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HIV-Specific CD4(+) and CD8(+) immune responses are generated with a gp120-depleted, whole-killed HIV-1 immunogen with CpG immunostimulatory sequences of DNA.

We examined the adjuvant effects of a synthetic CpG oligodeoxynucleotide immunostimulatory sequence (ISS) using a whole-killed, gp120-depleted HIV antigen (HIV-1 antigen) in a Lewis rat model. We hypothesized that HIV-1-specific CD4(+) T helper (Th) immune responses could be enhanced when an ISS was combined with an HIV-1 antigen in incomplete Freund's adjuvant (IFA). We also reasoned that if such Th responses were sufficient, such a combination might also induce HIV-specific CD8(+) T cell immune responses. Here we demonstrate that the HIV-1 antigen in IFA combined with ISS stimulates both CD4(+) and CD8(+) HIV-specific immune responses as measured by interferon-gamma (IFN-gamma) in the ELISPOT assay. A strong correlation between these CD4(+) and CD8(+) responses was demonstrated. Furthermore, we found that the HIV-1 antigen in IFA with ISS as an adjuvant stimulated strong antibody responses to core antigen (p24). These studies suggest that the combination of the whole-killed, gp120-depleted HIV-1 antigen in IFA with ISS may be an ideal candidate to test in nonhuman primates and in human studies as a preventive HIV-1 vaccine.

AIDS Vaccines↗

Comparison of an enzyme immunoassay to an indirect fluorescent immunoassay for the detection of antinuclear antibodies.

The standard method for detecting antinuclear antibodies (ANAs) is by immunofluorescence assay (IFA), a method that is labor intensive and subjective. In an attempt to overcome these limitations, several commercial enzyme immunoassays (EIAs) have been developed. We report the results of our evaluation of the ANA Microplate EIA (Sanofi Diagnostics Pasteur, Chaska, MN). For the evaluation, 808 serum samples were tested by EIA and IFA; 52 specimens were positive by both assays, 561 were negative by both assays, 91 were positive by EIA only, and 3 were positive by IFA only. Borderline results (not positive or negative) were obtained for 101 specimens, which were excluded when calculating the sensitivity, specificity, and positive and negative predictive values of this assay, which were 94.6%, 86.0%, 36.4%, and 99.5%, respectively. Because of its high negative predictive value, this assay can be used reliably to detect ANA-negative samples; however, the low positive predictive value indicates that EIA-positive specimens should be retested by an IFA to determine the final result.

Antibodies, Antinuclear↗

Reactivity of serologic tests for the detection of antibody specific to cytomegalovirus.

A total of 212 sera were assayed for antibody specific for cytomegalovirus by complement fixation (CF), indirect immunofluorescence (IFA Electro-Nucleonics Laboratory, Inc., Bethesda, MD), ELISA (Cordis Laboratories, Inc., Miami, FL) and the FIAX system (International Diagnostic Technology, Santa Clara, CA). Correlation of CF with IFA, ELISA, and FIAX was 61%, 78%, and 71%, respectively. Quantitative correlation between IFA and FIAX and ELISA was not possible because of the broad range of reaction intensity of the latter two tests in sera with a particular IFA titer.

Antibodies, Viral↗

Evaluation of Cytomegelisa immunoglobulin M assay and comparison with indirect fluorescent antibody testing of QAE-Sephadex A50-treated sera.

