bluuebunny/crossref_metadata_2025 huggingface dataset:
Emergence of a new virulent clone within the electrophoretic type 5 complex of serogroup B meningococci in Norway
Authors: Wedege E , Kolberg J , Delvig A , Høiby E A , Holten E , Rosenqvist E , Caugant D A
Published: January 2020

An increase in B:15:P1.12 meningococci among isolates from patients with Neisseria meningitidis infection in Norway in recent years led to further characterization of such strains. Between 1987 and 1992, B:15:P1.12 strains constituted 9.8% (24 strains) of B:15 isolates. The B:15:P1.12 strains belonged to the electrophoretic type 5 (ET-5) complex, but 17 (71%) strains were a new clone (ET-5c) not found elsewhere in the world. All but one strain of ET-5c were responsible for a localized outbreak of systemic meningococcal disease in western Norway. A novel monoclonal antibody (202,G-12), developed against the unknown variable region 2 on the class 1 protein of one of these strains, bound to 19 of the 15:P1.12 strains, 4 strains bound the subtype P1.13 reference monoclonal antibody MN24H10.75, and the remaining strain showed no reaction. Sequencing of porA genes demonstrated a series of nine threonine residues in the deduced variable region 2 of the latter strain, while four and five threonine residues were found in the corresponding regions of strains reacting with the monoclonal antibodies 202,G-12 and MN24H10.75, respectively. Epitope mapping with synthetic peptides showed that 202,G-12 bound to a sequence of 11 amino acids which included the four threonine residues specific for subtype P1.13a. Immunoglobulin G antibodies against the P1.7,16 subtype protein, induced in volunteers after vaccination with the Norwegian meningococcal vaccine, did not cross-react on immunoblots with the subtype protein of clone ET-5c. Thus, postvaccination class 1 protein antibodies, assumed to be protective, may not be effective against infection with the new clone.
Comparison of the effects of Ficoll-Hypaque separation and whole blood lysis on results of immunophenotypic analysis of blood and bone marrow samples from patients with hematologic malignancies
Authors: Tamul K R , Schmitz J L , Kane K , Folds J D
Published: January 2020

We compared flow cytometric immunophenotyping results obtained by using the lysed whole blood method of sample preparation with those obtained by using Ficoll-Hypaque-separated cells on 44 consecutive specimens from patients with various hematologic malignancies. When the samples were analyzed as a group, seven antigens (CD2, CD3, CD5, CD11c, CD20, CD22, and CD34) demonstrated significantly different percentages of positively staining cells. When the samples were grouped by disease, results for patients with acute lymphocytic leukemia were discordant for CD22 and HLA-DR and results for patients with hairy cell leukemia were discordant for CD34. Most of the differences, however, were not with antigens critical to the evaluation of the malignancy. Additionally, the most frequent reason for differences in the percentage of positive cells was due to isotype control-based placement of the quadrant markers and not an actual discrepancy in staining. However, analysis of the CD34 antigen yielded eight instances in which staining of Ficoll-Hypaque-separated cells was essentially negative, but a clearly positive population was evident with the lysed preparation. This finding has important implications because of the prognostic significance of this antigen. Further studies are needed to determine the cause of this phenomenon.
Intrathecal synthesis of immunoglobulin G and Mycobacterium tuberculosis-specific humoral immune response in tuberculous meningitis
Authors: Cho T Y , Park S C , Cho S N , Lee H R , Kim S K , Kim S K , Lee B I
Published: January 2020

Local synthesis of immunoglobulin G (IgG) in the central nervous system was investigated in 10 patients with tuberculous meningitis (TBM), 15 patients with aseptic meningitis (AM), and 15 patients with pulmonary tuberculosis only (PTBO). The IgG synthesis rate for patients with TBM was 56.4 +/- 18.9 mg/day (mean +/- standard deviation), which was significantly higher than that for patients with AM (8.0 +/- 6.7 mg/day, P < 0.001) and that for patients with PTBO (7.5 +/- 4.4 mg/day, P < 0.001). Therefore, the increased IgG synthesis rate in the central nervous system provided supporting evidence for differentiating the diagnosis of TBM from that of AM (sensitivity, 100%; specificity, 83.3%). Simultaneous measurement by enzyme-linked immunosorbent assay of IgG seroreactivity to lipoarabinomannan and purified protein derivative antigens in cerebrospinal fluid (CSF) demonstrated seropositivity in all 6 patients with TBM, 4 of 15 patients with AM, and 4 of 10 patients with PBTO. All patients showing false-positive reactivity in CSF demonstrated seropositivity in sera and normal ranges for IgG synthesis rates in CSF. Also, the semiquantitive measurement of IgG antibody (Ab) titers in these patients demonstrated higher IgG Ab titers in serum than in CSF except for one patient with a highly elevated albumin quotient, suggesting a leaky blood-brain barrier. The results strongly suggested that the Mycobacterium tuberculosis-specific IgG Abs were diffusible through the blood-brain barrier, which addresses the pitfall of serological tests for the early diagnosis of TBM. The serological detection of IgG Abs to lipoarabinomannan and purified protein derivative antigens in CSF could be misleading in the presence of simultaneously elevated of IgG Abs in serum.
Oligonucleotide primers designed to differentiate pathogenic pseudomonads on the basis of the sequencing of genes coding for 16S-23S rRNA internal transcribed spacers
Authors: Tyler S D , Strathdee C A , Rozee K R , Johnson W M
Published: January 2020

