The use of ferromagnetic dacron as solid-phase in enzyme immunoassays

Ferromagnetic dacron is proposed as an alternative solid-phase for magnetic enzyme immunoassays. Human serum albumin (HSA) was covalentlyimmobilized onto ferromagnetic dacron and as enzyme immunoassay was developed using anti-HSA rabbit sera. Peroxidase, o-phenylenediamine (OPD) and hydrogen peroxide were used anti-HSA rabbit sera. Peroxidase, o-phenylenediamine (OPD) and hydrogen peroxide were used as the enzymatic label and substrates, respectively. Best results were observed when particles of 63-100 µm (diameter) and 10 µg of immobilized antigen were used. Positive reactions were detected until dilutions of1:51200 of immune sera. Its reproducibility was similar to standard ELISA. Disruption of the immunocomplexes formed and recuperation of the immobilized antigen in other immunoassays also proved to be reliable.

Calomys callosus: an alternative model to study fibrosis in Schistosomiasis mansoni: the pathology of the acute phase

Twenty Calomys callosus, Rengger, 1830 (Rodentia-Cricetidae) were studied in the early stage of the acute schistosomal mansoni infection (42nd day). The same number of Swiss Webster mice were used as a comparative standard. Liver and intestinal sections, fixed in formalin-Millonig and embedded in paraffin, were stained with hematoxilin and eosin, PAS-Alcian Blue, pH = 1.0 and 2.5, Lennert's Giemsa, Picrosirius plus polarization microscopy, Periodic acid methanamine silver, Gomori's silver reticulin and resorcin-fuchsin. Immunohistological study (indirect immunofluorescence and peroxidase labeled extravidin-biotin methods) was done with antibodies specific to pro-collagen III, fibronectin, elastin, condroitin-sulfate, tenascin, alpha smooth muscle actin, vimentin and desmin. The hepatic granulomas were small, reaching only 27 of the volume of the hepatic Swiss Webster granuloma. They were composed mainly by large immature macrophages, often filled by schistosomal pigment, characterizing an exsudative-macrophage granuloma type. The granulomas were situated in the parenchyma and in the portal space. They were often intravascular, poor of extracellular matrix components, except fibronectin and presented, sometimes alpha smooth muscle actin and vimentin positive cells. The C. callosus intestinal granulomas were similar to Swiss Webster, showing predominance of macrophages. Therefore, the C. callosus acquire very well the Schistosoma mansoni infection, without developing strong hepatic acute granulomatous reaction, suggesting lack of histopathological signs of hypersensitivity.

The use of polyvinyl alcohol glutaraldehyde as solid-phase in ELISA for plague

Discs of polyvinyl alcohol cross-linked with glutaraldehyde were synthesized under acid catalysis (H2SO4). Then, the antigen F1 purified from Yersinia pestis was covalently linked to this modified polymer. Afterwards, an enzyme-linked immunosorbent assay (ELISA) was established for the diagnosis of plague in rabbit and human. The best conditions for the method were achieved by using 1.3 ¼g of F1 prepared in 0.067 M phosphate buffer, pH 7.2, containing 1 M NaCl (PBS); anti-IgG peroxidase conjugate diluted 6,000 times and as a blocking agent 3% w/v skim milk in PBS. The titration of positive rabbit serum according to this procedure detected antibody concentrations up to 1:12,800 times. The present method, the conventional ELISA and passive haemagglutination assay are compared.

A new murine model of persistent lung eosinophilic inflammation

We summarize here the main characteristics of a novel model of pulmonary hypersensitivity. Mice were immunized with a subcutaneous implant of a fragment of heat solidified chicken egg white and 14 days later challenged with ovalbumin given either by aerosol or by intratracheal instillation. This procedure induces a persistent eosinophilic lung inflammation, a marked bone marrow eosinophilia, and Th2-type isotypic profile with histopathological findings that resemble human asthma. Further, this model is simple to perform, reproducible in different strains of mice, does not require adjuvants nor multiple boosters. Based on these characteristics we propose it as a suitable murine model of allergic eosinophilic lung inflammation.

