bluuebunny/crossref_metadata_2025 huggingface dataset:
Time to peak serum antibody response to influenza vaccine
Authors: Rastogi S , Gross P A , Bonelli J , Dran S , Levandowski R A , Russo C , Weksler M E , Kaye D , Levison M , Abrutyn E
Published: January 2020

The time to the appearance of a peak serum antibody response to influenza virus vaccine is not clearly defined. We compared the most commonly used time intervals described in the literature--4 and 6 weeks after vaccination. We studied 118 elderly patients from three different geographic sites. The 1992 to 1993 trivalent inactivated influenza virus vaccine containing influenza virus A/Beijing/353/89 (H3N2), influenza virus A/Texas/36/91 (H1N1), and influenza virus B/Panama/45/90 was used. No statistically significant differences were found at the 4- and 6-week intervals after vaccination.
Stability of cytomegalovirus antibodies in plasma during prolonged storage of blood components
Authors: Pappin A , Grissom M , Mackay W , Huang Y , Yomtovian R
Published: January 2020

Cytomegalovirus (CMV) antibody testing is currently limited by manufacturers' guidelines to specimens stored for 7 days or less. We examined the stability of CMV antibodies in plasma from platelets and whole-blood units during storage using a rapid, automated, recombinant protein-based immunoassay which qualitatively detects total antibody to human CMV. Testing of single-donor apheresis platelets was performed on baseline serum and platelet-free plasma and on platelet-free plasma 8 days later. Indeterminate, positive, and negative CMV antibody results were maintained over time for 97% (75 of 77) of the platelet specimens. For whole-blood units, initial testing of donor serum and plasma obtained from erythrocyte segments took place within 7 days of phlebotomy. Indeterminate, positive, and negative CMV antibody results were maintained on subsequent analyses performed on erythrocyte segments at 2, 4, 6, and 8 weeks for 100% of whole-blood specimens. An important potential benefit of CMV antibody testing of stored platelets and blood is the elimination of a costly, dedicated, CMV-negative inventory. The study suggests that CMV antibody testing can be conveniently and reliably performed on blood components over the entire storage period.
Dilution assessment of cervicovaginal secretions obtained by vaginal washing for immunological assays
Authors: Bélec L , Meillet D , Lévy M , Georges A , Tévi-Bénissan C , Pillot J
Published: January 2020

Local immunological defense mechanisms in the cervicovaginal mucosa currently remain incompletely defined, especially from a quantitative point of view. Addition of an inert substance, lithium chloride (LiCl), into the washing buffer used to carry out the vaginal washing for collecting cervicovaginal secretions and measurement of its concentration with a flame absorption spectrophotometer, before and after the specimen is sampled, permits the quantification of the volume of cervicovaginal secretions collected and the approximation of the dilution factor of a soluble component introduced by the washing. Lithium, at a concentration of 10 mM, gives the best precision of measurement and has no effect on the results of the immunoassays. In a population of 27 nonpregnant women (age range, 18 to 45 years), the volume of cervicovaginal secretions collected by vaginal washing with 3 ml of LiCl-phosphate-buffered saline was 12% +/- 3.2% (mean +/- standard deviation) of the total volume and showed large interindividual variations (range, 5.6 to 18.8%); the mean dilution factor of a soluble component from the vaginal secretions was 9.9% +/- 2.8% (range, 6.3 to 18.8%). According to the date of the menstrual cycle, the mean volume of collected cervicovaginal secretions was significantly increased in the luteal phase in comparison with the follicular phase; conversely, the mean dilution factor of a soluble component was more important in the follicular than in the luteal phase. These features strengthen the need to quantify accurately the dilution factor introduced by vaginal washing when studying cervicovaginal immunity.
Cloning and sequence analysis of a newly identified Brucella abortus gene and serological evaluation of the 17-kilodalton antigen that it encodes
Authors: Hemmen F , Weynants V , Scarcez T , Letesson J J , Saman E
Published: January 2020

A thus far unknown gene encoding a Brucella abortus protein has been isolated from a lambda gt11 expression library probed with sera from Brucella-infected sheep. Sequence analysis of the cloned gene revealed the presence of an open reading frame of 158 amino acids encoding a protein of 17.3 kDa (calculated molecular mass). The recombinant B. abortus protein, expressed in Escherichia coli, and the corresponding Brucella melitensis protein migrated at the same apparent molecular masses as shown by Western blotting (immunoblotting). Among a series of serum samples from B. melitensis- or B. abortus-infected sheep and cows, 51 and 39%, respectively, showed a signal at 17 kDa on Western blot analysis of total protein extract from Brucella bacteria. These figures amount to 70 and 61% for sheep and cattle, respectively, in a competitive enzyme-linked immunosorbent assay with a specific monoclonal antibody. These data indicate that the 17-kDa antigen may be useful for serological diagnosis of Brucella infection.
Transport of recombinant human CD4-immunoglobulin G across the human placenta: pharmacokinetics and safety in six mother-infant pairs in AIDS clinical trial group protocol 146
Authors: Shearer W T , Duliege A M , Kline M W , Hammill H , Minkoff H , Ammann A J , Chen S , Izu A , Mordenti J
Published: January 2020