Universal primers targeting conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rRNA genes (rDNAs) were used to amplify the 16S-23S rDNA internal transcribed spacers (ITS) from eight species of pseudomonads which have been associated with human infections. Amplicons from reference strains of Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas gladioli, Pseudomonas mallei, Pseudomonas mendocina, Pseudomonas pickettii, Pseudomonas pseudomallei, and Xanthomonas maltophilia were cloned from each species, and sequence analysis revealed a total of 19 distinct ITS regions, each defining a unique sequevar with ITS sizes ranging from 394 (P. cepacia) to 641 (P. pseudomallei) bp. Five distinct ITS sequevars in P. cepacia, four in P. mendocina, three in P. aeruginosa, two each in P. gladioli and P. pseudomallei, and one each in P. mallei, P. pickettii, and X. maltophilia were identified. With the exception of one P. cepacia ITS, all ITS regions contained potential tRNA sequences for isoleucine and/or alanine. On the basis of these ITS sequence data, species-specific oligonucleotide primers were designed to differentiate P. aeruginosa, P. cepacia, and P. pickettii. The specificities of these primers were investigated by testing 220 clinical isolates, including 101 strains of P. aeruginosa, 103 strains of P. cepacia, and 16 strains of P. pickettii, in addition to 24 American Type Culture Collection (ATCC) Pseudomonas strains. The results showed that single primer pairs directed at particular ITSs were capable of specifically identifying the ATCC reference strains and all of the clinical isolates of P. aeruginosa and P. pickettii, but this was not the case with several ITS-based primer pairs tested for P. cepacia. This pathogen, on the other hand, could be specifically identified by primer pairs directed against the 23S rDNA.
Cytokine secretion induced by superantigens in peripheral blood mononuclear cells, lamina propria lymphocytes, and intraepithelial lymphocytes
Authors: Sperber K , Silverstein L , Brusco C , Yoon C , Mullin G E , Mayer L
Published: January 2020

Superantigens are potent inducers of T-cell proliferation and induce a broad range of cytokines, including tumor necrosis factor (TNF), gamma interferon, and interleukin 2 (IL-2). In the present study, we compared the abilities of different staphylococcal superantigens (staphylococcal enterotoxin B [SEB], staphylococcal enterotoxin E [SEE], and toxic shock syndrome toxin 1 [TSST-1]) to stimulate distinct cytokine profiles in peripheral blood mononuclear cells (PBMC), lamina propria lymphocytes (LPL), and intraepithelial lymphocytes (IEL). One million PBMC, LPL, and IEL were stimulated with various concentrations of superantigen (10 to 0.001 ng/ml) for 24, 48, and 72 h. Maximum cytokine production by PBMC, LPL, and IEL was observed for all three superantigens at 48 h at a concentration of 1 ng/ml. In PBMC, SEE and TSST-1 stimulated more IL-2 and gamma interferon than SEB. SEE and TSST-1 also stimulated more TNF and IL-4 production than SEB. In contrast, SEB stimulated more IL-6 than either SEE or TSST-1. In LPL, there was no SEE-induced IL-2 or IL-4 production, but IL-6, TNF, and gamma interferon were induced. SEB similarly induced no IL-2 or gamma interferon from the LPL, but IL-4, IL-6, and TNF were detected. TSST-1 stimulation of LPL resulted in IL-2 and TNF production but no IL-4, IL-6, or gamma interferon. In IEL, SEE induced no IL-2, IL-4, or gamma interferon but produced IL-6 and TNF, while SEB stimulation resulted in no IL-2 or gamma interferon but did result in detectable IL-4, IL-6, and TNF. Taken together, these data indicate that there are significant differences in the cytokine profiles induced by superantigens in LPL and IEL compared with those in PBMC, and these differences may relate to differences in activation requirements.
Distinct profiles of immunoglobulin G-binding-protein expression by invasive serotype M1 isolates of Streptococcus pyogenes
Authors: Raeder R , Boyle M D
Published: January 2020

Analysis of immunoglobulin G (IgG)-binding-protein expression by invasive group A streptococcal isolates of the M1 serotype collected as part of a Centers for Disease Control and Prevention surveillance study revealed two distinct phenotypes. One group of type M1 isolates expressed a surface protein reactive with all four human IgG subclasses (type IIo), while a second group expressed a surface protein demonstrating significant reactivity only with human IgG3 (type IIb). The functional forms of IgG-binding protein were antigenically related, and both were recognized by a rabbit polyclonal antiserum to serotype M1 but not by normal rabbit serum. While the quantities of antigenic M1 protein present in the extracts of representative isolates displaying each phenotype differed, the functional differences were found to be qualitative and not solely quantitative. The IgG-binding properties of these antigenically related M1 proteins could be readily distinguished from those of another IgG-binding protein, protein H. Type M1 isolates of the IIb phenotype differed from those of the IIo phenotype by secreting larger amounts of a casein-hydrolyzing protease into culture supernatants.
Analysis of immunoglobulin G-binding-protein expression by invasive isolates of Streptococcus pyogenes
Authors: Raeder R , Boyle M D
Published: January 2020

Invasive group A streptococcal isolates collected as part of a Centers for Disease Control and Prevention surveillance study were analyzed for expression of immunoglobulin G (IgG)-binding proteins. Two IgG-binding phenotypes of group A isolates of the M1 serotype were identified. The first group expressed a surface protein that bound all four human IgG subclasses (type IIo) and was recognized by rabbit anti-serotype M1-specific antiserum but not by normal rabbit serum. The second group expressed an IgG-binding protein that was also recognized by the anti-serotype M1 antiserum but demonstrated significant nonimmune reactivity only with human IgG3 (type IIb). Analysis of extracts of the isolates for reactivity with human IgA, fibrinogen, and albumin was also performed. The importance of the binding of human plasma proteins to pathogenic group A streptococci remains to be established.
Immunomodulatory effects of alpha interferon and thymostimulin in patients with neoplasias
Authors: Munno I , Marinaro M , Gesario A , Cannuscio B , Michel Y , Paulling E
Published: January 2020

