Islet antigen-specific T cells and regulatory T cells in type 1 diabetes

T cells are the key players in the development of type 1 diabetes (T1D), mediating autoimmune reactions leading to the destruction of insulin producing beta cells in the islets. We aimed to analyze the role of different T-cell subtypes in the autoimmunity and pathogenesis of T1D. The frequency of islet antigen-specific (GAD65-, proinsulin-, and insulin-specific) CD4+ T cells was investigated in vitro in T1D patients, at-risk individuals (diabetes-associated autoantibody positive), and in controls, using MHC class II tetramers. An overall higher frequency of CD4+ T-cells recognizing the GAD65 555−567 peptide was detected in at-risk individuals. In addition, increased CD4+ T-cell responses to the same GAD65 epitope displaying a memory phenotype were observed in at-risk and diabetic children, which demonstrate a previous encounter with the antigen in vivo. Avidity and phenotypic differences were also observed among CD4+ T-cell clones induced by distinct doses of GAD65 autoantigen. T-cell clones generated at the lowest peptide dose displayed the highest avidity and expressed more frequently the TCR Vβ5.1 chain than low-avidity T cells. These findings raise attention to the antigen dose when investigating the diversity of antigen-specific T cells. Furthermore, an increased regulatory response during the preclinical phase of T1D was also found in genetically at-risk children. Higher frequencies of regulatory T (Treg) cells (CD4+CD25_high HLA-DR-/CD69-) and natural killer T (NKT) cells (CD161+Vbeta11+) were observed in children with multiple autoantibodies compared to autoantibody-negative controls. Taken together, these data showed increased frequency of islet-specific CD4+ T-cells, especially to the GAD65 555-567 epitope, and Treg and NKT cell upregulation in children at-risk for T1D, suggesting their importance in T1D pathogenesis

REPRODUCTIVE SYSTEM AND POLLEN FLOW IN PROGENIES OF Qualea grandiflora Mart., A TYPICAL SPECIES OF THE BRAZILIAN CERRADO

This study analyzed the reproductive system and the pollen dispersion pattern of Qualea grandiflora progenies. This is a typical species from the Brazilian Cerrado about which there are not too many studies from the genetics point of view. The study was conducted in an area of 2.2 hectares located in the Conservation Unit managed by the Forest Institute of the state of São Paulo, Brazil (Assis State Forest). Total genomic DNA of 300 seeds from 25 plants (12 seeds from each plant) was extracted and amplified using specific primers to obtain microsatellite markers. Results showed that selfing is frequent among adults and progenies, and the species reproduces by outcrossing between related and unrelated individuals (0.913). The single-locus outcrossing rate was 0.632, which indicates that mating between unrelated individuals is more frequent than between related plants. The selfing rate was low (0.087), that is, the species is allogamous and self-fertilization is reduced. About 35% of the plants in the progenies were full-sibs, and about 57%, half-sibs. Besides, about 8% of the progenies were selfing siblings. The genetic differentiation coefficient within progenies was 0.139, whereas the fixation rate was about 27%. The estimate of the effective size revealed that the genetic representativeness of descent was lower than expected in random mating progenies: The analyzed samples corresponded to only 13.2 individuals of an ideal panmictic population. In environmental recovery programs, seeds, preferably from different fruits, should be collected from 95 trees to preserve the genetic diversity of the species.

