Polymorphism in the lymphotoxin-alpha gene, position +252 (rs909253), is not associated with preeclampsia development in Brazilian women

PURPOSE: To investigate the frequencies of polymorphic allele and genotypes for the LT-α gene, position +252 (rs909253), in Brazilian women with preeclampsia.METHODS: This is a case-control study, in which 30 women with preeclampsia, classified according to the criteria of the National High Blood Pressure Education Program, and 115 women in the control group, with at least two healthy pregnancies, were selected. Peripheral blood was collected, and DNA was extracted, followed by genotyping, using specific primers and restriction analysis. The genotypes obtained were AA, AG and GG. Statistical analysis was performed using the χ2association test. The Hardy-Weinberg Equilibrium was tested using the Haploview Program.RESULTS: The results showed no association between genotypes and preeclampsia development (χ2=2.0; p=0.4). When the AG and GG genotypes were grouped according to allele G presence or absence (genotype AA), the data showed that the presence of allele G was not significantly different between cases (women with preeclampsia) and controls (χ2=0.0; p=1.0). The LT-α gene polymorphism, position +252 (rs909253), seems not to be an important candidate for the development of preeclampsia. Other inflammatory genes should be researched, and studies involving gene-environment interactions should be performed, in order to reach a better understanding of the etiology of the preeclampsia.

Effect of medroxy-progesterone acetate on follicular growth and endometrial cycloxygenase-2 (COX-2) expression during the bovine estrous cycle

The objective of this study was to evaluate the effect of medroxy-progesterone acetate (MAP) with or without estradiol benzoate (EB) on follicular growth during the estrous cycle in cattle. In the first experiment, Hereford cows were synchronized with a synthetic analogue of PGF2 alpha and were treated with two different doses of MAP (250 or 500 mg) with or without EB for 7 days starting on day 8 of the estrous cycle. Follicular growth was inhibited (P<0.05) in all cows except controls and those receiving 250mg MAP without EB. Seventy-five percent of the animals (15/20) showed estrus on days 21 and 22 of the cycle rather than at MAP withdrawal, demonstrating that these treatments did not induce estrus. To determine whether the EB treatment altered endometrial sensitivity to oxytocin and thus the luteolytic cascade, multiparous pre-synchronized cows received 5 mg of EB followed 6 hours later with 50 IU of oxytocin (OT; n=9). Eight hours after EB injection, endometrial fragments were collected from the cows on days 4, 13 and 17 of the estrous cycle and COX-2 gene expression was measured by PCR. EB increased COX-2 mRNA levels only on day 17 of the estrous cycle (P<0.05). In conclusion, MAP alone or associated with EB is able to suppress bovine follicular growth. However, EB in the presence of MAP is not efficient to induce luteolysis in cows when injected on day 8 of the estrous cycle.

Muscarinic toxins : characterization of adrenoceptor binding and applicability of membrane anchoring via glycosylphosphatidylinositol tail

Adrenoceptors (ARs), G-protein coupled receptors (GPCRs) at the plasma membrane, respond to endogenous catecholamines noradrenaline and adrenaline. These receptors mediate several important physiological functions being especially important in the cardiovascular system and in the regulation of smooth muscle contraction. Impairments in the function of these receptors can thus lead to severe diseases and disorders such as to cardiovascular diseases and benign prostatic hyperplasia. The Eastern green mamba (Dendroaspis angusticeps) venom has been shown to contain toxins that can antagonize the functions of GPCRs. The most well-known are muscarinic toxins (MTs) targeting muscarinic acetylcholine receptors (mAChRs) with high affinity and selectivity. However, some reports have indicated that these toxins might also act on the α1- and α2-ARs which can be divided into various subtypes; the α1-ARs to α1A-, α1B- and α1D-ARs and α2-ARs to α2A-, α2B- and α2C-ARs. In this thesis, the interaction of four common MTs (MT1, MT3, MT7 and MTα) with the adrenoceptors was characterized. It was also evaluated whether these toxins could be anchored to the plasma membrane via glycosylphosphatidylinositol (GPI) tail. Results of this thesis reveal that muscarinic toxins are targeting several α-adrenoceptor subtypes in addition to their previously identified target receptors, mAChRs. MTα was found to interact with high affinity and selectivity with the α2B-AR whereas MT7 confirmed its selectivity for the M1 mAChR. Unlike MTα and MT7, MT1 and MT3 have a broad range of target receptors among the α-ARs. All the MTs characterized were found to behave as non-competitive antagonists of receptor action. The interaction between MTα and the α2B-AR was studied more closely and it was observed that the second extracellular loop of the receptor functions as a structural entity enabling toxin binding. The binding of MTα to the α2B-AR appears to be rather complex and probably involves dimerized receptor. Anchoring MTs to the plasma membrane did not interfere with their pharmacological profile; all the GPI-anchored toxins created retained their ability to block their target receptors. This thesis shows that muscarinic toxins are able to target several subtypes of α-ARs and mAChRs. These toxins offer thus a possibility to create new subtype specific ligands for the α-AR subtypes. Membrane anchored MTs on the other hand could be used to block α-AR and mAChR actions in disease conditions such as in hypertension and in gastrointestinal and urinary bladder disorders in a cell-specific manner and to study the physiological functions of ARs and mAChRs in vivo in model organisms.