In this report, we have evaluated the immunological effects following administration of alpha interferon (IFN-alpha) in combination with thymostimulin (TP-1), as well as of IFN-alpha and TP-1 alone in patients with neoplasias who underwent surgery and were subsequently treated with conventional chemotherapy. Data suggest that the combination of IFN-alpha and TP-1 is the most effective in the up-regulation of some immune parameters such as the CD4(+)-CD8+ cell-dependent antibacterial activity. Since this immune function plays an important role in the host protection against different targets such as invading microorganisms and/or neoplastic cells, the administration of TP-1-IFN-alpha is advisable for patients with neoplasias under chemotherapy.
Heterogeneity of VP4 neutralization epitopes among serotype P1A human rotavirus strains
Authors: Contreras J F , Menchaca G E , Padilla-Noriega L , Tamez R S , Greenberg H B , López S , Arias C F
Published: January 2020

We have used serotype-specific VP4 and VP7 neutralizing monoclonal antibodies (Nt-MAbs), as well as subgroup (SG)-specific MAbs, to characterize by enzyme immunoassay rotavirus strains isolated from diarrheic infants in the city of Monterrey, Mexico, from July 1993 to March 1994. Of a total of 465 children studied, 140 were rotavirus positive, including 3 patients infected with non-group A rotaviruses. The SG and VP7 (G) serotype specificities could be determined for 118 (84%) of the 140 rotavirus-positive stool specimens; 4 rotavirus strains were serotype G1 and SGII; 1 strain was serotype G2 and SGI+II; 112 strains were serotype G3 and SGII; 1 strain was serotype G3 and SGI; and none of the strains was serotype G4. Fifty-eight specimens, representing the 13 different group A rotavirus electropherotypes detected, were chosen for VP4 (P) serotyping. Of these, 48 (83%) strains reacted with the P1A serotype-specific Nt-MAb 1A10. None of the strains reacted with the serotype P2-specific Nt-MAbs tested. Not all viruses that reacted with Nt-MAb 1A10 were recognized by Nt-MAbs 2A3 and 2G1, which also recognize P1A strains, indicating heterogeneity of neutralization epitopes among serotype P1A human rotaviruses. This heterogeneity could be relevant for the specificity of the VP4-mediated neutralizing antibody immune response and indicates the need for antigenic characterization, in addition to genomic typing, of the VP4 proteins of circulating human rotavirus field strains.
Monoclonal rat antibodies directed against Toxoplasma gondii suitable for studying tachyzoite-bradyzoite interconversion in vivo
Authors: Gross U , Bormuth H , Gaissmaier C , Dittrich C , Krenn V , Bohne W , Ferguson D J
Published: January 2020

We previously reported the in vitro analysis of stage differentiation of Toxoplasma gondii in murine bone marrow-derived macrophages. The purpose of this study was to generate monoclonal rat antibodies that might be suitable for investigating tachyzoite-bradyzoite interconversion in vivo with the murine model. Immunization of Fischer rats with cysts of T. gondii NTE resulted in the generation of seven monoclonal antibodies of the immunoglobulin G2a, G2b, or M isotype, which were further characterized by the immunoblot technique, immunofluorescence assay, immunohistology, and immunoelectron microscopy. Immunoblots demonstrated specific reactivity of five monoclonal antibodies with proteins with molecular masses of 40, 52, 55, 60, 64, 65, and 115 kDa. One antibody (CC2) appeared to recognize a differently expressed antigen depending on the parasite stage, reacting with a 40-kDa molecule in tachyzoites and a 115-kDa antigen in bradyzoites and oocysts. Several other monoclonal antibodies were shown to be stage specific and to react in immunofluorescence assays or in immunoblots with either tachyzoites or bradyzoites. Kinetics of stage conversion in vitro could be monitored by immunofluorescence with two of these monoclonal antibodies. Preliminary immunohistological investigations of tissue sections from infected mice demonstrated the possible usefulness of these monoclonal antibodies for future in vivo studies on stage differentiation of T. gondii in the murine system.
A manual bead assay for the determination of absolute CD4+ and CD8+ lymphocyte counts in human immunodeficiency virus-infected individuals
Authors: Carella A V , Moss M W , Provost V , Quinn T C
Published: January 2020

CD4+ T lymphocytes are currently the most common surrogate marker indicating disease progression in individuals infected with human immunodeficiency virus (HIV). Since the cost of enumerating lymphocyte phenotypes is quite high, an inexpensive bead assay analyzed by light microscopy (cytosphere assay; Coulter Corporation, Hialeah, Fla.) was developed as an alternative method for counting CD4+ and CD8+ T lymphocytes. To evaluate the reliability of the cytosphere assay, heparinized blood was collected from 117 HIV-infected individuals and tested for both CD4+ and CD8+ lymphocytes by flow cytometry and the cytosphere assay. The Pearson correlation coefficient of the cytosphere assay compared with that of flow cytometry for CD4+ T lymphocytes was 0.93, with mean values +/- standard deviations of 534 +/- 509 by flow cytometry and 499 +/- 477 by the cytosphere assay. The correlation coefficient for CD8+ T lymphocytes was 0.86, with mean values of 831 +/- 543 by flow cytometry and 746 +/- 472 by the cytosphere assay. The sensitivity and specificity of the cytosphere assay in determining absolute CD4+ T-lymphocyte counts of less than 200/microliters were 97.6 and 94.7%, respectively. The positive predictive value was 90.9%, and the negative predictive value was 98.6%. The cytosphere assay was highly correlative to flow cytometry in determining CD4+ and CD8+ T-lymphocyte counts among HIV-infected patients. The ease and limited resources needed to perform this test make it ideal in developing countries and other areas where technology and finances are limited.
Blocking antibody assay for confirmation of urogenital Chlamydia infection
Authors: Nachamkin I , Riddle D L , O'Connor J
Published: January 2020

