Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules

In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24 h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured it, TCM plus FSH and 10% estrous cow serum. After fertilization. presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P < 0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless. there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis. (C) 2009 Elsevier Inc. All rights reserved.

Expression of transcription factors NF-kappa B and AP-1 in nasal polyposis

Background The treatment and prognosis of nasal polyposis (NP) may be influenced by transcription factors, but their expression is poorly understood. Objective To determine the expression of transcription factors [(nuclear factor-kappa B) NF-kappa B and (activator protein) AP-1], cytokines [IL-1 beta, TNF-alpha and (granulocytes and macrophage colony-stimulating factor) GM-CSF], growth factor (b-FGF), chemokine (eotaxin-2) and adhesion molecule (ICAM-1) in NP in comparison with nasal mucosa controls. Methods Cross-sectional study. Twenty biopsies of nasal polyps were compared with eight middle turbinate biopsies. p65, c-Fos, IL-1 beta, TNF-alpha, ICAM-1, b-FGF, eotaxin-2 and GM-CSF were analysed through RQ-PCR, and p65 and c-Fos were also analysed through Western blotting. Results NF-kappa B expression was increased in patients with NP when compared with control mucosa (P < 0.05), whereas AP-1 expression did not differ significantly between groups. Expressions of IL-1 beta, eotaxin-2 and b-FGF were also increased in patients with NP compared with controls (P < 0.05). Conclusions The transcription factor NF-kappa B is more expressed in NP than in control mucosa. This is important in NP because NF-kappa B can induce the transcription of cytokines, chemokines and adhesion molecules, which play an important role in the inflammatory process. Moreover, transcription factors influence the response to corticosteroids, which are the basis of NP treatment. Transcription factor AP-1 does not seem to have a significant role in the pathological process.

Interferon-gamma, interleukin-10, intercellular adhesion molecule-1, and chemokine receptor 5, but not interleukin-4, attenuate the development of periapical lesions

This study examines the role of Th1 (interferon-gamma [IFN-gamma]) and Th2 (interleukin-4 [IL-4] and IL-10) cytokines, an intercellular adhesion molecule (ICAM-1), and a chemokine receptor (CCR5) in the pathogenesis of periapical lesions at different stages of development in knockout mice. For lesion induction, the first molar was opened and inoculated with 4 bacterial strains and left open to the oral environment. After 21 and 42 days, the IFN-gamma, IL-10, ICAM-1, and CCR5 knockout animals presented periapical lesions larger than those of wild-type animals. There was no statistically significant difference between periapical lesions induced in IL-4 knockout and wild-type animals during the periods evaluated. Our findings suggest an important role for IFN-gamma, IL-10, ICAM-1, and CCR5 in the pathogenesis of experimentally induced pulp infection and bone destruction as endogenous suppressor of periapical lesion development, whereas IL-4 appears to present a nonsignificant effect on periapical lesion modulation.

alpha 1-acid glycoprotein and alpha 1-antitrypsin as early markers of treatment response in patients receiving the intensive phase of tuberculosis therapy

The identification of early markers that predict the response to anti-tuberculosis treatment would facilitate evaluation of new drugs and improve patient management. This study aimed to determine whether selected acute phase proteins and micronutrients measured at the time of diagnosis and during the first weeks of treatment could predict treatment responses during the 2-month standard intensive phase of therapy. For this purpose, alpha 1-antitrypsin, alpha 1-acid gtycoprotein, alpha 2-macroglobutin, C-reactive protein, C3, C4, zinc, copper and selenium concentrations were measured in Brazilian patients with smear-positive tuberculosis at the time of diagnosis and 1, 3, 5 and 8 weeks after initiation of therapy. Patients were classified into fast (n = 29), intermediate (n = 18) and slow responders (n = 10) if they were smear-negative at 3, 5 or 8 weeks of treatment. alpha 1-acid gtycoprotein on enrolment and 1 week of treatment, alpha 1-antitrypsin at week 1 and C-reactive protein and C3 after 3 weeks of therapy were higher in slow responders than in fast responders. alpha 1-antitrypsin and alpha 1-acid glycoprotein may be helpful in predicting treatment response at the time of initiation of therapy, and could be used as early markers to identify patients with an increased likelihood of treatment failure. (C) 2008 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Development of chronic cardiomyopathy in canine Chagas disease correlates with high IFN-gamma, TNF-alpha, and low IL-10 production during the acute infection phase

