bluuebunny/crossref_metadata_2025 huggingface dataset:
Method for Simultaneous Measurement of Antibodies to 23 Pneumococcal Capsular Polysaccharides
Authors: Biagini Raymond E. , Schlottmann Sonela A. , Sammons Deborah L. , Smith Jerome P. , Snawder John C. , Striley Cynthia A. F. , MacKenzie Barbara A. , Weissman David N.
Published: September 2003

We describe a fluorescent covalent microsphere immunoassay (FCMIA) method for the simultaneous (multiplexed) measurement of immunoglobulin G (IgG) antibodies to 23 pneumococcal capsular polysaccharide (PnPS) serotypes present in the pneumococcal polysaccharide vaccine (PPV23) licensed by the Food and Drug Administration, i.e., PnPSs 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. In addition, the assay incorporates an internal control that allows for contemporaneous evaluation of the effectiveness of pneumococcal cell wall polysaccharide (C-PS) preadsorption and a second control of PnPS 25 (which is not present in any polysaccharide or conjugate vaccine), which can be used to evaluate interassay reproducibility (useful for pre- versus postvaccination studies). The FCMIA was standardized with U.S. reference antipneumococcal serotype standard serum 89S-2. Preadsorption of 89S-2 with each PnPS and C-PS yielded homologous inhibition for serotypes 1, 6B, 9N, 9V, 11A, 12F,14, 15B, 18C, 19A, 19F, 20, 22F, 25, and 33F; heterologous inhibition for serotypes 9V, 10A, 11A, 12F, 15B, 17F, 20, and 23F; and neither homologous nor heterologous inhibition for serotypes 2, 3, 4, and 5. The minimum detectable concentrations for the 24 multiplexed (PnPS and C-PS) FCMIAs ranged from 20 pg/ml for PnPS 3 to 600 pg/ml for PnPS 14. The PnPS FCMIA method has numerous benefits over enzyme-linked immunosorbent assays commonly used to measure anti-PnPS-specific IgG levels, including increased speed, smaller sample volumes, equivalent or better sensitivity, and increased dynamic range.
Antigen-Presenting Cell Modulation Induces a Memory Response to p24 in Peripheral Blood Leukocytes from Human Immunodeficiency Virus-Infected Individuals
Authors: Kolber Michael A. , Saenz Maria O.
Published: September 2003

The accurate determination of human immunodeficiency virus type 1 (HIV-1)-specific proliferative responses is critically important when evaluating immune recovery after highly active antiretroviral therapy. Using a new assay to enhance proliferative responses to recall and HIV antigen, we addressed the questions of whether viral load affects cellular immunity and whether long-term viral load suppression results in loss of antigen-specific responder cells. This assay is based on the fact that lipopolysaccharide (LPS) can augment proliferative responses to antigen after monocyte adherence to a tissue culture plate. Twenty-six HIV-1-infected individuals donated peripheral blood leukocytes (PBL). Proliferation assays against p24, using LPS and cell adherence, were performed on all samples. Medical record abstraction provided information on CD4 cell nadir and time of viral load suppression. PBL from HIV-1-infected individuals with a viral load of <200 copies/ml had a significant proliferative response and a stimulation index of >5 to p24 (12 of 15) compared to those with a viral burden (2 of 11), using the LPS-adherence assay. Proliferative responses to p24 could be found in PBL from virally suppressed donors independent of the CD4 cell nadirs and in the majority of the donors who were virally suppressed for >10 months (7 of 10). The data presented here demonstrate that LPS and monocyte adherence provide a sensitive and specific way to boost proliferative responses to recall and HIV antigens.
Detection of
Trypanosoma cruzi
and
Trypanosoma rangeli
Infection by Duplex PCR Assay Based on Telomeric Sequences
Authors: Chiurillo Miguel Angel , Crisante Gladys , Rojas Agustina , Peralta Andreina , Dias Manuel , Guevara Palmira , Añez Néstor , Ramírez José Luis
Published: September 2003

We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of
Trypanosoma cruzi
and
Trypanosoma rangeli
in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of
T. cruzi
and 1/4th of
T. rangeli
cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all
T. cruzi
and
T. rangeli
strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.
Validation of Serological Correlate of Protection for Meningococcal C Conjugate Vaccine by Using Efficacy Estimates from Postlicensure Surveillance in England
Authors: Andrews Nick , Borrow Ray , Miller Elizabeth
Published: September 2003

Meningococcal C conjugate (MCC) vaccines were licensed on the basis of serological correlates of protection without efficacy data. The original correlate of protection was established by using a serum bactericidal antibody assay (SBA) with human complement (hSBA), with titers ≥4 predicting protection. However, the antibody data supporting licensure were largely generated by SBA with rabbit complement (rSBA), which gives higher titers than hSBA. While rSBA titers ≥128 reliably predict protection, as measured by hSBA, sera with rSBA titers in the range of 8 to 64 may not have hSBA titers ≥4. For rSBA titers in this equivocal range, a fourfold rise pre- to postvaccination with the MCC vaccine and/or a characteristic booster response to a polysaccharide challenge was proposed as a correlate of protection. To validate this proposed rSBA correlate, age-specific efficacy estimates for MCC vaccines obtained from postlicensure surveillance in England were compared with the efficacy predicted by the percentage of individuals in these age groups with rSBA titers above different cutoffs at 4 weeks and at 7 to 9 months after vaccination with the MCC vaccine. The average time since vaccination in the cohorts in whom efficacy was measured ranged from 8 to 10 months. The rSBA cutoff of ≥128 was shown to significantly underestimate efficacy, with rSBA cutoffs of ≥4 or ≥8 at 4 weeks postvaccination with the MCC vaccine being the most consistent with observed efficacy. When the levels obtained 7 to 9 months postvaccination with the MCC vaccine were used, all rSBA cutoffs significantly underestimated efficacy, suggesting that continuing protection is less dependent on the SBA level at the time of exposure but is more reliant on immunologic memory.
Effect of Probiotic Bacteria on Induction and Maintenance of Oral Tolerance to β-Lactoglobulin in Gnotobiotic Mice
Authors: Prioult Guénolée , Fliss Ismail , Pecquet Sophie
Published: September 2003