We evaluated the Syva MicroTrak Chlamydia Blocking Antibody Assay as a confirmatory assay for the detection of Chlamydia trachomatis in urogenital specimens. During a 5-month period, 109 positives were obtained (4.9%) by the MicroTrak II Enzyme Immunoassay as implemented with the Syva XL automated enzyme immunoassay instrument. Of 98 evaluable samples, 92 (93.9%) were confirmed as positive by the blocking assay, and 3 blocking-negative samples had organisms detected by direct fluorescent-antibody analysis (blocking sensitivity, 96.8%). We found that direct fluorescent-antibody analysis of samples with a specimen-to-cutoff absorbance ratio of < or = 2.0 was a reasonable confirmation alternative and was more cost-effective than the blocking assay.
Comparison of serum histamine-releasing activity and clinical manifestations in chronic idiopathic urticaria
Authors: Tanus T , Atkins P C , Zweiman B
Published: January 2020

We found increased serum histamine-releasing activity (HRA) in 27% of patients with chronic idiopathic urticaria. Patterns of clinical manifestations were similar in those with and without serum HRA, including responses to a standard treatment regimen. In HRA+ patients the degree of serum HRA generally correlated with clinical disease activity.
A sensitive and specific PCR method to detect Helicobacter felis in a conventional mouse model
Authors: Kong L , Smith J G , Bramhill D , Abruzzo G K , Bonfiglio C , Cioffe C , Flattery A M , Gill C J , Lynch L , Scott P M , Silver L , Thompson C , Kropp H , Bartizal K
Published: January 2020

Although many detection methods have been used to determine Helicobacter colonization in small animal models, the sensitivity and specificity of these detection methods are limited. To improve the Helicobacter felis conventional mouse model for accurate evaluation of therapeutic regimens, we developed a PCR for detection of, and a competitive PCR for quantitation of, H. felis in viral antibody-free (VAF) mice. The PCR was based on the H. felis 16S rRNA gene. An internal control DNA was used for competitive quantitation of the PCR. VAF conventional Swiss-Webster mice were infected with an H. felis culture by oral gavage. At various times after H. felis challenge and therapy, stomach mucosa was collected and evaluated by PCR. PCR detected approximately 50 to 100 H. felis cells per mouse stomach and showed no cross-reaction with other bacteria commonly found in mouse stomachs. Colonization of H. felis in the mouse stomach was confirmed by culture isolation from germfree mice and histological examination of VAF mice. Response to therapy in this H. felis model correlated well with results seen in human clinical trials with H. pylori. A model utilizing PCR detection which may be useful for discovering new antibiotics and/or vaccines against Helicobacter ulcer disease has been developed.
Interlaboratory study evaluating quantitation of antibodies to Haemophilus influenzae type b polysaccharide by enzyme-linked immunosorbent assay
Authors: Madore D V , Anderson P , Baxter B D , Carlone G M , Edwards K M , Hamilton R G , Holder P , Käyhty H , Phipps D C , Peeters C C , Schneerson R , Siber G R , Ward J I , Frasch C E
Published: January 2020

An interlaboratory study was conducted to determine whether an enzyme-linked immunosorbent assay (ELISA) with an antigen preparation composed of various-sized fragments of Haemophilus influenzae type b polysaccharide conjugated to human serum albumin could be standardized across laboratories and whether the ELISA-derived results from different laboratories are equivalent to those obtained by the standard radioactive antigen binding assay (RABA) for quantitation of anti-H, influenzae type b polysaccharide antibodies. Twenty coded human serum samples were quantitated by ELISA in 11 laboratories and by RABA in 5 laboratories. The mean RABA-derived values served as the basis for all comparisons. While the overall correspondence of antibody values between the two methods was good, significant differences were found among some of the 11 ELISA data sets and among the mean RABA values. Seven laboratories generated higher ELISA antibody values for low-titered sera. Four laboratories generated antibody concentrations that were not statistically different between the two assay methods. The results therefore indicate that the ELISA can tolerate substantial variations in protocol, such as the use of different plates and different antibody reagents, without affecting the quantitation of serum antibodies. However, attention should be focused on low-titered sera, as some assay conditions may yield spurious results. This ELISA is a serologic assay which can serve as an alternative to the RABA for quantitation of antibodies to H. influenzae type h polysaccharide.
Celiac disease and hypoparathyroidism: cross-reaction of endomysial antibodies with parathyroid tissue
Authors: Kumar V , Valeski J E , Wortsman J
Published: January 2020

