Ultrastructural and histological characteristics of the equine endometrium at day 5 post ovulation.

Rapid endometrial adaptations occur with the embryo entering the uterus to create a receptive uterine environment, which is essential for the conceptus' development. The aim of this study was to demonstrate ultrastructural and histological changes of the endometrium at day 5 after ovulation in cyclic and inseminated mares. Mares were daily examined by transrectal palpation and ultrasonographic examination of the reproductive tract until ovulation was detected. In the first cycle, endometrial biopsies from 10 cyclic mares (Cyclic group) were collected on day 5 post-ovulation. In the second cycle, the same mares were inseminated with fresh semen from a fertile stallion (Inseminated group). Intrauterine biopsies were collected on day 5 post-ovulation, and according to sampling moment, inseminated mares were subdivided into two subgroups, those sampled at day 5-5.5 (n = 5) and those sampled at day 5.5-6 (n = 5). Biopsy samples were analyzed through scanning electron microscopy and light microscopy. Inseminated group presented an increase in glandular diameter, decrease in ciliated cell population, and an increase in lymphocyte population, compared to Cyclic group. No differences were observed between both experimental groups in number of micro-ciliated polygonal cells, percentage of flat or protruded cells in the epithelium, amount of secretion over the epithelium, glandular density, glandular luminal diameter, height of the glandular epithelium, amount of intraglandular secretion, blood vessel diameter and number of eosinophils and neutrophils. No differences in any of the variables were detected between subgroups from inseminated mares. These facts lead to the hypothesis that there is some sort of signaling to prepare and adapt the uterus to maintain pregnancy even before embryo arrival. There is also evidence to support an alternative hypothesis suggesting that all of the above mentioned are inflammatory events, resulting from a previous inflammation due to residual seminal effects. The results here presented lead to the conclusion that significant ultrastructural and histological changes of the endometrium occur on day 5 post ovulation in inseminated mares.

Proteomic profile of histotroph during early embryo development in mares.

There is a complex cascade involving proteins during early embryo development and maternal recognition, which is very important for maintenance of a conceptus. The aim of this study was to compare proteomic profile of uterine fluid after ovulation in pregnant and cyclic mares. In the first cycle, samples of uterine fluid of 30 cyclic mares were collected on days 7 (n = 10), 10 (n = 10) and 13 (n = 10) post ovulation and constituted the Cyclic group. In the second cycle, the same mares were bred to a fertile stallion. At days 7, 10 and 13 uterine fluid samples were collected. Immediately after sample collection, the mare's uteri were flushed, and those with an embryo recovered were assigned to the Pregnant group. Of the 30 mares flushed embryos were recovered from 6 mares on day 7, 6 on day 10 and 6 on day 13. Samples from the mares without embryo recovery were excluded from both groups. The uterine fluid samples were processed by two-dimensional electrophoresis technique followed by matrix assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry for the identification of relevant protein spots. From a total of 677 detected spots 19 were identified, 13 more abundant in Pregnant group and 6 in Cyclic group. In summary, pregnant and cyclic mares showed proteins with different abundance. Identified proteins were related to the transport of lipids through the embryo capsule, uterine motility, ATP generation, maternal immunological tolerance, cell proliferation, differentiation, metabolism and angiogenesis. Changes in the proteomic profile of uterine fluid during early embryo development in mares were related with the conceptus presence, suggesting that these alterations may be important for conceptus development and maternal recognition of pregnancy.

Ultrastructural and histological characteristics of the endometrium during early embryo development in mares.