In this study, the effect ofLactobacillus paracasei(NCC 2461),Lactobacillus johnsonii(NCC 533) andBifidobacterium lactisBb12 (NCC 362) on the induction and maintenance of oral tolerance to bovine β-lactoglobulin (BLG) was investigated in mice. Germfree mice were monocolonized with one of the three strains before oral administration of whey protein to induce tolerance. Mice were then injected with BLG and sacrificed 28 or 50 days after whey protein feeding for humoral and cellular response measurement. Conventional and germfree mice were used as controls. Both humoral and cellular responses were better suppressed in conventional mice than in germfree and monoassociated mice throughout the experiment and better suppressed inL. paracasei-associated mice than in mice colonized withB. lactisorL. johnsonii.The latter two mono-associations suppressed humoral responses only partially and cellular responses not at all. This study provides evidence that probiotics modulate the oral tolerance response to BLG in mice. The mono-colonization effect is strain-dependant, the best result having been obtained withL. paracasei.
Antibody Responses to
Escherichia coli
O157 and Other Lipopolysaccharides in Healthy Children and Adults
Authors: Navarro Armando , Eslava Carlos , Hernandez Ulises , Navarro-Henze Jose Luis , Aviles Magali , Garcia-de la Torre Guadalupe , Cravioto Alejandro
Published: September 2003

In Mexico, diarrheal disease due to different serotypes of
Escherichia coli
is highly prevalent, with only sporadic isolation of O157 non-H7 strains. This could be due to exposure to the O157 or related
E. coli
lipopolysaccharide (LPS), such as O7 or O116, at an early age. By using enzyme-linked immunosorbent assay (ELISA) and Western blotting, the present study analyzed 605 serum samples from Mexican adults and infants without clinical symptoms of disease for the presence of antibodies to these three
E. coli
LPSs. The bactericidal activities of homologous and heterologous rabbit and human serum samples against O7, O116, and O157
E. coli
LPSs were also determined. By using a cutoff point of 0.7, it was found by the ELISAs that 28 of 562 (5%) of the serum samples from adolescents and adults and 2 of 43 (5%) of the serum samples from infants less than 1 year of age reacted with the O157 LPS. By using cutoff points between 0.4 and 0.699, the proportion of serum samples from both age groups that reacted with the O157 LPS increased to 20%. Western blotting analysis of selected serum samples that showed an intermediate response against the O157 LPS by the ELISAs showed that 61 of 88 (69%) reacted with the same LPS. A similar result was observed for maternal milk samples. The bactericidal activities of rabbit serum samples against the O7, O116, and O157 LPSs showed that they were positive for both homologous and heterologous antigens. Similar results were observed with the human serum samples. O157 non-H7 strains were identified in only 10% of the
E
.
coli
strains isolated from 263 Mexican children with and without diarrhea over the past 15 years. This absence of O157:H7 strains in Mexico may be associated with the presence of antibodies against O157 or related
E. coli
LPSs.
Effect of Bacterial Flora on Postimmunization Gastritis following Oral Vaccination of Mice withHelicobacter pyloriHeat Shock Protein 60
Authors: Yamaguchi Hiroyuki , Osaki Takako , Taguchi Haruhiko , Sato Noriko , Toyoda Atushi , Takahashi Motomichi , Kai Masanori , Nakata Noboru , Komatsu Akio , Atomi Yutaka , Kamiya Shigeru
Published: September 2003

In order to assess the efficacy of oralHelicobacter pyloriheat shock protein 60 (HSP60) as a vaccine, protection againstH. pyloriinfection in specific-pathogen-free (SPF) C57BL/6 and germfree (GF) IQI mice was examined. Prophylactic oral vaccination of these two strains of mice with eitherH. pyloriHSP60 orEscherichia coliGroEL inhibitedH. pyloricolonization by 90 to 95% at 3 weeks postinfection (p.i.). However, these mice were only partially protected because bacterial loads increased in all animals at 10 weeks p.i. Anti-H. pyloriHSP60 immunoglobulin G was detected in serum at 3 weeks p.i. in mice vaccinated with eitherH. pyloriHSP60 or GroEL. Significant increases in the gastritis scores were observed only in SPF mice immunized withH. pyloriHSP60. These results indicate that oral vaccination withH. pyloriHSP60 has partial protective effects on subsequentH. pyloriinfection but also induces postimmunization gastritis. However, GF mice immunized withH. pyloriHSP60 did not suffer from severe gastritis. Therefore, the presence of bacterial flora appears to contribute to the induction of postimmunization gastritis.
Immunity to Human Immunodeficiency Virus (HIV) in Children with Chronic HIV Infection Receiving Highly Active Antiretroviral Therapy
Authors: Weinberg Adriana , Pott Gregory B.
Published: September 2003