Celiac disease (CD) is a gluten-sensitive enteropathy characterized by the presence of serum antibodies to endomysial reticulin and gliadin antigens. CD has been associated with various autoimmune endocrine disorders, such as diabetes. We report a rare case of idiopathic hypoparathyroidism with coexistent CD characterized by the presence of serum autoantibodies. Studies were conducted to determine the specificities of these autoantibodies and to localize the antibody binding sites by indirect immunofluorescence and immunoelectron microscopy. Sera from a patient with idiopathic hypoparathyroidism and CD and from two patients with CD alone were tested by indirect immunofluorescence for autoantibodies to parathyroid and endomysial antigens. The specificities of the antibody reactions were determined by testing the sera before and after absorption with monkey stomach tissue. In addition, immunoelectron microscopic studies were performed to determine the localization of the endomysial antigen. Indirect-immunofluorescence studies on the patient's serum were positive with the parathyroid as well as the endomysial substrate. Similar reactions were also observed with the sera of endomysial antibody-positive patients with CD. Absorption of the sera with monkey stomach powder, which is known to have the endomysial antigen, abolished the antibody activities on both the endomysial substrate and the parathyroid tissue. Immunoelectron microscopic studies showed that endomysial antibody activity was associated with antigens localized on the myocyte plasma membrane and in the intercellular spaces. Thus, reactions of the patient's serum with the parathyroid tissue were due to endomysial antibodies and were not parathyroid specific as in patients with idiopathic hypoparathyroidism who did not have coexistent CD. In conclusion, indirect-immunofluorescence tests on parathyroid tissue detect not only tissue-specific antibodies but also cross-reactive antibodies, and this should be taken into consideration when these tests are performed.
An indirect double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using baculovirus-expressed antigen for the detection of antibodies to glycoprotein E of pseudorabies virus and comparison of the method with blocking ELISAs
Authors: Kimman T G , de Leeuw O , Kochan G , Szewczyk B , van Rooij E , Jacobs L , Kramps J A , Peeters B
Published: January 2020

Antibodies in porcine sera against glycoprotein E (gE) of pseudorabies virus (PRV) are usually measured in blocking enzyme-linked immunosorbent assays (ELISAs) with one or two murine monoclonal antibodies (MAbs) directed against gE. Our aim was to develop a confirmation assay which is based on another principle and which is able to detect antibodies directed against most potential binding sites on gE with high specificity. Therefore, we developed an indirect double-antibody sandwich assay (IDAS) using recombinant gE expressed by baculovirus (BacgE960). A fragment of the gE gene consisting of nucleotide positions +60 to +1020 of gE, coding for the major antigenic sites of gE but not the transmembrane region, was cloned behind the signal sequence of PRV gG and the p10 promoter in a baculovirus vector. Immunoblot analysis showed that the expressed protein reacted with MAbs directed against five of the six antigenic sites on gE. Although the conformation of some antigenic sites, notably antigenic sites E and C, was not identical to their natural conformation, the expressed protein bound gE-specific antibodies in porcine sera in Western blots (immunoblots) and ELISAs. For the IDAS, a coating MAb directed against the nonimmunodominant antigenic site A on gE was chosen. A major obstacle in binding ELISAs, such as the IDAS, appeared to be the high nonspecific binding activity observed in porcine sera. As a result, sera could be tested only in relatively high dilutions in the BacgE960 IDAS, in contrast to the testing of sera in blocking ELISAs. The sensitivity and specificity of the newly developed BacgE960 IDAS were evaluated and compared with those of five commercially available blocking ELISAs by using several sets of sera of known PRV disease history. The BacgE960 IDAS assay had a high diagnostic specificity and a moderate sensitivity. The five blocking ELISAs differed remarkably in sensitivity and specificity, thereby illustrating the need for standardization and confirmation. We conclude that the BacgE960 IDAS is a useful and specific additional (confirmatory) test for the detection of antibodies to gE.
Circulating complement proteins in patients with sepsis or systemic inflammatory response syndrome
Authors: Stöve S , Welte T , Wagner T O , Kola A , Klos A , Bautsch W , Köhl J
Published: January 2020

The systemic inflammatory response of the body to invading microorganisms, termed sepsis, leads to profound activation of the complement system. Pathophysiological concepts suggest that complement activation occurs very early in this syndrome. Thus, we discuss whether the determination of concentrations of the complement components C3a, C5a, and C3 in plasma as well as of the C3a/C3 ratio might be helpful to diagnose sepsis early. For this purpose, 33 patients from an intensive care unit were monitored for 10 days. In comparison with healthy donors, C3a levels and the C3a/C3 ratio of intensive-care-unit patients were significantly elevated (P < 0.0001) on admission. In contrast, C3 levels were significantly reduced (P < 0.0001) but increased during the study. C5a levels in the plasma of healthy donors and patients were identical. Twenty-two of 33 patients fulfilled microbiological and clinical criteria of sepsis. Eleven patients had signs of systemic inflammatory response syndrome but no microbiological evidence of sepsis. The groups could be differentiated from each other by their C3a levels or their C3a/C3 ratios during the first 24 h after the clinical onset of sepsis (P < 0.05). Septic patients in shock had higher C3a levels than normotensive septic patients, although the differences were not significant. Nonsurvivors had significantly higher C3a levels on admission than survivors (P = 0.0185). No differences were found between septic patients who developed adult respiratory distress syndrome and those who did not. Thus, determination of C3a concentrations in plasma may prove useful (i) to diagnose sepsis early, (ii) to differentiate between patients with sepsis and those with systemic inflammatory response syndrome, and (iii) to assess prognosis.
Detection of rubella virus-specific immunoglobulin M antibodies with a baculovirus-expressed E1 protein
Authors: Schmidt M , Lindqvist C , Salmi A , Oker-Blom C
Published: January 2020

