Preparation and study of an exoantigen of Histoplasma capsulatum for serological reactions.

The aim of this study was to determine the usefulness of a yeast-phase exo-antigen of Histoplasma capsulatum in standard serologic reactions. Three native strains of H.capsulatum which belong to Mycology Center collection were employed. They were maintained in their yeast-phase by weekly subcultures in 2% dextrose broth agar at 37 degrees C. After one week incubation yeast cells were suspended in distilled water containing thimerosal and phenylmethyl sulfonyl fluoride at a concentration of 1:5000. This suspension was left at room temperature for 72 h, then the supernatant was separated by centrifugation and it was lyophilized. Proteins and polysaccharides concentrations were determined. Immunodiffusion (ID) tests were carried out with an antigenic dilution containing 1.4 mg/ml of proteins. This exo-antigen was submitted to SDS-PAGE. Seven protein fractions were detected but only two of them showed antigenic activity against a pool of positive human sera; the molecular weights of these two proteins were 97 kDa and 66 kDa respectively. A metabolic antigen from the mycelial phase of H. capsulatum was used as control. A rabbit gammaglobulin anti-H. capsulatum was prepared and employed as positive control in serologic reactions. The antigenic capacity of ten batches of this exo-antigen was studied by ID and counterimmunoelectrophoresis (CIE) tests using serum samples of 20 hamsters experimentally infected by intracardiac inoculation of the yeast-phase of H. capsulatum. All tests presented positive results after three weeks of the infection. Fifty sera from patients suffering progressive histopasmosis were analyzed: ID, CIE and complement fixation (CF) tests were performed in all cases. HIV negative patients presented 7/7 (100%) positive reactions with the yeast-phase exoantigen and 5/7 (71.4%) with histoplasmin. In HIV positive patients CIE and CF were the most sensitive serologic tests, they gave positive results in 15/43 cases (34.8%) with the yeast-phase exo-antigen and in 7/43 cases (13.9%) with histoplasmin. Sera from 10 patients with paracoccidioidomycosis, aspergillosis and candidiasis respectively were studied by ID with the aim of detecting serologic cross reactions. No cross reaction was detected in these serum samples. This yeast-phase exo-antigen of H. capsulatum is more sensitive than and equally as specific as control histoplasmin.     

Tumor plasminogen activators and their clinical significance]

By means of a radio-immunological method the amount of plasminogen activators released by tumours was determined. The concentrations of tumour plasminogen activators was considerably higher in the venous blood of ovarial tumours than in the peripheral blood. In the uterine fluid the uterus cavity the concentration of plasminogen activator was increased in cases with neoplastic endometrium compared to those with normal endometrium.     

Leukocyte migration inhibition reaction in trophoblastic tumor patients]

Inhibition of leukocyte migration was studied in 40 patients with trophoblastic tumours under the effect of protein extract from chorionepithelioma. A marked inhibition of leukocyte migration was revealed in patients with signs of active tumour process before and during the treatment. Four patients in whom no inhibition was revealed either before or during the treatment were an exception. In the majority of the patients the inhibition effect was absent against the background of clinical well-being achieved as a result of surgical and chemotherapeutic, or chemotherapeutic treatment alone. Leukocytes of healthy donors failed to respond to the tumour extract in all 24 cases.     

Use of the Immunodiffusion Test in the Serodiagnosis of Aspergillosis

The diagnostic value of an immunodiffusion (ID) test with standardized precipitinogens derived from five Aspergillus species was determined with sera from 60 proven and 12 suspected cases of aspergillosis. The data demonstrated that the greatest number of aspergillosis cases were detected by the concurrent use of A. fumigatus and A. niger precipitinogens. With these precipitinogens, the ID test permitted the serodiagnosis of aspergillosis in 82% of the 60 proven cases and in 83% of the 12 suspected cases. The presence of one or more precipitins was indicative of aspergilloma, of allergic bronchopulmonary aspergillosis, or of invasive aspergillosis. Precipitins were detected in 93% of the sera from patients with aspergilloma, in 50% of the sera from patients with allergic bronchopulmonary aspergillosis, and in 88% of the sera from patients with invasive aspergillosis. Although the presence of one or two precipitin bands could indicate any form of aspergillosis, the presence of three or four was strong evidence of either aspergilloma or invasive aspergillosis. The ID test was found to be 100% specific in an evaluation of its effectiveness with 65 sera from individuals with other systemic mycotic infections, bacterial or neoplastic diseases, and from apparently normal humans. In diagnosed cases of aspergillosis, the examination of serial serum specimens provided information about the clinical course of the disease. A reduction in the number of precipitin bands and significant titer changes were noted as the patients responded to therapy.     

