Hybrid capture II and PapilloCheck® tests for detection of anal high-risk human papillomavirus

Introduction This study evaluated the level of concordance between hybrid capture II (HCII) and PapilloCheck® for the detection of high-risk human papillomavirus (HPV) in anal samples. Methods Anal cell samples collected from 42 human immunodeficiency virus (HIV)+ patients were analyzed. Results Considering only the 13 high-risk HPV types that are detectable by both tests, HCII was positive for 52.3% of the samples, and PapilloCheck® was positive for 52.3%. The level of concordance was 80.9% (Kappa = 0.61). Conclusions Good concordance was observed between the tests for the detection of high-risk HPV.

Detection of testicular cancer in men presenting with infertility

PURPOSE: Infertility is one of the less common presenting features associated with testicular tumors. We evaluated the histologic and biochemical findings, and pregnancy outcome in patients presenting with infertility who were found to have testicular tumors. METHODS: Seven patients with infertility were found to have testicular cancer over a 15-year period. All patients had a testicular ultrasound evaluation. The indications for the ultrasound were testicular pain in 2 patients, suspicious palpable mass in 4, and to rule out the presence of germ cell neoplasia in a patient with carcinoma in situ detected on a previous biopsy. Physical exam, histological findings, hormonal levels, tumor markers, and pregnancy outcome results were recorded from the patients medical charts. RESULTS: Two men had elevated serum follicle stimulant hormone and luteinizing hormone levels, 1 of them had an abnormally low serum testosterone level. Tumor markers were normal in all patients. In 4 patients the tumor was on the right side and in 3 on the left. The histological diagnoses were seminoma (n = 5), Leydig cell tumor (n = 1), and carcinoma in situ (n = 1). Of the 7 patients, 5 underwent adjuvant radiation therapy. Two patients had sperm cryopreserved. Follow up on fertility status was available in 6 cases. One patient has established a pregnancy and 5 did not achieve a pregnancy after treatment for their cancer. CONCLUSIONS: Most of the men who have testicular cancer and male infertility have a seminona. Therefore, men who present with infertility should be thoroughly investigated to rule out such serious, concomitant diseases along with their infertility.

Restriction in the repertoire of detectable autoantibodies in polyclonal B cell activations and the mimicry of autoantigens by idiotopes

Despite the existence of erythrocyte-autoreactive B cells in normal animals, erythrocyte-autoantibodies could not be detected during polyclonal B-cell activation (PBA) both in patients with visceral leishmaniasis and in bacterial lipopolysacharide (LPS) - injected mice. The failure to detect these autoantibodies in mice with PBA di not seem to be due to suppressor-cell activity, since (1) transfer of spleen cells from LPS-treated mice to naive recipients did not affect the erythrocyte-autoantibody response elicited by subsequent injections of rat erythrocytes and (2) low doses of X-radiation did no lead to erythrocyte-autoantibody detection in LPS-treated mice. The possibility that the detection of erytrocyte-autoantibodies could be affected by autoantibodies with idiotopes mimicring erythrocyte epitopes, the synthesis of which would also be triggerred in PBA, is discussed. Indirect evidence for the existence in normal animal of an expanded lymphocyte population with DNP-binding. Ia-mimicring antigen receptors is presented.

Improved enzyme-linked immunoadsorbent assay (ELISA) for the study of Trypanosoma cruzi-host cell interaction in vitro

We herein present an improved assay for detecting the presence of Trypanosoma cruzi in infected cultures. Using chagasic human sera (CHS), we were able to detect T. cruzi infection in primary cultures of both peritoneal macrophages and heart muscle cells (MHC). To avoid elevated background levels - hitherto observed in all experiments especially in those using HMC - CHS were preincubated with uninfected cells in monolayers or suspensions prior to being used for detection of T. cruzi in infected monolayers. Preincubation with cell suspensions gave better results than with monolayers, reducing background by up to three times and increasing sensitivity by to twenty times. In addition, the continous fibroplastic cell line L929 was shown to be suitable for preadsorption of CHS. These results indicate that the high background levels observed in previous reports may be due to the presence of human autoantibodies that recognize surface and/or extracellular matrix components in cell monolayers. We therefore propose a modified procedure that increases the performance of the ELISA method, making it an useful tool even in cultures that would otherwise be expected to present low levels of infection or high levels of background