The structural proteins of rubella virus (RV) were expressed in insect cells by using the baculovirus expression vector system. The recombinant E1 envelope glycoprotein was purified by immunoaffinity chromatography and used to detect RV-specific immunoglobulin M antibodies in a time-resolved fluoroimmunoassay. Correlation analysis between the reactivities of antibodies against this recombinant E1 and the reactivities against authentic RV antigen shows that purified E1 can detect RV antibodies of the immunoglobulin M type.
Peptide sequences of glycoprotein G-2 discriminate between herpes simplex virus type 2 (HSV-2) and HSV-1 antibodies
Authors: Levi M , Rudén U , Wahren B
Published: January 2020

The complete herpes simplex virus type 2 envelope glycoprotein G was represented by overlapping synthetic peptides. Herpes simplex virus type 2-specific human seroreactivities were mainly seen against three peptides, peptides G2-64, G2-69, and G2-70, located in the C-terminal part of glycoprotein G. This is, interestingly, a region which has strong homology between herpes simplex virus types 1 and 2. G2-69 was the most herpes simplex virus type 2-specific peptide, reacting with 93% (13 of 14) of herpes simplex virus type 2 immunoglobulin G-positive human serum samples (n = 14) and none of the type 2 negative serum samples (n = 16) tested. The epitope of peptide G2-69 was mapped to the amino acid sequence RYAHPS, and the antibody binding to G2-64 could be increased by chemical oxidation of the peptide. The three reactive peptides were used, alone or combined, together with herpes simplex virus type 2 antigen in an attempt to discriminate anti-herpes simplex virus type 2 from anti-herpes simplex virus type 1 immunoglobulin G. Inclusion of the herpes simplex virus type 2 glycoprotein G peptides increased the specificity compared with the use of glycoprotein G alone. Combinations of peptides with whole glycoprotein also showed a greater discriminative capacity than single peptides. We conclude that these synthetic glycoprotein G peptides may be useful for herpes simplex virus type 2 serology based on peptides or combinations of peptides and antigens. This is the first study describing the possibility of discriminative herpes simplex virus serology by using synthetic peptides combined with small amounts of whole glycoprotein G.
Cloning and expression of a putative alcohol dehydrogenase gene of Entamoeba histolytica and its application to immunological examination
Authors: Kimura A , Hara Y , Kimoto T , Okuno Y , Minekawa Y , Nakabayashi T
Published: January 2020

To clone and express the genes encoding major antigens of Entamoeba histolytica, we constructed a lambda gt11 cDNA library for E. histolytica HM1:IMSS and screened it with pooled sera from patients with amoebiasis. A 1,223-bp cDNA was cloned (clone 1223), and its nucleotide sequence was determined. The amino acid sequence predicted to be encoded by the open reading frame of clone 1223 consisted of 396 residues and showed 32.5 and 32.3% homology to the NADH-dependent butanol dehydrogenases I and II (bdhA and bdhB) of Clostridium acetobutylicum, respectively. In addition, 29 of the 34 consensus positions of bdhA and bdhB were also well conserved in clone 1223. The recombinant protein expressed from clone 1223 had an estimated molecular mass of 43.5 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigenicity and specificity of the recombinant protein were evaluated by an enzyme-linked immunosorbent assay using sera obtained from two clinical groups of patients with amoebiasis and a group of healthy controls. The recombinant protein had potent and specific antigenicity. In all, 53 serum samples (88.3%) from 60 patients with amoebiasis were positive for immunoglobulin G antibody against the recombinant protein, with a mean optical density value of 0.42. In contrast, 53 of 54 healthy control serum samples were negative, with only 1 positive serum sample showing the lower optical density value. These results suggested that clone 1223 is promising in terms of providing a useful antigen for the accurate serodiagnosis of amoebiasis and that the gene encodes a putative alcohol dehydrogenase of E. histolytica.
Recombinant Toxoplasma gondii surface antigen 1 (P30) expressed in Escherichia coli is recognized by human Toxoplasma-specific immunoglobulin M (IgM) and IgG antibodies
Authors: Harning D , Spenter J , Metsis A , Vuust J , Petersen E
Published: January 2020

The immunodominant surface antigen of Toxoplasma gondii, surface antigen 1 (SAG1), was expressed in Escherichia coli as a fusion protein containing a majority of the SAG1 protein supplied with six histidyl residues in the N-terminal end. The recombinant protein was purified on a Ni-chelate column and then on a fast-performance liquid chromatography column and was in a nonreduced condition. It was recognized by T. gondii-specific human immunoglobulin G (IgG) and IgM antibodies as well as by a mouse monoclonal antibody (S13) recognizing only nonreduced native SAG1. Antibodies induced in mice by the recombinant SAG1 recognized native SAG1 from the T. gondii RH isolate in culture. Recombinant SAG1 is suitable for use in diagnostic systems for detecting anti-SAG1-specific IgG and IgM antibodies.
Modification of the Mycobacterium bovis extracellular protein MPB70 with fluorescein for rapid detection of specific serum antibodies by fluorescence polarization
Authors: Lin M , Sugden E A , Jolley M E , Stilwell K
Published: January 2020

