H antigen presence in an Ascaris lumbricoides extract

Previous experiences have demonstrated the same ABO system and P system antigens in A. lumbricoides extracts and in their hosts. The aim was to show the behavior of an A. lumbricoides extract from an O Group patient against monoclonal antibodies of different specificities. Agglutination Inhibition Tests were carried out facing the extract against monoclonal antibodies (anti A 2.23; anti B 2.54; anti B 2.62; anti AB 2.39 and anti H 2.72) in optimal concentrations. Suspensions of O Group fresh red cells were used as revealing system. The extract only inhibited the agglutination of anti H 2.72 with O erythrocytes. The semiquantitative Agglutination Inhibition Test of the extract was made against two series of anti H 2.72 dilutions by using O Group fresh red cells as revealing system. A difference of five dilutions between the titers of both series has been observed and the presence of H Antigen in the extract has been significantly confirmed. The fact that the extract did not inhibit the agglutination against anti A, anti B and anti AB has corroborated our previous observations about absence of A and B epitopes in A. lumbricoides extracts from O Group patients. The results of the preceding studies and this experience have demonstrated the membrane glycoconjugated importance in A. lumbricoides. They could be involved in molecular mimicry for this parasite.

St. Louis encephalitis vírus: first isolation from a human in São Paulo state, Brasil

This paper reports the isolation of St. Louis encephalitis virus (SLEV) from a febrile human case suspected to be dengue, in São Pedro, São Paulo State. A MAC-ELISA done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected IgG antibody for flaviviruses. An indirect immunofluorescent assay done on the C6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. RNA extracted from the infected cell culture supernatant was amplified by RT-PCR in the presence of NS5 universal flavivirus primers and directly sequenced. Results of BLAST search indicated that this sequence shares 93% nucleotide similarity with the sequence of SLEV (strain-MSI.7), confirmed by RT-PCR performed with SLEV specific primers. Since SLEV was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of São Paulo and in Brazil. This finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. SLEV infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue.

An immunofluorescence test for diagnosis of ophthalmic herpes in a mouse corneal model

Herpes simplex virus type 1 (HSV-1) ophthalmic disease is the most common cause of corneal blindness in humans world-wide. Current culture techniques for HSV take several days and commercially available HSV laboratory based diagnostic techniques vary in sensitivity. Our study was conducted to evaluate the use of a quicker and simpler method to herpes ophthalmic diagnosis. Corneal smears were made by firm imprints of infected mouse eyes to glass slides, after smears were fixated with cold acetone, and an indirect immunofluorescence (IIF) method was performed using monoclonal antibodies in a murine model of ophthalmic herpes. Eye swabs from infected mice were inoculated in Vero cells for virus isolation. Cytology and histology of the eye were also performed, using hematoxylin-eosin routine. Mouse eyes were examined by slit-lamp biomicroscopy for evidence of herpetic disease at various times postinoculation. We made a comparative evaluation of sensitivity, specificity and speed of methods for laboratory detection of HSV. Our results indicate that this IIF method is quick, sensitive, specific and can be useful in the diagnosis of ophthalmic herpes as demonstrated in an animal model.

Detection of Arah1 (a major peanut allergen) in food using an electrochemical gold nanoparticle-coated screen-printed immunosensor

A gold nanoparticle-coated screen-printed carbon electrode was used as the transducer in the development of an electrochemical immunosensor for Ara h 1 (a major peanut allergen) detection in food samples. Gold nanoparticles (average diameter=32 nm) were electrochemically generated on the surface of screen-printed carbon electrodes. Two monoclonal antibodies were used in a sandwich-type immunoassay and the antibody–antigen interaction was electrochemically detected through stripping analysis of enzymatically (using alkaline phosphatase) deposited silver. The total time of the optimized immunoassay was 3 h 50 min. The developed immunosensor allowed the quantification of Ara h 1 between 12.6 and 2000 ng/ml, with a limit of detection of 3.8 ng/ml, and provided precise (RSD <8.7%) and accurate (recovery >96.6%) results. The immunosensor was successfully applied to the analysis of complex food matrices (cookies and chocolate), being able to detect Ara h 1 in samples containing 0.1% of peanut.

