bluuebunny/crossref_metadata_2025 huggingface dataset:
Hematological reference ranges among healthy Ugandans
Authors: Tugume S B , Piwowar E M , Lutalo T , Mugyenyi P N , Grant R M , Mangeni F W , Pattishall K , Katongole-Mbidde E
Published: January 2020

Reference values are essential for the interpretation of hematologic data in clinical practice and research studies. Symptom-free human immunodeficiency virus antibody-negative Ugandan adults (183 subjects, aged 15 to 74 years, 37.7% women and 62.3% men) were studied to establish hematological reference ranges. The central 95% areas under the distribution curves were 1,453 to 4,448 cells per microliters for the absolute lymphocyte count, 559 to 2,333 cells per microliters for the absolute CD4 count, 253 to 1,396 cells per microliters for the absolute CD8 count, and 0.68 to 4.4 for the CD4/CD8 ratio. Women had significantly higher mean absolute lymphocyte counts (2,826 versus 2,568/microliters), absolute CD4 counts (1,425 versus 1,154/microliters) and absolute CD4/CD8 ratios (2.58 versus 1.88) than did men. These reference ranges differ from those reported for populations outside Africa.
beta-2 Microglobulin values among human immunodeficiency virus (HIV)-negative, HIV-positive asymptomatic, and HIV-positive symptomatic Ugandans
Authors: Piwowar E M , Tugume S B , Grant R M , Lutalo T , Pattishall K , Katongole-Mbidde E
Published: January 2020

Mean serum beta-2 microglobulin levels among healthy human immunodeficiency virus-seronegative and asymptomatic and symptomatic human immunodeficiency virus-seropositive Ugandans were found to be 2.35, 3.75, and 5.06 mg/liter, respectively (P < 0.001). The upper limit of the normal range (3.5 mg/liter) is higher in this African population than that reported elsewhere.
Evaluation of two recombinant Maedi-visna virus proteins for use in an enzyme-linked immunosorbent assay for the detection of serum antibodies to ovine lentiviruses
Authors: Power C , Richardson S , Briscoe M , Pasick J
Published: January 2020

Defined segments of the gag polyprotein and transmembrane envelope glycoprotein from Maedi-visna virus were expressed as glutathione S-transferase fusion proteins in Escherichia coli and evaluated singly and in combination for use in an enzyme-linked immunosorbent assay (ELISA). Two hundred sixty field serum specimens from 15 sheep flocks were tested in parallel with recombinant and whole-virus antigens, and the relative sensitivities and specificities of the recombinant antigens were calculated. When the recombinant gag and transmembrane proteins were used in combination, a sensitivity of 97.4% and a specificity of 99.4% relative to whole-virus antigen were observed, indicating the utility of these proteins in diagnostic testing.
Nucleotide sequence analysis and seroreactivities of the 65K heat shock protein from Mycobacterium paratuberculosis
Authors: el-Zaatari F A , Naser S A , Engstrand L , Burch P E , Hachem C Y , Whipple D L , Graham D Y
Published: January 2020

Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. It has also been implicated as a possible cause of Crohn's disease, an inflammatory bowel disease of unknown etiology. The mycobacterial 65K heat shock proteins (hsp-65K) are among the most extensively studied mycobacterial proteins, and their immunogenic characteristics have been suggested to be the basis for autoimmunization in chronic inflammatory diseases. In this context, we isolated and sequenced the hsp-65K-encoding gene from our M. paratuberculosis PTB65K genomic library. A high degree of identity was found between the open reading frame (ORF) of the PTB65K gene and those of Mycobacterium tuberculosis (89.6%), Mycobacterium leprae (86.6%), and Mycobacterium avium 18 (98.8%). The amino acid sequence alignment of the PTB65K protein with the hsp-65K homologs revealed that the M. tuberculosis and M. leprae proteins each differed by 36 amino acid residues and that the M. avium 18 protein differed by 8 residues. We also investigated the humoral immune responses of animals with Johne's disease and patients with Crohn's disease against the recombinant PTB65K antigen. Immunoblot analysis showed that sera from only 3 of 10 clinically ill and 5 of 25 subclinically ill cows reacted with PTB65K. In addition, sera from two of two sheep and one of two goats with clinical symptoms of Johne's disease also reacted with PTB65K; 0 samples from 10 normal cows reacted. In humans, sera from 7 of 13 patients with Crohn's disease, 3 of 4 with tuberculosis, 5 of 6 with leprosy, 5 of 12 with non-inflammatory bowel disease, and 0 of 4 with ulcerative colitis reacted with the recombinant PTB65K antigen. These results indicate that this PTB65K heat shock protein is uninformative when used for serodiagnosis of Johne's disease in animals. However, in humans, the high intensity of antibody reactions of some sera from Crohn's disease patients compared with that from noninflammatory bowel disease patients showed a positive correlation with mycobacterial diseases.
Cloning of porcine cytokine-specific cDNAs and detection of porcine tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-1 beta gene expression by reverse transcription PCR and chemiluminescence hybridization
Authors: Vézina S A , Roberge D , Fournier M , Dea S , Oth D , Archambault D
Published: January 2020