Evaluation of Three Mycobacterium leprae Monoclonal Antibodies in Mucus and Lymph Samples from Ziehl-Neelsen Stain Negative Leprosy Patients and their Household Contacts in an Indian Community

Mucus and lymph smears collected from leprosy patients (9) and their household contacts (44) in the Caño Mochuelo Indian Reservation, Casanare, Colombia, were examined with monoclonal antibodies (MoAb) against Mycobacterium leprae. The individuals studied were: 5 borderline leprosy (BB) patients, 4 with a lepromatous leprosy (LL), all of whom were undergoing epidemiological surveillance after treatment and 44 household contacts: 21 of the LL and 23 contacts of the BB patients. The MoAb were reactive with the following M. leprae antigens: 65 kd heat shock protein, A6; soluble antigen G7 and complete antigen, E11. All the samples were tested with each of the MoAb using the avidin-biotin-peroxidase technique and 3,3 diaminobenzidine as chromogen. The patients and household contacts studied were all recorded as Ziehl-Neelsen stain negative. The MoAb which showed optimal reaction was G7, this MoAb permited good visualization of the bacilli. Five patients with BB diagnosis and one with LL were positive for G7; of the BB patients' household contacts, 9 were positive for G7; 7 of the LL patients' household contacts were positive for the same MoAb. MoAb G7 allowed the detection of bacillar Mycobacterium spp. compatible structures in both patients and household contacts. G7 permited the visualization of the complete bacillus and could be used for early diagnosis and follow-up of the disease in patients.

Reactive Nitrogen and Oxygen Intermediates in Patients with Cutaneous Leishmaniasis

The metabolisms of reactive nitrogen and oxygen intermediates (RNI and ROI) in patients with cutaneous leishmaniasis (CL) were investigated and compared with those of healthy subjects. To determine RNI metabolism, nitrite plus nitrate concentrations were measured spectrophotometrically. Nitrite concentration in plasma was determined directly by the Griess method. Nitrate levels in plasma were measured after reduction into nitrite by using copper-cadmium-zinc. ROI metabolism was evaluated by measuring erythrocyte superoxide dismutase, catalase and glutathione peroxidase activities. Plasma nitrite plus nitrate levels and erythrocyte superoxide dismutase activity were higher in the patient group than healthy subjects (p<0.01). In contrast, erythrocyte catalase and glutathione peroxidase activities were lower (p<0.05, p<0.01, respectively). ROI metabolism was altered in relation to hydrogen peroxide elevation in patients with CL. These alterations in ROI enable nitric oxide (NO) to amplify its leishmanicidal effect. The determination of ROI and RNI in patients with CL may be a useful tool to evaluate effector mechanisms of NO and clinical manifestations.

The identification and characterization of new immunogenic egg components: implications for evaluation and control of the immunopathogenic T cell response in schistosomiasis

In schistosomiasis, granuloma formation to parasite eggs signals the beginning of a chronic and potentially life-threatening disease. Granulomas are strictly mediated by CD4+ T helper (Th) cells specific for egg antigens; however, the number and identity of these T cell-sensitizing molecules are largely unknown. We have used monoclonal T cell reagents derived from egg-sensitized individuals as probes to track down, isolate and positively identify several egg antigens; this approach implicitly assures that the molecules of interest are T cell immunogens and, hence, potentially pathogenic. The best studied and most abundant egg component is the Sm-p40 antigen. Sm-p40 and its peptide 234-246 elicit a strikingly immunodominant Th-1-polarized response in C3H and CBA mice, which are H-2k strains characterized by severe egg-induced immunopathology. Two additional recently described T cell-sensitizing egg antigens are Schistosoma mansoni phosphoenolpyruvate carboxykinase (Sm-PEPCK) and thioredoxin peroxidase-1 (Sm-TPx-1). In contrast to Sm-p40, both of these molecules induce a more balanced Th-1/Th-2 response, and are relatively stronger antigens in C57BL/6 mice, which develop smaller egg granulomas. Importantly, Sm-p40 and Sm-PEPCK have demonstrated immunogenicity in humans. The findings in the murine model introduce the important notion that egg antigens can vary significantly in immunogenicity according to the host's genetic background. A better knowledge of the principal immunogenic egg components is necessary to determine whether the immune responses to certain antigens can serve as indicators or predictors of the form and severity of clinical disease, and to ascertain whether such responses can be manipulated for the purpose of reducing pathology.