The Cytomegelisa IgM assay (M. A. Bioproducts, Walkersville, MD) was evaluated as an alternative to indirect fluorescent antibody (IFA) testing of QAE-Sephadex A50-treated sera for detection of cytomegalovirus (CMV)-specific IgM. The Cytomegelisa assay was consistently more sensitive than IFA testing for detection of CMV-specific IgM. Of 101 rheumatoid factor-negative sera that were IFA positive before column separation, only 47 (46%) remained positive after column separation, while 72 (71%) were positive using Cytomegelisa. Nine transplant patients who had primary CMV infection develop were also evaluated. Two of the nine did not form sufficient IgM to remain IFA positive after column separation, however, all had IgM detected by Cytomegelisa. In all cases, Cytomegelisa provided laboratory evidence of CMV infection earlier or at the same time as IgG seroconversion or IgM testing by column/IFA. Comparison of both methods with 100 consecutive sera received for CMV testing yielded five sera positive for CMV-IgM with the column/IFA method and 20 using Cytomegelisa. The specificity of Cytomegelisa was evaluated with 56 RF-positive/CMV IgG-positive sera. Four of 56 (7%) appeared positive after column separation; however, of concern, 22 of 56 (39%) remained positive in the Cytomegelisa.

Antibodies, Viral↗

Diagnostic value of serum antibody and antigen detection in heroin addicts with systemic candidiasis.

The diagnostic value of antibody detection by indirect hemagglutination (IHA), indirect immunofluorescence (IFA), and counterimmunoelectrophoresis (CIE) and of Candida albicans mannan antigen detection by latex agglutination was studied in 36 cases of systemic candidiasis in heroin addicts. The IHA and IFA techniques were highly sensitive (97% and 91%, respectively), but their specificity was low (60% and 50%). When a titer of greater than or equal to 1:2,560 was used as a criterion for IHA positivity, the specificity of the test rose to 87%, with sensitivity at 75%. CIE had a high degree of specificity (96%) but a low degree of sensitivity (58%). A good correlation was found between clinical evolution of infection and serologic data. Two of 12 patients who could be followed for 9-16 months had a rise in antibody titer detected either by IFA or by IHA and CIE. These two patients had a persistent chondrocostal tumor and C. albicans endocarditis, respectively. All of the other patients, who were cured, had a decrease in titer detected by IHA and IFA and had negative CIE results at the end of follow-up. Serum mannan antigen was not found in any case. The detection of antibody to C. albicans may be useful for diagnosis and follow-up of such patients.

Adult↗

Rearrangement of genes located on homologous chromosomal segments in mouse and man: the location of genes for alpha- and beta-interferon, alpha-1 acid glycoprotein-1 and -2, and aminolevulinate dehydratase on mouse chromosome 4.

Gene mapping studies to determine the order of alpha- and beta-interferon (Ifa, Ifb), aminolevulinate dehydratase (Lv), and alpha-1 acid glycoprotein-1 and -2 (Orm-1, Orm-2) relative to each other and to the reference genes brown (b), B-cell maturation factor responsiveness (Bmfr-1), and major urinary protein-1 (Mup-1) are reported. The most likely order was Mup-1--Lv--b--Orm-1, Orm-2--Ifa, Ifb--Bmfr-1. This order suggested that two chromosomal segments located on chromosome 4 in the mouse and chromosome 9 in man have been conserved since divergence of lineages leading to man and mouse; these segments are marked by soluble aconitase-1 (Aco-1) and galactose-1 phosphate uridyl transferase (Galt) and by Lv and Orm-1. This order also demonstrated that, although genes located on opposite arms of chromosome 9 in man remain syntenic in the mouse, gene order has not been conserved; Ifa and Ifb are not located in their expected locations near Aco-1 and Galt. The position of Ifa and Ifb between Orm-1 and Bmfr-1 could not be determined with certainty because of apparent heterogeneity in recombination frequencies between crosses involving conventional laboratory strains of mice and crosses involving interspecific matings between laboratory mice and Mus spretus. This result suggests that caution must be exercised when using M. spretus in linkage crosses.

Animals↗

An enzyme-linked immunosorbent assay for detection of IgM antibodies to Toxoplasma gondii: use for diagnosis of acute acquired toxoplasmosis.