A reverse transcription PCR assay with porcine cytokine-specific primers was developed to clone cDNA fragments and generate cDNA probes that were specific for porcine tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and IL-1 beta. The specificities of the cDNA PCR products were confirmed by sequence analysis on the basis of known porcine cytokine gene sequences. The reverse transcription PCR assay was also used to study cytokine mRNA expression in lipopolysaccharide (LPS)-stimulated and control unstimulated porcine alveolar macrophages. The cDNA products were analyzed in ethidium bromide-stained agarose gels, and the transcription level of each cytokine was determined relative to the endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA level of each cytokine by measuring the intensity of the chemiluminescence hybridization signals by densitometric scanning. Various levels of cytokine mRNAs were detected in both LPS-stimulated and control unstimulated cells. Thus, TNF-alpha mRNA levels were enhanced in the cell cultures stimulated for 6 h with LPS compared with those in control cell cultures. No differences in TNF-alpha transcription levels between LPS-stimulated and control cells were observed after incubation for 24 or 55 h. Enhancements of IL-6 and IL-1 beta mRNA levels were also observed in the cultures stimulated with LPS for 6 and 24 h compared with the cytokine mRNA levels in control cell cultures. The presence of cytokine mRNA transcripts in the LPS-stimulated macrophage cultures correlated with the detection of these soluble cytokines by the bioassays. In contrast, no soluble cytokine was detected in control macrophage culture supernatants in the presence of cytokine mRNA transcripts.
Recombinant vaccinia viruses expressing hepatitis A virus structural polypeptides: detection of an anti-VP0 response in convalescent-phase sera
Authors: Karayiannis P , O'Rourke S , Watts R , Waters J , Hill V , Carman W F , Thomas H C
Published: January 2020

We have generated a number of recombinant vaccinia viruses which expressed the hepatitis A virus (HAV) structural polypeptides VP1, VP2, VP3, and VP4, either alone or in combination. The relevant sequences encoding these polypeptides were amplified from cloned cDNA by PCR and then cloned into the insertion vector pGS62. The presence of the HAV structural polypeptide-encoding sequences in the recombinant viruses was confirmed by Southern blot analysis, whilst their transcription and translation were demonstrated by Northern (RNA) blot analysis and immunodetection, respectively. Immunoprecipitation studies using these constructs have detected the presence of an anti-VP0 response in human convalescent sera following HAV infection. The significance of this finding and the usefulness of these constructs in studying cell-mediated immunity during recovery from HAV infection are discussed.
A comparison of electrochemiluminescence and flow cytometry for the detection of natural latex-specific human immunoglobulin E
Authors: Kobrynski L , Tanimune L , Pawlowski N A , Douglas S D , Campbell D E
Published: January 2020

In vitro correlates of type 1 hypersensitivity to natural latex (NL) proteins continue to be limited by both sensitivity and specificity. Methods which have detection limits in the picogram range, namely, radioallergosorbent assays (RAST) and enzyme-linked immunosorbent assays (ELISA), are inadequate for the identification of NL hypersensitivity in certain at-risk groups, such as health care workers. A flow cytometry assay (FCA), previously shown to be comparable to RAST and ELISA in the identification of NL-sensitized pediatric patients with spina bifida, was compared with electrochemiluminescence (ECL) in the evaluation of pediatric patients with spina bifida and NL-sensitized adult health care workers. As with RAST and ELISA, ECL is capable of detecting picogram amounts of specific analyte. The ECL assay detected NL-specific immunoglobulin E (NL-IgE) in three of six health care workers with strong histories of NL hypersensitivity. All six patients were negative by FCA. Further, 2 of 11 spina bifida patients found to be NL-IgE negative by FCA were NL-IgE positive by ECL. These findings suggest that in sensitivity the ECL assay is an improvement over the FCA for the identification of NL-sensitive individuals.
Neutralizing and enhancing activities of human respiratory syncytial virus-specific antibodies
Authors: Gimenez H B , Chisholm S , Dornan J , Cash P
Published: January 2020

The neutralizing and enhancing activities of respiratory syncytial virus (RSV)-specific antibodies were examined. These two biological activities were measured for a panel of six monoclonal antibodies (MAbs) specific to the RSV surface F and G glycoproteins. Four MAbs specific for the F protein possessed both neutralizing and enhancing activities. One MAb (11-2-D2), specific to the G protein, enhanced RSV infection of U937 cells, a human macrophage cell line, but did not neutralize virus infectivity. One MAb (11-3-A3), specific to the F protein, efficiently neutralized virus infectivity but did not enhance RSV infection of U937 cells. MAb 11-3-A3 neutralized representative strains of the two antigenic subtypes of RSV. Assays performed with mixtures of MAbs showed that high concentrations of MAb 11-3-A3 masked the enhancing activity of MAb 11-2-D2. The assay of mixtures of two MAbs possessing only enhancing activities demonstrated that this response was synergistic. The role of neutralizing and enhancing antibodies in determining the outcome of RSV infection was examined for infants from whom cord blood serum samples were collected at birth. There was no significant difference in the magnitude of the serum-enhancing activities between infants who were hospitalized with RSV infections and a group of age- and sex-matched control infants with no reported respiratory illness requiring hospitalization. However, the results indicated a possible correlation between RSV infection of the infants and the occurrence of in vitro antibody-dependent enhancement of the cord blood sera at a serum dilution of 10(-2). A significant inverse correlation was found between the plaque-neutralizing and enhancing activities of the cord blood sera from infants, irrespective of subsequent RSV infection. These data are discussed in relation to the possible contribution of antibody-dependent enhancement to the normal course of RSV pathology in vivo.
Development of mucosal and systemic lymphoproliferative responses and protective immunity to human group A rotaviruses in a gnotobiotic pig model
Authors: Ward L A , Yuan L , Rosen B I , Tô T L , Saif L J
Published: January 2020

