-ranging lions, relocation of seropositive animals by humans to seronegative lion populations must be considered very carefully.
Extracellular proteinase activity of Cryptococcus neoformans
Authors: Chen L C , Blank E S , Casadevall A
Published: January 2020

Extracellular proteinase activity was studied for eight strains of Cryptococcus neoformans var. neoformans and two strains of Cryptococcus neoformans var. gattii. Proteinase activity was measured by protein agar clearance, azoalbumin hydrolysis, gelatin liquefaction, and protein substrate polyacrylamide gel electrophoresis. All strains of C. neoformans produced extracellular proteolytic activity. Maximal extracellular proteinase activity in supernatants of C. neoformans cultures was associated with late logarithmic- and stationary-phase cultures. C. neoformans was able to utilize murine immunoglobulin G1, bovine immunoglobulin G, and human complement factor 5 for growth in media containing these proteins as the sole sources of carbon and nitrogen, suggesting a capacity to degrade immunologically important proteins. Protein substrate polyacrylamide gel electrophoresis revealed several bands with proteolytic activity at apparent molecular masses of 200, 100, and 50 kDa. The results confirm the existence of extracellular proteinase activity for C. neoformans.
Lymphocyte subset diversity in idiopathic CD4+ T lymphocytopenia
Authors: Tassinari P , Deibis L , Bianco N , Echeverría de Pérez G
Published: January 2020

The clinical and public health importance of CD4+ T lymphocytopenia without human immunodeficiency virus infection is still unclear. We describe herein two new human immunodeficiency virus-negative patients with low numbers of peripheral CD4+ T cells and opportunistic infections (cerebral toxoplasmosis and tuberculosis plus extrapulmonary histoplasmosis). The low numbers of CD4+ CD29+ memory cells, the high percentage of gamma delta T-cell receptor cells, and the recovery of CD4+ cells after treatment were remarkable.
Selective gamma-chain T-cell receptor gene rearrangements in a patient with Omenn's syndrome: absence of V-II subgroup (V gamma 9) transcripts
Authors: Mathioudakis G , Good R A , Chernajovsky Y , Day N K , Platsoucas C D
Published: January 2020

Only gamma-chain T-cell receptor transcripts utilizing V-1 subgroup gene segments were found in peripheral blood lymphocytes from a patient with Omenn's syndrome. gamma-Chain T-cell receptor transcripts utilizing the V gamma 9 (V-II subgroup) gene segment were absent in peripheral blood lymphocytes from this patient. V gamma 9 J gamma 1.2 C gamma 1 rearrangements are those primarily found in peripheral blood lymphocytes (70 to 85%) from normal donors.
Evidence for the presence of immunoglobulin E antibodies specific to the cell wall phosphomannoproteins of Candida albicans in patients with allergies
Authors: Kanbe T , Morishita M , Ito K , Tomita K , Utsunomiya K , Ishiguro A
Published: January 2020

To determine the major antigenic component of Candida albicans against immunoglobulin E (IgE) antibodies in the sera of patients with allergies who were positive for IgE antibodies to C. albicans crude antigen in a CAP system, phosphomannoproteins (CAMP/A or CAMP/B for serotype A or B strain, respectively) and their acid-stable portions (CAMP-S/A or CAMP-S/B) were isolated from beta-mercaptoethanol (2-ME) extracts of C. albicans cells of serotypes A and B, and IgE antibodies against these components were compared with those against protein complex and enolase (CAE) fractions isolated from C. albicans cells. The dot blot test, which was used to detect IgE antibodies to the C. albicans antigens, showed that IgE antibodies to the 2-ME extract and phosphomannoprotein fractions were present in the sera of 98.0% (2-ME extract), 96.8% (CAMP/A), 93.2% (CAMP-S/A), 97.2% (CAMP/B), and 81.5% (CAMP-S/B) of the patients, whereas IgE antibodies to the protein complex and CAE fractions were found in the sera of 73.6 and 48.8% of the patients, respectively. The extent of IgE binding to the 2-ME extract and phosphomannoproteins was well correlated with the fluorescence intensities estimated with the CAP system. Furthermore, the results obtained from the inhibition experiment with the CAP system indicated that the binding of IgE antibodies to Candida antigens is strongly inhibited by the phosphomannoprotein fraction and is an indication that the serum of the patients contained IgE antibodies specific to the cell wall phosphomannoproteins of C. albicans. Finally, an initial chemical analysis indicated that the epitopes for IgE antibodies on the phosphomannoproteins is a carbohydrate portion, since the ability of CAMP/A to inhibit the binding of IgE antibodies to the homologous CAMP/A was destroyed after oxidation by sodium periodate but not after digestion with proteinase K.
Studies of proliferative responses by long-term-cryopreserved peripheral blood mononuclear cells to bacterial components associated with periodontitis
Authors: Miller G A , Hickey M F , D'Alesandro M M , Nicoll B K
Published: January 2020