Comparison of a Newly Developed Latex Agglutination Test and an Immunodiffusion Test in the Diagnosis of Systemic Candidiasis

A newly developed latex agglutination (LA) test and a modified immunodiffusion (ID) test were evaluated. The antigen used was a homogenate of Candida albicans. A total of 167 antisera were employed in the evaluation. They included 36 sera from clinically well persons; 78 from patients with various clinical forms of candidiasis; 52 from patients with proven cases of aspergillosis, blastomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, nocardiosis, paracoccidioidomycosis, sporotrichosis, and tuberculosis; and one serum from a patient with toruloposis. Use of the LA test in conjunction with the ID test permitted the detection of more than 90% of 43 proven candidiasis cases. Of all the heterologous cases and normal human sera tested, LA reactions were noted with 3 of 10 cryptococcosis case specimens, 1 of 9 tuberculosis case specimens, and with the torulopsemia case serum. In contrast, the only heterologous serum reactive in the ID test was that from the patient with torulopsemia. Torulopsis glabrata and C. albicans antisera gave identical reactions in LA and ID tests with T. glabrata or C. albicans antigens. ID tests with selected antigens, however, permitted differentiation of rabbit and human T. glabrata antibody from that of C. albicans antibody. Six different precipitins were recognized with the C. albicans antigens. The occurrence of multiple precipitin lines and high LA titers was suggestive of severe candidiasis. The LA test, in contrast to the ID test, appeared to have prognostic value. Together, the LA and ID tests provided a simple, rapid, and accurate means of detecting and monitoring infections by species of Candida.     

Diagnosis of California (La Crosse) Encephalitis by Precipitin Techniques: a Prospective Study

Counterelectrophoresis (CEP) and immunodiffusion (ID) were evaluated prospectively as methods for the early and rapid laboratory diagnosis of California encephalitis (CE). CEP and ID studies were done on paired sera from 127 patients with acute central nervous system infections. After the precipitin tests were completed, conventional hemagglutination-inhibition, neutralizing, and complement fixing antibody titers were measured. The CEP system detected antibodies in 7 (41%) of 17 CE patients during their acute illness and in all 17 patients during convalescence. The ID method was less sensitive; 3 of 17 acute sera and 16 of 17 convalescent sera were ID positive. Comparative precipitin studies indicated that La Crosse virus was the infecting California group subtype in all 17 CE patients. Because CEP can be performed in 1.5 h, is at least as sensitive as hemagglutination-inhibition, neutralizing, and complement fixing tests, and can detect prospectively 41% of CE patients during their acute illness, it is recommended as a rapid diagnostic test for CE.     

Detection of trophoblastic beta 1-glycoprotein in tumor tissue and blood in ovarian neoplasms

Using immunochemical analysis methods (the reaction of precipitation in agar, immunoenzymatic method, immunofluorescence), trophoblastic beta 1-glycoprotein (TBG) concentration in tumour tissue and in the blood serum of patients with ovarian cancer was studied. By rabbits immunization with glycoprotein fraction of ovarian adenocarcinoma, dissolved in 0.6 M sulfosalicylic acid, the authors obtained antibodies to TBG. Immunoenzymatic method showed, that TBG level is raised during ovarian cancer (more than 3 micrograms/l): in 18% of tumour extracts, in 12.5% of blood sera samples and in 41.6% of cases in ascites fluid. Utilizing indirect immunofluorescence method morphological structures of trophoblastic type were identified in paraffin sections of ovarian adenocarcinoma. The authors suppose, that such structures may be responsible for TBG biosynthesis in ovarian tumours.     

An approach for immunodiagnosis of clinical cases of filariasis

In this communication an immunodiagnostic approach has been adopted for detection of antigen and antibody in amicrofilaeamic Mf(-) patients by countercurrent immuno electrophoresis (CCIE) and immunodiffusion (ID). Using Setaria cervi and Immune Complex (IC) antigens, out of fifteen clinical cases the number of positive patients in CCIE were twelve and ten respectively. Sixty percent of the Mf(-) cases were positive in antigen detection against both the homologous and heterologous antibody. In ID nine Mf(-) cases gave precipitin bands against S. cervi antigen while with IC antigens ten patients were positive. In similar experiments, it was found that out of fifteen Mf(-) cases nine and eleven patients were positive in antigen detection against microfilaraemic Mf(+) sera and S. cervi antibody respectively. All the Mf(+) cases were positive in both antibody and antigen detection. From the standpoint of immunodiagnosis the data were analysed by two-way analysis of variance study and a newly developed system using Binomial distribution. The sera from the control group were negative in all the immunodiagnostic tests.     