Gnotobiotic pigs were orally inoculated with virulent Wa strain (G1P1A[8]) human rotavirus (group 1), attenuated Wa rotavirus (group 2) or diluent (controls) and were challenged with virulent Wa rotavirus 21 days later. On various postinoculation or postchallenge days, virus-specific responses of systemic (blood and spleen) and intestinal (mesenteric lymph node and ileal lamina propria) mononuclear cells (MNC) were assessed by lymphoproliferative assays (LPA). After inoculation, 100% of group 1 pigs and 6% of group 2 pigs shed virus. Diarrhea occurred in 95, 12, and 13% of group 1, group 2, and control pigs, respectively. Only groups 1 and 2 developed virus-specific LPA responses prior to challenge. Group 1 developed significantly greater mean virus-specific LPA responses prior to challenge and showed no significant changes in tissue mean LPA responses postchallenge, and 100% were protected against virulent virus challenge. By comparison, both group 2 and controls had significantly lower LPA responses at challenge and both groups showed significant increases in mean LPA responses postchallenge. Eighty-one percent of group 2 and 100% of control pigs shed challenge virus, and both groups developed diarrhea that was similar in severity postchallenge. The virus-specific LPA responses of blood MNC mirrored those of intestinal MNC, albeit at a reduced level and only at early times postinoculation or postchallenge in all pigs. In a separate study evaluating antibody-secreting-cell responses of these pigs (L. Yuan, L.A. Ward, B.I. Rosen, T.L. To, and L.J. Saif, J. Virol. 70:3075-3083, 1996), we found that the magnitude of a tissue's LPA response positively correlated with the numbers of virus-specific antibody-secreting cells for that tissue, supporting the hypothesis that the LPA assesses T-helper-cell function. The magnitude of LPA responses in systemic and intestinal tissues also strongly correlated with the degree of protective immunity elicited by the inoculum (p = 0.81). We conclude that blood may provide a temporary "window" for monitoring intestinal T cells and that the LPA can be used to assess protective immunity to human rotaviruses.
Serological response to in vitro-shed antigen(s) of Tritrichomonas foetus in cattle
Authors: Bondurant R H , van Hoosear K A , Corbeil L B , Bernoco D
Published: January 2020

We developed a serological assay for detection of (l) an erythrocyte-adhering molecule(s) shed by the bovine venereal pathogen Tritrichomonas foetus and (II) serum antibodies to this antigen(s) in exposed cattle. Sera from exposed and unexposed cattle were tested for their ability to induce complement-mediated lysis of bovine erythrocytes that had been previously incubated overnight at room temperature in pH-adjusted supernatants of T. foetus culture media. Eight of 180 serum specimens from six groups of presumably unexposed cows or heifers showed a positive (> or = 1:2) hemolytic titer (specificity = 95.6%). Thirteen of 14 females in two experimentally infected groups showed a positive hemolytic titer following infection (sensitivity = 94%). In experimentally infected heifers, there was little correlation (r2 = 0.33) between serum hemolytic titers with respect to shed antigen and titers obtained in serum enzyme-linked immunosorbent assays in which whole T. foetus served as the antigen. Serum hemolytic titers rose 3 to 4 weeks sooner than did previously described vaginal mucus immunoglobulin G1 or immunoglobulin A titers with respect to whole-cell antigen or TF1.17 subunit antigen, respectively. Among 14 chronically infected bulls, only 6 (43%) showed a positive hemolytic titer. This study is the first, to our knowledge, to show a specific serological response in the host to an in vitro-shed antigen(s) of T. foetus and suggests a useful diagnostic test for potentially exposed herds.
Antigenic diversity of meningococcal outer membrane protein PorA has implications for epidemiological analysis and vaccine design
Authors: Feavers I M , Fox A J , Gray S , Jones D M , Maiden M C
Published: January 2020

The currently used serological subtyping scheme for the pathogen Neisseria meningitidis is not comprehensive, a proportion of isolates are reported as not subtypeable (NST), and few isolates are fully characterized with two subtypes for each strain. To establish the reasons for this and to assess the effectiveness of DNA-based subtyping schemes, dot blot hybridization and nucleotide sequence analyses were used to characterize the genes encoding antigenic variants of the meningococcal subtyping antigen, the PorA protein. A total of 233 strains, including 174 serologically NST and 59 partially or completely subtyped meningococcal strains, were surveyed. The NST isolates were chosen to be temporally and geographically representative of NST strains, isolated in England and Wales, and submitted to the Meningococcal Reference Unit in the period 1989 to 1991. The DNA-based analyses demonstrated that all of the strains examined possessed a porA gene. Some of these strains were serologically NST because of a lack of monoclonal antibodies against certain PorA epitopes; in other cases, strains expressed minor variants of known PorA epitopes that did not react with monoclonal antibodies in serological assays. Lack of expression remained a possible explanation for serological typing failure in some cases. These findings have important implications for epidemiological analysis and vaccine design and demonstrate the need for genetic characterization, rather than phenotypic characterization using monoclonal antibodies, for the identification of meningococcal strains.
Differential effects of interleukin-12, interleukin-15, and interleukin-2 on human immunodeficiency virus type 1 replication in vitro
Authors: Bayard-McNeeley M , Doo H , He S , Hafner A , Johnson W D , Ho J L
Published: January 2020

