Prevalence of Sarcocystis neurona and Neospora spp. infection in horses from Brazil based on presence of serum antibodies to parasite surface antigen

Sera from 961 horses from Brazil were tested for antibodies against the major surface antigens SnSAG4 and NhSAG1 to determine the seroprevalence of Sarcocystis neurona and Neospora hughesi, respectively. Antibodies against SnSAG4 were detected in 669 (69.6%) of the horses, while antibodies against NhSAG1 were detected in only 24 (2.5%) of the horses. These serologic results suggest that there is a high concentration of S. neurona in the environment of Brazil, which results in marked exposure of horses to this parasite. Additionally, the data further confirm that infection with Neospora spp. is relatively uncommon in horses. (c) 2005 Elsevier B.V. All tights reserved.

Transgenic lettuce seedlings carrying hepatitis B virus antigen HBsAg

The obtainment of transgenic edible plants carrying recombinant antigens is a desired issue in search for economic alternatives viewing vaccine production. Here we report a strategy for genetic transformation of lettuce plants (Lactuca sativa L.) using the surface antigen HBsAg of hepatitis B virus. Transgenic lettuce seedlings were obtained through the application of a regulated balance of plant growth regulators. Genetic transformation process was acquired by cocultivation of cotyledons with Agrobacterium tumefaciens harboring the recombinant plasmid. It is the first description of a lettuce Brazilian variety Vitória de Verão genetically modified.

Origins of sequence diversity in the malaria vaccine candidate merozoite surface protein-2 (MSP-2) in Amazonian isolates of Plasmodium falciparum

The recent evolution of Plasmodium falciparum is at odds with the extensive polymorphism found in most genes coding for antigens. Here, we examined the patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2), a major malarial repetitive surface antigen. We compared the msp-2 gene sequences from closely related clones derived from sympatric parasite isolates from Brazilian Amazonia and used microsatellite typing to examine, in these same clones, the haplotype background of chromosome 2, where msp-2 is located. We found examples of msp-2 sequence rearrangements putatively created by nonreciprocal recombinational events, such as replication slippage and gene conversion, while maintaining the chromosome haplotype. We conclude that these nonreciprocal recombination events may represent a major source of antigenic diversity in MSP-2 in P falciparum populations with low rates of classical meiotic recombination. (c) 2006 Elsevier B.V. All rights reserved.

Ultrastructural characterization of CD133(+) stem cells bound to superparamagnetic nanoparticles: possible biotechnological applications

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Safety of kidney transplantation using anti-HBc-positive donors

Background. Prospective studies evaluating the risk of hepatitis B virus (HBV) transmission in transplants of kidneys from hepatitis B core antibody (anti-HBc)-positive/ hepatitis B surface antibody (anti-HBs) negative donors are still lacking. The objective of this study was to assess the safety of kidney transplantation with the use of anti-HBc positive donors.Methods. This prospective case series study included 50 kidney transplant recipients from anti-HBc positive donors with or without anti-HBs positivity. Recipients were required to test positive for anti-HBs (titers >10 mUI/mL), regardless of anti-HBc status, and negative for hepatitis B surface antigen (HBsAg). Recipient and donor data were retrieved from medical records, databases, and organ procurement organization sheets. Liver function tests were performed at progressively increasing post-transplantation intervals. Complete serologic tests for HBV were performed before transplantation, 3 and 6 months after transplantation, and annually thereafter.Results. Six months after transplantation, all recipients were negative for HBsAg, HBeAg, anti-HBe, and anti-HBcIgM. No seroconversion was observed among the 20 patients who received kidneys from anti-HBc positive/anti-HBs negative donors. No patient showed elevated liver enzymes during follow-up.Conclusions. Kidney transplantation using organs from anti-HBeIgG positive donors (even when they are concurrently anti-HBs negative) in anti-HBs positive recipients is a safe procedure and may be considered as a way to expand the donor pool.

Distinct localization of T cell Agrin during antigen presentation - evidence for the expression of Agrin receptor(s) in antigen-presenting cells

Agrin is over-expressed by activated and autoimmune T cells, and synergizes with the T cell receptor (TCR) to augment cell activation. In the present study, we show that Agrin accumulates to distinct areas of the plasma membrane and that cell activation causes its redistribution. During antigen presentation, Agrin primarily accumulates to the periphery of the mature immunological synapse, mostly in lamellipodia-like protrusions that wrap around the antigen-presenting cell and, conversely, anti-Agrin sera induced a significant redistribution of TCR at the plasma membrane. We also provide evidence for the expression of Agrin receptors in peripheral blood monocytes, dendritic cells and a fraction of B cells. Interestingly, interferon-a treatment, which induces the expression of Agrin in T cells, also augmented Agrin binding to monocytes. Stimulation of monocytes with recombinant Agrin induced the clustering of surface receptors, including major histocompatibility complex class II, activation of intracellular signalling cascades, as well as enhanced dsRNA-induced expression of pro-inflammatory cytokines interleukin-6 and tumour necrosis factor-a. Collectively, these results confirm the location of Agrin at the immunological synapse between T cells and antigen-presenting cells and justify further characterization of its receptors in the immune system.