Freezing techniques provide a means for repeating and extending immunological assays with frozen aliquots of an individual's peripheral blood mononuclear cell fraction. Lymphocytes which are stored frozen for a limited time retain their ability to respond to polyclonal B-cell activators, mitogens, and antigens of dental interest. Our studies extend these previous findings by determining lymphocyte functional activity following frozen storage for up to 100 weeks. In addition, the autologous immune response was measured by spontaneous lymphocyte proliferation following 0, 1, 40, and 60 weeks of frozen storage. Peak responses for all individuals occurred at day 7 of incubation. The lymphocyte proliferative response to the superantigens toxic shock syndrome toxin-1 (TSST-1) and Staphylococcus enterotoxin A (SEA) were not changed after 100 weeks of frozen storage. Maximum responses varied among the individuals but occurred at equivalent stimulator concentrations. However, slopes generated from data obtained following 0, 4, 13, 20, 30, 50, 88, and 100 weeks of frozen storage showed no significant deviation from zero (P > 0.05) for all individuals tested. After 100 weeks of storage, the total changes in proliferative activity (counts per minute per week) were -2.1% +/- 16.8% and -5.5% +/- 17.0% for TSST-1 and SEA, respectively. The lymphocyte proliferative responses to pokeweed mitogen, concanavalin A, and sonicates of two periodontal pathogens (Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans) following frozen storage were similar to those with TSST-1 and SEA. These results indicate that peripheral blood mononuclear cells stored frozen may serve as appropriate controls to monitor changes in the disease state long-term periodontal treatment.
Parvovirus B19 in anemic liver transplant recipients
Authors: Ndimbie O K , Frezza E , Jordan J A , Koch W , van Thiel D H
Published: January 2020

Five hundred thirty-three liver transplant recipients were seen for follow-up care over a 6-month period. Of these, 23 (4.3%) had a hemoglobin level of < or = 9 g/dl, with 19 being eligible for inclusion in this study. The median hemoglobin level was 8.7 g/dl. Two patients had iron-deficiency anemia. All of the patients were on therapeutic drugs which can suppress erythropoiesis or shorten the lifespan of mature erythrocytes. Six patients (31.6%) were viremic for human parvovirus B19 but none was B19 immunoglobulin M seropositive. Two patients were immunoglobulin M seropositive for cytomegalovirus. The patients with circulating B19 DNA were not easily distinguished from those without the virus by their laboratory results. The absence of reticulocyte counts for these patients contributed to this inability to differentiate B19 from other causes of anemia, particularly drug myelotoxicity. The high likelihood of making a specific diagnosis with the increasing availability of PCR should spur the search for this virus in the liver transplant population.
Cytomegalovirus glycoprotein B-specific antibody analysis using electrochemiluminescence detection-based techniques
Authors: Ohlin M , Silvestri M , Sundqvist V A , Borrebaeck C A
Published: January 2020

An electrochemiluminescence technique was used to develop versatile and sensitive assay strategies for determination of seroreactivities against biologically important cytomegalovirus neutralization epitopes expressed on glycoprotein B. Indirect binding assays showed wide linear assay ranges and revealed that serum samples diluted in parallel with a monoclonal antibody-based standard, simplifying quantitative analytical assessments.
PCR-restriction fragment length polymorphism analysis of the ospC gene for detection of mixed culture and for epidemiological typing of Borrelia burgdorferi sensu stricto
Authors: Masuzawa T , Komikado T , Yanagihara Y
Published: January 2020

Restriction fragment length polymorphism (RFLP) analysis of the outer surface protein C (ospC) gene amplicon was used for rapid screening for genetic variability within Borrelia burgdorferi sensu stricto species and for detection of multiple borreliae in culture. Primers for the ospC gene amplified a fragment of about 600 bp from Borrelia cultures. After cleavage of the amplified products by MboI and DraI, eight different RFLP types were found among 13 B. burgdorferi sensu stricto strains from various sources and geographical areas, and three RFLP types were found among 10 representative isolates from skin biopsy specimens taken from patients residing on the eastern end of Long Island, New York (B. W. Berger, R. C. Johnson, C. Kodner, and L. Coleman, J. Clin. Microbiol. 30:359-361, 1992). These results suggested that the DNA organization of B. burgdorferi sensu stricto is heterogeneous not only globally but also within a localized geographical area and that the ospC-based typing approach could differentiate the B. burgdorferi sensu stricto. From the results obtained using mixed cultures of two different RFLP types of B. burgdorferi sensu stricto, contamination of at least 0.5% of different types of Borrelia cells in culture could be detected. This method could detect a multiple-B. burgdorferi sensu stricto infection in the bladders of mice experimentally infected with two different RFLP type strains. The present study showed that RFLP analysis of ospC-PCR products is a reliable method for epidemiological typing of B. burgdorferi sensu stricto and could be used for rapid detection of mixed Borrelia culture and multiple B. burgdorferi sensu stricto infections in animals, ticks, and patients.
Correlation of seroreactivity with response to antibiotics in pediatric Lyme borreliosis
Authors: Fawcett P T , Rosé C D , Gibney K M , Doughty R A
Published: January 2020