Detection of the peanut allergen Ara h 6 in foodstuffs using a voltammetric biosensing approach

A voltammetric biosensor for Ara h 6 (a peanut allergen) detection in food samples was developed. Gold nanoparticle-modified screen-printed carbon electrodes were used to develop a sandwich-type immunoassay using two-monoclonal antibodies. The antibody-antigen interaction was detected through the electrochemical detection of enzymatically deposited silver. The immunosensor presented a linear range between 1 and 100 ng/ml, as well as high precision (inter-day RSD ≤9.8 %) and accuracy (recoveries ≥96.7 %). The detection and quantification limits were 0.27 and 0.88 ng/ml, respectively. It was possible to detect small levels of Ara h 6 in complex food matrices.

Cytomegalovirus in colorectal cancer and idiopathic ulcerative colitis

Cytomegalovirus (CMV) is a genus in the family Herpesviridae that has been associated with gastrointestinal syndromes. In this work we looked for a possible association of CMV infection with colorectal cancer and ulcerative colitis (UC). Blood and enteric tissue samples of 14 patients with colorectal cancer and of 21 with UC were subjected to a nested-PCR that amplifies part of the gB gene of CMV and also to immunohistochemistry using a specific monoclonal antibody to IE 76kDa protein of CMV. CMV was detected by nested-PCR in the blood and/or the enteric tissue of nine (64.3%) colorectal cancer and 16 (76.2%) ulcerative colitis patients. In the immunohistochemistry it was observed that 12 (12/21, 57.1%) positive enteric tissue samples of patients with UC and none from patients with colorectal cancer (0/14) were positive to CMV. The positivity of CMV infections in the UC patient group (12/21, 57.1%) showed by both techniques, was significantly higher (p = 0.015) than that observed for colorectal cancer patients (2/14, 14.3%). These results suggest an association of ulcerative colitis with CMV infection of the enteric tissue.

Antigenic and genetic characterization of the first rabies virus isolated from the bat Eumops perotis in Brazil

Although the main transmitters of rabies in Brazil are dogs and vampire bats, the role of other species such as insectivorous and frugivorous bats deserves special attention, as the rabies virus has been isolated from 36 bat species. This study describes the first isolation of the rabies virus from the insectivorous bat Eumops perotis. The infected animal was found in the city of Ribeirão Preto, São Paulo. The virus was identified by immunofluorescence antibody test (FAT) in central nervous system (CNS) samples, and the isolation was carried out in N2A cell culture and adult mice. The sample was submitted to antigenic typing using a panel of monoclonal antibodies (CDC/Atlanta/USA). The DNA sequence of the nucleoprotein gene located between nucleotides 102 and 1385 was aligned with homologous sequences from GenBank using the CLUSTAL/W method, and the alignment was used to build a neighbor-joining distance-based phylogenetic tree with the K-2-P model. CNS was negative by FAT, and only one mouse died after inoculation with a suspension from the bat's CNS. Antigenic typing gave a result that was not compatible with the patterns defined by the panel. Phylogenetic analysis showed that the virus isolated segregated into the same cluster related to other viruses isolated from insectivorous bats belonging to genus Nyctinomops ssp. (98.8% nucleotide identity with each other).

C4d Presence in Kidney Allograft Biopsy: Sensitivity and Specifity of Immunoperoxidase vs Immunofluorescence