When infected with Trypanosoma cruzi, Beagle dogs develop symptoms similar to those of Chagas disease in human beings, and could be an important experimental model for a better understanding of the immunopathogenic mechanisms involved in chronic chagasic infection. This study evaluates IL-10, IFN-gamma and TNF-alpha production in the sera, culture supernatant, heart and cervical lymph nodes and their correlation with cardiomegaly, cardiac inflammation and fibrosis in Beagle dogs infected with T. cruzi. Pathological analysis showed severe splenomegaly, lymphadenopathy and myocarditis in all infected dogs during the acute phase of the disease, with cardiomegaly, inflammation and fibrosis observed in 83% of the animals infected by T. cruzi during the chronic phase. The data indicate that infected animals producing IL-10 in the heart during the chronic phase and showing high IL-10 production in the culture supernatant and serum during the acute phase had lower cardiac alterations (myocarditis, fibrosis and cardiomegaly) than those with high IFN-gamma and TNF-alpha levels. These animals produced low IL-10 levels in the culture supernatant and serum during the acute phase and did not produce IL-10 in the heart during the chronic phase of the disease. Our findings showed that Beagle dogs are a good model for studying the immunopathogenic mechanism of Chagas disease, since they reproduce the clinical and immunological findings described in chagasic patients. The data suggest that the development of the chronic cardiac form of the disease is related to a strong Th1 response during the acute phase of the disease, while the development of the indeterminate form results from a blend of Th1 and Th2 responses soon after infection, suggesting that the acute phase immune response is important for the genesis of chronic cardiac lesions. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Tunicamycin inhibition of N-glycosylation of alpha-glucosidase from Aspergillus niveus: partial influence on biochemical properties

Treatment of Aspergillus niveus with 30 mu g tunicamycin/ml did not interfere with alpha-glucosidase production, secretion, or its catalytic properties. Fully- and under-glycosylated forms of the enzyme had similar molecular masses, similar to 56 kDa. Moreover, the absence of N-glycans did not affect either pH optimum (6.0) or temperature optimum (65A degrees C). The K(m) and V(max) values of under- and fully-glycosylated forms of alpha-glucosidase were similar when assessed for hydrolysis of starch (similar to 0.6 mg/ml, similar to 350 mu mol glucose per min per ml), maltose (similar to 0.54 mu mol, similar to 330 mu mol glucose per min per ml) and p-nitrophenyl-alpha-d-glucopyranoside (similar to 0.54 mu mol, similar to 8.28 mu mol p-nitrophenol per min per ml). However, the under-glycosylated form was sensitive to high temperatures probably because, in addition to stabilizing the protein conformation, glycosylation may also prevent unfolded or partially folded proteins from aggregating. Binding assays clearly showed that the under-glycosylated protein did not bind to concanavalin A but has conserve its jacalin-binding property, suggesting that only O-glycans might be intact on the tunicamycin treated form of the enzyme.

13,14-Dihydro-15-Keto Prostaglandin F(2)alpha Release in Response to Oxytocin Challenge Early Post-Partum in Anoestrous Nelore Cows Submitted to Temporary Calf Removal and Progesterone Priming

The study evaluated, in early post-partum anoestrous Nelore cows, if the increase in plasma oestradiol (E2) concentrations in the pre-ovulatory period and/or progesterone priming (P4 priming) preceding ovulation, induced by hormonal treatment, reduces the endogenous release of prostaglandin PGF(2)alpha and prevents premature lysis of the corpus luteum (CL). Nelore cows were subjected to temporary calf removal for 48 h and divided into two groups: GPE/eCG group (n = 10) and GPG/eCG group (n = 10). Animals of the GPE/eCG group were treated with a GnRH agonist. Seven days later, they received 400 ID of eCG, immediately after PGF(2)alpha treatment, and on day 0, 1.0 mg of oestradiol benzoate (EB). Cows of the GPG/eCG group were similarly treated as those of the GPE/eCG group, except that EB was replaced with a second dose of GnRH. All animals were challenged with oxytocin (OT) 9, 12, 15 and 18 days after EB or GnRH administration and blood samples were collected before and 30 min after OT. Irrespective of the treatments, a decline in P4 concentration on day 18 was observed for cows without P4 priming. However, animals exposed to P4 priming, treated with EB maintained high P4 concentrations (8.8 +/- 1.2 ng/ml), whereas there was a decline in P4 on day 18 (2.1 +/- 1.0 ng/ml) for cows that received GnRH to induce ovulation (p < 0.01). Production of 13,14-dihydro-15-keto prostaglandin F(2)alpha (PGFM) in response to OT increased between days 9 and 18 (p < 0.01), and this increase tended to be more evident in animals not exposed to P4 priming (p < 0.06). In conclusion, the increase in E2 during the pre-ovulatory period was not effective in inhibiting PGFM release, which was lower in P4-primed than in non-primed animals. Treatment with EB promoted the maintenance of elevated P4 concentrations 18 days after ovulation in P4-primed animals, indicating a possible beneficial effect of hormone protocols containing EB in animals with P4 priming.