Response to treatment with antibiotics was compared with serologic reactivity and clinical symptoms in a pediatric population with presumptive diagnoses of Lyme borreliosis. The population analyzed for this study consisted of a subset of a larger Lyme clinic population being monitored as part of a prospective study on pediatric Lyme borreliosis. All patients resided in an area in which Ixodes scapularis and Borrelia burgdorferi are considered endemic. Serum from patients was tested by enzyme-linked immunosorbent assay and Western blotting. Response to antibiotics was evaluated by members of a pediatric Lyme clinic. Results showed that positive serologic test results correlate with a favorable response to antibiotics, as does the presence of erythema migrans (EM), regardless of serologic status. Seronegative patients without EM had chronic fatigue and arthralgia and/or myalgia as primary symptoms and did not respond to antibiotics, even when multiple courses of treatment were given. These results indicate that serologic tests designed to have high specificity can reliably rule out Lyme borreliosis in patients with chronic symptoms, thus preventing unnecessary treatment with antibiotics.
Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group
Authors: Maslanka S E , Gheesling L L , Libutti D E , Donaldson K B , Harakeh H S , Dykes J K , Arhin F F , Devi S J , Frasch C E , Huang J C , Kriz-Kuzemenska P , Lemmon R D , Lorange M , Peeters C C , Quataert S , Tai J Y , Carlone G M
Published: January 2020

A standardized serum bactericidal assay (SBA) is required to evaluate the functional activity of antibody produced in response to Neisseria meningitidis serogroup A and C vaccines. We evaluated assay parameters (assay buffer, target strains, growth of target cells, target cell number, complement source and concentration, and methods for growth of surviving bacteria) which may affect the reproducibility of SBA titers. The various assay parameters and specificity of anticapsular antibody to five serogroup A strains (A1, ATCC 13077, F8238, F9205, and F7485) and four serogroup C strains (C11, G7880, G8050, and 1002-90) were evaluated with Centers for Disease Control and Prevention meningococcal quality control sera. The critical assay parameters for the reproducible measurement of SBA titers were found to include the target strain, assay incubation time, and complement. The resulting standardized SBA was used by 10 laboratories to measure functional anticapsular antibody against serogroup A strains F8238 and serogroup C strain C11. In the multilaboratory study, SBA titers were measured in duplicate for 14 pairs of sera (seven adults and seven children) before and after immunization with a quadrivalent polysaccharide (A, C, Y, and W-135) vaccine. The standardized SBA was reliable in all laboratories regardless of experience in performing SBAs. For most sera, intralaboratory reproducibility was +/- 1 dilution; interlaboratory reproducibility was +/- 2 dilutions. The correlation between median titers (interlaboratory) and enzyme-linked immunosorbent assay total antibody concentrations was high for both serogroup A (r = 0.86; P < 0.001; slope = 0.5) and serogroup C (n = 0.86; P < 0.001; slope = 0.7). The specified assay, which includes the critical parameters of target strain, incubation time, and complement source, will facilitate interlaboratory comparisons of the functional antibody produced in response to current or developing serogroup A and C meningococcal vaccines.
Characterization of specific immunoglobulin G (IgG) and its subclasses (IgG1 and IgG2) against the 23-valent pneumococcal vaccine in a healthy adult population: proposal for response criteria
Authors: Rodrigo M J , Miravitlles M , Cruz M J , de Gracia J , Vendrell M , Pascual C , Morell F
Published: January 2020