Recombinant CD4-immunoglobulin G (rCD4-IgG) is a 98-kDa human immunoglobulin-like protein that is produced by fusing the gp120 binding domain of CD4 to the Fc portion of the human IgG1 heavy chain. This hybrid molecule was given to human immunodeficiency virus (HIV)-infected pregnant women at the onset of labor by intravenous bolus at 1 mg/kg of body weight (group A; n = 3) and 1 week prior to and at the onset of labor by the same route and at the same dose (group B; n = 3). In addition to pharmacokinetic studies, safety in the mothers and infants was determined through routine chemistries, hematology, and urinalysis; immunologic and HIV infection statuses in the infants were assessed through lymphocyte cultures, p24 antigen level determination, culture of HIV from plasma, PCR, lymphocyte subset enumeration, quantitative immunoglobulin analysis, and lymphocyte proliferation. Thirty minutes after the rCD4-IgG injection, concentrations in maternal serum were 12 to 23 micrograms/ml. These concentrations declined slowly, with initial and terminal half-lives (mean +/- standard deviation) of 9.95 +/- 3.23 and 47.6 +/- 22.3 h, respectively. Infants were born 2.6 to 46.5 h after rCD4-IgG administration; concentrations of rCD4-IgG in cord blood ranged from 28 to 107 ng/ml. The half-life of rCD4-IgG in infants ranged from 5 to 29 h. These data demonstrate that the transfer of rCD4-IgG from the mother to the fetus is rapid and that newborns do not appear to have any difficulty eliminating rCD4-IgG. No safety concerns in mothers or infants were encountered. Although the study did not address the question of efficacy, none of the infants was HIV type 1 infected 36 months later. In summary, these findings document that bifunctional immune molecules can be transported across the placenta, and this general approach may be used in the future to block vertical transmission of HIV type 1.
Infection with human T-lymphotropic virus types I and II results in alterations of cellular receptors, including the up-modulation of T-cell counterreceptors CD40, CD54, and CD80 (B7-1)
Authors: Dezzutti C S , Rudolph D L , Lal R B
Published: January 2020

To examine the phenotypic alterations associated with human T-lymphotropic virus types I and II (HTLV-I and -II) infection, long-term cell lines (n = 12 HTLV-I cell lines; n = 11 HTLV-II cell lines; n = 6 virus-negative cell lines) were analyzed for the cell surface expression of various lineage markers (i.e., myeloid, progenitor, and leukocyte), integrin receptors, and receptor-counterreceptor (R-CR) pairs responsible for cellular activation. As expected, all cell lines expressed the markers characterizing the leukocyte lineage (CD43, CD44, and CD53). Of the progenitor-myeloid markers examined (CD9, CD13, CD33, CD34, and CD63), only the percent expression of CD9 was significantly increased on HTLV-I and -II-infected cell lines as compared with that on virus-negative cell lines. Analysis of the beta 1 integrin subfamily (CD29, CD49b, CD49d, CD49e, and CD49f) showed no significant change, except that CD49e was significantly decreased on the HTLV-infected cell lines. For the beta 2 integrin subfamily, the cell surface density was increased for CD18 and CD11a, while the CD11c molecule was expressed exclusively on the HTLV-I- and HTLV-II-infected cell lines. Analysis of several R-CR pairs (CD2-CD58, CD45RO-CD22, CD5-CD72, CD11a-CD54, gp39-CD40, and CD28-CD80) demonstrated that comparable levels of expression of the Rs (CD2, CD45RO, CD5, and CD28) and of some of the CRs (CD58, CD22, and CD72) were in all cell lines; however, CD54, CD40, and CD80 were expressed constitutively on the HTLV-I- and HTLV-II-infected cell lines. Functionally, the expression of these R-CR pairs did not appear to affect the autologous proliferation since monoclonal antibodies to these R-CR pairs were not able to inhibit proliferation of the infected cell lines. Taken together, our results indicate that HTLV-I and -II can modulate the expression of several T-cell activation molecules and CRs normally expressed on alternate cell types.
Reversal of immunosuppression of lymphocyte proliferation caused by sera from persons with AIDS
Authors: Tomar R H , John P , Hinds P
Published: January 2020

Sera from persons with AIDS contain inhibitors of lymphocyte proliferation. Inhibitory activity can be detected before the development of AIDS in humans. There appear to be at least three distinct suppressive moieties, one of which is prostaglandin E2. We and others had previously shown that serum samples from subjects with AIDS contained antibody to the cell line HUT 102B2. We attempted to remove this antibody and to determine if that action would also remove the inhibitory activity present in human immunodeficiency virus-positive sera. We incubated sera from subjects with AIDS with HUT 102B2 cells and tested the resultant supernatants for inhibition of cell proliferation. We found that this procedure significantly reversed inhibition by serum. Other cells and cell lines were similarly tested, but only HUT 102B2 cells absorbed the inhibitory product(s). However, we determined that secretory material from another cell line, MLA 144, also reversed inhibition. The physical characteristics of the supernatant were investigated. Thus, two procedures and likely separate products, possibly cytokines, reverse immunosuppression by sera from persons with AIDS.
Differential effects of human immunodeficiency virus type 1 envelope protein gp120 on interferon production by mononuclear cells from adults and neonates
Authors: Nair M P , Chadha K C , Stadler I , Sweet A , Schwartz S A
Published: January 2020