Detection of intracytoplasmic cytokines by flow cytometry

Flow cytometry has been used as a powerful technique for studying cell surface antigen expression as well as intracellular molecules. Its capability of analyzing multiple parameters simultaneously on a single cell has allowed identification and studies of functional cell subsets within heterogeneous populations. In this respect, several techniques have been developed during the past few years to study cytokine-producing cells by flow cytometry in humans and several animal models.

Whole blood assay to access T cell-immune responses to Mycobacterium tuberculosis antigens in healthy Brazilian individuals

The production of interferon gamma (IFNgamma) guarantees effective T cell-mediated immunity against Mycobacterium tuberculosis infection. In the present study, we simply compare the in vitro immune responses to Mycobacterium antigens in terms of IFNg production in a total of 10 healthy Brazilian volunteers. Whole blood and mononuclear cells were cultivated in parallel with PPD, Ag85B, and M. bovis hsp65, and five-days supernatants were harvested for cytokine detection by ELISA. The inter-assay result was that the overall profile of agreement in response to antigens was highly correlated (r² = 0.9266; p = 0.0102). Potential analysis is in current progress to dictate the usefulness of this method to access the immune responses also in tuberculosis patients and its contacts.

Usefulness of a Nested-polymerase chain reaction for molecular diagnosis of human T-cell lymphotropic virus type I/II

This study aimed at implementing a Nested-polymerase chain reaction (Nested-PCR) for the molecular diagnosis of human T-cell lymphotropic virus type I/II (HTLV-I and HTLV-II) infections in peripheral blood mononuclear cells of infected subjects in Argentina. The sensitivity and specificity of the assay for the detection of regional strains were assessed by comparing them with the molecular assay of reference PCR-hybridization. The Nested-PCR detected 1 MT-2 cell (³ 8 proviral copies)/1x106 non-infected cells showing high sensitivity for provirus detection. While both molecular assays showed high specificity (100%) for HTLV-I and HTLV-II detection, the sensitivity values differed: 100% for Nested-PCR and 67% for PCR-hybridization assay. Moreover, this technique showed less sensitivity for the detection of DNA sequences of HTLV-II (33%) than for the detection of DNA sequences of HTLV-I (75%). The high sensitivity and specificity of the Nested-PCR for regional strains and its low costs indicate that this assay could replace the PCR-hybridization assay for the molecular diagnosis of HTLV-I/II infections. It will be interesting to assess the usefulness of this assay as a tool for the molecular diagnosis of HTLV-I/II infections in other developing countries. Other studies that include a greater number of samples should be conducted.

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

Detection of replication-defective hepatitis A virus based on the correlation between real-time polymerase chain reaction and ELISA in situ results

ELISA in situ can be used to titrate hepatitis A virus (HAV) particles and real-time polymerase chain reaction (RT-PCR) has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10(5) copies/mL) (p = 0.0002), while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001). At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.

Development of a molecular method for detection and identification of Xanthomonas campestris pv. viticola