The aim of the study was to standardize an enzyme-linked immunosorbent assay (ELISA) method for the quantification of immunoglobulin G (IgG) and its subclasses (IgG1 and IgG2) against the 23-valent pneumococcal vaccine and to establish the criteria for a normal response to the vaccine. Forty healthy individuals (20 women and 20 men; mean age, 29 years) were studied. All were vaccinated with the 23-valent pneumococcal vaccine; blood samples were drawn just prior to and 3 weeks after immunization. Quantification of specific IgG and its subclasses was performed by an ELISA with the vaccine as the antigen. The linearity of the ELISA method was demonstrated by the similar slopes of the linear regression lines generated from the titration of sera with different antibody concentrations. The specificity of the antibodies against the vaccine was demonstrated by (i) an absorption test with pneumococcal vaccine, (ii) a cross-reactivity experiment with Haemophilus influenzae type b polysaccharide, and (iii) affinity chromatography with protein A-Sepharose. Response to the vaccine was defined by using the lower level of the 90% probability interval (one-tailed) for postimmunization-specific IgG, IgG1, and IgG2. By using this cutoff, responders were considered to be those with an absolute increase in antibody titers higher than 395 arbitrary units/ml for IgG, 0.350 A450 units for IgG1, and 0.314 A450 units for IgG2. Overall, 20 (50%) subjects had IgG, IgG1, and IgG2 responses, 9 (22.5%) had IgG and IgG2 responses, 4 (10%) had IgG1 responses, 3 (7.5%) had IgG and IgG1 responses, and 4 (10%) were nonresponders. Ninety percent of our population responded to the 23-valent pneumococcal vaccine. Up to 10% of healthy individuals may respond to an IgG subclass without significant increases in total IgG titers. The ELISA method that is described may be useful for evaluating the specific antibody response against polysaccharides.
Distemper: not a new disease in lions and tigers
Authors: Myers D L , Zurbriggen A , Lutz H , Pospischil A
Published: January 2020

In light of recent canine distemper virus (CDV) epidemics, we set out to determine the historical significance of CDV infection in captive lions and tigers in Switzerland. The retrospective case material consisted of 42 lion and tiger necropsy cases from 1972 to 1992. Necropsy reports for all lions and tigers were reviewed. All existing paraffin tissues were immunohistochemically examined with a polyclonal antibody raised against CDV. The results for 19 of the 42 lions and tigers were classified as positive by immunohistochemistry; 23 results were negative or questionable. The results for four animals (three positive and one negative ) were further tested by in situ hybridization, and the results concurred with the immunohistochemistry findings. CDV infection of large cats is older and more widespread than previously thought. All large cats in captivity should be immunized even if canine distemper is not believed to be a problem for large cats in the area.
Comparison of antinuclear antibody testing methods: immunofluorescence assay versus enzyme immunoassay
Authors: Gniewek R A , Stites D P , McHugh T M , Hilton J F , Nakagawa M
Published: January 2020

Performances of anti-nuclear antibody testing by immunofluorescence assay (ANA-IFA) and enzyme immunoassay (ANA-EIA) were compared in relation to patient diagnosis. A total of 467 patient serum samples were tested by ANA-IFA (Kallestad; Sanofi) and ANA-EIA (RADIAS; Bio-Rad), and their age, sex, diagnosis, disease status, and medications were obtained through chart review. Reference ranges were established by testing 98 healthy blood donor samples. Eighty-six samples came from patients with diffuse connective tissue diseases, including systemic lupus erythematosus, discoid lupus erythematosus, or drug-induced lupus (n = 71); systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal motility abnormalities, sclerodactyly, and telangiectasia), or Raynaud's syndrome (n = 8); Sjögren's syndrome (n = 5); mixed connective tissue disease (n = 5); and polymyositis or dermatomyositis (n = 3). The sensitivity, specificity, positive predictive value, and negative predictive value for ANA-IFA were 87.2, 48.0, 29.1, and 93.9%, respectively, for the reference range of < 1:160. For ANA-EIA, they were 90.7, 60.2, 35.8, and 96.4%, respectively, for the reference range of < 0.9. ANA-EIA offers equivalent sensitivity and higher specificity compared to ANA-IFA.
Lymphocyte, monocyte, and natural killer cell reference ranges in postpartal women
Authors: Gennaro S , Fehder W , Gallagher P , Miller S , Douglas S D , Campbell D E
Published: January 2020

Normative values for immune-cell subsets in postpartal women, who are recovering from the relative immunosuppression of pregnancy, have not been established. Considerable differences in normative values for subsets of immune cells have been demonstrated based on sociodemographic factors, such as age and race. In order to make accurate clinical decisions about postpartal women, comparisons with normal reference ranges are necessary. Therefore, flow cytometric data for 51 healthy women at 4 months postpartum are presented and changes over the first 4 postpartal months are documented. The levels of some lymphocyte cell subsets, such as CD4+/CD45RA+ and Ia on lymphocytes, remained stable over time. The levels of other lymphocyte cell subsets, such as CD4+/CD29+, increased over the first 4 postpartal months, while those of other cell subsets, such as CD8 and CD11b, increased between delivery and 2 months postpartum and then dropped again by the fourth postpartal month. The levels of two natural killer cell subsets (CD3-/CD16+ and CD3-/CD57+) rose from delivery until 1 month postpartum and then plateaued. Comparisons were made with reference ranges of nonpostpartal groups provided in the literature and in a study of healthy women being conducted in the same laboratory, and postpartal women were found to have lower values of CD8, CD3-/CD16+, CD4+/CD45RA+, CD20, and CD11b than those reported in the literature.
Combined determination of Coxiella burnetii-specific immunoglobulin M (IgM) and IgA improves specificity in the diagnosis of acute Q fever
Authors: Devine P , Doyle C , Lambkin G
Published: January 2020