Cytokines may have clinical utility as therapeutic agents for human immunodeficiency virus type 1 (HIV-1) infection and as an adjuvant for vaccines. The effect of interleukin-12 (IL-12) and IL-15 on in vitro HIV-1 replication was investigated. IL-12 and IL-15 at doses up to 10 ng/ml had little effect on basal HIV-1 p24 antigen production by chronically HIV-infected T (ACH-2) and monocytic (U1) cell lines. For ACH-2 cells stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml), IL-12 and IL-15 significantly increased p24 antigen production by 20 and 30%, respectively (n = 6). In contrast, IL-12 and IL-15 (10 ng/ml) treatment of PMA-stimulated U1 cells decreased p24 antigen production by 16 and 15%, respectively (n = 6). We next studied the effect of IL-12 and IL-15 on HIV-infected peripheral blood mononuclear cells (PBMCs). In 10 HIV-seropositive patients' PBMCs cocultured with mitogen-activated HIV-seronegative donor cells, two patterns of p24 antigen production were observed in response to IL-2: low (p24 antigen production < 10(3) pg/ml; n = 8) and high (p24 antigen production > 10(3) pg/ml; n = 2) response. For the low-response pattern, IL-12 and IL-15 increased viral replication by 97-fold and 100-fold, respectively (P = 0.05 and 0.004, respectively). For the high-response pattern, both IL-12 and IL-15 suppressed HIV replication. The effect of IL-2, IL-12, and IL-15 on acute in vitro infection by HIV-1JRCSF was also examined. IL-12 did not increase p24 antigen production above basal levels while IL-2 and IL-15 significantly enhanced p24 antigen production (by approximately 2-fold). In conclusion, IL-12 and IL-15 may have differential effects on latent and acute HIV infection, and their ability to enhance HIV production may depend on cell activation. Thus, the use of these cytokines may be dictated by the clinical state of the patient.
Comparison of saliva and serum for human immunodeficiency virus type 1 antibody testing in Uganda using a rapid recombinant assay
Authors: Grant R M , Piwowar E M , Katongole-Mbidde E , Muzawalu W , Rugera S , Abima J , Stramer S L , Kataaha P , Jackson B
Published: January 2020

The accuracy and acceptability of saliva human immunodeficiency virus type 1 (HIV-1) antibody testing were compared with serum testing in a study of paired specimens from HIV-1-seropositive and HIV-1-seronegative Ugandan adults attending a clinic for sexually transmitted diseases. Saliva collection was performed with the Omni-sal device (Saliva Diagnostic Systems, Vancouver, Wash.), and antibody testing was performed by a rapid filter paper assay (Test-Pack; Abbott Laboratories, Abbott Park, Ill.). Relative to serum testing, the sensitivity of saliva testing was 95% (195 of 205) and the specificity was 99% (295 of 297). The sensitivity of saliva testing was higher for patients with elevated levels of beta-2 microglobulin in sera and greater numbers of HIV-1-related symptoms. Pre- and poststudy interviews indicated that saliva testing did not foster inordinate fears of saliva exposure. The development of saliva tests that are inexpensive and do not require electricity is needed.
Rates of tetanus protection and transplacental tetanus antibody transfer in pregnant women from different socioeconomic groups in Peru
Authors: Madico G , Salazar G , McDonald J , Checkley W , Calderón M , Verastegui M , Gilman R H
Published: January 2020

In developing countries, neonatal tetanus causes significant mortality. Using a new competitive enzyme-linked immunosorbent assay to measure anti-tetanus toxin antibody levels, we compared rates of protection, total antibody levels, and maternal-to-fetal antibody ratios between different socioeconomic groups in Peru. Upper-middle-class women 25 years and older had significantly lower rates of protection and mean anti-tetanus toxin antibody levels than did lower-class women of the same age. Nevertheless, the former had higher fetal-to-maternal antibody ratios, independent of maternal age, total antibody levels, or parity. We conclude that future vaccination programs in Latin America must target upper-middle-class as well as lower-class women.
Mycobacterium tuberculosis efpA encodes an efflux protein of the QacA transporter family
Authors: Doran J L , Pang Y , Mdluli K E , Moran A J , Victor T C , Stokes R W , Mahenthiralingam E , Kreiswirth B N , Butt J L , Baron G S , Treit J D , Kerr V J , Van Helden P D , Roberts M C , Nano F E
Published: January 2020

The Mycobacterium tuberculosis H37Rv efpA gene encodes a putative efflux protein, EfpA, of 55,670 Da. The deduced EfpA protein was similar in secondary structure to Pur8, MmrA, TcmA, LfrA, EmrB, and other members of the QacA transporter family (QacA TF) which mediate antibiotic and chemical resistance in bacteria and yeast. The predicted EfpA sequence possessed all transporter motifs characteristic of the QacA TF, including those associated with proton-antiport function and the motif considered to be specific to exporters. The 1,590-bp efpA open reading frame was G+C rich (65%), whereas the 40-bp region immediately upstream had an A+T bias (35% G+C). Reverse transcriptase-PCR assays indicated that efpA was expressed in vitro and in situ. Putative promoter sequences were partially overlapped by the A+T-rich region and by a region capable of forming alternative secondary structures indicative of transcriptional regulation in analogous systems. PCR single-stranded conformational polymorphism analysis demonstrated that these upstream flanking sequences and the 231-bp, 5' coding region are highly conserved among both drug-sensitive and multiply-drug-resistant isolates of M. tuberculosis. The efpA gene was present in the slow-growing human pathogens M. tuberculosis, Mycobacterium leprae, and Mycobacterium bovis and in the opportunistic human pathogens Mycobacterium avium and Mycobacterium intracellular. However, efpA was not present in 17 other opportunistically pathogenic or nonpathogenic mycobacterial species.
Novel compound tetra-, dinucleotide microsatellite polymorphism in the tumor necrosis factor/lymphotoxin locus
Authors: Greenberg S J , Fujihara K , Selkirk S M , Yu F , Du T L , Glenister N , Hohmann P , Rickert M H , Spence P O , Miller C E , Jacobs L D
Published: January 2020