Serology of paracoccidioidomycosis

The purposes of the present work were: i) to study the positivity indices and compare titers obtained with the indirect immunofluorescence (II), tube precipitation (TP), complement fixation (CF) and double immunodiffusion on agar gel (ID) tests in the sera of 196 patients with paracoccidioidomycosis before treatment, and ii) to compare the initial titers of II with those obtained 1 year or more after treatment. II was the most sensitive serologic reaction (85.2%), and the positivity indices for CF, ID and TP were 67.7%, 66.0% and 50.0%, respectively. The sera tended to show parallel mean titers in II, CF and TP tests. One year after treatment there was a fall in titers of II in 66.2% of patients. The data, taken as a whole, demonstrate the usefulness of the indirect immunofluorescent test and the importance of using 2 or more serologic tests for the diagnosis and monitoring of patients with paracoccidioidomycosis.     

Quantitative immunoenzyme technic in the evaluation of specificity of the beta 1-g-globulin test in trophoblastic tumors]

The method of guantitative immunoenzymatic determination of beta 1-G-globulin (TSG) in the blood serum has been developed. The sensitivity of the method is 6 ng/ml TSG. It has been shown that elevated levels (12-100 ng/ml and higher) are usually observed in trophoblastic tumours of the uterus. The TSG ectopic synthesis is found to proceed in some tumours of the gastrointestinal tract and testicular teratoblastomas.     

Detection and Identification of Diagnostic Histoplasma capsulatum Precipitates by Counterelectrophoresis

Studies were carried out to develop and evaluate a counterelectrophoresis (CEP) technique for the rapid and specific identification of the diagnostically important histoplasmosis H and M precipitin bands. Well-defined and centrally located precipitin bands were produced by using a discontinuous buffer system and a gel matrix composed of agarose and ionagar no. 2. A template was devised which allowed the selective identification of the H and M precipitins. Comparative evaluations were performed with the microimmunodiffusion (ID) and complement fixation tests. In 52 sera from persons with histoplasmosis, either the H or M precipitin, or both, were identified in 42 (81%) of the cases with the CEP technique and in 43 (83%) with the ID test. With sera from 28 persons with heterologous diseases, the CEP technique, like the ID test, failed to react. The specificity of the CEP technique was dependent upon the use of the identity template. The CEP technique is recommended for routine use in laboratories testing moderate numbers of sera. It provides accurate and reproducible results within 90 min, in contrast to the ID test, which requires 18 to 24 h.     

Route and method of delivery of DNA vaccine influence immune responses in mice and non-human primates.

BACKGROUND: In spite of the large number of studies that have evaluated DNA-based immunization, few have directly compared the immune responses generated by different routes of immunization, particularly in non-human primates. Here we examine the ability of a hepatitis B surface antigen (HBsAg)-encoding plasmid to induce immune responses in mice and non-human primates (rhesus monkeys: Macaca mulatta) after delivery by a number of routes. MATERIALS AND METHODS: Eight different injected [intraperitoneal (IP), intradermal (ID), intravenous (IV), intramuscular (IM), intraperineal (IPER), subcutaneous (SC), sublingual (SL), vaginal wall (VW)] and six noninjected [intranasal inhalation (INH), intranasal instillation (INS), intrarectal (IR), intravaginal (IVAG), ocular (Oc), oral feeding (oral)] routes and the gene gun (GG) were used to deliver HBsAg-expressing plasmid DNA to BALB/c mice. Sera were assessed for HBsAg-specific antibodies (anti-HBs, IgG, IgG1, IgG2a) and cytotoxic T lymphocyte (CTL) activity measured. Three of the most commonly used routes (IM, ID, GG) were compared in rhesus monkeys, also using HBsAg-expressing vectors. Monkeys were immunized with short (0-, 4- and 8-week) or long (0-, 12- and 24-week) intervals between boosts, and in the case of GG, also with different doses, and their sera were assessed for anti-HBs. RESULTS: In one study, anti-HBs were detected in plasma of mice treated by five of eight of the injected and none of the six noninjected routes. The highest levels of anti-HBs were induced by IM and IV injections, although significant titers were also obtained with SL and ID. Each of these routes also induced CTL, as did IPER and VW and one noninjected route (INH) that failed to induce antibodies. In a second study, GG (1.6 microg) was compared to ID and IM (100 microg) delivery. Significant titers were obtained by all routes after only one boost, with the highest levels detected by IM. Delivery to the skin by GG induced exclusively IgG1 antibodies (Th2-like) at 4 weeks and only very low IgG2a levels at later times; ID-immunized mice had predominantly IgG1 at 4 weeks and this changed to mixed IgG1/IgG2a over time. Responses with IM injection (in the leg or tongue) were predominantly IgG2a (Th1-like) at all times. IV injection gave mixed IgG1/IgG2a responses. In monkeys, in the first experiment, 1 mg DNA IM or ID at 0, 4, and 8 weeks gave equivalent anti-HB titers and 0.4 microg at the same times by GG induced lower titers. In the second experiment, 1 mg DNA IM or ID, or 3.2 microg by GG, at 0, 12, and 24 weeks, gave anti-HB values in the hierarchy of GG > IM > ID. Furthermore, high titers were retained after a single immunization in mice but fell off over time in the monkeys, even after boost. CONCLUSIONS: Route of administration of plasmid DNA vaccines influences the strength and nature of immune responses in mice and non-human primates. However, the results in mice were not always predictive of those in monkeys and this is likely true for humans as well. Optimal dose and immunization schedule will most likely vary between species. It is not clear whether results in non-human primates will be predictive of results in humans, thus additional studies are required. http://link.springer-ny.com/link/service/journals/00020/bibs /5n5p287. html     