The aim of this study was to evaluate ultrastructural and histological changes in the endometrium on days 7, 10 and 13 post-ovulation in pregnant and cyclic mares. Mares were routinely examined by transrectal palpation and ultrasonographic examination of the reproductive tract until estrus was detected. In the first cycle, endometrial biopsies from 30 cyclic mares (Cyclic group) were collected on days 7, 10 and 13 post-ovulation. In the second cycle, the same mares were bred by a fertile stallion. At days 7, 10 and 13 post-ovulation intrauterine biopsies were collected. Immediately after sample collection, the mare's uteri were flushed, and those mares with embryo recovery were assigned to the Pregnant group. From ovulation detection until day of uterine biopsy, blood samples to measure Progesterone concentrations were collected daily in cyclic and pregnant mares. A larger blood vessel caliber was observed in pregnant mares than in cyclic from day 7-13. On the 7th day of pregnancy a large loss of ciliated cells was evident in the group of pregnant mares in comparison with the Cyclic group and the superficial cells of the endometrium were more protruded, and a small amount of histotrophic material between the folds was observed. On the 10th day of pregnancy, the glandular histotrophic secretion and the secretion of luminal epithelium became more intense than the secretion of cyclic mares. On the 13th day of pregnancy, a very large amount of histotroph was observed within large glandular openings surrounded by ciliated cells. The concentrations of P4 were affected by day (P < 0.001), but were not affected by group. Changes occurred in the uterine environment thereupon the entry of the embryo into the uterus. In the stroma and in the lumen, these modifications may aid to provide the necessary nutrition for the initial development of the embryo and to promote changes at cellular structures that will interact in the embryonic signaling and future fixation, implantation and placentation.

The HLA-A, -B and -DRB1 polymorphism in a large dataset of South Brazil bone marrow donors from Rio Grande do Sul.

Human leukocyte antigen (HLA) genes are very informative in population genetics studies and their variability has been widely used to reconstruct the history of geographic and/or demographic expansions of human populations. The characterization of HLA diversity at the population level is also fundamental in clinical studies, particularly for bone marrow transplantation programs. In this study, we investigated the HLA molecular variation in Rio Grande do Sul, South Brazil, in order to identify possible regional differences across this state. More than 97,000 bone marrow donors were typed at the HLA- A, -B and -DRB1 loci and analyzed by considering two kinds of subdivisions based on both self-identified ethnicity and place of residence: (a) the official geographic subdivision defined by the Brazilian Institute of Geography and Statistics and (b) known information about the colonization history of the state. HLA allele and haplotype frequencies were estimated and compared among the defined subgroups. The results indicate a lack of correlation between genetic variation and geography and thus no clear HLA genetic structure based on geographic criteria. On the other hand, major differences were observed regarding ethnicity. In addition, local populations from Rio Grande do Sul were found to be genetically similar to their corresponding parental European populations from Germany, Italy and Portugal, as documented by historical data. Overall, this study provides a thorough characterization of the HLA genetic variation in Rio Grande do Sul and a better understanding of its demographic history, being most useful for the development of more efficient strategies in bone marrow donors' recruitment.

Human platelet antigen genotyping of platelet donors in southern Brazil.

Human platelet antigens (HPA) are immunogenic structures that result from single nucleotide polymorphisms (SNPs) leading to single amino acid substitutions. This study sought to determine the allele and genotype frequencies of HPA-1, HPA-2, HPA-3, HPA-4, HPA-5 and HPA-15 in platelet donors from the state of Rio Grande do Sul (RS), Brazil, and compare their allele frequencies to those observed in other populations. HPA genotyping was performed by PCR-SSP method. The study sample comprised 201 platelet donors (167 Caucasians and 34 non-Caucasians). Allele 'a' was that most commonly found for HPA-1 to 5 in both groups. The HPA-15ab genotype predominated over homozygous genotypes of this system. Fisher's exact test revealed statistically significant differences for the HPA-5 system, with a greater prevalence of the HPA-5b allele in non-Caucasians. The neighbour-joining method and principal components analysis revealed genetic proximity between our Caucasian group and European populations. We conclude that the allele frequencies of HPA-1 to 5 and HPA-15 found in our Caucasian sample are similar to those reported for European populations. These findings corroborate the ethnic makeup of the population of RS. The higher frequency of the HPA-5b allele found in the non-Caucasian group of our sample suggests the possibility of allosensitization in patients who receive platelet transfusions from genetically incompatible donors.

Proteomics of endometrial fluid after dexamethasone treatment in mares susceptible to endometritis.