High prevalence of alpha-thalassemia among individuals with microcytosis and hypochromia without anemia

In order to determine the contribution of alpha-thalassemia to microcytosis and hypochromia, 339 adult outpatients seen at Unicamp University Hospital (with the exception of the Clinical Hematology outpatient clinics), who showed normal hemoglobin (Hb) levels and reduced mean corpuscular volume and mean corpuscular hemoglobin, were analyzed. Ninety-eight were Blacks (28.9%) and 241 were Caucasians (71.1%). In all cases, Hb A2 and F levels were either normal or low. The most common deletional and nondeletional forms of alpha-thalassemia [-alpha3.7, -alpha4.2, --MED, -(alpha)20.5, alphaHphIalpha, alphaNcoIalpha, aaNcoI and alphaTSAUDI] were investigated by PCR and restriction enzyme analyses. A total of 169 individuals (49.9%) presented alpha-thalassemia: 145 (42.8%) were heterozygous for the -alpha3.7 deletion (-alpha3.7/aa) and 18 (5.3%) homozygous (-alpha3.7/-alpha3.7), 5 (1.5%) were heterozygous for the nondeletional form alphaHphIalpha (alphaHphIalpha/aa), and 1 (0.3%) was a --MED carrier (--MED/aa). Among the Blacks, 56 (57.1%) showed the -alpha3.7/aa genotype, whereas 12 (12.2%) were -alpha3.7/-alpha3.7 and 1 (1.0%) was an alphaHphIalpha carrier; among the Caucasians, 89 (36.9%) were -alpha3.7/aa, 6 (2.5%) had the -alpha3.7/-alpha3.7 genotype, 4 (1.7%) presented the nondeletional form (alphaHphIalpha/aa), and 1 (0.4%) was a --MED carrier. These results demonstrate that alpha-thalassemia, mainly through the -alpha3.7 deletion, is an important cause of microcytosis and hypochromia in individuals without anemia. These data are of clinical relevance since these hematological alterations are often interpreted as indicators of iron deficiency.

alpha-Smooth muscle actin and proliferating cell nuclear antigen expression in focal segmental glomerulosclerosis: functional and structural parameters of renal disease progression

The aim of the present study was to investigate the expression of alpha-smooth muscle actin (alpha-SM-actin) and proliferating cell nuclear antigen (PCNA) in renal cortex from patients with focal segmental glomerulosclerosis (FSGS) and their correlations with parameters of renal disease progression. We analyzed renal biopsies from 41 patients with idiopathic FSGS and from 14 control individuals. The alpha-SM-actin immunoreaction was evaluated using a score that reflected the changes in the extent and intensity of staining in the glomerular or cortical area. The PCNA reaction was quantified by counting the labeled cells of the glomeruli or renal cortex. The results, reported as median ± percentile (25th; 75th), showed that the alpha-SM-actin scores in the glomeruli and tubulointerstitium from the renal cortex were 2.0 (2.0; 4.0) and 3.0 (3.0; 4.0), respectively, in patients with FSGS, and 0.5 (0.0; 1.0) and 0.0 (0.0; 0.5) in the controls. The number of PCNA-positive cells per glomerulus and graded field of tubulointerstitium from the renal cortex was 0.2 (0.0; 0.4) and 1.1 (0.3; 2.2), respectively, for patients with FSGS, and 0.0 (0.0; 0.5) and 0.0 (0.0; 0.0) for controls. The present data showed an increase of alpha-SM-actin and PCNA expression in glomeruli and renal cortex from FSGS patients. The extent of immunoreaction for alpha-SM-actin in the tubulointerstitial area was correlated with the intensity of proteinuria. However, there was no correlation between the kidney expression of these proteins and the reciprocal of plasma creatinine level or renal fibrosis. These findings suggest that the immunohistochemical alterations may be reversible.