Immunoglobulin M (IgM) and IgA responses in patients with acute Q fever were compared by enzyme-linked immunosorbent assay. An increase in both IgM and IgA was observed in paired sera from all 19 patients with acute Q fever, and both IgM and IgA levels showed good correlation with complement fixation test titers. Paired sera from 23 patients with infections other than Q fever were also tested. IgM levels were elevated in three of these patients, while IgA levels were elevated in three different patients (87% specificity for either IgM or IgA). As no patients in the disease control group showed elevated levels of both IgM and IgA, definition of a positive result as elevated levels of both IgM and IgA improved specificity to 100% without a decrease in sensitivity. This study indicates that detection of specific IgA is a useful adjunct to that of IgM in the diagnosis of acute Q fever.
Immunoblot reactivity of polyclonal and monoclonal antibodies with periplasmic flagellar proteins FlaA1 and FlaB of porcine Serpulina species
Authors: Fisher L N , Duhamel G E , Westerman R B , Mathiesen M R
Published: January 2020

The periplasmic-flagellum (PF) proteins of Triton X-100-soluble and Triton X-100-insoluble sodium dodecyl sulfate-treated fractions from reference and field strains of Serpulina hyodysenteriae, Serpulina innocens, and Serpulina pilosicoli were characterized by Western blotting with a rabbit polyclonal antibody (PAb) specific for the 44-kDa PF sheath protein of S. hyodysenteriae (Z. Li, F. Dumas, D. Dubreuil, and M. Jacques, J. Bacteriol. 175:8000-8007, 1993) and a murine monoclonal antibody (MAb), designated 7G2, specific for the PF core FlaB proteins of S. hyodysenteriae. The MAb 7G2 reacted with a conserved epitope present in the 37-, 34-, and 32-kDa PF core FlaB proteins of all Serpulina species. This suggested that the core FlaB proteins are conserved among porcine Serpulina species. An immunoreactive band of approximately 44 kDa was present with all S. hyodysenteriae, S. innocens, and S. pilosicoli strains that were reacted with the PAb. The specificities of the PAb and the MAb for the FlaA1 and FlaB proteins of Serpulina species were confirmed by N-terminal amino acid sequencing of 44- and 37-kDa proteins, respectively, of S. hyodysenteriae and S. pilosicoli. Results from this study provide further evidence that the 44-kDa protein FlaA1 and the 37-, 34-, and 32-kDa FlaB proteins are conserved among porcine Serpulina species.
Evaluation of a dipstick enzyme-linked immunosorbent assay for detection of antibodies to dengue virus
Authors: Wu S J , Hanson B , Paxton H , Nisalak A , Vaughn D W , Rossi C , Henchal E A , Porter K R , Watts D M , Hayes C G
Published: January 2020

Accurate serological confirmation of dengue (DEN) infection is difficult, because simple reliable assays for the detection of DEN antibodies are not available. To address this problem, a dipstick enzyme-linked immunosorbent assay (ELISA) was evaluated. The dipstick contained dots of serially diluted DEN 2 antigen. To detect immunoglobulin G (IgG), the dipstick was processed through four reaction cuvettes containing test serum, enhancer, enzyme-conjugated anti-human IgG and IgM antibody, and substrate. Total assay time was 45 min. To detect IgM, the serum was passed through a protein G device to remove IgG. The dipstick was then processed as before, except that the incubation times were longer and enzyme-conjugated anti-human IgM was used. The total assay time was 3 h. The dipstick ELISA results were compared with results from microplate ELISA. The IgG dipstick ELISA showed a sensitivity of 95.2% and a specificity of 100% compared to an IgG microplate ELISA with serum samples from 125 individuals living in an area in which DEN is endemic. In tests with 75 serum samples from patients with clinically suspected acute DEN infections, the IgM dipstick ELISA showed a sensitivity of 97.9% and specificity of 100% compared to those of an IgM antibody capture microplate ELISA. These results showed that the dipstick ELISA was a sensitive and specific test for the detection of either DEN IgM or IgG in human serum. The dipstick ELISA was also shown to be useful for detecting seroconversions to DEN IgM or IgG in paired serum samples from 20 patients with virus isolation-confirmed acute DEN infections.
Detection of serum antibodies to CagA and VacA and of serum neutralizing activity for vacuolating cytotoxin in patients with Helicobacter pylori-induced gastritis
Authors: Donati M , Moreno S , Storni E , Tucci A , Poli L , Mazzoni C , Varoli O , Sambri V , Farencena A , Cevenini R
Published: January 2020