While considerable progress in examining the course of human immunodeficiency virus (HIV) infection in adults has been made, a better understanding of the natural history of perinatal HIV infection remains to be obtained. Dysregulation of the production and functions of various cytokines, especially the interferons (IFNs), during HIV infections has been reported. Using an in vitro model system, we examined the effects of the HIV type 1 envelope protein, gp120 (10, 50, and 100 ng/ml), on gamma IFN (IFN-gamma) and IFN-alpha production by lymphocytes from neonates and adults and also examined the potential regulatory effects of gp120 on phorbol 12-myristate acetate (PMA)- and Sendai virus-induced IFN-gamma and IFN-alpha production by lymphocytes. PMA at a concentration of 50 ng/ml plus 50 ng of calcium ionophore A23187 per ml was used to induce IFN-gamma, while 150 hemagglutinating units of Sendai virus was used to induce IFN-alpha production. The antiviral activity of both IFN-alpha and IFN-gamma in leukocyte culture supernatants was assayed on BG-9 cells by a dye uptake technique using vesicular stomatitis virus as a challenge virus. Placental cord blood leukocyte (CBL) samples from healthy, term infants and adult peripheral blood leukocytes (APBL) produced no IFN in response to gp120. However, CBL produced significantly decreased levels of IFN-gamma compared with APBL in response to PMA plus ionophore. gp120 significantly suppressed both Sendai virus-induced IFN-alpha and PMA-induced IFN-gamma production by both CBL and APBL in a dose-dependent manner. However, gp120-induced suppression of IFN-alpha and IFN-gamma was significantly greater with CBL than with APBL. Treatment of CBL and APBL with gp120 did not induce any phenotypic alteration of the CD45 RO+ subset. Increased suppression of IFN-alpha and IFN-gamma production by gp120 in neonates may partially explain their apparent increased susceptibility to the clinical progression of HIV infections compared with that of adults.
Prevalence of gca, a gene involved in synthesis of A-band common antigen polysaccharide in Pseudomonas aeruginosa
Authors: Currie H L , Lightfoot J , Lam J S
Published: January 2020

Two distinct forms of lipopolysaccharide are expressed by Pseudomonas aeruginosa. These forms are known as the A band and the B band. In an attempt to obtain a better understanding of A-band lipopolysaccharide synthesis, a previously isolated A-band gene known as the gca gene (GDP-D-mannose conversion protein for A-band common antigen polysaccharide) was sequenced and analyzed. Previous protein expression data from our laboratory, along with nucleotide sequence analysis from the present study, suggest that the Gca protein is encoded by the open reading frame ORF36.5. Amino acid homology reveals that this protein may be functioning as a dehydratase or as a bifunctional enzyme, facilitating the conversion of GDP-D-mannose to GDP-D-rhamnose. The distribution of this gca gene among the 20 P. aeruginosa O serotypes, clinical isolates, and other Pseudomonas species was also examined. Southern hybridization results revealed that the gca gene is present and conserved on a 1.6-kb KpnI fragment among all 20 O serotypes with the exception of serotype O12. In addition, the gca gene is not universally found among all pseudomonads; however, probe-reactive profiles are similar to that of P. aeruginosa when the gca gene is present. Primers were designed from the gca nucleotide sequence, and PCR amplification of a 700-bp product was found with each of the 20 O serotypes. Because of the conservation of this gene, gca may be useful as a diagnostic tool for detecting the presence of P. aeruginosa as well as other Pseudomonas species.
Characteristics and clinical significance of a stabilization assay to detect specific antibodies to reverse transcriptase of human immunodeficiency virus
Authors: Morita M , Suzuki T , Nakajima K , Shiozawa C , Gill M J , Hoshino H
Published: January 2020

Antibodies against reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) have been detected in seropositive subjects by immunoprecipitation, Western immunoblotting, and neutralization assay. Recently, we noticed that the antibodies against RT stabilized RT upon heat inactivation, and we have developed a stabilization assay of RT antibody. Briefly, the RT of HIV-1 is completely inactivated by incubation at 56 degrees C for 20 min, but this inactivation is inhibited in the presence of a specific antibody directed against this molecule. We examined the specificity and clinical significance of this stabilization assay. HIV-1 antibody-positive sera stabilized HIV-1 RT but not HIV-2 RT, whereas half of these sera cross-neutralized HIV-2 RT. Antibody titers against RT determined by the neutralization assay and the stabilization assay were compared with clinical characteristics. Antibodies against HIV-1 RT were much more frequently detected by the stabilization assay than by the neutralization assay. Statistically significant associations were found between stabilizing antibody titer and CD4+ cell number in peripheral blood of patients and also between antibody titer and CD4+/CD8+ ratios. These results indicate that our new stabilization assay to detect specific antibodies against RT of HIV-1 is useful as a clinical marker of infection and progress of the disease.
Adenosine deaminase isoenzyme levels in patients with human T-cell lymphotropic virus type 1 and human immunodeficiency virus type 1 infections
Authors: Tsuboi I , Sagawa K , Shichijo S , Yokoyama M M , Ou D W , Wiederhold M D
Published: January 2020