Metabolic markers following beta-adrenoceptor agonist infusion in footshock-stressed rats

Stress hormones can alter metabolic functions in adipose tissue and liver, as well as the sensitivity of rat white adipocytes and rat atrial responses to ß-adrenergic agonists. In this study, we examined the effects of three daily footshock stress sessions on the plasma corticosterone, glucose, glycerol and triacylglycerol levels of fed, conscious male rats, and on the plasma glucose, glycerol and triacylglycerol levels of the same rats following iv infusions of ß-adrenergic agonists (isoproterenol: 0.4 nmol kg-1 min-1, noradrenaline: 5.0 µg kg-1 day-1, and BRL 37344 ([±]-[4-(2-[(2-[3-chlorophenyl]-2-hydroxyethyl)amino]propyl)phenoxy]acetic acid), a selective ß3-adrenoceptor agonist: 0.4 nmol kg-1 min-1). Plasma corticosterone levels increased significantly after each stress session, while triacylglycerol levels increased after the first session and glucose increased after the second and third sessions. Glycerol levels were unaltered after stress. These results suggest that repeated footshock stress may induce a metabolic shift from triacylglycerol biosynthesis to glucose release by hepatic tissue, with glycerol serving as one of the substrates in both pathways. Stressed rats were more sensitive to infusion of noradrenaline plus prazosin and to infusion of isoproterenol, with elevated plasma glucose, glycerol and triacylglycerol levels. The higher sensitivity of stressed rats to isoproterenol and noradrenaline was probably related to the permissive effect of plasma corticosterone. Only BRL 37344 increased plasma glycerol levels in stressed rats, probably because ß3-adrenoceptors are not involved in hepatic triacylglycerol synthesis, thus allowing glycerol to accumulate in plasma.

Bone mineral density in young women of the city of São Paulo, Brazil: correlation with both collagen type I alpha 1 gene polymorphism and clinical aspects

Osteoporosis is a multifactorial disease with great impact on morbidity and mortality mainly in postmenopausal women. Although it is recognized that factors related to life-style and habits may influence bone mass formation leading to greater or lower bone mass, more than 85% of the variation in bone mineral density (BMD) is genetically determined. The collagen type I alpha 1 (COLIA1) gene is a possible risk factor for osteoporosis. We studied a population of 220 young women from the city of São Paulo, Brazil, with respect to BMD and its correlation with both COLIA1 genotype and clinical aspects. The distribution of COLIA1 genotype SS, Ss and ss in the population studied was 73.6, 24.1 and 2.3%, respectively. No association between these genotypes and femoral or lumbar spine BMD was detected. There was a positive association between lumbar spine BMD and weight (P<0.0001), height (P<0.0156), and body mass index (BMI) (P<0.0156), and a negative association with age at menarche (P<0.0026). There was also a positive association between femoral BMD and weight (P<0.0001), height (P<0.0001), and BMI (P<0.0001), and a negative correlation with family history for osteoporosis (P<0.041). There was no association between the presence of allele s and reduced BMD. We conclude that a family history of osteoporosis and age at menarche are factors that may influence bone mass in our population.

Kinetics of TNF-alpha and IFN-gamma mRNA expression in islets and spleen of NOD mice

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of pancreatic ß cells. Non-obese diabetic (NOD) mice spontaneously develop diabetes similar to the human disease. Cytokines produced by islet-infiltrating mononuclear cells may be directly cytotoxic and can be involved in islet destruction coordinated by CD4+ and CD8+ cells. We utilized a semiquantitative RT-PCR assay to analyze in vitro the mRNA expression of TNF-alpha and IFN-gamma cytokine genes in isolated islets (N = 100) and spleen cells (5 x 10(5) cells) from female NOD mice during the development of diabetes and from female CBA-j mice as a related control strain that does not develop diabetes. Cytokine mRNAs were measured at 2, 4, 8, 14 and 28 weeks of age from the onset of insulitis to the development of overt diabetes. An increase in IFN-gamma expression in islets was observed for females aged 28 weeks (149 ± 29 arbitrary units (AU), P<0.05, Student t-test) with advanced destructive insulitis when compared with CBA-j mice, while TNF-alpha was expressed in both NOD and CBA-j female islets at the same level at all ages studied. In contrast, TNF-alpha in spleen was expressed at higher levels in NOD females at 14 weeks (99 ± 8 AU, P<0.05) and 28 weeks (144 ± 17 AU, P<0.05) of age when compared to CBA-j mice. The data suggest that IFN-gamma and TNF-alpha expression in pancreatic islets of female NOD mice is associated with ß cell destruction and overt diabetes.