IMMUNOCYTOCHEMICAL DIFFERENTIATION OF REACTIVE HYPERPLASIA FROM FOLLICULAR LYMPHOMA USING MONOCLONAL-ANTIBODIES TO CELL-SURFACE AND PROLIFERATION-RELATED MARKERS

We tested the hypothesis that a panel of antibodies to cell surface, cytoplasmic, and nuclear antigens could reliably distinguish the cells composing reactive germinal centers from those composing follicular lymphoma. Immunocytochemistry was performed on deparaffinized sections of methacarn-fixed lymph node and tonsil (15 cases of reactive hyperplasia and 14 cases of follicular lymphoma) using antibodies to the nerve growth factor receptor (NGFR5), bcl-2 protein (124), proliferating cell nuclear antigen (PCNA; 19A2), and CD45RA (MT2). In 100% of cases of reactive hyperplasia, both MT2 and 124 showed positive immunostaining of mantle zone and scattered interfollicular lymphocytes, but in all cases there was a sharply demarcated absence of immunostaining of germinal center cells. However, diffuse immunostaining of follicular centers with MT2 (64%) and 124 (93%) and scattered intervening cells were seen in follicular lymphoma. The combination of antibodies to CD45RA and bcl-2 yielded positive immunostaining of follicular center cells in 93% of follicular lymphomas. The germinal center cells of reactive hyperplasia showed >75% nuclear positivity with antibodies to PCNA, in contrast to the follicular lymphoma cells, which showed variable PCNA indices ranging from 25 to >75%. A minority of follicular lymphoma cases (29%) showed PCNA indices comparable with those seen in cases of reactive hyperplasia. Antibodies to NGFR were positive in all cases of reactive hyperplasia and in 79% of cases of follicular hyperplasia, although the immunostaining intensity was generally decreased in follicular hyperplasia. In summary, antibodies to bcl-2 appear to be superior to those to CD45RA in distinguishing reactive hyperplasia from follicular lymphoma. Reactive hyperplasia cannot be discriminated from follicular hyperplasia using antibodies to PCNA or to nerve growth factor receptor.

Medial Septal Area Vasopressin Receptor Subtypes in the Regulation of Urine and Sodium Excretion in Rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Proliferating cell nuclear antigen (PCNA) in non-Hodgkin's lymphomas: Correlation with working formulation and Kiel classification in formalin-fixed paraffin-embedded material

PCNA is a 36-KD proliferating cell nuclear antigen associated with the cell cycle. The immunocytochemical detection of PCNA represents a useful tool for the study of tumor proliferation activity. This study documents the detection of PCNA, using antibody PC 10 in formalin-fixed, paraffin-embedded tissue, and correlates the proliferative activity of the non-Hodgkin's lymphomas (NHL) with histological grading assessed by the International Working Formulation (WF) and Kiel classification. In 92 cases of NHLs we found a strong correlation between the PCNA index and lymphoma grading. Statistically significant differences were also found between the proliferative index (PI) in low and high grade lymphomas according to the Kiel classification (t = 9.519; p < 0.001) and between low, intermediate and high grade lymphomas according to the WF classification (F = 79.01; p < 0.001). In the Kiel classification the mean of low grade lymphomas was 39.5% and of high grade 75.7%. In the WF the average of low grade lymphomas was 29.7%, intermediate 53.1% and high 75.1%. Although the differences among the groups had been significant, we found variations inside each histological subgroup in both classifications. The intermediate lymphomas were the most heterogeneous group, with PI inside the same histologic subtypes coincident with low and high grade lymphomas. Since PCNA may be used as a marker of cell proliferation in clinical studies to estimate the biological aggressiveness of lymphomas, its determination in intermediate grade NHL could be very useful to evaluate individual cases in this group and determine prognosis and probably the appropriate therapy.