Thirty patients with dyspepsia, with histological diagnosis of gastritis, and with endoscopic diagnosis of peptic ulcer disease (PUD) (n = 13) or nonulcer dyspepsia (NUD) (n = 17) were admitted to the study. Helicobacter pylori vacuolating cytotoxin-producing strains (Tox+) were isolated from 14 (46.7%) patients, whereas non-cytotoxin-producing (Tox-) H. pylori strains were isolated from the remaining patients. Of 30 patients studied, 20 (66.7%) had serum cytotoxin neutralizing activity in vitro. Fourteen patients with Tox+ H. pylori strains showed serum cytotoxin neutralizing activity and serum immunoglobulin G (IgG) and IgA antibodies reactive with both 87-kDa H. pylori vacuolating cytotoxin (VacA) and 128-kDa cytotoxin-associated gene product (CagA) by immunoblotting using native enriched preparations of VacA and CagA proteins from H. pylori culture supernatants as the antigens. A 94-kDa antigen cross-reacting with the 87-kDa VacA protein could be demonstrated in culture supernatant with immune sera from humans and animals. All patients (n = 10) lacking serum neutralizing activity were also negative for IgG or IgA against VacA antigen, whereas 6 of the 10 patients showed IgG serum antibody responses against CagA antigen. The prevalence of antibodies to VacA and CagA antigens was significantly (P < 0.001) higher in patients with gastritis (20 and 26 patients for VacA and CagA, respectively, of 30 patients) than in H. pylori culture-negative controls (0 of 27 for both VacA and CagA) and in randomly selected blood donors (17 and 21 for VacA and CagA, respectively, of 120 subjects). All patients with PUD had antibodies to CagA, whereas 13 of 17 (76.5%) patients with NUD had anti-CagA antibodies. Serum IgG antibodies to VacA were present in 9 (69.2%) patients with PUD of 13 patients and in 11 (64.7%) patients with NUD of 17 patients. Anti-CagA antibodies seemed to correlate better with PUD than anti-VacA antibodies.
Production of polyclonal antibodies to feline tumor necrosis factor
Authors: Otto C M , Niagro F , McGraw R A , Rawlings C A
Published: January 2020

Two 13-amino-acid peptides were synthesized based on the putative feline tumor necrosis factor (FeTNF) sequence. The synthesized peptides were conjugated to keyhole limpet hemocyanin, emulsified in complete Freund's adjuvant, and injected into rabbits. The gene for FeTNF was cloned into the FLAG (International Biotechnologies Inc. [IBI], Kodak, New Haven, Conn.) fusion protein expression vector. The expressed fusion protein was purified by using the M-1 anti-FLAG octapeptide monoclonal antibody (IBI, Kodak). The fusion protein was emulsified in complete Freund's adjuvant and injected into chickens. The immune sera generated to the synthetic peptides and the fusion protein recognized the recombinant FeTNF fusion protein on Western or dot blot assay. The preimmune and immune sera were incubated with naturally occurring FeTNF (supernatants from lipopolysaccharide-stimulated cultured feline peritoneal exudate or peripheral mononuclear cells). The antibody raised to the recombinant FeTNF fusion protein and N-terminal synthetic peptide neutralized bioactivity of native FeTNF and recombinant human TNF. Preimmune sera did not have any neutralizing activity. The polyclonal antibodies were not specific for FeTNF, since both porcine and human recombinant TNF were neutralized by the fusion protein antibodies. The synthetic peptide antibodies recognized recombinant feline and equine TNF on a Western blot.
A new competitive enzyme-linked immunosorbent assay demonstrates adequate immune levels to rabies virus in compulsorily vaccinated Japanese domestic dogs
Authors: Sugiyama M , Yoshiki R , Tatsuno Y , Hiraga S , Itoh O , Gamoh K , Minamoto N
Published: January 2020

A competitive enzyme-linked immunosorbent assay (c-ELISA) was developed as an alternative to the viral neutralization (VN) test for rapid and simple detection of antibodies to rabies virus. The competitor antibody in the c-ELISA was a biotinylated monoclonal antibody to the nucleoprotein of rabies virus. Initial comparisons showed a high correlation between titers obtained with the VN test and the c-ELISA (n = 88, r = 0.90), indicating that the c-ELISA could be used as a reliable substitute for the VN test. To evaluate the immune status of Japanese dogs to rabies virus, a total of 1,019 serum samples were collected from domestic dogs in 1994 and tested for antibodies with the c-ELISA. Overall, 84.8% of the dogs had antibodies against rabies virus, indicating that the vaccination strategy for preventing rabies outbreaks in domestic dogs is probably sufficient in Japan. Dogs receiving final vaccinations a year or more previously were 48.3 and 90.3% positive for antibodies when vaccinated once only or two or more times, respectively. This suggests that almost all dogs vaccinated twice or more remain seropositive for over 1 year in Japan.
Interleukin-1 receptor mRNA expression in activated bovine leukocytes in vitro
Authors: Yu P W , Schuler L A , Czuprynski C J
Published: January 2020