High concentrations of KCl release noradrenaline from noradrenergic neurons in the rat anococcygeus muscle

The aim of the present study was to investigate the effects of high concentrations of KCl in releasing noradrenaline from sympathetic nerves and its actions on postsynaptic alpha-adrenoceptors. We measured the isotonic contractions induced by KCl in the isolated rat anococcygeus muscle under different experimental conditions. The contractile responses induced by KCl were inhibited by alpha-adrenoceptor antagonists in 2.5 mM Ca2+ solution. Prazosin reduced the maximum effect from 100 to 53.9 ± 10.2% (P<0.05) while the pD2 values were not changed. The contractile responses induced by KCl were abolished by prazosin in Ca2+-free solution (P<0.05). Treatment of the rats with reserpine reduced the maximum effect induced by KCl as compared to the contractile responses induced by acetylcholine from 339.5 ± 157.8 to 167.3 ± 65.5% (P<0.05), and increased the pD2 from 1.57 ± 0.01 to 1.65 ± 0.006 (P<0.05), but abolished the inhibitory effect of prazosin (P<0.05). In contrast, L-NAME increased the contractile responses induced by 120 mM KCl by 6.2 ± 2.3% (P<0.05), indicating that KCl could stimulate the neurons that release nitric oxide, an inhibitory component of the contractile response induced by KCl. Our results indicate that high concentrations of KCl induce the release of noradrenaline from noradrenergic neurons, which interacts with alpha1-adrenoceptors in smooth muscle cells, producing a contractile response in 2.5 mM Ca2+ (100%) and in Ca2+-free solution, part of which is due to a direct effect of KCl on the rat anococcygeus muscle.

Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns), the alpha-globin genes are duplicate (alpha2 and alpha1) and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP). Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene) submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystem™ and GenePhor™, Amersham Biosciences), different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystem™ and 20% gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.

Increased production of tumor necrosis factor-alpha in whole blood cultures from children with primary malnutrition

Because low tumor necrosis factor-alpha (TNF-alpha) production has been reported in malnourished children, in contrast with high production of TNF-alpha in experimental protein-energy malnutrition, we reevaluated the production of TNF-alpha in whole blood cultures from children with primary malnutrition free from infection, and in healthy sex- and age-matched controls. Mononuclear cells in blood diluted 1:5 in endotoxin-free medium released TNF-alpha for 24 h. Spontaneously released TNF-alpha levels (mean ± SEM), as measured by enzyme immunoassay in the supernatants of unstimulated 24-h cultures, were 10,941 ± 2,591 pg/ml in children with malnutrition (N = 11) and 533 ± 267 pg/ml in controls (N = 18) (P < 0.0001). TNF-alpha production was increased by stimulation with lipopolysaccharide (LPS), with maximal production of 67,341 ± 16,580 pg/ml TNF-alpha in malnourished children and 25,198 ± 2,493 pg/ml in controls (P = 0.002). In control subjects, LPS dose-dependently induced TNF-alpha production, with maximal responses obtained at 2000 ng/ml. In contrast, malnourished patients produced significantly more TNF-alpha with 0.02-200 ng/ml LPS, responded maximally at a 10-fold lower LPS concentration (200 ng/ml), and presented high-dose inhibition at 2000 ng/ml. TNF-alpha production a) was significantly influenced by LPS concentration in control subjects, but not in malnourished children, who responded strongly to very low LPS concentrations, and b) presented a significant, negative correlation (r = -0.703, P = 0.023) between spontaneous release and the LPS concentration that elicited maximal responses in malnourished patients. These findings indicate that malnourished children are not deficient in TNF-alpha production, and suggest that their cells are primed for increased TNF-alpha production.