In serum, the enzyme adenosine deaminase (ADA) is known to be divided into two isoenzymes, ADA1 and ADA2, which have different molecular weights and kinetic properties. The present study investigated ADA isoenzyme levels in the sera of patients infected with retroviruses associated with adult T-cell leukemia (ATL), human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy (HAM), and AIDS, ADA isoenzyme activities were found to be significantly (P < 0.001) higher in the sera of patients with ATL, HAM, and AIDS than in the sera of healthy controls. In the case of the ADA subtypes in the sera of patients with ATL, ADA1 activity was significantly (P < 0.001) elevated in patients with the acute and lymphoma types of ATL compared with that in patients with the chronic and smoldering types of ATL. ADA2 activity was significantly elevated in the sera of patients with the acute, lymphoma, and chronic types of ATL (P < 0.001) compared with that in patients with smoldering ATL and HTLV-1 carriers. In the case of patients with human immunodeficiency virus type 1 (HIV-1) infection, ADA1 and ADA2 activities in the sera of patients with AIDS and HIV-1 antibody-positive individuals were significantly (P < 0.001) higher than those in the sera of HIV-1 antibody-negative individuals. A significant elevation in ADA2 activity was also seen in the sera of AIDS patients (P < 0.01) compared with that in the sera of HIV-1 antibody-positive individuals. These results suggest that the magnitude of elevation of ADA isoenzyme levels in serum correlates well with the clinical conditions of the patients with these diseases. Measurement of the activities of ADA isoenzymes may therefore provide an additional parameter for distinguishing the subtypes of ATL and may prove to be useful as prognostic and therapeutic monitors in diseases associated with HTLV-1 and HIV-1 infections.
Serum antibody to lipopolysaccharide antigens of Shigella species among U.S. military personnel deployed to Saudi Arabia and Kuwait during Operations Desert Shield and Desert Storm
Authors: Hyams K C , Malone J D , Bourgeois A L , Hawkins R , Hale T L , Murphy J R
Published: January 2020

During Operations Desert Shield and Desert Storm, U.S. troops were at high risk of diarrheal disease due to Shigella spp., particularly Shigella sonnei. In order to better understand the serologic response to Shigella infection, 830 male U.S. combat troops were evaluated before and after the deployment to Saudi Arabia and Kuwait for immunoglobulin A (IgA) and IgG anti-Shigella lipopolysaccharide (LPS) (antibody to S. sonnei form I and Shigella flexneri serotypes 1a, 2a, and 3a) in serum. Just before deployment, 10.3% of the subjects were seropositive for IgA and 18.3% were positive for IgG anti-Shigella LPS. IgA and IgG anti-LPS antibody levels in serum prior to deployment were significantly associated with nonwhite race and ethnicity, birth outside the United States, and antibody to hepatitis A virus and Helicobacter pylori. During the deployment, which lasted for a mean of 131 days, 60% of the subjects reported at least one episode of diarrhea and 15% reported an episode of diarrhea with feverishness; also, 5.5% of the subjects exhibited IgA seroconversion to Shigella LPS and 14.0% exhibited IgG seroconversion. A significant association between the development of diarrheal symptoms and either positive predeployment anti-LPS antibody or seroconversion was not found. These data indicate that in this population of U.S. Desert Storm troops who were at high risk of Shigella infection, there was no apparent relation between IgA or IgG anti-Shigella LPS in serum and diarrheal disease.
Natural killer cell cytotoxicity in elderly humans after influenza immunization
Authors: Kutza J , Gross P , Kaye D , Murasko D M
Published: January 2020

Previous studies have reported that human natural killer (NK) cytotoxicity can be augmented by either in vitro stimulation with influenza virus antigens or in vivo administration of killed influenza vaccine. The study demonstrating the latter conclusion reported an increase in NK cytotoxicity lasting for 4 weeks postvaccination in young subjects. We initiated our study to determine if a similar increase in NK activity was observed in an elderly population after immunization with the 1992-1993 influenza vaccine. NK activity of 34 elderly (mean age, 77.3 years) was determined at 3 time points: prevaccination, 4 to 6 weeks postvaccination, and 5 to 6 months after vaccination. In contrast to the results of the previous study, the NK cytotoxicity of our elderly subjects was not augmented by the influenza vaccine at any time tested. We also determined the number of CD56+ cells in whole-blood samples at each of the time points and found that there is no change in NK cell number after influenza vaccination.
Diversity of host species and strains of Pneumocystis carinii is based on rRNA sequences
Authors: Shah J S , Pieciak W , Liu J , Buharin A , Lane D J
Published: January 2020

We have amplified by PCR Pneumocystis carinii cytoplasmic small-subunit rRNA (variously referred to as 16S-like or 18S-like rRNA) genes from DNA extracted from bronchoalveolar lavage and induced sputum specimens from patients positive for P. carinii and from infected ferret lung tissue. The amplification products were cloned into pUC18, and individual clones were sequenced. Comparison of the determined sequences with each other and with published rat and partial human P.carinii small-subunit rRNA gene sequences reveals that, although all P. carinii small-subunit rRNAs are closely related (approximately 96% identity), small-subunit rRNA genes isolated from different host species (human, rat, and ferret) exhibit distinctive patterns of sequence variation. Two types of sequences were isolated from the infected ferret lung tissue, one as a predominant species and the other as a minor species. There was 96% identity between the two types. In situ hybridization of the infected ferret lung tissue with oligonucleotide probes specific for each type revealed that there were two distinct strains of P. carinii present in the ferret lung tissue. Unlike the ferret P. carinii isolates, the small-subunit rRNA gene sequences from different human P. carinii isolates have greater than 99% identity and are distinct from all rat and ferret sequences so far inspected or reported in the literature. Southern blot hybridization analysis of PCR amplification products from several additional bronchoalveolar lavage or induced sputum specimens from P. carinii-infected patients, using a 32P-labeled oligonucleotide probe specific for human P. carinii, also suggests that all of the human P. carinii isolates are identical. These findings indicate that human P. carinii isolates may represent a distinct species of P. carinii distinguishable from rat and ferret P. carinii on the basis of characterization of small-subunit rRNA gene sequences.
Secretion of the mycobacterial 19-kilodalton protein by Escherichia coli, a novel method for the purification of recombinant mycobacterial antigens
Authors: Hewinson R G , Harris D P , Whelan A , Russell W P
Published: January 2020