Interleukin-1 (IL-1) is a key player in inflammation and the immune response. To better understand the complex interactions of IL-1 and its receptors in inflammation, we need to investigate how type I and type II IL-1 receptors (IL-1RI and IL-1RII) are regulated by cytokines and other mediators. Using semiquantitative reverse transcriptase PCR and Northern analysis, we examined the regulation of IL-1RI and IL-1RII mRNA levels in bovine polymorphonuclear leukocytes (PMNs) (i.e., neutrophils) and peripheral blood mononuclear cells (PBMCs) in vitro. IL-1RI mRNA levels were up-regulated in PBMCs by recombinant bovine IL-1beta (rBoIL-1beta), recombinant bovine granulocyte-macrophage colony-stimulating factor (rBoGM-CSF), rBoIL-4, recombinant bovine gamma interferon (rBoIFN-gamma), and dexamethasone. IL-1RI mRNA was increased in bovine PMNs exposed to rBoGM-CSF, rBoIL-4, and dexamethasone but was down-regulated by rBoIL-1beta and rBoIFN-gamma. IL-1RII mRNA was increased in bovine PBMCs and PMNs after exposure to rBoIL-1beta, rBoGM-CSF, rBoIL-4, and dexamethasone. In contrast, rBoIFN-gamma down-regulated the expression of bovine IL-1RII mRNA in PBMCs. These findings suggest that the expression of bovine IL-1RI and IL-1RII mRNAs is regulated differently by certain soluble stimuli (e.g., IFN-gamma) in PMNs and PBMCs.
Clinical field site evaluation of the FACSCount for absolute CD3+, CD3+ CD4+, and CD3+ CD8+ cell count determinations in Thailand
Authors: Young N L , Ponglertnapakorn P , Shaffer N , Srisak K , Chaowanachan T , On-Thern V , Kittinunvorakoon C , Bunwattanakul A , Suksaweang S , Pobkeeree V , Punnotok J , Mastro T D
Published: January 2020

The FACSCount flow cytometer absolute CD3+, CD3+ CD4+, and CD3+ CD8+ cell counts measured at a field site hospital laboratory in Thailand were compared to FACScan absolute counts obtained at a nearby research laboratory. Correlation coefficients for 208 samples were > or = 0.95. The FACSCount was accurate, and it was easier and less expensive to operate than the FACScan. Additionally, the FACScan-generated lymphocyte percentage value was accurate for use with the FACScan SimulSET software.
Altered expression of Fc gammaRIII (CD16) on polymorphonuclear neutrophils from individuals with human immunodeficiency virus type 1 disease and pulmonary tuberculosis
Authors: Meddows-Taylor S , Martin D J , Tiemessen C T
Published: January 2020

Fc gammaRIII (CD16) expression on the surfaces of polymorphonuclear neutrophils was significantly reduced in human immunodeficiency virus type 1-seropositive patients with pulmonary tuberculosis compared to that in individuals with either disease alone or in healthy blood donors. This downregulation of Fc gammaRIII may contribute to the enhanced susceptibility of coinfected individuals to opportunistic infections.
Expression and purification of Shiga-like toxin II B subunits
Authors: Acheson D W , De Breucker S A , Jacewicz M , Lincicome L L , Donohue-Rolfe A , Kane A V , Keusch G T
Published: January 2020

Shiga-like toxins (SLTs), which are produced by certain strains of Escherichia coli, are composed of enzymatically active A and B subunit multimers responsible for the toxin's binding. We have previously purified large amounts of the SLT-I B subunit by using a hyperexpression vector in Vibrio cholerae under the control of the trc promoter. In this study we examined various expression vectors to maximize yields of the SLT-II B subunit. The SLT-II B subunit has been expressed by using both the T7 promoter and the tac promoter in E. coli. When expressed from a plasmid containing the structural gene for SLT-II B deleted of the leader sequence, SLT-II B was able to form multimers when cross-linked, although SLT-II B production from this plasmid was unreproducible. SLT-II B expressed in all three systems appeared to form unstable multimers, which did not readily bind to a monoclonal antibody which preferentially recognizes B subunit multimers. SLT-II B expression was not increased by moving any of the plasmids into V. cholerae. Polyclonal antibodies raised to SLT-II B in rabbits recognized B subunit in SLT-II holotoxin yet were poorly neutralizing. SLT-II B was also expressed as a fusion protein with maltose-binding protein and could be cleaved from maltose-binding protein with factor Xa. Although the expression vectors were able to make large amounts of SLT-II B, as determined by Western blotting (immunoblotting), the levels of purified SLT-II B subunit were low compared with those obtained previously for SLT-I B subunit, probably because of instability of the multimeric SLT-II B subunit.
Immunization of cattle with recombinant Babesia bovis merozoite surface antigen-1
Authors: Hines S A , Palmer G H , Jasmer D P , Goff W L , McElwain T F
Published: January 2020