Elevated progesterone concentrations enhance prostaglandin F(2 alpha) synthesis in dairy cows

The objective was to evaluate the influence of varying plasma progesterone (P(4)) concentrations throughout the luteal phase in dairy cows on PGF(2 alpha) production (assessed as plasma concentrations of 13,14-dihydro-15-keto-PGF(2 alpha); PGFM) following treatment with estradiol-17 beta (E(2)) or oxytocin (OT). In all experiments, time of ovulations was synchronized with the OvSynch protocol and Day 0 corresponded to day of second GnRH injection. In Experiment 1, non-lactating dairy cows on Day 6 remained non-treated (n = 9), received 20 mg LH (n = 7), or had ovarian follicles larger than 6 mm aspirated (n = 8). In Experiment 2, cows on Day 6 were untreated (n = 9) or received 5000 IU hCG (n = 10). In Experiments 1 and 2, all cows received 3 mg E(2) on Day 17, and blood samples were collected every 30 min from 2h before to 10h after E(2). Experiment 3 was conducted in two periods, each from Days 0 to 17 of the estrous cycle. At the end of Period 1, animals switched treatments in a crossover arrangement. Animals in Group 2/8 (n = 4) received 2 kg/d of concentrate in the first period and 8 kg/d in the second period. Animals in Group 8/2 (n = 7) received the alternate sequence. Blood was collected daily for measurement Of P(4) 4 h after concentrate feeding. On Day 17, blood was collected from 1 h before to 1 h after a 100 IU OT injection. In Experiment 1, both plasma P(4) and release Of PGF(2 alpha) were similar between LH-treated and control cows (P > 0.10). In Experiment 2, plasma P4 was elevated to a greater extent on Day 17 in cows treated with hCG (P < 0.05) and plasma PGFM was also greater in hCG-treated animals (treatment x time interaction; P < 0.05). In Experiment 3, there was a group x period interaction (P < 0.01) for plasma P(4), indicating that less concentrate feeding was associated with greater plasma P(4). Release of PGF(2 alpha) in response to OT was greater for cows receiving less concentrate (group x period interaction; P < 0.05). In conclusion, dairy cows with more elevated blood P(4) concentrations released more PGF(2 alpha) in response to E(2) or OT. (c) 2008 Elsevier B.V. All rights reserved.

Sequence variation of the alpha-lactalbumin gene in Holstein and Nellore cows

The alpha-lactalbumin is a subunit of lactose-synthase, an enzyme responsible for lactose production, a disaccharide that influences milk production. Sequence variations of bovine alpha-lactalbumin have been associated with differences in milk yield. This study aimed to analyze allelic frequency differences at position-1689 (g. AG) and+15 (g. AG) of the alpha-lactalbumin gene in Holstein (Bos taurus) and Nellore (Bos indicus) cows. Blood samples were analyzed from 34 Holstein, 104 Nellore, and 99 Dairy Nellore cows using PCR-RFLP. The different RFLP patterns were sequenced and a novel sequence variation on nucleotide-46 was identified. An adenine at this position was designated as the A allele and a guanine was designated B allele. The frequencies of alleles A-1689, A-46, and A+15 differed between Holstein and both Nellore breeds. The results show that differences in alpha-lactalbumin allelic variants in the 5`-flanking and the 5`-UTR region might be associated with differences in milk production between Holstein cows and cows from Nellore breeds. However, the lack of difference between Nellore and Dairy Nellore suggests that other sequence variantions that regulate milk production might be responsible for the selection of Dairy Nellore cows with superior milk production.