In an enzyme-linked immunosorbent assay (ELISA) for detection of IgM antibodies to Toxoplasma gondii, sera from individuals with negative results in the Sabin-Feldman dye test (DT) and sera from individuals with chronic toxoplasma infection all yielded negative results, even when tested undiluted. In contrast, of sera obtained from individuals with recently acquired toxoplasmosis, all sera that gave positive results in both the DT and the IgM-immunofluorescent antibody (IgM-IFA) test and 92.8% of sera that gave negative results in the IgM-IFA test yielded strongly positive results in the IgM-ELISA. Thus, the IgM-ELISA is more sensitive than the IgM-IFA test in the diagnosis of recently acquired infection with T. gondii. Moreover, sera that gave negative results in the DT but that contained either antinuclear antibodies or rheumatoid factor and thus caused false-positive results in the IgM-IFA test all yielded negative results in the IgM-ELISA, probably because serum IgM and IgG fractions were separated during the initial step.

Acute Disease↗

Concomitant cases of acquired toxoplasmosis in children of a single family: evidence of reinfection.

During 1979, three brothers had antibody titers for toxoplasmosis of 1:1,024, 1:64, and 1:16, respectively, by IgG indirect immunofluorescent antibody (IgG-IFA) test. The first child also had a fever and lymphadenopathy. In August 1980 the three children had lymphadenopathy and IgG-IFA test titers between 1:4,096 and 1:16,000. Two other brothers, first examined at that time, had IgG-IFA test titers between 1:1,024 and 1:4,096, one with ascending titers and the other with IgM antibodies to Toxoplasma gondii. The latter had lymphadenopathy, fever, and hepatosplenomegaly. Clinical and serologic examinations during March, April, and September 1981 revealed good health and decreasing IgG-IFA test titers in most of the brothers. The simultaneous increase of antibody titers during August 1980 in the three initial patients lead to the consideration of a probable reinfection. A simultaneous reactivation of the disease was considered less probable because acute toxoplasmosis occurred in two other brothers at the same time.

Adolescent↗

Enzyme-linked immunosorbent assay for the diagnosis of clinical and subclinical melioidosis.

An enzyme-linked immunosorbent assay (ELISA) for the detection of specific IgG and IgM antibody to Pseudomonas pseudomallei was developed. The IgG-ELISA was compared with the indirect fluorescence assay for IgG antibody (IgG-IFA) and the indirect hemagglutination (IHA) test in studies with serum specimens from persons from endemic areas for melioidosis and from persons from nonendemic areas of Australia. The sensitivity and specificity of the IgG-ELISA were 90% and 99%, respectively, comparable to those obtained with the IgG-IFA. The IgG-ELISA was more sensitive than the IHA test (74%) and was more suitable than the IgG-IFA as a serologic screening test for melioidosis. The IgM-ELISA was compared with the IgM-IFA as a marker of disease stage in patients with melioidosis. There was good diagnostic agreement between the tests; 92% of patients with active disease gave IgM-ELISA titers greater than or equal to 1:5,120 and 93% of patients with subclinical melioidosis had IgM-ELISA titers less than or equal to 1:1,280. Of the overlap group of patients with a borderline IgM-ELISA titer of 1:2,560, approximately 33% were clinical cases. An uncommon disease stage consisting of a self-limited, short-term, flu-like, pyrexial illness accompanied by elevated serum IgM-ELISA titers (greater than or equal to 1:5,120) was seen in a small number of patients residing in endemic Australia.

Adolescent↗

Prevalence of antibody to Borrelia burgdorferi by indirect fluorescent antibody assay, ELISA, and western immunoblot in healthy adults in Wisconsin and Arizona.