Our objective was to describe the CD4-mediated human immunodeficiency virus (HIV)-specific cell-mediated immunity (CMI) and its virologic and immunologic correlates in children with chronic HIV infection on highly active antiretroviral therapy (HAART). Twelve HIV-infected children on stable antiretroviral therapy with a median level of CD4
+
lymphocytes (CD4%) of 25.5% and a median viral load (VL) of 786 HIV RNA copies/ml were enrolled in this study. Nine of these children were also cytomegalovirus (CMV) seropositive. Blood mononuclear cells, stimulated with HIV and CMV antigens, were used to measure lymphocyte proliferation and to enumerate gamma interferon (IFN-γ)-producing CD4
+
cells. HIV CMI and CMV CMI were detected in similar proportions of patients and correlated with each other, although the HIV responses were less robust. HIV lymphocyte proliferation significantly increased with lower HIV VL and showed a trend to increase with higher CD4% and longer time on HAART. The in vitro IFN-γ response to HIV or CMV was not affected by CD4%, VL, or HAART. Pediatric patients with established HIV infection on HAART frequently exhibit HIV CMI despite undetectable HIV replication. We concluded that the association between HIV CMI and CMV CMI indicates that the same factors govern responsiveness to either antigen.
Enzyme-Linked Immunoassay Using Recombinant
trans
-Sialidase of
Trypanosoma cruzi
Can Be Employed for Monitoring of Patients with Chagas' Disease after Drug Treatment
Authors: Pereira-Chioccola Vera Lucia , Fragata-Filho Abilio Augusto , Levy Antonio Marcos de Apparecida , Rodrigues Mauricio M. , Schenkman Sergio
Published: September 2003

trans
-Sialidase is an enzyme present on the surface of
Trypanosoma cruzi
and is an important antigen recognized by sera from patients with Chagas' disease. In the present study we investigated whether the benznidazole treatment of patients with Chagas' disease induced changes in the reactivity of serum toward a recombinant form of
trans-
sialidase in order to develop an assay for monitoring of patients after treatment for Chagas' disease, which is needed at Chagas' disease control centers. By using an enzyme-linked immunosorbent assay containing a recombinant protein corresponding to the catalytic domain of
trans-
sialidase, we found that the antigen had a high specificity for sera from untreated patients with Chagas' disease. Sera from healthy individuals or patients with active visceral leishmaniasis minimally cross-reacted with the antigen. Anti-
trans
-sialidase immunoglobulin was detected in 98% of 151 untreated patients with Chagas' disease. Of these, 124 patients were treated for 60 days with benznidazole (5 mg/kg of body weight/day), and their sera were assayed for reactivity with the recombinant
trans-
sialidase. By using this methodology, three groups of patients could be established. The first group (60 patients), which was considered to have been successfully treated, showed no reactivity after treatment. The second group (46 patients) still showed signs of infection, and after treatment their sera recognized
trans-
sialidase, but with reduced titers. The third group (18 patients) was considered to be resistant to drug treatment, and their sera presented identical reactivities before and after treatment. These results suggest that determination of the absence of antibodies to recombinant
trans-
sialidase in treated patients by the present assay is indicative of treatment success, while the presence of antibodies may indicate the persistence of infection. Therefore, this method may be useful for the diagnosis and monitoring of patients undergoing benznidazole treatment.
Competitive Binding Inhibition Enzyme-Linked Immunosorbent Assay That Uses the Secreted Aspartyl Proteinase ofCandida albicansas an Antigenic Marker for Diagnosis of Disseminated Candidiasis
Authors: Morrison Christine J. , Hurst Steven F. , Reiss Errol
Published: September 2003

The secreted aspartyl proteinases (Saps) ofCandida albicanshave been implicated as virulence factors associated with adherence and tissue invasion. The potential use of proteinases as markers of invasive candidiasis led us to develop a competitive binding inhibition enzyme-linked immunosorbent assay (ELISA) to detect Sap in clinical specimens. Daily serum and urine specimens were collected from rabbits that had been immunosuppressed with cyclophosphamide and cortisone acetate and infected intravenously with 107C. albicansblastoconidia. Disseminated infection was confirmed by organ culture and histopathology. Although ELISA inhibition was observed when serum specimens from these rabbits were used, more significant inhibition, which correlated with disease progression, occurred when urine specimens were used. Urine collected as early as 1 day after infection resulted in significant ELISA inhibition (mean inhibition ± standard error [SE] compared with preinfection control urine, 15.7% ± 2.7% [P< 0.01]), and inhibition increased on days 2 through 5 (29.4% ± 4.8% to 44.5% ± 3.5% [P< 0.001]). Urine specimens from immunosuppressed rabbits infected intravenously withCandida tropicalis,Candida parapsilosis,Candida krusei,Cryptococcus neoformans,Aspergillus fumigatus, orStaphylococcus aureuswere negative in the assay despite culture-proven dissemination. Nonimmunosuppressed rabbits receiving oral tetracycline and gentamicin treatment were given 2 × 108C. albicansblastoconidia orally or intraurethrally to establish colonization of the gastrointestinal tract or bladder, respectively, without systemic dissemination; urine specimens from these rabbits also gave negative ELISA results. Dissemination to the kidney and spleen occurred in one rabbit challenged by intragastric inoculation, and urine from this rabbit demonstrated significant inhibition in the ELISA (mean inhibition ± SE by day 3 after infection, 32.9% ± 2.7% [P< 0.001]). The overall test sensitivity was 83%, the specificity was 92%, the positive predictive value was 84%, the negative predictive value was 91%, and the efficiency was 89% (166 urine samples from 33 rabbits tested). The specificity, positive predictive value, and efficiency could be increased to 97, 95, and 92%, respectively, if at least two positive test results were required for a true positive designation. The ELISA was sensitive and specific for the detection of Sap in urine specimens from rabbits with disseminatedC. albicansinfection, discriminated between colonization and invasive disease, reflected disease progression and severity, and has the potential to be a noninvasive means to diagnose disseminated candidiasis.
Detection of Anti-LeishmaniaImmunoglobulin G Antibodies in Urine Specimens of Dogs with Leishmaniasis
Authors: Solano-Gallego L. , Rodríguez A. , Iniesta L. , Arboix M. , Portús M. , Alberola J.
Published: September 2003