Corticotherapy is a common treatment in mares susceptible to endometritis. Isoflupredone improves pregnancy rates and affects the protein profile of endometrial fluid in comparison to untreated mares. Dexamethasone decreases postbreeding fluid accumulation and uterine edema; however, its effects on the protein profile of the endometrial fluid have not yet been studied. The aim of the present study was to verify the effect of dexamethasone on the protein profile of endometrial fluid, in the presence or absence of infection, from mares susceptible to persistent postbreeding endometritis. Nine susceptible mares aged between 7 and 18 years were used. After checking for signs of estrus, mares were subjected to four treatments: C: mares received no treatment and served as control; D: mares received 40-mg dexamethasone at breeding, with collection of samples after 6 hours; I-6 and I-24: intrauterine infusion of 1 × 10(9)Streptococcus zooepidemicus/mL and samples collected after 6 and 24 hours; I/D-6 and I/D-24: intrauterine infusion of 1 × 10(9)S zooepidemicus/mL and 40-mg dexamethasone, collecting the sample after 6 and 24 hours. All mares were subjected to all treatments. Samples were collected and subjected to two-dimensional electrophoresis and mass spectrometry for the identification of relevant protein spots. Corticotherapy altered the protein profile of the endometrial fluid of susceptible mares, characterized by an increase and/or decrease in the optical density of inflammatory acute-phase proteins. We conclude that the use of dexamethasone in mares with and without infection alters the protein profile of endometrial fluid of susceptible mares.

Recombinant expression and purification of the bovine acidic Seminal Fluid Protein

The acidic Seminal Fluid Protein (aSFP), a 12.9 kDa protein is a maker for bovine semen freezability possibly due to its antioxidant activity and effect on sperm mitochondrial function. However, its precise function on sperm preservation during freezing thaw is poorly understood. The use of recombinant DNA technology allows new approaches on the study of function and structure of proteins, and its production in procaryote systems offers several advantages. The present work describes the recombinant expression of the bovine aSFP and its binding properties. A cDNA library from the bovine seminal vesicle was used as template for amplification of the aSFP coding region. The amplicon was cloned into a pET23a (+) vector and transformed into E.coli BL21 pLysS strain. The recombinant expression was obtained in E coli. One step ion immobilized affinity chromatography was performed, resulting in high yield of purified protein. To determine the bioactivity of the r aSFP, the protein was incubated in different concentrations with 10 7 spermtozoa at 37°C for 5 h. Western blotting and fluorescence microscopy analyses showed the ability of the recombinant aSFP to attach to the spermatozoa. Based on our results, the described method can be used to obtain mg levels of recombinant aSFP.

DEFB1 gene 5' untranslated region (UTR) polymorphisms are marginally involved in inflammatory bowel disease in south Brazilians.

The possible association of three DEFB1 gene polymorphisms with susceptibility to develop ulcerative colitis (UC) and Crohn's disease (CD) was investigated in Brazilian patients and controls. Although a clear and strong association between functional 5'-UTR DEFB1 SNPs and susceptibility/protection to IBDs cannot be drawn, our results suggest a possible involvement of DEFB1 gene in inflammatory bowel diseases, especially with the colonic localization of Crohn's disease.