The principle of fluorescence polarization described by Perrin (F. Perrin, J. Phys. Radium 7:390-401, 1926) was applied to the development of a novel assay that used fluorescein-labeled Mycobacterium bovis secretory protein MPB70 for rapid detection of anti-MPB70 antibodies in selected sera from three M. bovis-infected species (elk, Ilama, and bison). Labeling of purified MPB70 with fluorescein isothiocyanate resulted in the incorporation of 0.96 +/- 0.08 (mean +/- standard deviation; n = 3) fluorescein group per MPB70 molecule. The labeled protein fluoresced strongly with an emission maximum at 518 nm when excited with light of a wavelength near 493 nm, and its immunoreactivity with anti-MPB70 monoclonal antibody 4C3/17 was not altered by modification with fluorescein. The fluorescence polarization assay protocol was optimized for analysis of serum samples by incorporating into the assay buffer 0.05% lithium dodecyl sulfate, which prevents the occurrence of some nonspecific interactions. Sera from M. bovis-infected animals, selected on the basis of exhibiting the presence of anti-MPB70 antibodies, as detected by enzyme-linked immunosorbent assay (ELISA), reacted with fluorescein-labeled MPB70, resulting in an increase in polarization of up to 330 milli-polarization units, in contrast to the values for noninfected sera (167 to 178 mP), which were close to that obtained in the absence of specific antibodies (164.7 +/- 3.3 mP; n = 6). These results demonstrated the feasibility of using fluorescein-labeled MPB70 to detect anti-MPB70 antibodies by fluorescence polarization and suggested that the assay described here can be an alternative to ELISA or other antibody assay systems. The advantages of this original methodology and its general applicability to the diagnosis of infectious diseases are discussed.
Effect of gamma interferon on expression of Fc gamma receptors in monocytes of newborn infants and adults
Authors: Fairchild K D , Hudson R G , Douglas S D , McKenzie S E , Polin R A
Published: January 2020

Fc gamma receptors provide an essential link between cellular and humoral immunity, and little is known about their expression in monocytes of newborn infants. We compared baseline and gamma interferon (IFN-gamma)-induced expression of Fc gamma RI and Fc gamma RII protein and Fc gamma RI mRNA in monocytes from healthy, term infants and adults. Fluorescence-activated cell sorter analysis demonstrated that baseline expression of monocyte Fc gamma RI in newborn infants was not significantly different from that in adults, while Fc gamma RII protein expression in monocytes derived from newborns was significantly higher than that for adults (mean channel fluorescence [MCF] for newborns and adults, 5.53 and 4.50, respectively [P = 0.039]). In vitro treatment with recombinant IFN-gamma increased the expression of Fc gamma RI in monocytes of newborns and adults to the same extent (2.4- and 2.2-fold increase in MCF in newborns and adults, respectively, at 42 h). We developed a semiquantitative fluorescence reverse transcriptase PCR which demonstrated a significant increase in mRNA for Fc gamma RI in monocytes of newborns and adults with in vitro IFN-gamma exposure, indicating that IFN-gamma acts by increasing the transcription or transcript stability of Fc gamma RI mRNA. While there was no significant effect of IFN-gamma treatment on Fc gamma RII expression in monocytes from adults, there was a 20% increase in Fc gamma RII in monocytes from newborns (P = 0.009). Monocytes from healthy, term newborns and adults exhibit comparable baseline and IFN-gamma-induced levels of expression of Fc gamma RI and higher baseline and IFN-gamma-induced levels of expression of Fc gamma RII.
Serum antibody response to outer membrane proteins of Moraxella (Branhamella) catarrhalis in patients with bronchopulmonary infection
Authors: Christensen J J , Hansen N Q , Bruun B
Published: January 2020

A Western blot (immunoblot) method for detecting antibodies against outer membrane protein (OMP) epitopes of Moraxella (Branhamella) catarrhalis was evaluated. Paired serum samples from patients suspected of M. catarrhalis (n = 38) and non-M. catarrhalis (n = 25) bronchopulmonary infection were examined for the presence of antibodies of the immunoglobulin M (IgM), IgG, and IgA classes to OMPs from M. catarrhalis by a gel electrophoresis-immunoperoxidase technique (Western blotting); sera from 40 healthy adult blood donors were also included. A significantly (P = 0.004) more frequent occurrence of IgM-class antibodies and/or an increase in the number of IgG-class antibodies against different M. catarrhalis OMPs from acute- to convalescent-phase serum samples was found for patients with M. catarrhalis (79%) than for patients without M. catarrhalis (40%). IgM-class antibodies against OMPs of M. catarrhalis were found in acute- and/or convalescent-phase serum samples form 58% of patients with M. catarrhalis and 32% of patients without M. catarrhalis. Fifty percent of patients with M. catarrhalis and 16% of patients without M. catarrhalis had, from acute- to convalescent-phase serum samples, an increased number of IgG-class antibodies directed against different OMPs. A total of 34% of patients with M. catarrhalis and 4% of patients without M. catarrhalis had, from acute- to convalescent-phase serum samples, an increased number of IgA-class antibodies directed against different OMPs. The present study indicates that M. catarrhalis is one of the bacteria involved in acute exacerbations of chronic bronchitis.
A semiquantitative assay for CD8+ T-cell-mediated suppression of human immunodeficiency virus type 1 infection
Authors: Chen Y , Rinaldo C , Gupta P
Published: January 2020

A reproducible, semiquantitative assay was developed to measure the level of the anti-human immunodeficiency virus type 1 (anti-HIV-1) suppressive activity of CD8+ T cells. The assay had a wide dynamic range and could be applied to a relatively small number of fresh and cryopreserved peripheral blood mononuclear cells and purified CD8+ T cells. The suppressive activity was not due to cytolytic activity and was not major histocompatibility complex class I restricted. Suppression of HIV-1 infection by CD8+ T cells was consistently demonstrable with both endogenously infected autologous CD4+ T cells and exogenously infected allogeneic CD4+ T cells. This assay can be used to monitor the level of antiviral activity of CD8+ T cells in a retrospective and prospective manner in studies of the natural history of HIV-1 infection and of subjects receiving anti-HIV-1 therapy and vaccines.
Diagnosis of human immunodeficiency virus type 1 infection in infants by immune complex dissociation p24 assay
Authors: Paul M O , Toedter G , Hofheinz D , Tetali S , Pelton S , Marecki M , Brena A , Abrams E J , Landesman S , Pahwa S
Published: January 2020