For years, anti-Leishmaniaimmunoglobulin G (IgG) antibodies have been detected in the sera of dogs living in areas of leishmaniasis endemicity. They have also been found in the aqueous humor and cerebrospinal fluid. In contrast, a review of the literature failed to identify the detection of anti-Leishmaniaantibodies in urine samples from dogs with leishmaniasis. Ninety-five dog urine samples were examined for the presence of anti-Leishmaniaantibodies by using a protein A enzyme-linked immunosorbent assay (ELISA). Twenty additional urine samples were collected from healthy dogs as controls. An IgG2 ELISA was performed on 26 urine samples found positive by the protein A ELISA. Twenty-three urine samples found positive to anti-Leishmaniaantibodies were tested for the local production of anti-Leishmaniaantibodies in the urinary tract by means of the urine antibody coefficient. Ten urine samples (and the corresponding serum samples) were compared by Western blot (WB) analysis. Thirty-five out of the 95 urine samples were found positive, 57 were found negative, and 3 were found inconclusive for antibody detection by the protein A ELISA. A high correlation between protein A and IgG2 levels was found in positive urine samples. Anti-Leishmaniaantibodies were present in the urine of dogs that had leishmaniasis, urinary protein/creatinine (U P/C) ratios of greater than one, and normal urinary sediment. A statistically significant correlation was observed between the U P/C ratios and the levels of anti-Leishmaniaantibodies in positive urine samples. In general, WB analysis and the urine antibody coefficient suggested that the presence of anti-Leishmaniaantibodies in urine was the consequence of an impairment of filtration of the glomerular barrier. However, in some dogs, WB analysis could be interpreted as suggesting that the presence of anti-Leishmaniaantibodies was caused, to a lesser extent, by local antibody production in the urinary tract. Antibody detection in urine could be a noninvasive method for leishmaniasis diagnosis and prognosis in dogs with glomerulonephropathies.
Distinct Elevation of Levels of Anti-
Caenorhabditis elegans
Antibody in Sera of Patients with Inflammatory Bowel Disease
Authors: Oshitani Nobuhide , Hato Fumihiko , Kitagawa Seiichi , Watanabe Kenji , Fujiwara Yasuhiro , Higuchi Kazuhide , Matsumoto Takayuki , Arakawa Tetsuo
Published: September 2003

Dysregulation of immune responses to intestinal exogenous antigens contributes to the pathogenesis of inflammatory bowel disease, but the specific antigen responsible for the pathogenesis of inflammatory bowel disease is unknown. We measured serum antibody titers against
Caenorhabditis elegans
antigens. Immunoglobulin G (IgG) and IgG subclass anti-
C. elegans
antibodies in serum samples from 29 patients with ulcerative colitis, 30 patients with Crohn's disease, 7 patients with intestinal Behçet's disease, and 11 healthy controls were measured by enzyme-linked immunosorbent assay. Serum IgG and IgG2 antibody titers against
C. elegans
were significantly higher in patients with inflammatory bowel disease than in controls. Antibody levels were not affected by age, gender, disease activity, extent of disease, or small bowel involvement. The anti-
C. elegans
antibody titer was significantly lower in patients with Crohn's disease taking mesalazine or sulfasalazine than in patients not taking these drugs. The increased immune responses to
C. elegans
found in patients with inflammatory bowel disease reflect dysregulated immune responses to enteric antigens, which might play a role in the pathogenesis of inflammatory bowel disease.
Detection of Serum Antibodies to Ovine Progressive Pneumonia Virus in Sheep by Using a Caprine Arthritis-Encephalitis Virus Competitive-Inhibition Enzyme-Linked Immunosorbent Assay
Authors: Herrmann Lynn M. , Cheevers William P. , Marshall Katherine L. , McGuire Travis C. , Hutton Melinda M. , Lewis Gregory S. , Knowles Donald P.
Published: September 2003

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol.10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [35S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity.
Immune Responses to Differentiated Forms ofHelicobacter pyloriin Children with Epigastric Pain
Authors: Ng Bee Ling , Quak Seng Hock , Aw Marion , Goh Kee Tai , Ho Bow
Published: September 2003