IgG Anti-IgE Autoantibodies in Visceral Leishmaniasis

Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 ± 0.43) than sera from healthy individuals (OD = 1.35 ± 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 ± 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera.

The ue of polysiloxane/polyvinyl alcohol beads as solid phase in IgG anti-Toxocara canis detection using a recombinant antigen

Immunodetection of human IgG anti-Toxocara canis was developed based on ELISA and on the use of polysiloxane/polyvinyl alcohol (POS/PVA) beads. A recombinant antigen was covalently immobilized, via glutaraldehyde, onto this hybrid inorganic-organic composite, which was prepared by the sol-gel technique. Using only 31.2 ng antigen per bead, a peroxidase conjugate dilution of 1:10,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. This procedure is comparable to that which utilizes the adsorption of the antigen to conventional PVC plates. However, the difference between positive and negative sera mean absorbances was larger for this new glass based assay. In addition to the performance of the POS/PVA bead as a matrix for immunodetection, its easy synthesis and low cost are additional advantages for commercial application.

Genitourinary changes in hamsters infected and reinfected with Trypanosoma cruzi

Authors describe genitourinary changes in male hamsters infected and reinfected with Trypanosoma cruzi. Changes in genital organs have been described in human and in experimental chagasic infection. Genital dysfunctions in chronic chagasic patients affect ejaculation, libido and sexual potency, and testis biopsies may show arrested maturation of germ cells, oligozoospermia and azoospermia. Sixty-five male hamsters were inoculated and reinoculated with 2x10³ trypomastigotes of T. cruzi VIC strain, and 22 non-infected animals constituted the control group. Animals were necropsied and fragments from testis, epididymis, seminal vesicle and bladder were collected and stained with hematoxylin-eosin. Peroxidase anti-peroxidase procedure was utilized to detect tissue parasitism. T. cruzi nests were found in testis, epididymis and seminal vesicle of these hamsters. Such parasitism plays a role in the origin of genital lesions observed in humans and laboratory animals during chronic chagasic infection.

Vaccination with antioxidant enzymes confers protective immunity against challenge infection with Schistosoma mansoni

Schistosoma mansoni, an intravascular parasite, lives in a hostile environment in close contact with host humoral and cellular cytotoxic factors. To establish itself in the host, the parasite has evolved a number of immune evasion mechanisms, such as antioxidant enzymes. Our laboratory has demonstrated that the expression of antioxidant enzymes is developmentally regulated, with the highest levels present in the adult worm, the stage least susceptible to immune elimination, and the lowest levels in the larval stages, the most susceptible to immune elimination. Vaccination of mice with naked DNA constructs containing Cu/Zn cytosolic superoxide dismutase (CT-SOD), signal-peptide containing SOD or glutathione peroxidase (GPX) showed significant levels of protection compared to a control group. We have further shown that vaccination with SmCT-SOD but not SmGPX results in elimination of adult worms. Anti-oxidant enzyme vaccine candidates offer an advance over existing vaccine strategies that all seem to target the larval developmental stages in that they target adult worms and thus may have therapeutic as well as prophylactic value. To eliminate the potential for cross-reactivity of SmCT-SOD with human superoxide dismutase, we identified parasite-specific epitope-containing peptides. Our results serve as a basis for developing a subunit vaccine against schistosomiasis.

Sativa seeds against Schistosoma mansoni different stages

The schistosomicidal properties of Nigella sativaseeds were tested in vitro against Schistosoma mansoni miracidia, cercariae, and adult worms. Results indicate its strong biocidal effects against all stages of the parasite and also showed an inhibitory effect on egg-laying of adult female worms. In the present work we also studied the effects of crushed seeds on some antioxidant enzymes; which have a role in protection of adult worms against host oxidant killing; as well as some enzymes of glucose metabolism; which have a crucial role in the survival of adult worms inside their hosts. The data revealed that the used drug induce an oxidative stress against adult worms which indicated by a decrease in the activities of both antioxidant enzymes, superoxide dismutase, glutathione peroxidase, and glutathione reductase and enzymes of glucose metabolism, hexokinase and glucose-6-phosphate dehydrogenase. Disturbing of such enzymes of adult worms using N. sativa seeds could in turn render the parasite vulnerable to damage by the host and may play a role in the antischistosomal potency of the used drug.