The prevalence of antibody to Borrelia burgdorferi in healthy adults from Wisconsin and Arizona was determined by indirect fluorescent antibody assay (IFA), ELISA, and Western immunoblotting. A total of 301 sera from adult volunteer blood donors were collected from three areas of Wisconsin and compared with 49 consecutive anonymous adult volunteer donor sera from Tucson, Arizona, an area without reported Lyme borreliosis. Regional differences in seropositivity were found for Western immunoblotting (34[11%] of 301 from Wisconsin and none of 49 from Tucson; P less than .01) but not IFA or ELISA. No correlation was found among Western immunoblotting and IFA or ELISA results. For persons living in Madison or Milwaukee, Wisconsin (cities not endemic for Lyme borreliosis), 19 (86%) of 22 with a positive Western blot, but only 12 (48%) of 25 with a positive IFA or ELISA, had a significant exposure risk to B. burgdorferi-infected Ixodes dammini (odds ratio, 6.9; 95% confidence interval, 1.4-43.5). Western blot results were consistent with epidemiologic exposure to B. burgdorferi and implied frequent asymptomatic infection among healthy adults living in or visiting areas endemic for Lyme borreliosis.

Adult↗

Effectiveness of biweekly versus daily iron-folic acid administration on anaemia status in preschool children.

Three-quarters of preschool children in India are anaemic. With the aim of identifying a cost-effective strategy for iron supplementation, the study objective was to assess the effectiveness of daily versus biweekly iron-folic acid (IFA: 20 mg elemental iron and 0.1 mg folic acid/tablet) on change in haemoglobin (Hb) levels of preschool children (3-6 years). This was a rural community-based effectiveness study in Uttar Pradesh, North India. IFA was given in two schedules: biweekly (2 tablets/dose) and daily (1 tablet/dose) for 1 year with fortnightly monitoring for adherence. A total of 400 and 403 children were enrolled in daily and biweekly regimes, respectively, of which 57.32 per cent and 50.25 per cent were anaemic (Hb <11 g/dl) in each group. Adherence in biweekly and daily regimes was 89.05 per cent vs. 63.5 per cent. After 1 year, the mean Hb rise in daily and biweekly regime was 1.063 g/dl (SD: 1.6; p = 0.000) and 1.053 g/dl (SD: 1.73; p = 0.001), respectively. Reduction in point prevalence of anaemia was 65.7 per cent daily vs. 56.1 per cent in biweekly regimen (p = 0.0047). We conclude that biweekly as well as daily IFA administration is effective in raising Hb levels and decreasing community prevalence of anaemia significantly. However, since there is better adherence and lower drug costs associated with biweekly IFA administration, this can be considered for programme use.

Analysis of Variance↗

Incremental flip angle snapshot FLASH MRI of hepatic lesions: improvement of signal-to-noise and contrast.

Incremental flip angle (IFA) snapshot fast low angle shot (FLASH) is a new modification of inversion recovery snapshot FLASH MR imaging. The method changes the flip angle incrementally from low to high during data acquisition and was applied in the evaluation of 16 focal hepatic lesions in 10 patients. Sequence comparisons were performed with a fixed flip angle inversion recovery snapshot FLASH sequence (standard), a T1- and T2-weighted spin-echo (SE) sequence, and a T1-weighted breath-hold FLASH sequence. Whereas snapshot FLASH images in both pulse sequences were free from physiological motion artifacts, SE and FLASH images showed respiratory artifacts in some patients. Quantitative analysis of IFA snapshot FLASH images at low hepatic and low lesion signal revealed both superior lesion-liver signal-difference-to-noise ratio (SD/N) and superior contrast compared with standard snapshot FLASH without additional artifacts. Unless motion artifacts were evident, SE and FLASH images showed a higher anatomic resolution but lower SD/N and lower contrast than IFA snapshot images. Because of its superior SD/N and contrast, IFA snapshot FLASH will likely widen the application of fast MR imaging techniques.

Humans↗

Comparison of nasal brush and nasopharyngeal aspirate techniques in obtaining specimens for detection of respiratory syncytial viral antigen by immunofluorescence.