Effects of Ethanol on Synthesis of Prostaglandin F2 alpha in Bovine Females

Ethanol stimulates the production of prostaglandins in many species. The purpose of this study was to verify the effect of ethanol on the production of prostaglandin F2 alpha (PGF2 alpha) and luteolysis in bovine females. In the first experiment, Holstein cows at day 17 of the oestrous cycle were treated with 100% ethanol (0.05 ml/kg of body weight, IV; n = 5), saline (0.05 ml/kg of body weight, IV; n = 4) or synthetic prostaglandin (150 mu g of D-cloprostenol/cow, IM; n = 4). The plasma concentrations of 13, 14-dihydro-15-keto PGF2 alpha (PGFM; the main metabolite of PGF2 alpha measured in the peripheral blood) were assessed by radioimmunoassay (RIA). There was an acute release of PGFM in response to ethanol comparing to other treatments (p <= 0.05). However, only cows treated with PGF2 alpha underwent luteolysis. In the second experiment, endometrial explants of cross-bred beef cows (n = 4) slaughtered at day 17 of the oestrous cycle were cultured for 4 h. During the last 3 h, the explants were cultured with medium supplemented with 0, 0.1, I, 10 or 100 mu l of 100% ethanol/ml. Medium samples were collected at hours 1 and 4 and concentrations of PGF2 alpha were measured by RIA. Ethanol did not induce PGF2 alpha production by the endometrium. In conclusion, ethanol does not cause luteolysis in cows because it stimulates production of PGF2 alpha in extra-endometrial tissues.

Alpha lipoic acid in burning mouth syndrome - a randomized double-blind placebo-controlled trial

The burning mouth syndrome (BMS) is a chronic condition characterized by oral burning pain in the absence of clinical abnormalities and without established therapy. The purpose of this study was to evaluate the effectiveness of alpha lipoic acid (ALA) in the management of BMS symptoms through a randomized double-blind placebo-controlled trial. Thirty-eight patients (34 women and four men, median age 62.9 years, range 36-78) were included and 31 completed the study. The patients were randomized into two cycles of treatment: one with alpha lipoic acid and one with placebo both administered in identical capsules. These cycles were separated by a washout period of 20 days. The oral symptoms and the treatment response were assessed using a 100-mm visual analog scale before and after each cycle and the global perceived effect score, using a 5-point scale after each treatment cycle. The level of reduction on burning was significant for both treatments (paired t-test: P < 0.05; rp = 0.011; ral < 0.001). Considering the two cycles together, 22 patients reported at least some improvement after ALA use and 23 patients after placebo. Comparison of the oral assessment scores of the two cycles failed to demonstrate the effectiveness of ALA over placebo (t-test: P > 0.05; r = 0.75).

Chemokines and chemokine receptors coordinate the inflammatory immune response in human cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) includes different clinical manifestations displaying diverse intensities of dermal Inflammatory infiltrate Diffuse CL (DCL) cases are hyporesponsive and lesions show very few lymphocytes and a predominance of macrophages In contrast localized CL (LCL) cases are responsive to leishmanial antigen and lesions exhibit granulocytes and mononuclear cell infiltration in the early phases changing to a pattern with numerous lymphocytes and macrophages later in the lesion Therefore different chemokines may affect the predominance of cell infiltration in distinct clinical manifestations In lesions from LCL patients we examined by flow cytometry the presence of different chemokines and their receptors in T cells and we verified a higher expression of CXCR3 in the early stages of LCL (less than 30 days of infection) and a higher expression of CCR4 in the late stages of disease (more than 60 days of infection) We also observed a higher frequency of T cells producing IL-10 in the late stage of LCL Using immunohistochemistry we observed a higher expression of CCL7 CCL17 in lesions from late LCL as well as CCR4 suggesting a preferential recruitment of regulatory T cells in the late LCL Comparing lesions from LCL and DCL patients we observed a higher frequency of CCL7 in DCL lesions These results point out the Importance of the chemokines defining the different types of cells recruited to the site of the infection which could be related to the outcome of infection as well as the clinical form observed (C) 2010 American Society for Histocompatibility and Immunogenetics Published by Elsevier Inc All rights reserved

The Role of Toll-Like Receptor 2 in the Recognition of Aggregatibacter actinomycetemcomitans

Background: Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) is a Gram-negative bacterium present in the oral cavity and is usually associated with localized aggressive periodontitis. Isolated antigens from A. actinomycetemcomitans can activate innate immune cells through Toll-like receptors (TLRs), which are molecules that recognize structural components conserved among microorganisms. In this study, we evaluate the role of TLR2 in the recognition of A. actinomycetemcomitans. Methods: Macrophages and neutrophils from knockout mice with targeted disruption of TLR2 (TLR2(-/-) mice) and wild-type mice were collected and used for the subsequent assays. The production of cytokines and chemokines was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of apoptotic cells was determined by flow cytometry. In addition, the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR2(-/-) mice were examined. Results: The results show that TLR2-deficient mice developed more severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly higher bone loss and inflammatory cell migration to periodontal tissues. The inflammatory cell influx into the peritoneal cavities of TLR2(-/-) mice was three-fold lower than that observed for the littermate controls. A significantly diminished production of the cytokines tumor necrosis factor-alpha and interleukin-1 beta as well as the chemokine CC-ligand-5 in the peritoneal cavities of TLR2(-/-) mice was observed. In addition, a high frequency of apoptotic cells in the inflammatory exudates from TLR2(-/-) mice was observed. Phagocytosis and nitric oxide production was diminished in cells from TLR2(-/-) mice, facilitating the dissemination of the pathogen to the spleen. Conclusion: The results of this study highlight the involvement of TLR2 in recognizing A. actinomycetemcomitans and its essential role in controlling A. actinomycetemcomitans infection. J Periodontot 2009,80:2070-2019.