A polymorphic (TGCG)n, tetranucleotide repeat was discovered juxtaposed to the (GT)n dinucleotide repeat that comprises the tumor necrosis factor a microsatellite (TNF) located telomeric to the tumor necrosis factor/lymphotoxin gene cluster. The degree of complexity of this compound tetra-,dinucleotide microsatellite consists of 16 potential alleles of combined length ranging from 24 to 54 bp. The pattern of frequencies of individual alleles belonging to the compound TNFa microsatellite was established from 52 healthy volunteers and was found to be highly heterogeneous. The data diverges significantly from previously published statistics that recognized only a simple variable dinucleotide tandem repeat. The newly recognized compound tetra-, dinucleotide TNFa microsatellite polymorphism establishes a more accurate genetic basis to explore potential linkage with disease susceptibility genes located within this region of the class III major histocompatibility complex. In addition, variable tumor necrosis factor and lymphotoxin production may reflect the more complex polymorphic nature of this microsatellite region. Finally, compound microsatellites probably exist elsewhere, throughout the human genome. Recognition of their presence may have a considerable impact on the validity of past and future microsatellite-based genetic analyses.
Depletion of endogenous interleukin-10 augments interleukin-1 beta secretion by Mycobacterium bovis BCG-reactive human cells
Authors: Méndez-Samperio P , Garcia-Martinez E , Hernandez-Garay M , Solis-Cardona M
Published: January 2020

In this study, we found evidence that the interleukin-10 (IL-10) protein is functionally relevant in Mycobacterium bovis BCG-induced cytokine synthesis, as neutralization of endogenously synthesized IL-10 in human cells activated with BCG resulted in a two- to threefold increase in the level of IL-1 beta. When exogenous recombinant human IL-10 was added to human mononuclear cells, a significant reduction of BCG-induced IL-1 beta secretion was observed. This inhibitory effect was not attributed to a cytotoxic effect, since trypan blue exclusion studies indicated no loss of cell viability in the presence of IL-10, and it was specific, as it was completely abolished in the presence of anti-IL-10 neutralizing monoclonal antibody while an irrelevant antibody used as a control had no effect. Taken together, these are the first studies that demonstrate that the depletion of endogenous IL-10 via anti-IL-10 antibody results in a very significantly enhanced BCG-induced IL-1 beta secretion and that the addition of exogenous IL-10 to human mononuclear cells stimulated with BCG inhibits IL-1 beta production. Further experimental work is needed to determine if the neutralization of IL-10 activity via anti-IL-10 antibody indeed enhances cytokine synthesis in vivo. However, the present results may be of importance, since the use of anti-IL-10 antibody could presumably contribute to the protective immunity induced by BCG against tuberculosis via an increase in cytokine synthesis that would amplify antimicrobial systems.
Monoclonal yeast killer toxin-like candidacidal anti-idiotypic antibodies
Authors: Polonelli L , Séguy N , Conti S , Gerloni M , Bertolotti D , Cantelli C , Magliani W , Cailliez J C
Published: January 2020

Rat monoclonal yeast killer toxin (KT)-like immunoglobulin M (IgM) anti-idiotypic antibodies (KT-IdAbs) were produced by idiotypic vaccination with a mouse monoclonal antibody (MAb; MAb KT4) that neutralized a Pichia anomala KT characterized by a wide spectrum of antimicrobial activity. The characteristics of the KT-IdAbs were demonstrated by their capacity to compete with the KT to the idiotype of MAb KT4 and to interact with putative KT cell wall receptors (KTRs) of sensitive Candida albicans cells. The internal-image properties of KT-IdAbs were proven by their killer activity against KT-sensitive yeasts. This lethal effect was abolished by prior adsorption of KT-IdAbs with MAb KT4. These findings stressed the potential importance of antibody-mediated immunoprotection against candidiasis and suggested a feasible experimental approach for producing antimicrobial receptor antibodies without purifying the receptor. KT-IdAbs might represent the basis for producing engineered derivatives with a high potential for effective therapeutic antifungal activity.
Phage display and bacterial expression of a recombinant Fab specific for Pseudomonas aeruginosa serotype O6 lipopolysaccharide
Authors: Tout N L , Lam J S
Published: January 2020

Immunotherapy with antibodies (Abs) against the lipopolysaccharide (LPS) of Pseudomonas aeruginosa remains an alternative to serotype-specific LPS-based vaccines due to their limited use and to antibiotics due to the intrinsic resistance to antimicrobials observed in P. aeruginosa. We have chosen a monoclonal Ab (MAb), MF23-1, that binds to the O antigen of the most clinically relevant serotype, IATS O6, for producing a recombinant antibody. Heavy (H) and light (L) chain genes were isolated from MF23-1 to form a functional Fab molecule in the periplasm of Escherichia coli and on the surface of phage by using phagemid vector pComb3. The entire kappa L chain gene was used, but the H chain gene was amplified to 2 amino acids past cysteine 128 which is involved in interchain disulfide bond formation with the L chain. The truncated H chain associated with the L chain in the periplasm of E. coli to form a functional Fab molecule that bound in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay to O6 LPS. Therefore, the remainder of the CH1 past cysteine 128 is not essential for stable formation of the Fab portion of MF23-1. This recombinant Fab (r-Fab) was shown to be specific for the LPS of the most predominant clinical isolate, serotype O6, while no cross-reactivity was detected to the LPS of the other 19 remaining serotypes. This r-Fab was also expressed on the surface of filamentous phage upon addition of helper phage to recombinant E. coli containing phagemid. Recombinant phage from clones MT13 and MT24 bound specifically to O6 LPS in ELISA. These results represent an important step toward the design of therapeutic Abs to be used against P. aeruginosa infections.
Function and phenotype of immature CD4+ lymphocytes in healthy infants and early lymphocyte activation in uninfected infants of human immunodeficiency virus-infected mothers
Authors: Rich K C , Siegel J N , Jennings C , Rydman R J , Landay A L
Published: January 2020