OBJECTIVES: Evaluate the sensitivity/specificity of immunoperoxidase method in comparison with the standard immunofluorescence. MATERIAL AND METHODS: Retrospective review of 87 biopsies made for allograft dysfunction. Immunofluorescence (IF) was performed in frozen allograft biopsies using monoclonal antibody anti-C4d from Quidel®. The indirect immunoperoxidase (IP) technique was performed in paraffin-embebbed tissue with polyclonal antiserum from Serotec®. Biopsies were independently evaluated by two nephropathologist according Banff 2007 classification. RESULTS: By IF, peritubular C4d deposition were detected in 60 biopsies and absent in 27 biopsies. The evaluation of biopsy by IP was less precise due to the presence of background and unspecific staining. We find 13.8% (12/87) of false negative and Banff classification concordance in 79.3% (69/87) of cases (table1). The ROC curve study reveal a specificity of 100% and sensitivity of 80.0 % of IP method in relation to the gold standard (area under curve:0.900; 95% Confidence interval :0.817-0.954; p=0.0001). Banff Classification C4d Cases Immunofluorescence Immunoperoxidase n =87 Diffuse Negative 3 (3.4%) Focal Negative 9 (10.3%) Negative Negative 27 (31.0%) Diffuse Diffuse 33 (37.9%) Focal Focal 9 (10.3%) Diffuse Focal 6 (6.9%) CONCLUSION: The IP method presents a good specificity, but lesser sensitivity to C4d detection in allograft dysfunction. The evaluation is more difficult, requiring more experience of the observer than IF method. If frozen tissue is unavailable, the use of IP for C4d detection is acceptable.

Decreased Vascular Endothelial Growth Factor (VEGF) Expression in Focal Segmental Glomeruloesclerosis Lesions of Patients Under Sirolimus

Background: Proteinuria (PT) with SRL appears not only after conversion from a calcineurin inhibitor (CI), but also in de novo patients. The PT may be related to a hemodynamic effect of CI withdrawal or to a direct effect of SRL in glomerulus (GL). Recently an association between PT in SRL patients and FSGS lesions has been described. It is also known that SRL decrease VEGF synthesis and experimental data suggest that VEGF is essential to podocyte survival and differentiation. Aim: To determine if glomerular lesions and PT in SRL patients could be related to altered glomerular VEGF expression. Material and methods: We evaluated glomerular VEGF expression in 10 biopsies: A-allograft kidney in backtable (n=3); B-native normal kidney (n=1); C-native kidney with FSGS lesions (n=2); D-allograft kidney with FSGS lesions from proteinuric patients under SRL after conversion from CI (n=3); E-allograft kidney in proteinuric patient under SRL with a membranous glomerulonephritis (n=1). We employed indirect immunohistochemistry in paraffin-embedded sections using a mouse monoclonal antibody against human VEGF-C1 (Santa Cruz). Results: The controls biopsies (A; B) showed normal global VEGF expression, with strong podocyte staining. The VEGF expression in the group C was similar to the controls, although no FSGS lesions were observed in the stained GL. The group D showed normal VEGF expression in the apparently normal GL, hypertrophied podocytes with reduction of VEGF in anomalous GL, and no staining in slcerotic lesions. We observed a gradual reduction of VEGF expression with progressive dedifferentiation of podocytes. In the group E the VEGF was globally reduced, with some hypertrophied podocytes expressing decreased VEGF. Conclusion: We confirmed the diminished VEGF expression in injured podocytes of SRL patients.This decreased expression may result from a direct effect of SRL and precede the appearance of FSGS lesions and PT. Further studies are needed with greater number of cases and controls, including early biopsies of patients under SRL.

Characterization of a Hepatitis B virus strain in southwestern Paraná, Brazil, presenting mutations previously associated with anti-HBs Resistance

The present study investigated if hepatitis B virus (HBV) mutants circulate in the southwestern region of the State of Paraná, Brazil, by analyzing samples from children who received immunoprophylaxis but were born to HBV carrier mothers. Samples from 25 children were screened for HBV serum markers and for HBV DNA by PCR. Only one sample was positive for HBsAg, anti-HBs and HBV DNA, although the child had been vaccinated. Analysis of the S gene sequence of this sample showed the presence of a proline at position 105, a serine at position 114, three threonines at positions 115, 116 and 140, and a glutamine at position 129. The presence of these amino acids, except for serine at position 114, has been related to monoclonal or polyclonal therapy with anti-HBs after liver transplantation, whereas the presence of threonine at position 116 has been described in immunized children from Singapore. This finding demonstrates the possible circulation of HBV strains resistant to hepatitis B immunoprophylaxis in southwestern Paraná, Brazil. The genotype of the sample was identified as genotype D, which is frequently found in the region studied. Since 36% of the children had received incomplete or no immunoprophylaxis, more extensive follow-up of children born to HBsAg-positive mothers is needed.