Genetic relatedness of Plasmodium falciparum isolates and the origin of allelic diversity at the merozoite surface protein-1 (MSP-1) locus in Brazil and Vietnam

Background: Despite the extensive polymorphism at the merozoite surface protein-1 (MSP-1) locus of Plasmodium falciparum, that encodes a major repetitive malaria vaccine candidate antigen, identical and nearly identical alleles frequently occur in sympatric parasites. Here we used microsatellite haplotyping to estimate the genetic distance between isolates carrying identical and nearly identical MSP-1 alleles. Methods: We analyzed 28 isolates from hypoendemic areas in north-western Brazil, collected between 1985 and 1998, and 23 isolates obtained in mesoendemic southern Vietnam in 1996. MSP-1 alleles were characterized by combining PCR typing with allele-specific primers and partial DNA sequencing. The following single-copy microsatellite markers were typed: Polyα, TA42 (only for Brazilian samples), TA81, TA1, TA87, TA109 (only for Brazilian samples), 2490, ARAII, PfG377, PfPK2, and TA60. Results: The low pair-wise average genetic distance between microsatellite haplotypes of isolates sharing identical MSP-1 alleles indicates that epidemic propagation of discrete parasite clones originated most identical MSP-1 alleles in parasite populations from Brazil and Vietnam. At least one epidemic clone propagating in Brazil remained relatively unchanged over more than one decade. Moreover, we found no evidence that rearrangements of MSP-1 repeats, putatively created by mitotic recombination events, generated new alleles within clonal lineages of parasites in either country. Conclusion: Identical MSP-1 alleles originated from co-ancestry in both populations, whereas nearly identical MSP-1 alleles have probably appeared independently in unrelated parasite lineages.

The effect of CpG-ODN on antigen presenting cells of the foal

Background: Cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) has been used successfully to induce immune responses against viral and intracellular organisms in mammals. The main objective of this study was to test the effect of CpG-ODN on antigen presenting cells of young foals. Methods: Peripheral blood monocytes of foals (n = 7) were isolated in the first day of life and monthly thereafter up to 3 months of life. Adult horse (n = 7) monocytes were isolated and tested once for comparison. Isolated monocytes were stimulated with IL-4 and GM-CSF (to obtain dendritic cells, DC) or not stimulated (to obtain macrophages). Macrophages and DCs were stimulated for 14-16 hours with either CpG-ODN, LPS or not stimulated. The stimulated and non-stimulated cells were tested for cell surface markers (CD86 and MHC class II) using flow cytometry, mRNA expression of cytokines (IL-12, IFNα, IL-10) and TLR-9 using real time quantitative RT-PCR, and for the activation of the transcription factor NF-κB p65 using a chemiluminescence assay. Results: The median fluorescence of the MHC class II molecule in non-stimulated foal macrophages and DCs at birth were 12.5 times and 11.2 times inferior, respectively, than adult horse cells (p = 0.009). That difference subsided at 3 months of life (p = 0.3). The expression of the CD86 co-stimulatory molecule was comparable in adult horse and foal macrophages and DCs, independent of treatment. CpG-ODN stimulation induced IL-12p40 (53 times) and IFNα (23 times) mRNA expression in CpG-ODN-treated adult horse DCs (p = 0.078), but not macrophages, in comparison to non-stimulated cells. In contrast, foal APCs did not respond to CpG-ODN stimulation with increased cytokine mRNA expression up to 3 months of age. TLR-9 mRNA expression and NF-kB activation (NF-kB p65) in foal DCs and macrophages were comparable (p > 0.05) to adult horse cells. Conclusion: CpG-ODN treatment did not induce specific maturation and cytokine expression in foal macrophages and DCs. Nevertheless, adult horse DCs, but not macrophages, increased their expression of IL-12 and IFNα cytokines upon CpG-ODN stimulation. Importantly, foals presented an age-dependent limitation in the expression of MHC class II in macrophages and DCs, independent of treatment. © 2007 Flaminio et al; licensee BioMed Central Ltd.

Role of TLR-2 and fungal surface antigens on innate immune response against Sporothrix schenckii

Sporotrichosis is an infection caused by the dimorphic fungus Sporothrix schenckii. Toll-like receptors (TLRs) play an important role in immunity, since they bind to pathogen surface antigens and initiate the immune response. However, little is known about the role of TLR-2 and fungal surface antigens in the recognition of S. schenckii and in the subsequent immune response. This study aimed to evaluate the involvement of TLR-2 and fungal surface soluble (SolAg) and lipidic (LipAg) antigens in phagocytosis of S. schenckii and production of immune mediators by macrophages obtained from WT and TLR-2 -/- animals. The results showed that TLR-2-/- animals had had statistical lower percentage of macrophages with internalized yeasts compared to WT. SolAg and LipAg impaired phagocytosis and immunological mediator production for both WT and TLR-2-/-. The absence of TLR-2 led to lower production of the cytokines TNF, IL-1β, IL-12 and IL-10 compared to WT animals. These results suggest a new insight in relation to how the immune system, through TLR-2, recognizes and induces the production of mediators in response to the fungus S. schenckii. Copyright © Informa Healthcare USA, Inc.