Free Prostate-specific Antigen Forms and Kallikrein-related Peptidase 2: Tools for Prostate Cancer Diagnostics

Prostate-specific antigen (PSA) is a marker that is commonly used in estimating prostate cancer risk. Prostate cancer is usually a slowly progressing disease, which might not cause any symptoms whatsoever. Nevertheless, some cases of cancer are aggressive and need to be treated before they become life-threatening. However, the blood PSA concentration may rise also in benign prostate diseases and using a single total PSA (tPSA) measurement to guide the decision on further examinations leads to many unnecessary biopsies, over-detection, and overtreatment of indolent cancers which would not require treatment. Therefore, there is a need for markers that would better separate cancer from benign disorders, and would also predict cancer aggressiveness. The aim of this study was to evaluate whether intact and nicked forms of free PSA (fPSA-I and fPSA-N) or human kallikrein-related peptidase 2 (hK2) could serve as new tools in estimating prostate cancer risk. First, the immunoassays for fPSA-I and free and total hK2 were optimized so that they would be less prone to assay interference caused by interfering factors present in some blood samples. The optimized assays were shown to work well and were used to study the marker concentrations in the clinical sample panels. The marker levels were measured from preoperative blood samples of prostate cancer patients scheduled for radical prostatectomy. The association of the markers with the cancer stage and grade was studied. It was found that among all tested markers and their combinations especially the ratio of fPSA-N to tPSA and ratio of free PSA (fPSA) to tPSA were associated with both cancer stage and grade. They might be useful in predicting the cancer aggressiveness, but further follow-up studies are necessary to fully evaluate the significance of the markers in this clinical setting. The markers tPSA, fPSA, fPSA-I and hK2 were combined in a statistical model which was previously shown to be able to reduce unnecessary biopsies when applied to large screening cohorts of men with elevated tPSA. The discriminative accuracy of this model was compared to models based on established clinical predictors in reference to biopsy outcome. The kallikrein model and the calculated fPSA-N concentrations (fPSA minus fPSA-I) correlated with the prostate volume and the model, when compared to the clinical models, predicted prostate cancer in biopsy equally well. Hence, the measurement of kallikreins in a blood sample could be used to replace the volume measurement which is time-consuming, needs instrumentation and skilled personnel and is an uncomfortable procedure. Overall, the model could simplify the estimation of prostate cancer risk. Finally, as the fPSA-N seems to be an interesting new marker, a direct immunoassay for measuring fPSA-N concentrations was developed. The analytical performance was acceptable, but the rather complicated assay protocol needs to be improved until it can be used for measuring large sample panels.

Evaluation of a recombinant p24 antigen for the detection of Feline Immunodeficiency Virus-specific antibodies

Feline Immunodeficiency Virus is a worldwide infection and is considered a significant pathogen. The diagnosis of FIV infections is mainly based on commercially available rapid tests that are highly expensive in Brazil, hence it is rarely performed in the country. Furthermore, lentiviruses grow slowly and poorly in tissue cultures, making the production of viral antigen by classic means and thus the establishment of FIV immunodiagnosis impracticable. In order to deal with this, recombinant DNA techniques were adopted to produce the protein p24, a viral capsid antigen. The protein's reactivity evaluation analyzed by Western blot indicated that this recombinant antigen can be a useful tool for the immunodiagnostic of FIV infections.

Detection and quantification of Duffy antigen on bovine red blood cell membranes using a polyclonal antibody

Babesiosis is one of the most important diseases affecting livestock agriculture worldwide. Animals from the subspecies Bos taurus indicus are more resistant to babesiosis than those from Bos taurus taurus. The genera Babesia and Plasmodium are Apicomplexa hemoparasites and share features such as invasion of red blood cells (RBC). The glycoprotein Duffy is the only human erythrocyte receptor for Pasmodium vivax and a mutation which abolishes expression of this glycoprotein on erythrocyte surfaces is responsible for making the majority of people originating from the indigenous populations of West Africa resistant to P. vivax. The current work detected and quantified the Duffy antigen on Bos taurus indicus and Bos taurus taurus erythrocyte surfaces using a polyclonal antibody in order to investigate if differences in susceptibility to Babesia are due to different levels of Duffy antigen expression on the RBCs of these animals, as is known to be the case in human beings for interactions of Plasmodium vivax-Duffy antigen. ELISA tests showed that the antibody that was raised against Duffy antigens detected the presence of Duffy antigen in both subspecies and that the amount of this antigen on those erythrocyte membranes was similar. These results indicate that the greater resistance of B. taurus indicus to babesiosis cannot be explained by the absence or lower expression of Duffy antigen on RBC surfaces.