The function and phenotypes of CD4+ lymphocytes in infants are different than in adults and are modulated by maturational changes and exposure to environmental antigens. Infants of non-human immunodeficiency virus (HIV)-infected mothers and uninfected infants of HIV-infected mothers, 0 to 6 months of age, were examined for CD4+ lymphocyte function by in vitro interleukin-2 (IL-2) production and for CD4+ phenotypes by three-color flow cytometry. A minority of these uninfected infants (28%) had functional responses similar to those of healthy adult women (IL-2 production in response to anti-CD3, alloantigen, and mitogen), while the remainder were capable of responding to alloantigen and mitogen but not to anti-CD3. We did demonstrate reduced phytohemagglutinin-stimulated IL-2 production in uninfected infants born to HIV-seropositive mothers compared to that in infants from seronegative mothers. The proportions of CD3+ CD4+, CD4+ HLA-DR- CD38+, and CD4+ CD45RA+ RO- (naive) lymphocytes were much higher in infants than in adults, and the proportions of CD4+ CD45RA- RO+ (memory) and CD4+ CD25+ (IL-2 receptor-bearing) lymphocytes were lower in infants than in adults. The proportions of activated (CD4+ HLA-DR+ CD38+) and memory (CD4+ CD45RA- RO+) lymphocytes were increased in uninfected infants of HIV-infected mothers compared to infants of uninfected mothers. Therefore, T-helper-cell function is immature in many infants, but the CD4+ lymphocytes of some HIV-exposed, uninfected infants have been stimulated by antigen at an early age.
Development and application of genetic probes for detection of Enterocytozoon bieneusi in formalin-fixed stools and in intestinal biopsy specimens from infected patients
Authors: Carville A , Mansfield K , Widmer G , Lackner A , Kotler D , Wiest P , Gumbo T , Sarbah S , Tzipori S
Published: January 2020

The microsporidium Enterocytozoon bieneusi is closely linked to wasting and diarrhea in a high proportion of individuals with AIDS. However, its relative contribution to disease is uncertain because diagnosis until recently depended on procedures involving endoscopy. A sensitive PCR technique which amplifies a fragment of the small-subunit rRNA gene of E. bieneusi from formalin-fixed stool samples was developed. Of 80 formalin-fixed stool samples collected from 74 Zimbabweans and 6 U.S. patients who were human immunodeficiency virus positive, 50% tested positive for E. bieneusi by PCR, whereas 24% tested positive for E. bieneusi by light microscopy of trichrome-stained fecal smears. In addition, we describe an in situ hybridization technique which detected and identified E. bieneusi as the causative agent in all six intestinal biopsy specimens tested. Both the PCR and in situ hybridization procedures are sensitive diagnostic tools which will complement currently available techniques and enable the differentiation of E. bieneusi from other microsporidia to be made.
Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells
Authors: Romero-Steiner S , Libutti D , Pais L B , Dykes J , Anderson P , Whitin J C , Keyserling H L , Carlone G M
Published: January 2020

Host protection against pneumococcal disease i primarily mediated by phagocytosis. We developed and standardized an opsonophagocytic assay using HL-60 cells (human promyelocytic leukemia cells). Fifty-five serum samples were analyzed for the presence of functional antibody against seven pneumococcal serogroups or serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by using differentiated HL-60 cells (granulocytes) and peripheral blood leukocytes (PBLs). Six of the 55 serum samples were from unvaccinated adult volunteers, 31 serum samples were from adults who received one dose of the 14-valent or the 23-valent polysaccharide vaccine, and 18 serum samples were from 16-month-old infants who received four doses of an investigational 7-valent polysaccharide-protein conjugate vaccine. The results of an opsonophagocytic assay with HL-60 cells correlated highly with those of an assay with PBLs as effector cells (median r for seven serotypes = 0.87: P < 0.01). Opsonophagocytic titers were compared with the immunoglobulin G antibody concentrations determined by enzyme-linked immunosorbent assay (ELISA). The r values for serogroups or serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F were 0.61, 0.60, 0.67 0.90, 0.61, 0.39, and 0.57, respectively, when HL-60 cells were used as effector cells and 0.56, 0.47, 0.61, 0.90, 0.71, 0.31, and 0.62, respectively, when PBLs were used. The assay requires small amounts of serum (40 microliters per serotype), making this test suitable for assaying infant sera. Culturable cells aid in assay standardization and likely reduce donor-to-donor variability. This standardized assay, in combination with the standardized ELISA, can be used to evaluate current and developing pneumococcal vaccines, in which functional opsonophagocytic antibody activity may correlate with protection against pneumococcal disease.
Expression, characterization, and immunoreactivities of a soluble hepatitis E virus putative capsid protein species expressed in insect cells
Authors: Zhang Y , McAtee P , Yarbough P O , Tam A W , Fuerst T
Published: January 2020