Cattle immunized with a recombinant merozoite surface antigen-1 molecule (MSA-1) produced high-titered antibody that reacted with the surface of the parasite and neutralized merozoite infectivity in vitro. However, recombinant MSA-1 immunization did not confer protection against challenge with virulent Babesia bovis. These results indicate that antibody-mediated neutralization of merozoite infectivity in vitro, at least for MSA-1-specific antibody, does not reflect in vivo protective immunity to babesiosis.
Proteolytic activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases
Authors: Gordon V M , Klimpel K R , Arora N , Henderson M A , Leppla S H
Published: January 2020

Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activated by proteolytic cleavage at specific amino acid sequences. Previously, it was shown that PA and DT can be activated by furin. In Chinese hamster ovary (CHO) cells, wild-type (RKKR) and cleavage site mutants of PA, each administered with a modified form of anthrax toxin lethal factor (the N terminus of lethal factor fused to PE domain III), had the following potencies: RKKR (wild type) (concentration causing 50% cell death [EC50] = 12 ng/ml) > or = RAAR (EC50 = 18 ng/ml) > FTKR (EC50 = 24 ng/ml) > STRR (EC50 = 49 ng/ml). In vitro cleavage of PA and cleavage site mutants of PA by furin demonstrated that native PA (RKKR) and PA with the cleavage sequence RAAR are substrates for furin. To characterize eukaryotic proteases that play a role in activating bacterial toxins, furin-deficient CHO cells were selected after chemical mutagenesis. Furin-deficient cells were resistant to PE, whose cleavage site, RQPR, constitutes a furin recognition site and to all PA cleavage site mutants, but were sensitive to DT (EC50 = 2.9 ng/ml) and PA (EC50 = 23 ng/ml), whose respective cleavage sites, RKKR and RVRR, contain additional basic residues. Furin-deficient cells that were transfected with the furin gene regained sensitivity to PE and PA cleavage site mutants. These studies provide evidence that furin can activate the three toxins and that one or more additional proteases contribute to the activation of DT and PA.
The group A streptococcal virR49 gene controls expression of four structural vir regulon genes
Authors: Podbielski A , Flosdorff A , Weber-Heynemann J
Published: January 2020

Within a genomic locus termed the vir regulon, virR genes of opacity factor-nonproducing (OF-) group A streptococci (GAS) are known to control the expression of the genes encoding M protein (emm) and C5a peptidase (scpA) and of virR itself. Within the corresponding genomic locus, opacity factor-producing (OF+) GAS harbor additional emm-related genes encoding immunoglobulin G- and immunoglobulin A-binding proteins (fcrA and enn, respectively). The virR gene region of the OF+ GAS M-type 49 strain CS101 was amplified by PCR, and 2,650 bp were directly sequenced. An open reading frame of 1,599 bp exhibited 76% overall homology to published virR sequences. By utilizing mRNA analysis, the 5' ends of two specific transcripts were mapped 370 and 174 bp upstream of the start codon of this open reading frame. The deduced sequences of the corresponding promoters and their locations differed from those of previously reported virR promoters. Transcripts from wild-type fcrA49, emm49, enn49, and scpA49 genes located downstream of virR49 were characterized as being monocistronic. The transcripts were quantified and mapped for their 5' ends. Subsequently, the virR49 gene was inactivated by specific insertion of a nonreplicative pSF152 vector containing recombinant virR49 sequences. The RNA from the resulting vir-mut strain did not contain transcripts of virR49, fcrA49, emm49, or enn49 and contained reduced amounts of the scpA49 transcript when compared with wild-type RNA. The mRNA control from the streptokinase gene was demonstrated not to be affected. When strain vir-mut was rotated in human blood, it was found to be fully sensitive to phagocytosis by human leukocytes. Thus, the present study provides evidence that virR genes in OF+ GAS could be involved in the control of up to five vir regulon genes, and their unaffected regulatory activity is associated with features postulated as crucial for GAS virulence.