Using immune complex dissociation (ICD), we retrospectively examined serum and plasma of 206 infants aged 0 to 4 months who were perinatally exposed to human immunodeficiency virus (HIV). All samples were analyzed in a blinded manner. Infection status was determined based on the results of HIV culture and Centers for Disease Control and Prevention classification. The overall diagnostic sensitivity of the assay was 59% (93 samples, 73 infants), and specificity was 100% (160 samples, 133 infants). When the samples were analyzed according to age, sensitivity was highest at age 1 to 2 months (17 of 21 infants, 81%). Sensitivities at other ages were 53% at < 1 month, 55% at 2 to 3 months, and 48% at 3 to 4 months (9 of 17, 11 of 20, and 12 of 25 cases, respectively). In 11 evaluable cases there was a possible correlation of p24 antigen quantitation (in picograms per milliliter) with disease progression. We conclude that, as determined in this study, the ICD p24 is a rapid diagnostic assay for HIV infection with a sensitivity of >80% at 1 to 2 months of age and 100% specificity, as evaluated, up to 4 months of age.
Reconstruction of the immune system after unrelated or partially matched T-cell-depleted bone marrow transplantation in children: functional analyses of lymphocytes and correlation with immunophenotypic recovery following transplantation
Authors: Kook H , Goldman F , Giller R , Goeken N , Peters C , Comito M , Rumelhart S , Holida M , Lee N , Trigg M
Published: January 2020

Reconstitution of the immune system following T-cell-depleted bone marrow transplantation (BMT) in children has yet to be fully elucidated. Thus, we prospectively studied the recovery of immune function in 64 children who underwent T-lymphocyte-depleted marrow transplants using either matched family member donors or matched unrelated donors. We measured in vitro posttransplantation proliferative responses to phytohemagglutinin (PHA), concanavalin A, pokeweed mitogen, and Candida albicans antigen and assessed unidirectional allogeneic mixed-lymphocyte culture (MLC) responses at various times. A total of 129 healthy individuals served as normal controls for these assays. Responses to T-cell mitogens normalized within 12 months posttransplantation, while MLC responses normalized by 9 months. The presence of graft-versus-host disease (grade II or greater) and cytomegalovirus infection was associated with delays in immune function recovery. Importantly, immune function recovery correlated temporally with a rise in peripheral lymphocyte count. In contrast, the CD4/CD8 ratio was not predictive of immune recovery. Knowledge of immune function recovery may guide clinicians in devising strategies to minimize the risk of infection post-BMT.
Evaluation of Chlamydia immunoglobulin M (IgM), IgG, and IgA rELISAs Medac for diagnosis of Chlamydia pneumoniae infection
Authors: Kutlin A , Tsumura N , Emre U , Roblin P M , Hammerschlag M R
Published: January 2020

Chlamydia pneumoniae is an important pathogen responsible for a variety of respiratory diseases in humans. Cell culture remains the most specific method for C. pneumoniae diagnosis, but it is labor-intensive and time-consuming. Thus, serology, particularly microimmunofluorescence (MIF) testing, is frequently utilized. However, the MIF test has a significant subjective component. We evaluated a new serological test: Chlamydia Immunoglobulin M (IgG, IgA, and IgM rELISAs Medac, based on a recombinant Chlamydia-specific lipopolysaccharide (LPS) fragment, for the diagnosis of C. pneumoniae infection. The results of this study demonstrated that the use of rELISAs Medac with single sera does not appear to be sensitive or specific for diagnosis of C. pneumoniae infection compared to culture. In children, sensitivities of the rELISAs compared to culture did not exceed 34.2%, and the specificities ranged from 68.4% (IgG) to 91.2% (IgA). In adults, the sensitivities of the rELISAs were slightly higher, up to 77.8% (IgA or IgG), but the specificities ranged from a very low 20.8% for IgA or IgG to 81.1% for IgM. When multiple sera were tested, the results of the rELISAs Medac correlated with culture results in five of eight (62.5%) patients. However, this offers only a retrospective diagnosis, which makes it difficult to manage these patients prospectively.
Immune suppression in calves with bovine immunodeficiency virus
Authors: Zhang S , Wood C , Xue W , Krukenberg S M , Chen Q , Minocha H C
Published: January 2020

The present study was designed to evaluate the effect of bovine immunodeficiency virus (BIV) infection on immune functions and possible interactions between BIV and other bovine viruses in calves. Ten calves were inoculated intravenously with BIV, and five served as controls. An increased lymphocyte proliferation to BIV gag protein was demonstrated 2 to 6 weeks after BIV inoculation (P < 0.05). Lymphocyte subset differentiation revealed a decreased CD4/CD8 ratio (P < 0.05) during weeks 2 to 7, suggesting a possible immune dysfunction in BIV-infected calves. When the calves were inoculated with bovine herpesvirus type 1 (BHV-1), the antibody response to BHV-1 in BIV-infected calves was delayed and the antibody titers were significantly lower (P < 0.05). Injection of bovine viral diarrhea virus vaccine also elicited a lower neutralizing antibody response in BIV-infected calves. The results indicated that immune suppression occurred in BIV-infected calves.