Development of an indirect ELISA to detect Corynebacterium pseudotuberculosis specific antibodies in sheep employing T1 strain culture supernatant as antigen

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a chronic disease that affects goats and sheep, characterized by granuloma formation in subcutaneous and internal lymph nodes. CLA causes significant economic losses to commercial goat herds. In this study, we aimed to test secreted antigens secreted from T1 strain bacteria grown in brain heart infusion (BHI) broth in an indirect ELISA system to determine the presence of specific immunoglobulins against C. pseudotuberculosis. We analyzed the BHI antigen electrophoretic profile and the recognition pattern by infected sheep sera samples. The ELISA results were compared with multiplex PCR assay and IFN-gamma production. The ELISA was able to discriminate between negative and positive animals, with a sensitivity of 89% and a specificity of 99%, using microbiological isolation as gold standard. When this assay was compared with multiplex PCR and specific IFN-gamma quantification, six discrepant results were found among thirty-two samples. We concluded that the ELISA using antigens secreted from C. pseudotuberculosis T1 strain growth in BHI broth culture can be used for the serodiagnosis of CLA in sheep.

Storage substances in the androgametogenesis and mature pollen grain of Ilex paraguariensis St. Hil. (Aquifoliaceae)

Storage substances such as starch grains, proteins and lipids were studied during the male gametogenesis and in the mature pollen grain of Ilex paraguariensis St. Hil. (Aquifoliaceae). There are two cycles of amylogenesis and amylolyse. The first cycle lasts until the vacuolated stage when the starch is hydrolyzed and amorphous proteins are stored inside the single vacuole. The next cycle begins after mitosis with the formation of the vegetative and generative cells. At this point, the young vegetative cell stores many starch grains that are bigger than in the first cycle. During the maturation of the male gametophyte, the starch is hydrolyzed and it is absent in the mature pollen grain. Small lipid droplets surround the young generative cell after the mitosis of the androspore and are dispersed in the vegetative cytoplasm during its maturity. The relationship between the pollen storage substances and the ontogeny of the layers in the sporoderm, formation of the generative cell, and the male germ unit were discussed.

Nectar and pollen production in pumpkin (Cucurbita pepo L.)

Cucurbitaceae species depend on pollination by honey bees for fruit production. The overall objective of this work was to evaluate the potential of C. pepo for pollen and nectar production, that could help maintain colonies placed in the field. Plants of pumpkin were cultivated in field, in 1996 and 1997. Before anthesis, male flowers were covered to prevent visits by bees and other insects. After anthesis the flowers were uncovered and the following parameters were evaluated: 1) nectar production; 2) total sugar concentration in the nectar; 3) nectar replacement; and 4) production of pollen and flowers during the crop cycle. Nectar production varied from 18 to 79 µL flower-1 and increased progressively from 7h to 13h. The sugar concentration, measured at 7h, 9h and 11h, did not vary, averaging 50.5% ± 0.5% in 1996 and 40.5% ± 0.6% in 1997. At 13h the concentration decreased to 42% in 1996 and to 35% in 1997. Total daily nectar production was not influenced by removing nectar several times per day, indicating that nectar secretion is not stimulated or inhibited by frequent removal. The number of pollen grains did not differ in the two years, with an average of 43,669 ± 1,382 grains per flower. The peak rate of male and female flowers occurred from 60 to 66 days after planting (DAP) with 34.6 male flowers and 2.2 female flowers per plant, respectively. Cucurbita pepo has a potential for honey and pollen production of about 105 and 160 kg per hectare per season, respectively, which is enough to sustain, at least, five honeybee colonies.