Helicobacter pyloriinfection affects human populations of all ages. This gastric bacterium exists in spiral form and the reported viable but nonculturable coccoid form. The present study aims to examine the probable role of the coccoid form inH. pyloriinfection by comparing the seroprevalences of the spiral and the coccoid forms in children with epigastric pain. Four hundred eighty-nine children (mean age, 8.5 years) with epigastric pain formed the basis of this study. Five hundred ninety-nine schoolchildren of comparable ages and with no record of dyspepsia served as controls. The seroprevalence of antigens prepared from both morphological forms was examined by enzyme-linked immunosorbent assay. The results showed that 65 (13.3%) and 273 (55.8%) of 489 symptomatic children were seropositive for antigens of theH. pylorispiral and coccoid forms, respectively. In contrast, only 7.0% of the control group had elevated levels of immunoglobulin G antibodies against the spiral form, while 26.5% were positive for antibodies against the coccoid form. There were no significant differences between genders or among ethnic groups. The study showed a rise in seroprevalence corresponding with age: 7.1% for those ≤5 years to 21.4% for those ≥11 years. The seroprevalence of antigens of theH. pylorispiral and coccoid forms in children with epigastric pain was twofold higher than that in the control subjects. Interestingly, there was a fourfold increase in seropositivity for coccoid-form antigen compared to that for the spiral-form antigen among the symptomatic pediatric patients as well as the control group, indicating a possible infective role of the coccoid form ofH. pyloriin the pediatric patients with epigastric pain.
Lymphocyte Modulation in a Baboon Model of Immunosenescence
Authors: Jayashankar Lakshmi , Brasky Kathleen M. , Ward John A. , Attanasio Roberta
Published: September 2003

The age-related modulation of lymphocyte number and function was assessed in a nonhuman primate model consisting of healthy olive baboons (Papio cynocephalus anubis) of ages encompassing the entire life span of this species. The objectives of this study were to characterize an animal model of immunosenescence and to assess whether or not age should be considered when designing studies for the evaluation of vaccine candidates in baboons. Specifically the following parameters were assessed in baboons from 6 months to 26 years of age: relative numbers of B lymphocytes, CD4+and CD8+T lymphocytes, and T lymphocytes expressing CD28, CD25, and phytohemagglutinin-stimulated lymphoproliferative activity; and concentrations of total immunoglobulin, soluble interleukin-2 receptor α, and soluble CD30 in serum. There was a statistically significant effect of age on lymphocyte numbers. As age increased, relative B-cell numbers (ranging from 6 to 50%) decreased (P< 0.001) and relative T-cell numbers (ranging from 28 to 80%) increased (P< 0.001). The increase in T-cell numbers involved both the CD4+and CD8+subsets. In addition, there was a significant negative correlation of age with levels of soluble interleukin-2 receptor α in serum. Modulation of lymphocyte numbers appears to occur gradually during the entire baboon life span, thus suggesting the presence of an age-related developmentally regulated process. These findings indicate that baboons represent a potentially useful model to study selected phenomena related to immunosenescence. These findings also indicate that, when using the baboon model for vaccine or other experimental protocols requiring the assessment of immune responses, it would be appropriate to take into account the age of the animals in the study design.
Serum Glucan Levels Are Not Specific for Presence of Fungal Infections in Intensive Care Unit Patients
Authors: Digby Justin , Kalbfleisch John , Glenn Andy , Larsen Angie , Browder William , Williams David
Published: September 2003

Fungal infections in the critically ill patient are difficult to diagnose and are associated with a high mortality rate. A major obstacle to managing fungal infection is the lack of a reliable clinical assay that will rapidly identify patients with fungal sepsis. Glucans are polymers of glucose that are found in the cell wall of fungi and certain bacteria. Glucans are also released from the fungal cell wall into the extracellular milieu. Several studies have reported that detection of fungal glucan in serum or plasma is useful in the diagnosis of mycoses. However, recent studies have questioned the clinical utility of this assay. In this study, we examined serum glucan levels in intensive care unit (ICU) patients and attempt to correlate serum glucan levels with the presence of fungal infection. Following attainment of informed consent, serum was harvested from 46 ICU patients with confirmed fungal infections, confirmed bacterial infections, or no evidence of infection. Sera from eight healthy volunteers served as control. Serum glucan was assayed with a glucan-specificLimulusassay. Serum glucan levels were increased (69.6 ± 17 pg/ml;P< 0.001) in ICU patients versus the normal (11.5 ± 1.3 pg/ml) and noninfected ICU (27.4 ± 17 pg/ml) controls. However, serum glucan levels were not different in patients with confirmed fungal infections versus those with confirmed bacterial infections. Thus, serum glucan levels did not show a correlation with the presence of fungal infections and do not appear to be specific for fungal infections. However, the assay may be useful as a negative predictor of infection.
Group A Streptococcal Antibodies in Subjects with or without Rheumatic Fever in Areas with High or Low Incidences of Rheumatic Fever
Authors: Ayoub Elia M. , Nelson Beverly , Shulman Stanford T. , Barrett Douglas J. , Campbell J. Douglas , Armstrong George , Lovejoy John , Angoff Gerald H. , Rockenmacher Sol
Published: September 2003