Expression of oestrogen receptor-beta in invasive breast tumours

Steroid hormone receptors are used routinely to predict endocrine responsiveness in patients with breast cancer. Two oestrogen receptors (ERs): ER alpha and ER beta have been identified. Although ER alpha and ER beta genes share a large degree of homology, it is generally thought that their distribution and function are substantially different in many tissues. Both of them may be expressed in normal and neoplastic tissues of the breast. While much is known about ER alpha, the role of ER beta is still undefined, especially at the protein level. Recent development of reliable antibodies to ER beta has provided opportunity to test immunohistochemical reactions detecting ER beta in archival breast tumours. The aim of our study was to learn more about the cellular mechanisms underlying the relationship of ER beta and progesterone receptor (PR) in breast cancer tissues, discriminating between hormone-dependent and hormone-independent tumours. ER alpha and PR content of tumour tissues of 154 patients with breast cancer were tested by in situ indirect immunohistochemical method parallel with ligand binding biochemical assay. ER beta was detected in 8 ER alpha-/PR+ breast carcinomas by immunohistochemical method too. Steroid hormone receptor content was analysed comparing to the histologic type and grading of the tumours. CONCLUSIONS: A considerable part of breast carcinomas belongs to the ER+/PR+ and ER-/PR- groups. About 1-2% of the tumours is expected to be ER alpha-/ER beta+/PR+ type. In such cases ER alpha negative reaction together with PR positivity can signal the necessity of the immunohistochemical detection of ER beta in routine histopathological practice, presenting the precise steroid hormone receptor status for the most effective endocrine therapy of the patients.     

Studies on changes in lymphocyte HL-A antigens during the course of malignant diseases]

The presence of HL-A antigens 1, 2, 5, 7, 8 and 12 on the lymphocytes of 26 patients with blood diseases and malignant tumours was examined by means of the two-step microcytotoxicity test. The studies carried out several times in the course of the disease and with 4 to 5 sera of the same specificity. Two types of the serological modifications were found: 1. Transient loss of HL-A antigens in 6 patients, 2. Evidence of the polyreactivity of lymphocytes in 3 patients. The polyreactivity was later changed to the loss of HL-A antigens. In one patient, the destruction of lymphocytes in the course of the testing was proved. The importance of the results is discussed. The additional serological, morphological and clinical investigations seems to be necessary.     

Cellular thiols in rat liver cell lines possessing different growth characteristics

Thiol levels were measured in three cell lines derived from rat hepatocytes with different growth rates and degrees of tumorigenicity: IAR20 having normal epithelial morphology and no tumour forming ability; IAR6.1 being a chemically-transformed malignant cell line; and IAR6.1RT7 derived from an epithelial tumour obtained after injection of IAR6.1 cells into a syngenic animal. The mean levels of GSH, GSSG, low molecular weight thiols (LMWT), macromolecular thiols (MT) and total reactive protein sulphur (TRPS), expressed as nmoles-SH mg-1 protein, were found to be 25.5, 7.5, 50.1, 114.5 and 143.6 respectively for IAR20; 37.6, 3.9, 65.4, 126.8 and 148.4 for IAR6.1; 17.2, 4.4, 52.3, 141.0 and 168.2 for IAR6.1RT7. Cultures were treated with D,L-buthionine-S,R-sulphoximine (BSO) to cause greater than 70 per cent depletion of GSH and the measurements of cellular thiols repeated. Although treatment with BSO caused a substantive decrease in the LMWT fraction, there were no major changes in macromolecular thiols or in total reactive protein sulphur. The respective mean values for LMWT, MT and TRPS (expressed as nmoles-SH mg-1 protein) were 19.4, 109.8, 136.3 for IAR20; 17.2, 119.3, 143.6 for IAR6.1; 21.6, 150.7 and 163.5 for IAR6.1RT7. It is concluded that significant differences in thiol levels exist between the three rat liver cell lines studied. However, severe acute depletion of GSH is not reflected by changes in the levels of macromolecular thiols which suggests that there is only a slow equilibrium between these two major thiol pools.     