We have reported previously (R.G. Hewinson and W.P. Russell, J. Gen. Microbiol. 139:1253-1259, 1993) the secretion by Escherichia coli of a recombinant form of the immunogenic protein MPB70 of Mycobacterium bovis when the protein is translated from its native initiation codon. N-terminal sequence analysis of the purified protein revealed that the signal peptide of MPB70 was cleaved by an endopeptidase of E. coli at the same cleavage site as reported for the protein in M. bovis. Since both the B- and T-cell antigenicities of the purified recombinant protein were similar to that of the native protein, the 19-kDa antigen of M. bovis was used as a model to test whether the signal peptide of MPB70 could direct the secretion of heterologous proteins in E. coli and whether antigen produced in this way retained antigenicity superior to that of recombinant protein produced as a fusion to glutathione-S-transferase. A chimeric protein was produced in which the signal peptide of MPB70 was fused to the 19-kDa antigen of M. bovis at amino acid residue 23. This chimeric protein was found to be secreted into the periplasm and culture medium of recombinant E. coli, and the signal peptide was cleaved by an endopeptidase of E. coli during secretion. Secretion of the 19-kDa antigen facilitated purification of the antigen by two-stage preparative electrophoresis which gave yields of 2.5 mg of purified, soluble 19-kDa antigen from 2.5 g (wet weight) of E. coli. Antigen purified in this way retained both B- and T-cell antigenicities. Moreover, the nonspecific mitogenic activity of the purified 19-kDa antigen was low, while the magnitude of the T-cell response induced by the purified antigen was considerably higher than that observed with purified antigen produced as a fusion protein with glutathione-S-transferase.
Predictors of survival in human immunodeficiency virus type 1-seropositive intravenous drug users
Authors: Page J B , Lai S , Fletcher M A , Patarca R , Smith P C , Lai H C , Klimas N G
Published: January 2020

In an ongoing prospective study of street-recruited intravenous drug users (IDUs) in Miami, Fla., 116 human immunodeficiency virus type 1 (HIV-1)-infected IDUs were monitored for up to 7 years. This provided an opportunity to evaluate baseline immunological parameters as potential predictors of survival among HIV-1-infected IDUs. As expected, HIV-1-infected IDUs who had an advanced stage of the disease (Centers for Disease Control and Prevention classification III or IV); p24 antigenemia; human T-cell leukemia virus type 1/2 seropositivity; low CD4 counts (< or = 200); low hemoglobin (< or = 14), high serum immunoglobulin A (IgA) (> 500 mg/dl), or high serum IgG (> or = 3,500 mg/dl) levels; or low proliferative responses to pokeweed mitogen (< or = 1,500 cpm) and to phytohemagglutinin (< or = 80,000 cpm) at baseline had worse survival rates. Results from multivariate Cox's models of survival showed that the baseline serum IgG level, serum IgA level, and CD4 count independently predict survival in HIV-1-infected IDUs. Cross-validation procedures verified the above-mentioned findings. These findings support the routine consideration of serum immunoglobulin levels in addition to CD4 count, especially in early evaluation of disease stage, as these evaluations may modify application of prophylaxis and treatment for HIV-1-infected IDUs. We recommend consideration of use of serum IgG and IgA as immunological markers for long-range prediction of survival in HIV-1-infected IDUs. These determinations are less onerous and more appropriate for use in field studies and financially less favored settings.
Antibody response to Prevotella spp. in patients with ventilator-associated pneumonia
Authors: Grollier G , Doré P , Robert R , Ingrand P , Gréjon C , Fauchere J L
Published: January 2020

Although anaerobic bacteria are frequently isolated from the oropharyngeal flora, their potential pathogenic role in ventilator-associated pneumonia (VAP) has been poorly investigated. In order to evaluate the pathogenic role of Prevotella spp. isolated from protected specimen brushes, we investigated the systemic humoral response with the enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot) in 13 patients who developed a VAP associated with Prevotella species (group I). The antigen used was a mixture of whole-cell proteins taken from four reference Prevotella strains. We compared the antibody levels observed in these patients with those measured in 30 patients who developed a VAP unrelated to anaerobic bacteria (group II), in 27 patients with dental stumps (group III), and in 30 healthy patients (group IV) who had Prevotella species on dental plaque. The ELISA levels obtained in the four groups showed significant differences between group I and each of the three control groups (P < 0.05). The antibody profiles obtained by Western blot showed an intensity of response roughly superposable over levels obtained by ELISA and a species specificity. These findings suggested that colonization of these patients with Prevotella species may have been associated with an infectious process leading to a systemic humoral response and that these bacteria could play a role in VAP.
Evaluation of immunoassays for detection of antibodies to human herpesvirus 7
Authors: Black J B , Schwarz T F , Patton J L , Kite-Powell K , Pellett P E , Wiersbitzky S , Bruns R , Müller C , Jäger G , Stewart J A
Published: January 2020