The levels of streptococcal antibody titers in populations with or without rheumatic fever from an area with a relatively high incidence of rheumatic fever and an area with a low incidence of this disease were compared. Streptococcal antibody titers were determined for two populations, each of which included children without rheumatic fever (nonrheumatic children) and rheumatic fever patients. The two populations were derived from two separate geographic areas, one with a high incidence of rheumatic fever (Grenada) and another with a low incidence of this disease (central Florida). The results revealed an absence of consistent differences in the geometric mean antibody titers between the nonrheumatic subjects and the rheumatic fever patients from Grenada. In the population from Grenada, the mean anti-streptolysin O and anti-DNase B titers were higher in the nonrheumatic controls (Pof 0.085 and 0.029, respectively). However, the mean titer of the antibody to the group A streptococcal cell wall carbohydrate was higher in the rheumatic fever patients than in the nonrheumatic controls (P= 0.047). This finding contrasted with the finding that the means of all three streptococcal antibody titers in the patients with rheumatic fever were significantly higher than those in the nonrheumatic subjects from Florida (P= 0.01-<0.001). The reason for this paradoxical finding became evident when the streptococcal antibody titers of the nonrheumatic subjects from Grenada and Florida were compared, revealing significantly higher levels of all three antibodies in the nonrheumatic subjects from Grenada than in the nonrheumatic subjects from Florida (P< 0.001). These results suggest that nonrheumatic individuals in an area with a high incidence of rheumatic fever have inordinately elevated levels of streptococcal antibodies in serum. The presence of elevated streptococcal antibody titers in such a population, which probably reflects a high background prevalence of streptococcal infections, should be taken into consideration when evaluating the role of the group A streptococcus in nonpurulent complications of infections.
Clinical Manifestations, Epidemiology, and Laboratory Diagnosis of Human Monocytotropic Ehrlichiosis in a Commercial Laboratory Setting
Authors: Olano Juan P. , Hogrefe Wayne , Seaton Brent , Walker David H.
Published: September 2003

Clinical, epidemiological, and laboratory diagnostic issues of human monocytotropic ehrlichiosis (HME) were investigated in a retrospective case study conducted at a national reference laboratory (Focus Technologies, formerly MRL Reference Laboratory), and at the University of Texas Medical Branch at Galveston, Texas, during 1997 and 1998. Standard questionnaires were sent to physicians for each laboratory-diagnosed patient 2 days to 2 weeks after immunofluorescent antibody assay results were available. Among the 41 cases for which data were obtained, 32 (78%) were definite cases of HME, and 9 (22%) were probable cases of HME. Tick bite or exposure to ticks was recorded in more than 97% of cases. The most prominent clinical findings were fever, abdominal tenderness, and regional lymphadenopathy. There was an association between age and severity of illness. The main laboratory findings included leukopenia, thrombocytopenia, and elevated aspartate aminotransferase and alanine aminotransferase. Clinical and laboratory findings were nonspecific and were not good predictors of the severity of illness. The 90% of patients who received doxycycline treatment underwent rapid clinical improvement with a favorable outcome. The usual duration of effective treatment with doxycycline was 7 to 10 days. This retrospective study is unique because it was based in a commercial reference laboratory setting that receives specimens from different geographic locations. The clinical and laboratory information from 41 patients provides insight into the epidemiological, clinical, and laboratory characteristics of HME.
Detection by Two Enzyme-Linked Immunosorbent Assays of Antibodies toEhrlichia ruminantiumin Field Sera Collected from Sheep and Cattle in Ghana
Authors: Bell-Sakyi Lesley , Koney Enoch B. M. , Dogbey Otilia , Sumption Keith J. , Walker Alan R. , Bath Alasdair , Jongejan Frans
Published: September 2003

Two serological tests for detection of antibodies toEhrlichia(previouslyCowdria)ruminantium, the causative agent of heartwater, were compared by using field sera collected from sheep and cattle as part of serosurveys in Ghana. Sera selected as either negative or positive by a new polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) were tested by the indirect MAP1-B ELISA. Cutoff values of 14 percent positivity (14 PP) for both ruminant species were obtained for the MAP1-B ELISA by using preseroconversion Ghanaian sera and were compared with previously recommended cutoff values of 29 PP for sheep and 38 PP for cattle. With the 14-PP cutoff, of 151 sheep sera which tested negative by PC-ELISA, 89% were also negative by MAP1-B ELISA, while of 419 sheep sera positive by PC-ELISA, 98% were also positive by MAP1-B ELISA. Of 261 bovine sera negative by PC-ELISA, 82% were also negative by MAP1-B ELISA. Of 511 bovine sera positive by PC-ELISA, only 47% were positive by MAP1-B ELISA; these included 168 sera collected from cattle following first seroconversion as detected by both tests, with 125 of these sera positive by PC-ELISA but only 59 and 5 positive by MAP1-B ELISA with the 14- and 38-PP cutoff levels, respectively. These results indicate that both assays are highly sensitive and specific for detection ofE. ruminantiumexposure in sheep but that the MAP1-B ELISA lacks sensitivity for postseroconversion bovine sera in comparison to the PC-ELISA. Both tests confirmE. ruminantiumseroprevalence of at least 70% in Ghanaian sheep; levels of exposure amongAmblyomma variegatum-infested Ghanaian cattle are likely to be higher than the seroprevalence value of 66% obtained with the PC-ELISA.
Cloning and Expression of Mitochondrial Heat Shock Protein 70 ofTrypanosoma congolenseand Potential Use as a Diagnostic Antigen
Authors: Bannai Hiroshi , Sakurai Tatsuya , Inoue Noboru , Sugimoto Chihiro , Igarashi Ikuo
Published: September 2003