In order to develop a molecular method for detection and identification of Xanthomonas campestris pv. viticola (Xcv) the causal agent of grapevine bacterial canker, primers were designed based on the partial sequence of the hrpB gene. Primer pairs Xcv1F/Xcv3R and RST2/Xcv3R, which amplified 243- and 340-bp fragments, respectively, were tested for specificity and sensitivity in detecting DNA from Xcv. Amplification was positive with DNA from 44 Xcv strains and with DNA from four strains of X. campestris pv. mangiferaeindicae and five strains of X. axonopodis pv. passiflorae, with both primer pairs. However, the enzymatic digestion of PCR products could differentiate Xcv strains from the others. None of the primer pairs amplified DNA from grapevine, from 20 strains of nonpathogenic bacteria from grape leaves and 10 strains from six representative genera of plant pathogenic bacteria. Sensitivity of primers Xcv1F/Xcv3R and RST2/Xcv3R was 10 pg and 1 pg of purified Xcv DNA, respectively. Detection limit of primers RST2/Xcv3R was 10(4) CFU/ml, but this limit could be lowered to 10² CFU/ml with a second round of amplification using the internal primer Xcv1F. Presence of Xcv in tissues of grapevine petioles previously inoculated with Xcv could not be detected by PCR using macerated extract added directly in the reaction. However, amplification was positive with the introduction of an agar plating step prior to PCR. Xcv could be detected in 1 µl of the plate wash and from a cell suspension obtained from a single colony. Bacterium identity was confirmed by RFLP analysis of the RST2/Xcv3R amplification products digested with Hae III.

Detection of illegal estrogen administration through immunohistochemical markers in the bovine prostate

The immunodetection of diverse cell markers was evaluated in prostatic samples from bullocks, and bullocks showing epithelial hyperplasia-metaplasia, with oestrogen-induced changes, and in experimental samples from bullocks inoculated with dietylstilbestrol (DES). Antigen-retrieval procedures allowed the use of tissues that had been fixed in formalin for long periods. Three tissue markers were chosen for the study: cytokeratins 13 and 16, vimentin and desmin. Monoclonal antibody K8.12 (specific for cytokeratins 13 and 16) stained basal cells and hyperplastic-metaplastic epithelium; monoclonal antivimentin, and desmin, allowed the definition of fibromuscular changes.

Analysis of hematologic and serum chemistry values of Spheniscus magellanicus with molecular detection of avian malarial parasites (Plasmodium spp.)

Magellanic penguins (Spheniscus magellanicus) routinely migrate from their breeding colonies to Southern Brazil often contracting diseases during this migration, notably avian malaria, which has been already reported in Brazil and throughout the world. Detection of Plasmodium spp. in blood smears is the routine diagnostic method of avian malaria, however it has a low sensitivity rate when compared to molecular methods. Considering the negative impact of avian malaria on penguins, the aim of this study was to detect the presence of Plasmodium spp. in Magellanic penguins using Polymerase Chain Reaction (PCR) and by verifying clinical, hematological, and biochemical alterations in blood samples as well as to verify the likely prognosis in response to infection. Blood samples were obtained from 75 penguins to determine packed cell volume (PCV), red blood cell (RBC) and white blood cell (WBC) counts, mean corpuscular volume (MCV), uric acid, total protein, albumin, globulin and aspartate aminotransferase (AST) activity levels. Whole blood samples were used for PCR assays. Plasmodium spp. was detected in 32.0% of the specimens using PCR and in 29.3% using microscopic analyses. Anorexia, diarrhea and neurological disorders were more frequent in penguins with malaria and a significant weight difference between infected and non-infected penguins was detected. PCV and MCV rates showed no significant difference. RBC and WBC counts were lower in animals with avian malaria and leukopenia was present in some penguins. Basophil and lymphocyte counts were lower in infected penguins along with high monocyte counts. There was no significant difference in AST activities between infected and non-infected animals. There was a significant increase in uric acid values, however a decrease in albumin values was observed in infected penguins. Based on this study, we concluded that Plasmodium spp. occurs in Magellanic penguins of rehabilitation centers in Southeastern Brazil, compromising the weight of infected animals with clinical alterations appearing in severe cases of this disease. It was also noted that, although the hematological abnormalities presented by these animals may not have been conclusive, leukopenia, monocytosis and the decrease of basophils and lymphocytes revealed an unfavorable prognosis, and Plasmodium spp. infections may progress with elevated uric acid concentration and low albumin levels.