First isolation of dengue 4 in the state of São Paulo, Brazil, 2011

We report the first isolation of Dengue virus 4 (DENV-4) in the state of São Paulo, from two patients - one living in São José do Rio Preto and the other one in Paulo de Faria, both cities located in the Northwest region of the state. The virus isolations were accomplished in the clone C6/36 Aedes albopictus cell line, followed by indirect immunofluorescence assays, performed with type-specific monoclonal antibodies that showed positive reactions for DENV-4. The results were confirmed by Nested RT-PCR and Real-Time RT-PCR assays. The introduction of DENV-4 in a country that already has to deal with the transmission of three other serotypes increases the possibility of the occurrence of more severe cases of the disease. The importance of early detection of dengue cases, before the virus spreads and major outbreaks occur, should be emphasized.

Infliximab in Recalcitrant Generalized Pustular Arthropatic Psoriasis

Generalized pustular psoriasis is an unstable inflammatory type of psoriasis, with widespread areas of erythema and sterile pustules, associated with fever and systemic symptoms. Infliximab is a monoclonal antibody with anti-TNFalpha activity, approved for use in psoriasis. We describe a male patient with a long history of stable arthropathic psoriasis, hospitalized with a generalized pustular psoriasis and acute exacerbation of articular complaints. The disease was resistant to multiple therapies (acitretin, methotrexate and corticosteroids), so the patient was started on infliximab, with a very rapid response of both cutaneous and articular symptoms. He had complete clearing of lesions at week 12, and marked improvement of the articular symptoms. No recurrence occurred at 8 months of follow-up with infliximab every 8 weeks. Infliximab had an extremely rapid therapeutic action response on a recalcitrant generalized pustular psoriasis. The articular response was also excellent, with significant improvement of quality of life.

PARACOCCIDIOIDOMYCOSIS: CHALLENGES IN THE DEVELOPMENT OF A VACCINE AGAINST AN ENDEMIC MYCOSIS IN THE AMERICAS

SUMMARYParacoccidioidomycosis (PCM), caused by Paracoccidioides spp, is an important endemic mycosis in Latin America. There are two recognized Paracoccidioides species, P. brasiliensis and P. lutzii, based on phylogenetic differences; however, the pathogenesis and disease manifestations of both are indistinguishable at present. Approximately 1,853 (~51,2%) of 3,583 confirmed deaths in Brazil due to systemic mycoses from 1996-2006 were caused by PCM. Antifungal treatment is required for patients with PCM. The initial treatment lasts from two to six months and sulfa derivatives, amphotericin B, azoles and terbinafine are used in clinical practice; however, despite prolonged therapy, relapses are still a problem. An effective Th1-biased cellular immune response is essential to control the disease, which can be induced by exogenous antigens or modulated by prophylactic or therapeutic vaccines. Stimulation of B cells or passive transference of monoclonal antibodies are also important means that may be used to improve the efficacy of paracoccidioidomycosis treatment in the future. This review critically details major challenges facing the development of a vaccine to combat PCM.

Quantum dots: synthesis, functionalization and bioconjugation for biological applications

Dissertation presented to obtain the Ph.D degree in Engineering Sciences and Technology

Do You Know This Syndrome?

POEMS syndrome is a unique clinical entity, the diagnosis of which is made when polyneuropathy and monoclonal gammopathy occur together, associated with other changes such as organomegaly, endocrinopathy, skin changes and papilledema. Cutaneous manifestations are heterogeneous, with diffuse cutaneous hyperpigmentation, hemangiomas and hypertrichosis occurring more frequently. We report the case of a 65- year-old female patient with this syndrome, diagnosed after 15 years of disabling peripheral neuropathy.