Positivity of recombinant immunoblot assay (RIBA 2) for detection of antibodies to Hepatitis C virus (anti-HCV) is usually associated with HCV viremia. The significance of an indeterminate RIBA ...result defined by reactivity to only one HCV antigen is more problematic. The aim of this study was to determine: 1) the prevalence of RIBA indeterminate subjects, and 2) the correlation between RIBA indeterminate result and viremia according to the presence of normal or increased alanine aminotransferase (ALT) levels. In all RIBA 2 indeterminate subjects HCV RNA was detected by reverse transcription polymerase chain reaction (RT-PCR), and serum ALT was measured. An indeterminate RIBA 2 was found in 30 (15.15%) of subjects. Comparing the distribution of individual reactivity to HCV antigens (c22-3, c33c, c100-3 and 5-1-1) we found the reactivity to c22-3 to be the most frequent (p<0.001). Of 30 RIBA indeterminate subjects HCV RNA was detected in 19 (63,3%) of which 15 (78,9%) were anti-c22-3-positive. Analysing of RNA positivity according to ALT levels, we found that all the patients with elevated ALT and 15.3% of those with normal ALT levels were HCV RNA positive. We have found a significant correlation between viremia and elevated ALT levels. This study demonstrated that HCV viremia was frequent in subjects with indeterminate RIBA, especially in those with increased ALT levels.
Essential mixed cryoglobulinemia is frequently associated with chronic hepatitis. This report presents four cases of cryoglobulinemia with vasculitis associated with chronic hepatitis related to ...hepatitis C virus infection. Hepatitis C virus infection was ascertained in the four patients by both the presence in the serum of anti-HCV antibodies detected by the four-antigen recombinant immunoblot assay and of HCV RNA detected by polymerase chain reaction. In two patients tested, anti-HCV antibodies were not detected after centrifugation in the purified cryoglobulin but were detected in the supernatant. HCV RNA was detected in the purified cryoglobulin in all four patients and was detected in the supernatant in three patients. In one patient receiving recombinant interferon alfa, serum aminotransferases normalized and cryoglobulin disappeared; in another patient receiving recombinant interferon alfa, serum aminotransferases remained high and the cryoglobulin persisted. The presence of HCV RNA in the cryoglobulin and the parallelism of the changes of the cryoglobulinemia and of the serum aminotransferases during recombinant interferon alfa administration suggest that HCV infection is responsible for the production of cryoglobulinemia and vasculitis. It is proposed that HCV infection is a cause of cryoglobulinemia associated with chronic hepatitis.
The presence of pituitary adenylate cyclase activating polypeptide (PACAP) was previously demonstrated in the anterior pituitary by radioimmunoassay, immunohistochemistry, and reverse ...transcript-polymerase chain reaction (RT-PCR). With the use of cell immunoblot assay (CIBA), when the pituitary cells were cultured on nitrocellulose membrane, the release of PACAP by individual anterior pituitary cells was observed. The released peptide, trapped by the nitrocellulose membrane forming a blot around the cells, was demonstrated by immunocytochemistry. Double labeling revealed that a part of PACAP-immunoreactive cells can release LH as well. With the use of sandwich enzyme immunoassay (S-EIA), it was found that the concentration of PACAP in the anterior pituitaries is 10
−10 M. In cell culture in a similar concentration, PACAP stimulated the LH release from female gonadotropes, but did not influence it from male ones. The stimulated release of LH was indicated by the enhancement in the diameter of LH blots compared to the untreated control cultures. We concluded that PACAP may be released from the anterior pituitary cells in a concentration which would be able to influence LH release not only in vitro but under in vivo conditions as well. The effect of PACAP on LH release was different in female and male pituitary cultures.