The ability to use mitochondrial heat shock protein 70 (MTP) ofTrypanosoma congolenseas a diagnostic antigen was examined. One cDNA clone was obtained by immunoscreening of aT. congolenseprocyclic form (PCF) cDNA library with monoclonal antibody (MAb) 10F9. The cDNA clone contained an open reading frame of 1,977 bp encoding a polypeptide consisting of 659 amino acids. Southern blotting analysis indicated that there were at least three copies of theMTPgene in the haploid genome. Interference of the MTP RNA resulted in complete inhibition, which indicated that MTP is essential at the PCF stage. Northern and Western blotting analyses revealed that MTP is expressed both in the bloodstream form (BSF) and in PCF. The B-cell epitope recognized by MAb 10F9 was located within 206 amino acids from the C terminus. Depending on the conditions of protein extraction, MTP was cleaved into smaller polypeptides by endogenous proteases. However, the C-terminal epitope of MTP was preserved with a high degree of antigenicity, even after cleavage. Antibody detection by enzyme-linked immunosorbent assay with the truncated recombinant MTP revealed that anti-MTP antibodies exist in experimentally infected mouse sera. Thus, MTP may be useful as an antigen for the serodiagnosis of primaryT. congolenseinfection.
Detection of Antiendothelial Cell Antibodies by an Enzyme-Linked Immunosorbent Assay Using Antigens from Cell Lysate: Minimal Interference with Antinuclear Antibodies and Rheumatoid Factors
Authors: Drouet Christian , Nissou Marie-France , Ponard Denise , Arvieux Josiane , Dumestre-Pérard Chantal , Gaudin Philippe , Imbert Bernard , Massot Christian , Sarrot-Reynauld Françoise
Published: September 2003

The objective of the present work was to set up a routine test adapted to screening for antiendothelial cell antibodies (AECAs) in serum samples with minimal interference from antinuclear antibodies (ANAs) or rheumatoid factors (RFs). We compared the titers of AECAs titrated following two enzyme-linked immunosorbent assays (ELISAs): (i) an ELISA with ethanol-fixed EA.hy926 monolayers as the antigenic substrate and (ii) an ELISA with nucleus-depleted lysates prepared from EA.hy926 cells and normalized for protein (1.0 to 1.7 mg/ml) and DNA (≤0.1 μg/ml) contents as a surrogate substrate (postnuclear supernatant ELISA [PNS-ELISA]). The AECA titers in 51 serum samples, including 28 samples containing ANAs, were compared. A significantly positive correlation (
r
= 0.77;
P
< 0.001) between the two series was shown only for the ANA-negative serum samples. Conversely, ANAs or RFs in samples were shown not to interfere in tests for AECAs by the PNS-ELISA. AECAs recognize their antigenic targets in postnuclear supernatants, which is representative of the endothelial antigenic content, with improvement of the reliability of the assay, a prerequisite to application of the assay for their evaluation in clinical practice.
Effects of Hypothermia on Mortality and Inflammatory Responses to Endotoxin-Induced Shock in Rats
Authors: Taniguchi Takumi , Kanakura Hiroko , Takemoto Yasuhiro , Yamamoto Ken
Published: September 2003

We studied the effects of hypothermia on mortality rate, concentrations of tumor necrosis factor alpha and interleukin 6 in plasma, and the end products of nitric oxide (NO) in endotoxemia. It was found that moderate and mild hypothermia improved the mortality rate and attenuated cytokine responses and the elevation of the end products of NO after endotoxin injection and that these beneficial effects were similar for moderate and mild hypothermia.
Effects of Ovarian Steroids on Immunoglobulin-Secreting Cell Function in Healthy Women
Authors: Lü Fabien X. , Ma Zhongmin , Moser Susie , Evans Thomas G. , Miller Christopher J.
Published: September 2003

To determine the effect of the ovarian hormone cycle on immunity, immunoglobulin-secreting cell (ISC) frequency and lymphocyte subsets were examined in the blood of healthy women. We found that immunoglobulin A (IgA)-secreting cells (IgA-ISC) were fourfold more frequent than IgG-ISC in peripheral blood mononuclear cells (PBMC). Further, the ISC frequency in PBMC was highest (P< 0.05) during the periovulatory stage of the menstrual cycle. Thus, endogenous ovarian steroids regulate the ISC frequency and this may explain why women are more resistant to viral infections and tend to have more immune-mediated diseases than men do.
Immunological Characterizations of a Cloned 13.1-Kilodalton Protein from Pathogenic
Naegleria fowleri
Authors: Cho Myoung-Soo , Jung Suk-Yul , Park Sun , Kim Kyongmin Hwang , Kim Hyung-Il , Sohn Seonghyang , Kim Han-Jip , Im Kyung-Il , Shin Ho-Joon
Published: September 2003