The hepatitis E virus (HEV) open reading frame-2 (ORF-2) is predicted to encode a 71-kDa putative capsid protein involved in virus particle formation. When insect Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus containing the entire ORF-2 sequence, two types of recombinant proteins were produced; an insoluble protein of 73 kDa and a soluble protein of 62 kDa. The 62-kDa species was shown to be a proteolytic cleavage product of the 73-kDa protein. N-terminal sequence analysis of the 62-kDa protein indicated that it lacked the first 111 amino acids that are present in the full-length 73-kDa protein. A soluble 62-kDa protein was produced without the proteolytic processing by inserting the coding sequence of amino acids 112 to 660 of ORF-2 in a baculovirus expression vector and using the corresponding virus to infect Sf9 cells. The two recombinant 62-kDa proteins made by different mechanisms displayed immunoreactivities very compatible to each other. The 62-kDa proteins obtained by both proteolytic processing and reengineering demonstrated much higher sensitivities in detecting anti-HEV antibodies in human sera than the antigens made from bacteria, as measured by enzyme-linked immunosorbent assay. The data suggest that the soluble 62-kDa protein made from insect cells contains additional epitopes not present in recombinant proteins made from bacteria. Therefore, the 62-kDa protein may be useful for HEV diagnostic improvement and vaccine development. The reengineered construct allows for the consistent large-scale production of the soluble 62-kDa protein without proteolytic processing.
Inhibition of Prevotella and Capnocytophaga immunoglobulin A1 proteases by human serum
Authors: Frandsen E V , Kjeldsen M , Kilian M
Published: January 2020

Oral Prevotella and Capnocytophaga species, regularly isolated from periodontal pockets and associated with extraoral infections, secret specific immunoglobulin A1 (IgA1) proteases cleaving human IgA1 in the hinge region into intact Fab and Fc fragments. To investigate whether these enzymes are subject to inhibition in vivo in humans, we tested 34 sera from periodontally diseased and healthy individuals in an enzyme-linked immunosorbent assay for the presence and titers of inhibition of seven Prevotella and Capnocytophaga proteases. All or nearly all of the sera inhibited the IgA1 protease activity of Prevotella buccae, Prevotella oris, and Prevotella loescheii. A minor proportion of the sera inhibited Prevotella buccalis, Prevotella denticola, and Prevotella melaninogenica IgA1 proteases, while no sera inhibited Capnocytophaga ochracea IgA1 protease. All inhibition titers were low, ranging from 5 to 55, with titer being defined as the reciprocal of the dilution of serum causing 50% inhibition of one defined unit of protease activity. No correlation between periodontal disease status and the presence, absence, or titer of inhibition was observed. The nature of the low titers of inhibition in all sera of the IgA1 proteases of P. buccae, P. oris, and P. loescheii was further examined. In size exclusion chromatography, inhibitory activity corresponded to the peak volume of IgA. Additional inhibition of the P. oris IgA1 protease was found in fractions containing both IgA and IgG. Purification of the IgG fractions of five sera by passage of the sera on a protein G column resulted in recovery of inhibitory IgG antibodies against all three IgA1 proteases, with the highest titer being for the P. oris enzyme. These finding indicate that inhibitory activity is associated with enzyme-neutralizing antibodies.
Search for cytomegalovirus-specific immunoglobulin M: comparison between a new western blot, conventional western blot, and nine commercially available assays
Authors: Lazzarotto T , Brojanac S , Maine G T , Landini M P
Published: January 2020

We tested 101 serum samples obtained from pregnant women for the presence of human cytomegalovirus (HCMV)-specific IgM with nine different commercially available kits and with two Western blotting (WB) tests previously developed in our laboratory. The conventional WB test contains viral structural proteins separated on a gel from purified HCMV particles and transferred to nitrocellulose. The new WB test contains viral structural proteins and three recombinant proteins which contain the significant immunogenic portions of pp150 (ppUL32), pp52 (ppUL44), and p130 (pUL57). The results obtained indicate that the new WB test combines high specificity (92.5%) with high sensitivity (95.0%), characteristics that, in combination, have not been obtained with any of the other tests.
Antibody response against a Leishmania donovani amastigote-stage-specific protein in patients with visceral leishmaniasis
Authors: Ghedin E , Zhang W W , Charest H , Sundar S , Kenney R T , Matlashewski G
Published: January 2020

The antibody response against an amastigote-specific protein (A2) from Leishmania donovani was investigated. Sera from patients with trypanosomiasis and various forms of leishmaniasis were screened for anti-A2 antibodies. Sera from patients infected only with L. donovani or Leishmania mexicana specifically recognized the A2 recombinant protein. These results were consistent with karyotype analyses which revealed that the A2 gene is conserved in L. donovani and L. mexicana strains. The potential of this antigen in diagnosis was further explored by screening a series of sera obtained from patients in regions of the Sudan and India where L. donovani is endemic. The prevalence of anti-A2 antibodies was determined by Western blotting for all samples. Enzyme-linked immunosorbent assay (ELISA) and an immunoprecipitation assay were also performed on some of the samples. Anti-A2 antibodies were detected by ELISA in 82 and 60% of the samples from individuals with active visceral leishmaniasis (kala-azar) from the Sudan and India, respectively, while the immunoprecipitation assay detected the antibodies in 92% of the samples from India. These data suggest that the A2 protein may be a useful diagnostic antigen for visceral leishmaniasis.
Dilution assessment of cervicovaginal secretions collected by vaginal washing to evaluate mucosal shedding of free human immunodeficiency virus
Authors: Mohamed A S , Becquart P , Hocini H , Métais P , Kazatchkine M , Bélec L
Published: January 2020

A 10 mM concentration of lithium does not interfere with reverse transcription (RT) or PCR. Sampling of cervicovaginal fluid by vaginal washing, with lithium (10 mM) in the washing buffer as a marker of dilution, may be utilized to accurately determine in HIV-infected women, by quantitative RT-PCR, the genital shedding of acellular HIV RNA at the level of the mucosa itself.
Expression cloning and humoral immune response to the nucleocapsid and membrane proteins of equine arteritis virus
Authors: Kheyar A , Martin S , St-Laurent G , Timoney P J , McCollum W H , Archambault D
Published: January 2020