Interleukin-10 gene promoter and NFKB1 promoter insertion/deletion polymorphisms in systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrotic, immunological and vascular abnormalities. Nuclear factor-kB (NFKB), as a key transcription factor involved in the regulation of immune responses, appears to be a good candidate for studies on the pathogenesis of autoimmune diseases, as well as the interleukin-10 (IL-10) polymorphism, which other studies have suggested an association with SSc. Our objective was to study the association of NFKB and IL-10 gene polymorphisms with SSc. One hundred and fifty-one SSc patients and 147 healthy bone marrow donors were enrolled in a case-control study. Blood was collected for DNA extraction; typing of IL-10 genes was made by polymerase chain reaction with sequence-specific primers (PCR-SSP), and NFKB gene typing was made by restriction fragment length polymorphism (RFLP). Patients underwent clinical evaluation, serology, Doppler echocardiography and chest high-resolution computed tomography. The frequency of IL-10 (-1082) GG genotype was found to be significantly higher in SSc patients (36.4%) as compared to healthy controls (22.4%) (P = 0.012). The frequency of heterozygous genotype GA was significantly lower (P = 0.004) in patients (38.4%) in comparison with control subjects (55.8%). A predominance of the high-producing IL-10 phenotype (GCC(+) /GCC(+) ) was observed in SSc patients compared with healthy controls (37.7% versus 24.5%, respectively; OR: 1.87, 95% CI: 1.10-3.19, P = 0.019). No significant difference was found in the allelic and genotype distribution of the NFKB promoter polymorphism between patients and controls. No statistically significant associations were found between IL-10 or NFKB polymorphisms clinical and laboratory features of SSc. Our results confirmed the association of the high-producing phenotype (GCC(+) /GCC(+) ) with increased risk for SSc, but found no correlation with NFKB polymorphisms.

Analysis of KIR gene frequencies and HLA class I genotypes in prostate cancer and control group.

Prostate cancer is the second most common cancer in men, with a significant increase in incidence and mortality in men over 50 years of age. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer cell immunoglobulin-like receptors (KIR). The aim of our study is to evaluate the association between the KIR genes and HLA alleles in patients with prostate cancer and healthy controls. Two hundred patients with prostate cancer and 185 healthy controls were typed for HLA class I and KIR genes by PCR-SSP. When both groups were compared, no significant differences were found for HLA-C group 1 and group 2, HLA-Bw4, HLA-A3 and A11. No difference was seen either in KIR frequency between patients with prostate cancer and controls. In conclusion, our data suggest no potential role for the KIR gene system in prostate cancer.

Effect of corticotherapy on proteomics of endometrial fluid from mares susceptible to persistent postbreeding endometritis.

The objective was to determine the effects of corticotherapy, in the presence and absence of uterine inflammation, on proteomics of endometrial fluid from mares susceptible to endometritis. In 11 mares, estrus was induced seven times with 5 mg PGF(2α) given at 14-day intervals. The first estrus was a control (no treatment). During the third estrus, mares received glucocorticoid (GC) treatment (20 mg isoflupredone acetate) every 12 h, for three consecutive days. The fifth estrus was the Infected treatment (intrauterine infusion of 1 × 10(9) colony-forming unit/mL Streptococcus equi subspecies zooepidemicus). Finally, the seventh was a combination of GC + Infected treatment (infusion of bacteria 24 h after the first GC treatment). At 12 h after the end of each treatment, uterine samples were collected and submitted to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein separation and mass spectrometry. Both GC treatment and uterine lumen infection induced proteomic alterations in the endometrial fluid of susceptible mares, characterized by an increase, decrease, or both in the relative optic density and/or frequency of inflammatory acute phase proteins (APP), with major alterations occurring when corticotherapy was applied in the presence of an infectious process. Corticotherapy in the presence of infection increased α(1)-antitrypsin (AAT), transthyretin (TT), and actin, but reduced immunoglobulin G, whereas intrauterine infection increased haptoglobin (Hp) and apolipoprotein A-1 (ApoA-1) and decreased transferrin (TF). Infection reduced levels of α(1)-antitrypsin and transthyretin, whereas corticotherapy in the presence of infection increased their frequency. We concluded that GC influenced the immune response, not only as suppressors, but also as enhancers of local defense mechanisms, through an immunomodulatory action. Short-term corticotherapy could be beneficial for treatment of uterine infectious processes in the mare.

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their relation with semen freezability.