Pollen morphology of species of Hortia (Rutaceae)

A palinological study covering all 10 species of the Neotropical genus Hortia Vand. (Rutaceae) was conducted. Pollen grains were collected from herbarium exsiccates, acetolysed and mounted in glycerine jelly on glass slides. The grains were studied under light and scanning electron microscopy, including measurements of polar and equatorial diameters, shape of the grains, number and shape of apertures, and thickness of the exine. The results demonstrated that the genus is stenopalynous, with pollen grains in monads, subprolate to prolate, 3-colporate, with very thick exine and a psilate-perforate pattern of ornamentation. Pollen grains of all species revealed a great similarity, with few variations in the pattern of ornamentation of exine, number and form of apertures and measurements. Although well-characterized palinologycally, pollen features did not furnish relevant information on the position of Hortia into an intrafamilial phylogeny.

Multivariate analysis of pollen frequency of the native species Escallonia pulverulenta (Saxifragaceae) in Chilean honeys

The aim of this work was the identification of geographic zones suitable for the production of honeys in which pollen grains of Escallonia pulverulenta (Ruiz & Pav.) Pers. (Saxifragaceae) can be detected. The analysis of botanical origin of 240 honey samples produced between La Serena and Puerto Mont (the IV and X Administrative Regions of Chile), allowed the detection of pollen grains of E. pulverulenta in 46 Chilean honeys. The geographic distribution of the honeys studied is presented together with their affinities, through factor analysis and frequency tables. The study was based on the presence of E. pulverulenta pollen. Escallonia pulverulenta pollen percentages oscillated between 0.24% and 78.5%. Seventeen of the studied samples were designated as unifloral - i.e. samples showing more than 45% pollen of a determined plant species. Two of these corresponded to E. pulverulenta (corontillo, madroño or barraco) honeys. The remaining unifloral honeys correspond to 8 samples of Lotus uliginosus Schkuhr (birdsfoot trefoil), 2 samples of Aristotelia chilensis (Molina) Stuntz (maqui) and 1 sample of Escallonia rubra (Ruiz & Pav.) Pers. (siete camisas), Eucryphia cordifolia Cav. (ulmo or muemo), Weinmannia trichosperma Cav. (tineo), Rubus ulmifolius Schott (blackberry) and Brassica rapa L. (turnip). Honeys with different percentages of E. pulverulenta pollen - statistically analyzed through correspondence analysis - could be associated and assigned to one of three geographic types, defined on the basis of this analysis. The geographical type areas defined were the Northern Mediterranean Zone (samples from the IV Region), Central Mediterranean Zone (samples from the V to the VIII regions including two samples of unifloral Escallonia pulverulenta honey), and Southern Mediterranean Zone (samples from the IX Region).

Pollen analysis of honey and beebread derived from Brazilian mangroves

Pollen analyses were performed on honey and beebread from hives in apiaries located in two distinct mangrove areas dominated by Laguncularia racemosa (L.) C.F. Gaernt. One apiary was located at the edge of Guanabara Bay, Rio de Janeiro State, and the other near Maranguá Bay, Bahia State, Brazil. We investigated the contribution of nectar and pollen from mangrove vegetation to Apis mellifera L. honey and beebread stocks. Intensive visitation to this plant species by honeybees and the presence of its pollen grains in honey and beebread confirmed the importance of Laguncularia racemosa as a polliniferous and nectariferous species.

Two point deterministic model for acquisition of in vitro pollen grain androgenetic capacity based on wheat studies

This article discusses, from the standpoint of cellular biology, the deterministic and indeterministic androgenesis theories. The role of the vacuole and of various types of stresses on deviation of the microspore from normal development and the point where androgenetic competence is acquired are examined. Based on extensive literature review and data on wheat studies from our laboratory, a model for androgenetic capacity of pollen grain is proposed. A two point deterministic model for in vitro androgenesis is our proposal for acquisition of androgenetic potential of the pollen grain: the first switch point would be early meiosis and the second switch point the uninucleate pollen stage, because the elimination of cytoplasmatic sporophytic determinants takes place at those two strategic moments. Any abnormality in this process allowing the maintenance of sporophytic informational molecules results in the absence of establishment of a gametophytic program, allowing the reactivation of the embryogenic process