Diagnosis of respiratory syncytial virus by antigen detection is dependent on obtaining adequate respiratory epithelial cells. Two specimen collection methods, nasopharyngeal aspiration (NPA) and nasal brushing (NB), were compared. Thirty-two pediatric patients with presumed viral pneumonia or bronchiolitis (34 episodes) had both NPA and NB performed. Of 34 specimens 15 were culture-positive for respiratory syncytial virus. Of these 12 NPA samples and 10 NB samples had viral inclusions by immunofluorescent antibody staining (IFA). Of culture-negative samples, 1 of 17 NB was positive by IFA. One specimen obtained by NB had too few cells to read by the IFA method. Sensitivity and specificity were 80 and 100% for NPA and 67 and 94% for NB. Total respiratory cells and IFA-positive cells (classified as few, moderate, or many) were greater with NPA; however, NB was also an effective procedure and was better tolerated by children, less expensive and easier to perform.

Antigens, Viral↗

Adenovirus and respiratory syncytial virus-adenovirus mixed acute lower respiratory infections in Chilean infants.

BACKGROUND: In Chile respiratory syncytial virus (RSV) and adenovirus (AD) are the principal viruses detected in acute lower respiratory infections (ALRI) in infants. An overview of AD pneumonia in Chile to detect annual trends and to compare the severity of single AD or mixed RSV-AD infections is presented. METHODS: Surveillance in 4927 infants hospitalized for ALRI has been performed from 1989 to 2001 using immunofluorescence assay (IFA) and viral isolation. Clinical features in 117 infants with single genotyped AD and 81 infants with mixed RSV-AD infections were analyzed. RESULTS: Adenovirus cases declined from 20% annually in the early 1990s to approximately 5% in the 2000 decade. Genotype 7h showed increasing prevalence in hospitalized cases. The mean annual burden of hospitalizations caused by AD in Santiago was estimated to be 0.6%. No difference was observed in duration of fever, oxygen requirement and hospital stay between groups. Lung consolidation was more frequent in AD cases than mixed cases (P < 0.01); interstitial pattern and hyperinflation prevailed in the mixed cases (P < 0.01). No child died. AD diagnosis was confirmed on admission by IFA in 17% of cases of RSV-AD and in 43% of cases of single AD ALRI. AD cases diagnosed early by IFA had worse clinical outcome than those diagnosed later by virus isolation (P < 0.05). CONCLUSIONS: AD cases declined since 1989. Mixed RSV-AD infections were not more severe than single AD etiology. AD cases admitted with positive IFA had worse prognoses than AD infections diagnosed later by virus isolation.

Adenovirus Infections, Human↗

Single injection of insulin delays the recurrence of diabetes in syngeneic islet-transplanted diabetic NOD mice.

BACKGROUND: Insulin has been implicated in the pathogenesis of type 1 diabetes and oral administration of insulin has been shown to delay the onset of diabetes in NOD mice. In this study we determined whether a single footpad injection of insulin will protect syngeneic islet grafts from autoimmune destruction when placed under the kidney capsule of diabetic NOD mice. METHODS: Five hundred islets were transplanted under the kidney capsule of diabetic female NOD mice in conjunction with a single footpad injection of either pork insulin in saline or mixed with incomplete Freund's adjuvant (IFA). Control groups received either IFA or saline alone. RESULTS: Seven of 11 animals (63.6%) given insulin in IFA exhibit long-term graft survival (>75 days; mean +/- SEM >85.4+/-16.1) whereas only 3 of 12 animals (25.0%) in the IFA group had graft survival longer than 75 days (mean +/- SEM >41.9+/-12.8 days). In contrast, none of the animals that received insulin in saline (17.3+/-2.5 days) and saline only (16.1+2.0 days) exhibit prolonged graft survival. CONCLUSION: These results suggest that a single footpad injection of insulin can protect the islet graft from immune attack in NOD mice.

Animals↗

Kaposi's sarcoma in Uganda: risk factors for human herpesvirus 8 infection among blood donors.