The objective was to evaluate protein profiles of equine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine whether any of these proteins were related to semen freezability. Seminal plasma was collected from 10 stallions, of high and low semen freezability, housed at the State Stud of Lower Saxony, and routinely used in AI programs. Twenty-five protein spots were identified from the two-dimensional gel (12%), seven of which were present in all samples (all proteins were identified by MALDI-MS). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. Of the 25 proteins identified, two spots had greater relative content (P < 0.05) in seminal plasma samples collected from stallions with high semen freezability: spot 5 (80-85 kDa, isoelectric point [pI] 7.54), identified as CRISP-3; and spot 45 (18.2 kDa, pI 5.0-5.2), identified as HSP-2. Conversely, protein content was greater (P < 0.05) in seminal plasma samples from stallions with low semen freezability: spot 7 (75.4 kDa, pI 6.9-7.4), identified as lactoferrin; spot 15 (26.7 kDa, pI 5.51), identified as kallikrein; spot 25 (25 kDa, pI 7.54), identified as CRISP-3; and spot 35 (13.9 kDa, pI 3.8-4.2), identified as HSP-1. In conclusion, there were differences in the seminal plasma protein profile from stallions with high and low semen freezability. Furthermore, CRISP-3 and HSP-2 were potential seminal plasma markers of high semen freezability.

Killer cell immunoglobulin-like receptor (KIR) genes in systemic sclerosis.

A previous study has suggested that the combination KIR2DS2(+)/KIR2DL2(-) was related to increased risk for systemic sclerosis (SSc), while others have failed to reproduce this finding. Our objective was to study this matter further and test the association of other KIR genes with SSc. One hundred and ten SSc patients and 115 healthy bone marrow donors were enrolled in a case-control study. Blood was collected for DNA extraction; typing of 15 KIR genes and human leucocyte antigen-C (HLA-C) was made by polymerase chain reaction with sequence specific primers (PCR-SSP), followed by electrophoresis on agarose gel. Patients underwent clinical evaluation, serology, Doppler echocardiography and chest high-resolution computed tomography. The frequency of the inhibitory KIR2DL2 was significantly lower in patients [29.1% versus 65.2% in controls, P < 0.0001; odds ratio (OR) = 0.22, 95% confidence interval 0.12-0.40]. When combinations of activating and inhibitory KIR genes were analysed, the presence of KIR2DS2 in the absence of KIR2DL2 (KIR2DS2(+)/KIR2DL2(-)) was more frequent in patients than in controls (25.5% versus 1.7%, respectively; P < 0.0001; OR = 19.29, 4.24-122.26). However, the presence of both KIR2DS2 and KIR2DL2 (KIR2DS2(+)/KIR2DL2(+)) was more frequent in controls (57.4%) than in patients (28.2%, P < 0.0001), suggesting a preponderant protective effect of KIR2DL2 over KIR2DS2. Stratification for HLA-C1 status did not change these results. No statistically significant associations were found between KIR phenotypes and clinical and laboratory features of SSc. Our results suggest a protective role of KIR2DL2(+) phenotype and confirmed the association of the combination KIR2DS2(+)/KIR2DL2(-) with increased risk for SSc.

Killer cell immunoglobulin-like receptor gene diversity in a Caucasian population of southern Brazil.

Killer immunoglobulin-like receptors (KIR) regulate the activity of natural killer and T cells through an interaction with specific human leucocyte antigen (HLA) class I molecules on target cells. Diversity in KIR gene content, KIR allelic and haplotype polymorphism has been observed between different ethnic groups. However, most population studies on KIR variability have focused on Europe and Asia, while Americas, Oceania and Africa remain poorly studied. The aim of this study was to analyse the variability of KIR genes in 200 healthy nonrelated individuals from the Southern Brazilian population. KIR genes and HLA-A, -B and -Cw were genotyped using polymerase chain reaction-sequence-specific primers. Southern Brazilian population demonstrated several similarities to states that are closer geographically and distinct differences with Northern Brazil in the frequency of genes KIR2DS1, 2DS2, 2DS3, 2DS5, 3DL1, 3DS1, 2DL1 and 2DL2. The activating gene KIR2DS5 was the least frequent locus found in our group. Interaction of KIR/HLA was more common in the 2DS1-/2DL1+/C2+ association. This study demonstrated the diversity of KIR genes and of KIR/HLA association in a Caucasian group of Southern Brazil, establishing differences and similarities to other different populations.