Hantavirus cardiopulmonary syndrome (HCPS) is an acute, life threatening viral illness that was first recognized in the spring of 1993 during an outbreak in the Four Corners region of the southwest ...United States. The etiologic agent, Sin Nombre (SN) virus, is a zoonotic infection that is enzootic in deer mice (
Peromyscus maniculatus). Both human and rodent infections lead to specific antibody responses, and detection of such antibodies is the mainstay of diagnosis of infection in both species. More than 300 cases of HCPS have been confirmed in 30 states and four Canadian provinces since 1993 outbreak. Forty percent have been fatal. Conventional methods for the detection of hantavirus antibodies require 4–9 h. Since HCPS can rapidly progress to death, there is a clear need for a rapid diagnostic test. Rapid diagnostic tests for the detection of hantavirus infection should be ideally be (a) quick (b) simple to carry out, (c) allowing if possible for use in field conditions. A field-deployable test would have a particular advantage for diagnosis of infection in wild rodents, such as for risk assessments around case-households, as well as potential human diagnostic applications in battlefield situations. A prototype amperometric immunosensor based on highly dispersed immunoelectrode is developed for operation in field conditions on the importance of rapid and sensitive assay for hantavirus infection in mouse blood serum. This study was initiated to simplify and develop effective procedures for screening rodent serum samples in remote areas. A sandwich scheme immunoassay has been developed and used. Naphthol formed as a result of enzymatic reduction of naphthyl phosphate in the presence of alkaline phosphatase label has been detected amperometrically. The performance of the biosensor device is evaluated by testing mice blood samples in the wild under field conditions. The results were compared with the results obtained using the strip immunoblot assay (SIA) and that obtained from Centers for Disease Control and Prevention (CDC). The overall time of analysis is as low as 25 min and statistical analysis of the data indicated promising results. The device can be easily adapted for fast analysis of other microorganisms under non-laboratory conditions, in remote locations and field operations.
The catabolite control protein CcpA from
Bacillus megaterium was overproduced as a fusion protein to a 6xhis affinity tag and purified to homogeneity. Polyclonal antibodies of high affinity and ...specificity were raised against the purified protein. The serum did not crossreact with purified Lac repressor despite the fact that CcpA and LacI belong to the same protein family. Using this antiserum we identified proteins that share antigenic determinants with CcpA in many Gram-positive bacteria, including bacilli, staphylococci, streptococci, lactic acid bacteria, and some actinomycetes.
Immunocytochemical staining and cell immunoblot assay (CIBA) were performed in adenoma tissue from five patients with Cushing's disease and three patients with clinically silent corticotroph ...adenomas. All five patients with Cushing's disease showed hypersecretion of ACTH (130, 190, 331, 120, and 130 pg/ml), high levels of serum cortisol (26.6-44.0 μg/dl), and symptoms of Cushing's disease. All three patients with silent corticotroph adenoma showed hypersecretion of ACTH (110, 140, and 160 pg/ml) and normal levels of serum cortisol (11.4-26.8 μg/dl). The size of the pituitary adenoma on magnetic resonance imaging was smaller in patients with Cushing's disease (mean 8.2 mm) than in patients with silent corticotroph adenoma (mean 26.7 mm) (p=0.001). Transsphenoidal surgery was performed to totally resect the adenoma tissue. Immunostaining for ACTH showed diffuse ACTH-immunopositive cells in all eight adenomas. CIBA technique showed a good correlation between percentage of ACTH-immunopositive cells and level of plasma ACTH in patients with Cushing's disease but no correlation between the two parameters in patients with silent corticotroph adenoma. The percentage of ACTH-secreting cells and the amount of hormone secreted by a single cell are too low in silent corticotroph adenomas to cause an increase in plasma ACTH level corresponding to the large tumor size.