To provide a convenient and sensitive method for the detection of equine arteritis virus (EAV)-specific serum antibodies, we developed an immunoblot assay employing the EAV nucleocapsid (N) and membrane (M) proteins expressed in a procaryotic expression vector (pMAL-c2) for the production of recombinant maltose-binding (MBP) fusion proteins (MBP-N and MBP-M). The antigenic reactivity of the recombinant fusion proteins and their Xa factor cleavage EAV products was confirmed by immunoblot using horse antisera to EAV. Some horse sera, however, showed immune reactivity to the MBP fusion partner protein. Based on a total of 32 horse sera analyzed for the presence of EAV antibodies by immunoblot, using the MBP-N or -M fusion proteins and the Xa factor cleavage EAV products, and in the serum neutralization test, there was 100% concordance between the assays. Sera from horses experimentally infected with EAV were reactive in the immunoblot test with both the MBP-N and the MBP-M fusion proteins by day 14 after EAV exposure. The reactivity continued to the end of the experiment at day 145 after infection. This immune reactivity correlated with the detection of neutralizing antibodies in the serum samples. Based on these findings, the recombinant N and M proteins might be useful for serodetection of EAV-infected animals.
Cloning and characterization of two recombinant Neospora protein fragments and their use in serodiagnosis of bovine neosporosis
Authors: Louie K , Sverlow K W , Barr B C , Anderson M L , Conrad P A
Published: January 2020

Bovine neosporosis causes fetal abortion and/or congenital neurologic disease in cattle. For the serodiagnosis of this parasitic disease, two immunodominant clones from a bovine Neospora lambda gt11 library were identified, characterized, and expressed as recombinant proteins for the development of an enzyme-linked immunosorbent assay (ELISA). These two clones, designated N54 and N57, were 29 and 20 kDa, respectively, when expressed as histidine fusion proteins from the pRSET expression vector. Antibodies to recombinant protein N54 recognized five major bands from a Neospora tachyzoite lysate with molecular masses of 97, 87, 77, 67, and 64 kDa. Antibodies to recombinant protein N57 recognized four primary bands with molecular masses of 34, 31, 30, and 28 kDa. When a defined "gold standard" panel of bovine sera from confirmed Neospora-positive and Neospora-negative cattle were characterized by immunoblotting, 57 of the 60 Neospora-positive serum samples recognized proteins with the molecular masses of the N54 heptuplet. Binding to the N57 quadruplet was more variable. The same gold standard panel was used to evaluate and compare an N54-based ELISA, an N57-based ELISA, and a whole-tachyzoite lysate-based ELISA. The sensitivities and specificities were 95 and 96% (N54 ELISA), 82 and 93% (N57 ELISA), and 74 and 93% (lysate ELISA). Thus, compared to the whole-tachyzoite lysate-based ELISA, both recombinant-protein-based ELISAs had higher sensitivities and higher or the same specificities and can be used to replace the whole-tachyzoite lysate ELISA for the serodiagnosis of bovine neosporosis.
Fecal lactoferrin, interleukin-1beta, and interleukin-8 are elevated in patients with severe Clostridium difficile colitis
Authors: Steiner T S , Flores C A , Pizarro T T , Guerrant R L
Published: January 2020

Twenty-two patients with Clostridium difficile colitis as determined by positive enzyme immunoassay for toxin A were evaluated for fecal inflammatory markers and their relationship to the severity of illness. Fourteen of 22 specimens were positive for fecal lactoferrin (FLF), with titers from 1:50 to 1:800. Nine of 10 stools tested had ratios of interleukin-1beta (IL-1beta) to IL-1 receptor antagonist (IL-1ra) of >0.01. Seventeen of 22 specimens also had elevated IL-8 concentrations, and 12 of 14 had elevated IL-1beta concentrations. A review of the 18 available patient records revealed that fecal IL-8 concentrations, IL-1beta/IL-1ra ratios, and FLF titers were significantly higher in patients with moderate to severe disease than in patients with mild disease. These findings suggest that the proinflammatory effects of C. difficile may directly influence clinical characteristics of human disease.
Rapid diagnosis of pertussis in young infants: comparison of culture, PCR, and infant's and mother's serology
Authors: Grimprel E , Njamkepo E , Bégué P , Guiso N
Published: January 2020

The contribution of maternal pertussis serology comparing prepartum serum to serum collected during the infant's disease to the diagnosis of pertussis in infants was evaluated for 28 pairs of young infants with pertussis syndrome and their mothers and was compared to those of culture and PCR. Infants had a nasopharyngeal aspiration tested by PCR, and acute and convalescent sera were collected during their disease. Mothers had a first acute serum collected concomitantly with the infant's acute serum, and both acute sera were compared to a prepartum serum. Sera were analyzed by immunoblotting for the detection of anti-pertussis toxin (PT) antibodies. Serological evidence of pertussis in infants was assessed as either an increase in anti-PT antibody levels between the mother's prepartum and acute sera or the presence of antibodies in the infant's acute serum and their absence in both the mother's acute and prepartum sera. Culture and PCR sensitivity were 43 and 89%, respectively. Most infants (18 of 24) had no pertussis antibody detectable in their acute sera, confirming a delayed immune response at this age. A comparison of infant's and mother's serology, using prepartum serum, rapidly confirmed the diagnosis in 57% of the cases. Although less sensitive than PCR, this serological method should be used for a rapid diagnosis of pertussis in young infants when culture and PCR are either not available or negative.