An enzyme immunoassay (EIA), an immunoblot assay (IB), and an indirect immunofluorescence assay were developed for detection of human herpesvirus 7 (HHV-7) antibodies in human serum. Cross-absorption studies with EIA or IFA using HHV-7 and human herpesvirus 6 (HHV-6) antigens indicated that most human sera contain cross-reactive HHV-6 and HHV-7 antibodies and that the degree of cross-reactivity varies between individual serum specimens. Inhibition of homologous antibody activity by absorption with heterologous virus ranged from 0 to 57% by EIA. However, for every sample tested, absorption with homologous virus removed more activity than did heterologous virus. An 89-kDa protein was identified as an HHV-7-specific serologic marker by IB. Activity to this protein was not removed by absorption with HHV-6 antigen. Of the three assays, the EIA was the most sensitive (94%), while the IB was the most specific (94%). Approximately 80% of specimens collected from German adults and children older than 2 years were positive for HHV-7 antibodies by these assays.
Alterations in lymphocyte phenotype and function in children with shigellosis who develop complications
Authors: Azim T , Sarker M S , Hamadani J , Khanum N , Halder R C , Salam M A , Albert M J
Published: January 2020

This study was designed to see whether alterations occur in peripheral blood mononuclear cell phenotype and function in children with Shigella dysenteriae 1 infection with complications (leukemoid reaction and/or hemolytic-uremic syndrome) and whether there are any alterations prior to the development of complications. The following groups of children (ages, 12 to 60 months) were compared: children without any infection (n = 51), children with uncomplicated shigellosis (n = 65), children admitted with complicated shigellosis (leukemoid reaction and/or hemolytic-uremic syndrome) (n = 29), and children with shigellosis who developed complications after enrollment (subsequently complicated shigellosis) (n = 12). Tests for the peripheral blood mononuclear cell phenotype (CD3, CD4, CD8, CD57 [corrected], CD20, and CD25), spontaneous proliferation, and the proliferative response to phytohemagglutinin, pokeweed mitogen, and the lipopolysaccharide of S. dysenteriae 1 were performed, as were skin tests for delayed-type hypersensitivity (DTH). Children who subsequently developed complications differed from other groups of children as follows: (i) the numbers of CD3+ and CD4+ cells were lower than in uninfected children (P < 0.05), (ii) the CD4/CD8 ratio was lower than in children with uncomplicated shigellosis (P < 0.05) and in uninfected children (P < 0.05), and (iii) the levels of spontaneous proliferation of peripheral blood mononuclear cells were higher and DTH responses were lower than those in children with uncomplicated shigellosis (P < 0.05 and P < 0.017, respectively). Children with complications differed by having (i) increased numbers of CD3- CD57- [corrected] CD20- cells (P < 0.05) compared with those in other groups of children and (ii) lower CD4/CD8 ratios (P < 0.05), higher levels of spontaneous proliferation (P < 0.05), and lower DTH responses (P = 0.005) than children with uncomplicated shigellosis. Three to five days after enrollment, the number of CD4+ cells increased in children who subsequently developed complications (P = 0.025), i.e., when they developed complications and at this time their CD4+ cell number was similar to that of other groups of children. Thus, lymphocyte phenotype and function are altered prior to the development of complications in children with shigellosis, and once complications develop, the pattern of alterations changes. Whether these alterations have a role in precipitating complications or whether they reflect early events underlying the development of complications remains to be elucidated.
Antibody against human immunodeficiency virus type 1 (HIV-1) Tat protein may have influenced the progression of AIDS in HIV-1-infected hemophiliac patients
Authors: Re M C , Furlini G , Vignoli M , Ramazzotti E , Zauli G , La Placa M
Published: January 2020

Retrospective analysis of serum samples from a group of hemophiliac patients who became infected with human immunodeficiency virus type 1 (HIV-1) between 1984 and 1985 has shown that, at variance with other HIV-1-infected patients, at the onset, or at least at a very early phase of HIV-1 infection, they constantly have elevated levels of antibodies against HIV-1-transactivating Tat protein and an absent or barely detectable p24 antigenemia. Anti-Tat antibodies in initial serum samples from hemophiliac patients were probably the consequence of the passive administration of immunoglobulins present in low- or intermediate-purity clotting factor concentrates prepared from HIV-1-infected blood. Furthermore, the analysis of serial serum samples obtained during the course of the disease, in which passively acquired anti-Tat antibodies were substituted by actively produced antibodies, demonstrated an inverse relationship between anti-Tat antibody and p24 anti-genemia levels throughout the observation period. These data seem to suggest that anti-Tat antibody may have some influence on the course of HIV-1 infection.
Immunological characterization of a recombinant 27-kilodalton antigenic protein from Paracoccidioides brasiliensis
Authors: Ortiz B L , Garcia A M , Restrepo A , McEwen J G
Published: January 2020

We report the expression in Escherichia coli of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. When analyzed by immunoblotting, this recombinant antigenic protein was recognized by antibodies present in the sera of 40 of the 44 paracoccidioidomycosis patients studied. No cross-reactions were observed with sera from patients with other mycoses (histoplasmosis, aspergillosis, cryptococcosis, sporotrichosis, and chromoblastomycosis) or with tuberculosis.
Detection of candidal antigens in autoimmune polyglandular syndrome type I
Authors: Peterson P , Perheentupa J , Krohn K J
Published: January 2020