A study of the killer cell immunoglobulin-like receptor gene KIR2DS1 in a Caucasoid Brazilian population with psoriasis vulgaris.

Psoriasis is a chronic inflammatory skin disease whose pathogenesis and genetic background remain unclear. Considering that previous studies have suggested an association of psoriasis vulgaris (PV) and killer cell immunoglobulin-like receptors (KIRs), we typed 15 KIR genes and human leukocyte antigen (HLA)-Cw in 79 Brazilian Caucasoid patients with PV and 110 healthy controls by polymerase chain reaction (PCR) using sequence-specific oligonucleotides and sequence-specific primers. We did not observe a relevant increase in the frequency of the activating KIR2DS1 gene in the PV group [KIR2DS1, 46 of 79 cases (58.2%) vs 40 of 110 controls (36.4%)]. However, an association of KIR2DS1 with Cw*0602+ in 26.5% of PV patients was observed, while it was present in only 5.4% of controls. These results suggest that activating KIR2DS1 gene may not confer susceptibility to PV, and an association of KIR2DS1 gene with the HLA-Cw*0602+ was observed in these patients.

BSP A1/A2-like proteins in ram seminal plasma.

The objective of this study was to assess the protein profile of ovine seminal plasma using 2D-PAGE and verify if BSP A1/A2 are present in ovine seminal plasma. Seminal plasma was collected from three mature rams and pooled to eliminate individual differences. Seminal plasma samples were submitted to 2D-PAGE using 12% acrylamide gels. The image analysis software identified 21 protein spots on the air-dried gel, with molecular weight ranging from 15 to 115 kDa and pI 3.2 to 8.7. The most prominent spots were those <30 kDa. The most intensely stained spots were: 3 (18-19 kDa, pI 4.8-5.0), 5 (17-18 kDa, pI 5.0-5.2), 7 (15-16 kDa, pI 6.2-6.4), and 23 (105-108 kDa, pI 6.8-7.0). Three of these spots (spots 3, 5 and 7, respectively) accounted for 41.1% of the relative intensity of the spots of the gels, based on the intensity of the Comassie blue staining. Western blot analysis indicated that spots 3 and 5 were similar to BSP A1/A2 (16.5, pI 4.7-5.0 and 16 kDa, pI 4.9-5.2) identified in Manjunath's studies [Manjunath P, Sairam MR. Purification and biochimical characterization of three major acid proteins (BSP A1, BSP A2 and BSP A3) from bovine seminal plasma. Biochem J 7 (1987) 685-92.], based on the specific reaction of the polyclonal antibody to those spots.

Two-dimensional polyacrylamide gel electrophoresis of bovine seminal plasma proteins and their relation with semen freezability.

The objective of this study was to evaluate the low weight (10-30 kDa) protein profile of bovine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine if any of these proteins was associated with semen freezability. Seminal plasma was collected from 16 bulls of high or low semen freezability. Twelve protein spots were identified from the 2D gel (15%); six of these were present in all samples. Of the 12 proteins found, three spots, present in all samples, 3 (15-16 kDa), 5 (16-17 kDa), and 7 (10-12 kDa) had nonsignificant variation among bulls, regardless of their freezability classification. Four proteins were more abundant (P<0.05) in seminal plasma samples collected from bulls with high semen freezability than in samples of bulls with low semen freezability: the spots 3 (15-16 kDa, pI 4.7-5.2), 7 (11-12 kDa, pI 4.8-4.9), 11 (13-14 kDa, pI 4.0-4.5), and 23 (20-22 kDa, pI 4.8-5.2). On the other hand, spot 25 (25-26 kDa, pI 6.0-6.5) was more abundant (P<0.05) on seminal plasma samples from bulls with low semen freezability. The N-terminus sequence of protein 7 was identical to the acidic seminal fluid protein (aSFP). Protein 23 (after trypsin digestion) had structural similarity to bovine clusterin. We concluded that there were differences in the seminal plasma protein profile from bulls with low and high semen freezability; aSFP, clusterin, proteins 3 and 11 may be used as semen freezability markers; and protein 25 was related to low semen freezability.