Human herpesvirus 8 (HHV-8) is etiologically linked to Kaposi's sarcoma, a common cancer in Uganda. The authors assessed HHV-8 seroprevalence, risk factors for infection, and HHV-8 assays in a cross-sectional study of Ugandan blood donors. Of 3,736 specimens, the authors selected 203 reactive for HIV, hepatitis B surface antigen (HBsAg), or syphilis, and, randomly, 203 nonreactive specimens. For HHV-8 testing, the authors used two peptide-based enzyme-linked immunosorbent assays (EIAs), ORFK8.1 and ORF65, and an immunofluorescence assay (IFA). Specimens reactive in at least two assays or on IFA alone were considered HHV-8-seropositive. Prevalence estimates were weighted to account for the sampling scheme. Overall HHV-8 seroprevalence was 40%. HHV-8 seroprevalence was higher among HBsAg-positive donors (53%) than HBsAg-negative donors (39%; p =.02) and higher among HIV-positive donors (63%) than HIV-negative donors (39%; p <.001). HHV-8 seroreactivity showed no trend with age. Kappa values for assay concordances were 0.68 (ORFK8.1 EIA and IFA), 0.37 (ORF65 EIA and K8.1 EIA), and 0.29 (ORF65 EIA and IFA). The association between HHV-8 and HBsAg positivity and the lack of association between HHV-8 and age point to primarily nonsexual HHV-8 transmission during childhood. The association with HIV indicates sexual transmission may also occur. The role of ORF65 EIA in testing specimens from Africa warrants further evaluation.

Adolescent↗

Type-C virus antigen detection in co-cultures of human leukaemic bone marrow and dog cells.

Bone marrow of leukaemic patients, non-leukaemic patients and normal individuals were co-cultivated with the canine cell line A7573. These co-cultures were screened for retrovirus antigens by means of the indirect cytoplasmic immunofluorescence assay (IFA). Rabbit antisera directed against the major structural protein (p30) of woolly monkey (simian) sarcoma leukaemia virus (grown in human lymphoid cells) and Rauscher murine leukaemia virus were used for testing. After 2 months in culture, 6 of 17 co-cultures containing cells from leukaemic patients showed positive staining in the IFA with the anti-simian virus serum. In control dog cells fluorescence was never observed. Five of the six positive cultures were derived from leukaemic children. One of 12 co-cultures of the non-leukaemic group and one of nine normal bone marrow co-cultures were positive with the simian virus antiserum. None of the 38 co-cultures stained positive in the IFA with Rauscher virus antiserum. Absorption of the simian virus antiserum with calf serum or mouse mammary tumour virus had no dramatic effect in the IFA on positive control cells or on cells of a positive co-culture. However, absorption with purified simian virus (grown in rat cells) completely abolished these fluorescence reactions. The results provide evidence that simian sarcoma-leukaemia virus related information was present in the original bone marrow samples and that co-cultivation with permissive mammalian cells enabled the detection of virus footprints.

Adult↗

The use of a nested polymerase chain reaction for detecting Pneumocystis carinii from lung and blood in rat and human infection.

A nested polymerase chain reaction (PCR) assay was developed to detect both rat- and human-derived Pneumocystis carinii DNA. The nested PCR product was 125 bp long and was representative of part of the gene coding for the large subunit of mitochondrial ribosomal RNA. Twenty serial blood samples and 24 tissues from six immunosuppressed Sprague-Dawley rats were examined by nested PCR. All lung samples were positive by PCR and Toluidine blue O staining. Buffy coat samples and all the other tissues were PCR-negative during up to 6 weeks of immunosuppression. Thirty-five clinical bronchoalveolar lavage, induced sputum or tracheal aspirate samples from human patients were tested. Twelve of 35 were positive by both PCR and indirect fluorescence assay (IFA) and 19 of 35 were both PCR- and IFA-negative. Four of 35 were IFA-negative but PCR-positive and there were good responses in these patients to specific therapy, indicating that PCR may be more useful than IFA in clinical samples. P. carinii DNA was not detected in three blood samples. The nested PCR is a sensitive and specific DNA amplification method suitable for the routine diagnosis of P. carinii in human respiratory samples.

Animals↗