Autoimmune polyglandular syndrome type I (APS I) is associated with chronic mucocutaneous candidiasis. To characterize the antibody responses in this subgroup of Candida albicans infections, we screened a candidal cDNA expression library with patient sera and found four cDNA clones encoding the immunopositive proteins enolase, heat shock protein 90, pyruvate kinase, and alcohol dehydrogenase. The reactivity to these antigens was studied further by immunoprecipitation assays with in vitro-transcribed and -translated proteins. Analysis of sera from 44 APS I patients showed that the highest antibody reactivity was found with enolase (80% of patients reactive), but significant serological responses were also found with heat shock protein 90 (67%), pyruvate kinase (62.5%), and alcohol dehydrogenase (64%). Overall, 95.5% of patients had detectable antibodies to at least one of these proteins. The cDNAs of enolase and heat shock protein 90 were also expressed in Escherichia coli and studied by immunoblotting. Again, 84% of sera reacted with enolase, whereas 44% of sera reacted with heat shock protein 90. A good correlation between the two methods was found for both enolase (r = 0.86; n = 58; P < 0.001) and heat shock protein 90 (r = 0.71; n = 56; P < 0.001). Our results indicate that the four abundant candidal proteins are the major antigens and can be used as accurate markers of candidiasis in APS I patients. The immunoprecipitation assay described here is particularly useful for the rapid analysis of a large number of samples.
Determination of natural killer cell function by flow cytometry
Authors: Kane K L , Ashton F A , Schmitz J L , Folds J D
Published: January 2020

Natural killer cells (NK cells) are a subset of peripheral blood lymphocytes that mediate non-major histocompatibility complex-restricted cytotoxicity of foreign target cells. The "gold standard" assay for NK cell activity has been the chromium release assay. This method is not easily performed in the clinical laboratory because of difficulties with disposal of radioactive and hazardous materials, short reagent half-lives, expense, and difficulties with assay standardization. We describe a flow cytometric assay for the clinical measurement of NK cell activity. This study compared the chromium release assay and the flow cytometric assay by using clinically relevant specimens. There were no significant differences between the two assays in the measurement of lytic activity for 17 peripheral blood specimens or in reproducibility in repeated samplings of healthy individuals. We also established a normal range of values for NK activity in healthy adults and identified a small cluster of individuals who have exceptionally high or low levels of NK activity. The flow cytometric assay was validated by testing specimens from subjects expected to have abnormally low levels of NK activity (pregnant women) and specimens from healthy individuals in whom the activity of NK cells was enhanced by exposure to interleukin-2 or alpha interferon. Treatment with these agents was associated with a significant increase in NK activity. These results confirm and extend those of others, showing that the flow cytometric assay is a viable alternative to the chromium release assay for measuring NK cell activity.
Detection of pulmonary and extrapulmonary tuberculosis patients with the 38-kilodalton antigen from Mycobacterium tuberculosis in a rapid membrane-based assay
Authors: Zhou A T , Ma W L , Zhang P Y , Cole R A
Published: January 2020

A rapid membrane-based serologic assay using the 38-kDa antigen from Mycobacterium tuberculosis for the diagnosis of tuberculosis (TB) was evaluated with 201 patients with pulmonary TB, 67 patients with extrapulmonary TB, 79 Mycobacterium bovis BCG-vaccinated healthy controls, and 77 non-TB respiratory patients. The overall sensitivities, specificities, and positive and negative predictive values were, respectively, 92, 92, 84, and 96% for sputum-positive TB patients; 70, 92, 87, and 79% for sputum-negative TB patients; and 76, 92, 80, and 90% for extrapulmonary-TB patients. Only 2% (1 of 44) of the healthy control BCG-vaccinated subjects gave weak positive signals in the assay, indicating that this rapid serological assay is a valuable aid in clinical diagnosis for both pulmonary and extrapulmonary TB.
Time to peak serum antibody response to influenza vaccine in the elderly
Authors: Gross P A , Russo C , Teplitzky M , Dran S , Cataruozolo P , Munk G
Published: January 2020

The earliest time at which serum antibody peaks following administration of influenza virus vaccine in elderly persons is not clearly defined. We compared the time intervals of 2 and 4 weeks after vaccination. A commercial trivalent vaccine containing the hemagglutinins of influenza viruses A/Texas/36/91(H1N1), A/Shangdong/9/93(H3N2), and B/Panama/45/90 was used. The hemagglutination inhibition antibody titers at 2 weeks after vaccination were identical to the hemagglutination inhibition antibody titers at 4 weeks for all three vaccine components.
Alcohol inhibits lipopolysaccharide-induced tumor necrosis factor alpha gene expression by peripheral blood mononuclear cells as measured by reverse transcriptase PCR in situ hybridization
Authors: Nair M P , Kumar N M , Kronfol Z A , Greden J F , Lwebuga-Mukasa J S , Schwartz S A
Published: January 2020