Molecular mimicry between cardiac myosin and Trypanosoma cruzi antigen B13: identification of a B13-driven human T cell clone that recognizes cardiac myosin

Previous reports from our group have demonstrated the association of molecular mimicry between cardiac myosin and the immunodominant Trypanosoma cruzi protein B13 with chronic Chagas' disease cardiomyopathy at both the antibody and heart-infiltrating T cell level. At the peripheral blood level, we observed no difference in primary proliferative responses to T. cruzi B13 protein between chronic Chagas' cardiopathy patients, asymptomatic chagasics and normal individuals. In the present study, we investigated whether T cells sensitized by T. cruzi B13 protein respond to cardiac myosin. T cell clones generated from a B13-stimulated T cell line obtained from peripheral blood of a B13-responsive normal donor were tested for proliferation against B13 protein and human cardiac myosin. The results showed that one clone responded to B13 protein alone and the clone FA46, displaying the highest stimulation index to B13 protein (SI = 25.7), also recognized cardiac myosin. These data show that B13 and cardiac myosin share epitopes at the T cell level and that sensitization of a T cell with B13 protein results in response to cardiac myosin. It can be hypothesized that this also occurs in vivo during T. cruzi infection which results in heart tissue damage in chronic Chagas' disease cardiomyopathy

Interruption of recently induced immune responses by oral administration of antigen

Interest in oral tolerance has been renewed in the last few years as a possibility of intervention in human autoimmune diseases. An obstacle in this direction is that, although easily induced in animals virgin of contact with the antigen, oral tolerance becomes hard to induce in previously immunized animals. The present results show that there is an early period after primary immunization in which prolonged oral exposure to the antigen may arrest ongoing immune responses. Beyond this period, oral exposures to the antigen become ineffective and may actually boost immune responses. The end of the susceptible period coincides with the emergence of free specific antibodies in serum. However, the previous administration of purified anti-ovalbumin antibodies (40 µg) was unable to block the induction of oral tolerance to ovalbumin in normal mice

Modulation of Signaling Molecules by Human Leucocyte Antigen B27; Special Reference to STAT1

Reactive arthritis (ReA) is an inflammatory joint disease, which belongs to the group of Spondyloarthritis (SpA). It may occur after infections with certain gram-negative bacteria such as Salmonella and Yersinia. SpAs are strongly associated with the human leucocyte antigen (HLA)-B27. Despite active research, the mechanism by which HLA-B27 causes disease susceptibility is still unknown. However, HLA-B27 has a tendency to misfold during assembly. It is possible that the misfolding of HLA-B27 could alter signaling pathways and/or molecules involved in inflammatory response in cells. We have earlier discovered that in HLA-B27-positive cells the interaction between the host and causative bacteria is disturbed. Our recent studies indicate that the expression of HLA-B27 may alter certain signaling molecules by disturbing their activation. The aim of this study was to investigate whether the expression of HLA-B27 disturbs the signaling molecules, especially the phosphorylation of transcription factor STAT1. STAT1 is an important mediator of inflammatory responses. Our results show that the phosphorylation of the STAT1 is significantly altered in HLA-B27-expressing U937 monocytic cells compared with control cells. STAT1 tyrosine 701 is more strongly phosphorylated in HLAB27- expressing cells; whereas the phosphorylation of STAT1 serine 727 is prolonged. Phosphorylation of STAT1 was discovered to be dependent on protein kinase PKR. Furthermore, we found out that the expression of posttranscriptional gene regulator HuR was altered in HLA-B27-expressing cells. We also detected that HLA-B27-positive cells secrete more interleukin 6, which is an important mediator of inflammation. These results help to understand how HLA-B27 may confer susceptibility to SpAs.