Differential Production of Macrophage Inflammatory Protein-1 alpha, Stromal-Derived Factor-1, and IL-6 by Human Cultured Periodontal Ligament and Gingival Fibroblasts Challenged With Lipopolysaccharide From P. gingivalis

Background: Fibroblasts are considered important cells in periodontitis. When challenged by different agents, they respond through the release of cytokines that participate in the inflammatory process. The aim of this study is to evaluate and compare the expression and production of macrophage inflammatory protein (MIP)-1 alpha, stromal-derived factor (SDF)-1, and interleukin (IL)-6 by human cultured periodontal ligament and gingival fibroblasts challenged with lipopolysaccharide (LPS) from Porphyromonas gingivalis. Methods: Fibroblasts were cultured from biopsies of gingival tissue and periodontal ligament of the same donors and used on the fourth passage. After confluence in 24-well plates, the culture medium alone (control) or with 0.1 to 10 mu g/ml of LPS from P. gingivalis was added to the wells, and after 1, 6, and 24 hours, the supernatant and the cells were collected and analyzed by enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively. Results: MIP-1 alpha, SDF-1, and IL-6 protein production was significantly greater in gingival fibroblasts compared to periodontal ligament fibroblasts. IL-6 was upregulated in a time-dependent manner, mainly in gingival fibroblasts (P<0.05), which secreted more MIP-1 alpha in the lowest concentration of LPS used (0.1 mu g/ml). In contrast, a basal production of SDF-1 that was inhibited with the increase of LPS concentration was detected, especially after 24 hours (P<0.05). Conclusion: The distinct ability of the gingival and periodontal ligament fibroblasts to secrete MIP-1 alpha, SDF-1, and IL-6 emphasizes that these cells may differently contribute to the balance of cytokines in the LPS-challenged periodontium. J Periodontol 2010;81:310-317.

Alpha-oestrogen and progestin receptor expression in the hypothalamus and preoptic area dopaminergic neurones during oestrous in cycling rats

A secretory surge of prolactin occurs on the afternoon of oestrous in cycling rats. Although prolactin is regulated by ovarian steroids, plasma oestradiol and progesterone levels do not vary during oestrous. Because prolactin release is tonically inhibited by hypothalamic dopamine and modulated by dopamine transmission in the preoptic area (POA), the present study aimed to evaluate whether oestrogen receptor (ER)-alpha and progestin receptor (PR) expression in the dopaminergic neurones of arcuate (ARC), periventricular, anteroventral periventricular (AVPe) and ventromedial preoptic (VMPO) nuclei changes during the day of oestrous. Cycling rats were perfused every 2 h from 10-20 h on oestrous. Brain sections were double-labelled to ER alpha or PR and tyrosine hydroxylase (TH). The number of TH-immunoreactive (ir) neurones did not vary significantly in any area evaluated. ER alpha expression in TH-ir neurones increased at 14 and 16 h in the rostral-ARC and dorsomedial-ARC, 14 h in the caudal-ARC and 16 h in the VMPO, whereas it was unaltered in the ventrolateral-ARC, periventricular and AVPe. PR expression in TH-ir neurones of the periventricular and rostral, dorsomedial, ventrolateral and caudal-ARC decreased transitorily during the afternoon, showing the lowest levels between 14 and 16 h; but it did not vary in the AVPe and VMPO. Plasma oestradiol and progesterone concentrations were low and unaltered during oestrous, indicating that the changes in receptors expression were probably not due to variation in ligand levels. Thus, our data suggest that variations in ER alpha and PR expression may promote changes in the activity of medial basal hypothalamus and POA dopaminergic neurones, even under unaltered secretion of ovarian steroids, which could facilitate the occurrence and modulate the magnitude of the prolactin surge on oestrous.