We recently showed that alcohol significantly suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) production by whole blood and total mononuclear cells from healthy subjects as measured by bioassay. In the current study, we further examined the effect of alcohol on LPS-induced TNF-alpha gene expression by semiquantitative solution PCR and in situ reverse transcriptase PCR (RT-PCR) hybridization methods. Peripheral blood mononuclear cells were cultured with LPS (10 micrograms/ml) for 4 to 8 h with or without different concentrations of ethanol (0.1, 0.2, and 0.3% [vol/vol]). Total RNA from treated and untreated cultures was extracted and used for solution PCR analysis. Treated and untreated cells were subjected to both conventional in situ hybridization and RT-PCR in situ hybridization. In solution RT-PCR in vitro analysis, alcohol significantly suppressed TNF-specific message. In conventional in situ hybridization, the effect of alcohol on TNF-alpha gene expression was poorly detected. However, when cells were subjected to RT-PCR prior to in situ hybridization, cells treated with alcohol significantly suppressed expression of the message for TNF-alpha. These studies confirm our earlier finding that alcohol suppressed the production of TNF-alpha by LPS-induced whole blood cells and peripheral blood mononuclear cells. Furthermore, these studies also demonstrate that the RT-PCR in situ technique is a powerful tool for detecting and amplifying specific genes in whole cells when limited numbers of cells are available for RNA extraction.
Immunoblot analysis of anti-Ureaplasma urealyticum antibody in pregnant women and newborn infants
Authors: Cunningham C K , Bonville C A , Hagen J H , Belkowitz J L , Kawatu R M , Higgins A M , Weiner L B
Published: January 2020

The purpose of the present study was to determine the frequency in which antibody reactive to Ureaplasma urealyticum could be detected in a population of pregnant women and newborn infants. Serum samples from a prospective cohort of 80 healthy, U. urealyticum culture-positive and culture-negative pregnant women and a retrospective cohort of 522 infants born at between 25 and 42 weeks of gestation were studied by immunoblot analysis. Cultures of specimens from the lower genital tract were positive for U. urealyticum for 83% of the pregnant women, and serum immunoglobulin G (IgG) antibody which reacted to U. urealyticum was detectable in 93% of the pregnant women. Samples from five women (8%) had increases in the number of anti-U. urealyticum IgG bands over the course of the pregnancy. Samples from four of these five women had corresponding increases in the number of antibody bands present in IgA immunoblots. Six of the 522 samples from newborns or cord blood (1.1%) were positive for anti-U. urealyticum IgA; 5 of these 6 samples were also positive for IgM. The six anti-U. urealyticum IgA-positive infants were distributed as follows; 3 of 67 (4.5%) infants were delivered at 25 to 30 weeks of gestation, 3 of 176 (1.7%) infants were delivered at 31 to 34 weeks of gestation, and 0 of 279 infants were delivered at > or = 35 weeks of gestation. An antibody response to U. urealyticum can be detected in pregnant women and preterm infants and may serve as a marker of infection.
Unusually high seroprevalence of Borna disease virus in clade E human immunodeficiency virus type 1-infected patients with sexually transmitted diseases in Thailand
Authors: Auwanit W , Ayuthaya P I , Nakaya T , Fujiwara S , Kurata T , Yamanishi K , Ikuta K
Published: January 2020

The seroprevalence of Borna disease virus (BDV) in human immunodeficiency virus type 1-infected individuals in Thailand was examined by using recombinant BDV p24. A high (38 to 48%) rate of seroprevalence of BDV was observed in clade E-infected patients with sexually transmitted diseases, compared with those in clade E-infected prostitutes (8.3%), pregnant women (0%), clade B-infected intravenous-drug users (0%), and human immunodeficiency virus type 1-negative blood donors (1.9%).
A rapid and gentle method for isolation of genomic DNA from pathogenic Nocardia spp
Authors: Torres R D , Oletta C A , Zlotnik H
Published: January 2020

The lack of simple and efficient methods for extraction of DNA from Nocardia spp. has hampered molecular manipulation of the DNA for diagnostic purposes. In the present study, a method for the rapid extraction of undegraded genomic nocardial DNA was established. Briefly, 14 pathogenic Nocardia strains were grown at 37 degrees C for 3 to 5 days in Sauton broth containing 0.05% Tween 80. Subsequently, the cultures were treated for 48 h with 1.2 mg of cycloserine per ml (final concentration). Cells were then harvested by centrifugation and treated with a lysis solution containing 3 mg of lysozyme per ml. This was followed by the addition of proteinase K and sodium dodecyl sulfate to final concentrations of 0.2 mg/ml and 0.5%, respectively, and incubation for 1 h at 50 degrees C. DNA was precipitated with isopropanol after phenol-chloroform-isoamyl alcohol extractions and RNase treated before being quantitated and analyzed by agarose gel electrophoresis. The average undegraded DNA yields obtained were 101 micrograms for Nocardia brasiliensis and 121 micrograms for N. asteroides. This DNA was suitable for restriction endonuclease digestion and PCR amplification, which are methods being applied to the characterization and diagnosis of slowly growing organisms such as Nocardia spp.
False-positive serologic test resulting from a probable yeast infection in a chimpanzee
Authors: Heller T , Yin S , Tsarev S A , Purcell R H , Emerson S U
Published: January 2020

Sera used to identify putative hepatitis E viral proteins expressed in Pischia pastoris produced a false-positive reaction because of antibodies to a yeast protein. This report illustrates a potential problem when serological reagents are used in combination with recombinant proteins expressed in yeast.