Characterization of ndh gene of isoniazid resistant and susceptible Mycobacterium tuberculosis isolates from Brazil

Resistance in Mycobacterium tuberculosis to isoniazid (INH) is caused by mutations in the catalase-peroxidase gene (katG) , and within the inhA promoter and/or in structural gene. A small percentage (~ 10%) of INH-resistant strains do not present mutations in both of these loci. Other genes have been associated with INH resistance including the gene encoding for NADH dehydrogenase (ndh) . Here we report the detection of two ndh locus mutations (CGT to TGT change in codon 13 and GTG to GCG change in codon 18) by analyzing 23 INH-resistant and in none of 13 susceptible isolates from Brazilian tuberculosis patients. We also detected two isolates without a mutation in ndh, or any of the other INH resistance-associated loci examined, suggesting the existence of additional, as yet to be described, INH resistance mechanisms.

The role of platelet and plasma markers of antioxidant status and oxidative stress in thrombocytopenia among patients with vivax malaria

Malaria remains an important health problem in tropical countries like Brazil. Thrombocytopenia is the most common hematological disturbance seen in malarial infection. Oxidative stress (OS) has been implicated as a possible mediator of thrombocytopenia in patients with malaria. This study aimed to investigate the role of OS in the thrombocytopenia of Plasmodium vivax malaria through the measurement of oxidant and antioxidant biochemical markers in plasma and in isolated platelets. Eighty-six patients with P. vivax malaria were enrolled. Blood samples were analyzed for total antioxidant and oxidant status, albumin, total protein, uric acid, zinc, magnesium, bilirubin, total thiols, glutathione peroxidase (GPx), malondialdehyde (MDA), antibodies against mildly oxidized low-density lipoproteins (LDL-/nLDL ratio) and nitrite/nitrate levels in blood plasma and GPx and MDA in isolated platelets. Plasma MDA levels were higher in thrombocytopenic (TCP) (median 3.47; range 1.55-12.90 µmol/L) compared with the non-thrombocytopenic (NTCP) patients (median 2.57; range 1.95-8.60 µmol/L). Moreover, the LDL-/nLDL autoantibody ratio was lower in TCP (median 3.0; range 1.5-14.8) than in NTCP patients (median 4.0; range 1.9-35.5). Finally, GPx and MDA were higher in the platelets of TPC patients. These results suggest that oxidative damage of platelets might be important in the pathogenesis of thrombocytopenia found in P. vivax malaria as indicated by alterations of GPx and MDA.

Modification of oxidative status in Plasmodium berghei-infected erythrocytes by E-2-chloro-8-methyl-3-[(4'-methoxy-1'-indanoyl)-2'-methyliden]-quinoline compared to chloroquine

E-2-chloro-8-methyl-3-[(4'-methoxy-1'-indanoyl)-2'-methyliden]-quinoline (IQ) is a new quinoline derivative which has been reported as a haemoglobin degradation and ß-haematin formation inhibitor. The haemoglobin proteolysis induced by Plasmodium parasites represents a source of amino acids and haeme, leading to oxidative stress in infected cells. In this paper, we evaluated oxidative status in Plasmodium berghei-infected erythrocytes in the presence of IQ using chloroquine (CQ) as a control. After haemolysis, superoxide dismutase (SOD), catalase, glutathione cycle and NADPH + H+-dependent dehydrogenase enzyme activities were investigated. Lipid peroxidation was also assayed to evaluate lipid damage. The results showed that the overall activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were significantly diminished by IQ (by 53.5% and 100%, respectively). Glutathione peroxidase activity was also lowered (31%) in conjunction with a higher GSSG/GSH ratio. As a compensatory response, overall SOD activity increased and lipid peroxidation decreased, protecting the cells from the haemolysis caused by the infection. CQ shared most of the effects showed by IQ; however it was able to inhibit the activity of isocitrate dehydrogenase and glutathione-S-transferase. In conclusion, IQ could be a candidate for further studies in malaria research interfering with the oxidative status in Plasmodium berghei infection.