We previously cloned an antigenic gene (named
nfa1
) from a cDNA library of
Naegleria fowleri
by immunoscreening. The
nfa1
gene had a coding nucleotide sequence consisting of 357 bases and produced a recombinant 13.1-kDa protein (Nfa1). In this study, to get more information regarding the recombinant Nfa1 protein (rNfa1), we produced an anti-Nfa1 polyclonal antibody from mice immunized with rNfa1 and used a peroxidase staining method to carry out immunocytochemistry experiments. In addition, we observed the effect of the presence of an anti-Nfa1 antibody on the in vitro cytotoxicity of
N. fowleri
against Chinese hamster ovary (CHO) cells. Trophozoites of
N. fowleri
in cultivation reacted strongly with a peroxidase-labeled anti-Nfa1 antibody. In inflammatory and necrotic regions of brain tissue infected with
N. fowleri
, labeled trophozoites that were stained brown were also observed. When examined using a transmission electron microscope, the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of
N. fowleri
. When examined using a light microscope, CHO cells grown in cocultures with
N. fowleri
trophozoites (group I) for 48 h showed morphologically severe destruction but CHO cells grown in cocultures with
N. fowleri
trophozoites and an anti-Nfa1 polyclonal antibody (group II) showed less destruction. The results of a lactate dehydrogenase release assay showed that group I CHO cells exhibited 81% cytotoxicity and group II CHO cells exhibited 13.8% cytotoxicity.
Diagnostic Implications of Antigen-Induced Gamma Interferon, Nitric Oxide, and Tumor Necrosis Factor Alpha Production by Peripheral Blood Mononuclear Cells fromMycobacterium bovis-Infected Cattle
Authors: Waters W. R. , Palmer M. V. , Whipple D. L. , Carlson M. P. , Nonnecke B. J.
Published: September 2003

Bovine tuberculosis in the United States has proven costly to cattle producers as well as to government regulatory agencies. While in vivo responsiveness to mycobacterial antigens is the current standard for the diagnosis of tuberculosis, in vitro assays are gaining acceptance, especially as ancillary or complementary tests. To evaluate in vitro indices of cellular sensitization, antigen-induced gamma interferon (IFN-γ), nitric oxide (NO), and tumor necrosis factor alpha (TNF-α) responses by blood mononuclear cells fromMycobacterium bovis-infected cattle were quantified and compared. Using an aerosol model of infection, two doses of each of two strains ofM. bovis(95-1315 and HC-2045T) were used to induce a range of IFN-γ, NO, and TNF-α responses. Infection-specific increases in NO, but not in IFN-γ or TNF-α, were detected in nonstimulated cultures at 48 h, a finding that is indicative of nonspecific activation and spontaneous release of NO. The infective dose ofM. bovisorganisms also influenced responses. At 34 days postinfection, IFN-γ, NO, and TNF-α responses in antigen-stimulated cells from cattle receiving 105CFU ofM. bovisorganisms were greater than responses of cells from cattle infected with 103CFU ofM. bovisorganisms.The NO response, but not the IFN-γ and TNF-α responses, was influenced by infective strains ofM. bovis. The TNF-α, NO, and IFN-γ responses followed similar kinetics, with strong positive associations among the three readouts. Overall, these findings indicate that NO and TNF-α, like IFN-γ, may prove useful as indices for the diagnosis of bovine tuberculosis.
Modified Direct Agglutination Test for Simplified Serologic Diagnosis of Leishmaniasis
Authors: Gómez-Ochoa P. , Castillo J. A. , Lucientes J. , Gascón M. , Zarate J. J. , Arbea J. I. , Larraga V. , Rodriguez C.
Published: September 2003

Human leishmaniasis is a severe health problem in many countries around the world. Hence, a cheap, reliable, and accurate diagnostic test is required to fight this disease. Perhaps the direct agglutination test (DAT) meets these criteria, but antigen elaboration involves many difficulties. We have developed a new antigen elaboration method, the EasyDAT method, that avoids the problems associated with the DAT. In this study, we compared the traditional DAT antigen method with our EasyDAT antigen method by using canine sera. The sensitivities (100%) and specificities (98.7%) were the same for both methods; we therefore concluded that the EasyDAT
Leishmania
antigen method simplifies serologic diagnosis, making this method easier and cheaper to use.
Serotyping Isolates of
Anaplasma phagocytophilum
by Using Monoclonal Antibodies
Authors: Inokuma Hisashi , Brouqui Philippe , Dumler J. Stephen , Raoult Didier
Published: September 2003

Ten mouse monoclonal antibodies (MAbs) that react with
Anaplasma phagocytophilum
(the human granulocytic ehrlichiosis agent) Webster isolates were developed. Seven different isolates of
A. phagocytophilum
were subtyped with these MAbs. Western blot analysis revealed that these MAbs reacted mainly with 41- to 46-kDa Msp2 proteins. Six MAbs reacted with all isolates. Four other MAbs reacted with human isolates from Wisconsin, but not with human isolates from New York or with animal isolates. Three different serotypes were identified. These features may lead to the development of other specific MAbs in order to provide tools for antigenic characterization of human isolates of
A. phagocytophilum
.
Differential Recognition of
Plasmodium falciparum
Merozoite Surface Protein 2 Variants by Antibodies from Malaria Patients in Brazil
Authors: Kanunfre Kelly A. , Leoratti Fabiana M. S. , Hoffmann Erika H. E. , Durlacher Rui R. , Ferreira Antônio W. , Moraes-Ávila Sandra L. , Ferreira Marcelo U.
Published: September 2003

Four variants of merozoite surface protein 2 (MSP-2) of
Plasmodium falciparum
were used in serology to examine whether changes in repeat units affect its recognition by antibodies during infection with parasites of known MSP-2 types. The results indicate that variation in MSP-2 repeats may represent a mechanism for parasite immune evasion.