Levels of some cytokines and antibodies to hepatitis C virus in patients with chronic hepatitis C

The comparison of the levels of some cytokines (tumor necrosis factor alpha (TNF-alpha), IL-1beta, IL-2, IL-4) in the blood serum of patients with chronic hepatitis C (CHC) having different antibody spectrum was carried out. In CHC patients increased levels of the serum cytokines IL-1beta, TNF-alpha under study in comparison with cytokine levels in donor sera was noted. In patients with detected antiNS5 and antiHCV IgM and antiNS5 HCV the level of IL-1beta was significantly higher than that in CHC patients without antibodies in sera. A change in the levels of proinflammatory and anti-inflammatory cytokines in the blood sera of CHC patients may be of significant diagnostic and prognostic importance.     

Use of solid-phase immunoenzyme analysis for the serodiagnosis of pseudotuberculosis

The diagnostic test system based on the solid-phase enzyme immunoassay (EIA) for the detection of antibodies to Yersinia pseudotuberculosis in the sera of patients with the use of Soviet-made preparations and reagents has been developed. The test has been performed in microchambers for immunological reactions, thus making it possible to decrease the consumption of reagents 10-20 times in comparison with the traditional technique with the use of plates. The results of the titration of 42 sera in EIA and in the passive hemagglutination test (PHAT) are indicative of the presence of positive correlation (r = 0.78; p less than 0.05) between antibody titers in EIA and PHAT. A fourfold or greater increase in antibody titers has been determined by means of EIA in 80% of cases and with the use of PHAT in 55% of cases. The minimum diagnostic titer yielded by EIA has been determined: 1:256.     

Comparative Evaluation of Five Serological Methods for the Diagnosis of Sporotrichosis

The diagnosis of sporotrichosis can be time consuming. Serological procedures could facilitate the rapid and accurate diagnosis of this disease. A slide latex agglutination (SLA) test for sporotrichosis was developed and compared with the tube agglutination (TA), complement fixation (CF), and immunodiffusion (ID) tests in the serological study of 80 proven human cases of sporotrichosis representing the cutaneous, subcutaneous, and extracutaneous forms of the disease. In addition, the indirect fluorescent antibody (IFA) technique was applied to 61 case sera. In the SLA test, latex particles sensitized with culture filtrate antigens from the yeast form of Sporothrix schenckii (B 959) detected 94% of the cases, as compared to 96% of the cases detected by the TA test, 68% by the CF test, and 56% by the ID test. The IFA test detected 90% of the 61 cases. The SLA and ID tests were specific, showing no reactions with sera from 86 persons with no disease or with diseases other than sporotrichosis. Because of its sensitivity, specificity, ease of performance, and ability to provide results in 5 min, the SLA test is highly recommended for routine use in the clinical laboratory.     

Cathepsin B/cystatin C complex levels in sera from patients with lung and colorectal cancer

A sandwich-type ELISA has been developed for quantification of the complex between the cysteine proteinase cathepsin B (CB) and its reversible tight-binding inhibitor cystatin C (CC) in normal and pathological sera. The assay is based on a combination of catching Ab (3E1), raised against CB, and a horseradish peroxidase-labelled detection Ab (1A2), raised against CC. Only the CB/CC complex is able to evoke a signal in this assay. The detection limit of the assay was 15.5 nM and the working range between 31.3-200 nM. The within and between-run coefficients of variance (CV) varied from 4.7% to 9.4% and 11% to 12.8%, respectively, demonstrating satisfactory reproducibility of the method. The concentration of the CB/CC complex was determined in sera from 90 healthy controls, 32 patients with non-cancerous lung diseases, 148 patients with lung and 32 patients with colorectal cancer. The CB/CC complex was significantly less abundant in sera of patients bearing malignant lung tumours than in those with non-cancerous lung diseases or healthy controls (p<0.001). In colorectal cancer sera its level was significantly lower in advanced stages C and D than in early Dukes' stages A and B (p=0.02). Our results show that the increased levels of CB in malignant sera are not impaired effectively by CC and support the hypothesis of hindered inhibitory capability during cancer progression.