Patch-clamp detection of macromolecular translocation along nuclear pores

The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal 'gigaseal' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability to form a gigaseal in nucleus-attached configurations does not mean that only the activity of channels from the outer membrane of the nuclear envelope can be detected. Instead, we show that, in the presence of fully operational nuclear pores, it is likely that the large-conductance ion channel activity recorded derives from the nuclear pores. We conclude the technical section with the suggestion that the best way to demonstrate that the nuclear pores are responsible for ion channel activity is by showing with fluorescence microscopy the nuclear translocation of ions and small molecules and the exclusion of the same from the cisterna enclosed by the two membranes of the envelope. Since transcription factors and mRNAs, two major groups of nuclear macromolecules, use nuclear pores to enter and exit the nucleus and play essential roles in the control of gene activity and expression, this review should be useful to cell and molecular biologists interested in understanding how patch-clamp can be used to quantitate the translocation of such macromolecules into and out of the nucleus

Detection and analysis of apoptosis in peripheral blood cells from breast cancer patients

Apoptosis is a well-known specific process of cell death that normally occurs in physiological situations such as tissue or organ development and involution. During tumor growth there is a balance between proliferation and cell death which involves apoptotic mechanisms. In the present study genomic DNAs from 120 breast tumor biopsies were analyzed by agarose gel electrophoresis and none of them presented the fragmentation pattern characteristic of the apoptosis process. However, 33% of the 105 breast cancer patients clearly showed the apoptotic pattern when DNA from blood cells was analyzed. None of the DNAs from healthy volunteer blood cells showed any trace of apoptosis. Since the breast cancer patients were not receiving chemo- or hormone therapy, the possible relationship between blood cortisol levels and the apoptotic pattern found in patient blood cells was investigated. Using a chemoluminescence immunodetection assay, similar cortisol levels were observed in breast cancer patient sera presenting or not apoptotic blood cells and in healthy volunteer sera. Analysis of the clinical data obtained from 60 of these patients showed that patients bearing tumors of smaller size (under 20 mm) were more susceptible to the apoptotic effect in blood cells. According to the Elston grade, it was observed that 7 of 12 patients with grade III tumors (58%) presented apoptotic peripheral blood cells, in contrast to 10 of 48 patients with grade I and grade II tumors. These observations may reflect the immunosuppression characteristic of some breast cancer patients, which may contribute to tumor growth. Therefore, further studies are necessary to elucidate the factor(s) involved in such massive blood cell death.

Detection of specific IgE antibodies in parasite diseases

Activation of Th1 or Th2 cells is associated with production of specific immunoglobulin isotypes, offering the opportunity to use antibody measurement for evaluation of T cell function. Schistosomiasis and visceral leishmaniasis are diseases associated with Th2 activation. However, an IgE response is not always detected in these patients. In the present study we evaluated specific IgE antibodies to S. mansoni and L. chagasi antigens by ELISA after depletion of serum IgG with protein G immobilized on Sepharose beads or RF-absorbent (purified sheep IgG antibodies anti-human IgG). In schistosomiasis patients, specific IgE to SWAP antigen was demonstrable in only 10 of 21 patients (48%) (mean absorbance ± SD = 0.102 ± 0.195) when unabsorbed serum was used. Depletion of IgG with protein G increased the number of specific IgE-positive tests to 13 (62%) and the use of RF-absorbent increased the number of positive results to 20 (95%) (mean absorbances ± SD = 0.303 ± 0.455 and 0.374 ± 0.477, respectively). Specific IgE anti-L. chagasi antibodies were not detected in unabsorbed serum from visceral leishmaniasis patients. When IgG was depleted with protein G, IgE antibodies were detected in only 3 (11%) of 27 patients, and the use of RF-absorbent permitted the detection of this isotype in all 27 visceral leishmaniasis sera tested (mean absorbance ± SD = 0.104 ± 0.03). These data show that the presence of IgG antibodies may prevent the detection of a specific IgE response in these parasite diseases. RF-absorbent, a reagent that blocks IgG-binding sites and also removes rheumatoid factor, was more efficient than protein G for the demonstration of specific IgE antibodies.