The cardiomyopathic Syrian hamster develops a progressive cardiomyopathy characterized by cellular necrosis, hypertrophy, cardiac dilatation, and congestive heart failure. This study aimed to ...identify alterations in cardiac mechanical function and in the cellular content of sarcoplasmic reticulum (SR) Ca(2+)-release channels (ryanodine receptors, RyR) in the heart of the UM-X7.1 cardiomyopathic hamster during the development of heart failure. Experimental and healthy control hamsters were examined at 8, 18, and 28 wk of age. The UM-X7.1 hamsters had developed left ventricular (LV) hypertrophy at 8 wk and a marked LV dilatation at 18-28 wk. During the latter stage, the UM-X7.1 hamster hearts showed global hypokinesis. Equilibrium binding assays of high-affinity sites for 3Hryanodine were performed in ventricular homogenate preparations. There was no significant difference between the two groups in the maximum number of 3Hryanodine binding sites (Bmax) at either 8 or 18 wk of age, although the cardiac pump function was impaired in UM-X7.1 hamsters at 18 wk of age. By 28 wk, Bmax was significantly lower in the UM-X7.1 hamsters. Quantitative immunoblot assay revealed that the content of RyR protein in cardiomyopathic hearts, which was increased at the early stage, declined to below normal as heart failure advanced. These results suggest that the number of RyR in the UM-X7.1 cardiomyopathic hamsters was preserved at both the hypertrophic and early stages of heart failure with a possibly compensatory increase in the level of protein expression, although the cardiac function already showed a tendency to be impaired.
We compared saline (S) and sodium dodecyl sulphate (SDS) extracts from Taenia solium (homologous species – HO) and Taenia crassiceps (heterologous species – HE) metacestodes in order to detect IgG by ...ELISA and immunoblot assay (IBA) in cerebrospinal fluid (CSF) for the diagnosis of human neurocysticercosis (NC). CSF samples were obtained from 93 patients. Of these, 40 had NC, five had a diagnosis of probable NC, nine had central nervous system schistosomiasis or strongyloidiasis and 39 had other neurological alterations. Samples were analysed by ELISA and the results were compared with IBA in all samples with confirmed and probable NC diagnosis, in all samples with other central nervous system parasitic infection, and in 10 of those with another neurological alterations. ELISA sensitivity was 100%, 85%, 95% and 87.5% for the S‐HO, S‐HE, SDS‐HO and SDS‐HE extracts, respectively, and ELISA specificity was 100% for S‐HO, S‐HE, SDS‐HO extracts and 97.9% for SDS‐HE antigen. Immunodominant peptides detected by IBA were, by decreasing percentage of recognition: 64–68 and 45 kDa for S‐HO; 108–114, 92–95, 64–68, 83 and 88 kDa for S‐HE; 64–68, 108–114, 77 and 86 kDa for SDS‐HO; and 108–114, 88 and 92–95 kDa for SDS‐HE. Overall the homologous antigenic extracts showed higher sensitivity than the heterologous extracts in the diagnosis of NC in CSF samples. The heterologous extracts contained most of the immunodominant peptides presented in the homologous extracts, which are recognized by IgG antibodies in CSF samples.
Antibodies to hepatitis C virus (HCV) may decrease or disappear after viral clearance in treated or spontaneously resolved infection. We evaluated the usefulness of serial antibody assays in ...predicting the antiviral treatment responses. One hundred and four chronic hepatitis C patients who received 24 weeks of interferon and ribavirin combination therapy were assayed with a third generation enzyme immunoassay anti‐HCV. The mean titre of anti‐HCV decreased by more than 50% (from 89.5 ± 10.8 to 43.6 ± 17.5) at 48 weeks post‐treatment in patients with a sustained virological response, while in nonsustained virological responders and nonresponders, the titres remained over 85% of the pretreatment level at 48 weeks post‐treatment. There was a divergence of anti‐HCV titres between sustained and nonsustained virological responders during 6–9 months. By using the ratio of 9‐month to 6‐month titres as an index and receiver operator characteristic curve analysis with the cut‐off point set at 90%, we could differentiate sustained virological responders from nonsustained virological responders with a sensitivity and specificity of 91.7% and 90.9%, respectively, 3 months after treatment. The titre of this third generation anti‐HCV decreased progressively in sustained virological responders and this assay may be used to monitor and predict antiviral treatment responses.
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