Effect of D-alpha-tocopherol on tubular nephron acidification by rats with induced diabetes mellitus

The objective of the present study was to determine if treatment of diabetic rats with D-alpha-tocopherol could prevent the changes in glomerular and tubular function commonly observed in this disease. Sixty male Wistar rats divided into four groups were studied: control (C), control treated with D-alpha-tocopherol (C + T), diabetic (D), and diabetic treated with D-alpha-tocopherol (D + T). Treatment with D-alpha-tocopherol (40 mg/kg every other day, ip) was started three days after diabetes induction with streptozotocin (60 mg/kg, ip). Renal function studies and microperfusion measurements were performed 30 days after diabetes induction and the kidneys were removed for morphometric analyses. Data are reported as means ± SEM. Glomerular filtration rate increased in D rats but decreased in D + T rats (C: 6.43 ± 0.21; D: 7.74 ± 0.45; D + T: 3.86 ± 0.18 ml min-1 kg-1). Alterations of tubular acidification observed in bicarbonate absorption flux (JHCO3) and in acidification half-time (t/2) in group D were reversed in group D + T (JHCO3, C: 2.30 ± 0.10; D: 3.28 ± 0.22; D + T: 1.87 ± 0.08 nmol cm-2 s-1; t/2, C: 4.75 ± 0.20; D: 3.52 ± 0.15; D + T: 5.92 ± 0.19 s). Glomerular area was significantly increased in D, while D + T rats exhibited values similar to C, suggesting that the vitamin prevented the hypertrophic effect of hyperglycemia (C: 8334.21 ± 112.05; D: 10,217.55 ± 100.66; D + T: 8478.21 ± 119.81µm²). These results suggest that D-alpha-tocopherol is able to protect rats, at least in part, from the harmful effects of diabetes on renal function.

Concentration of CCL11, CXCL8 and TNF-alpha in sputum and plasma of patients undergoing asthma or chronic obstructive pulmonary disease exacerbation

Asthma and chronic obstructive pulmonary disease (COPD) are common respiratory illnesses characterized by chronic inflammation of the airways. The characterization of induced or spontaneously produced sputum is a useful technique to assess airway inflammation. In the present study, we compared the concentrations of CCL2, CCL11, CXCL8, and tumor necrosis factor-alpha (TNF-alpha) in plasma and induced sputum of patients with severe asthma or COPD and correlated the levels of these mediators with inflammatory cells in sputum. Asthmatic patients had elevated levels of eosinophils (40.1 ± 6.24%) in sputum whereas neutrophils (63.3 ± 4.66%) predominated in COPD patients. The levels of the chemokine CCL11 were markedly increased in sputum (708.7 ± 330.7 pg/ml) and plasma (716.6 ± 162.2 pg/ml) of asthmatic patients and correlated with the percentage of eosinophils in induced sputum. The concentrations of CXCL8 (817.0 ± 105.2 pg/ml) and TNF-alpha (308.8 ± 96.1 pg/ml) were higher in sputum of COPD patients and correlated with the percentage of neutrophils in induced sputum. There was also an increase in the concentrations of CXCL8 (43.2 ± 6.8 pg/ml) in sputum of asthmatic patients. These results validate that sputum is a suitable method to assess chemokines and cytokines associated with asthma and COPD. Moreover, the mechanisms involved in the synthesis of CCL11 and CXCL8/TNF-alpha would be helpful to better understand the inflammatory profile associated with asthma and COPD, respectively.

Effect of thymosin alpha-1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro

Thymosin alpha 1 (Tα1) has been shown to have beneficial effects on numerous immune system parameters, but little is known about the effects of Tα1 on patients with gastric carcinoma. The objective of this study was to determine the effect of Tα1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro, and to evaluate its efficacy as an immunoregulatory factor in patients with gastric carcinoma. We compared the effect of Tα1 on the frequency of CD4+ and CD8+ T cells, especially the CD4+CD25+Foxp3+ Tregs in peripheral blood mononuclear cells (PBMCs) from gastric carcinoma patients (N = 35) and healthy donors (N = 22). We also analyzed the changes in the proliferation of PBMCs in response to treatment with Tα1, and examined the production of Th1, Th2, and Th17 cytokines by PBMCs and tumor-infiltrating lymphocytes. The treatment of PBMCs from gastric cancer patients, with Tα1 (50 µg/mL) alone increased the percentage of CD4+CD25+Foxp3+ (suppressive antitumor-specific Tregs) from 1.68 ± 0.697 to 2.19 ± 0.795% (P < 0.05). Our results indicate that Tα1 increases the percentage of Tregs and IL-1β, TNF-α, and IL-6 in vitro.

Proteomic data show an increase in autoantibodies and alpha-fetoprotein and a decrease in apolipoprotein A-II with time in sera from senescence-accelerated mice

We evaluated changes in levels by comparing serum proteins in senescence-accelerated mouse-prone 8 (SAMP8) mice at 2, 6, 12, and 15 months of age (SAMP8-2 m, -6 m, -12 m, -15 m) to age-matched SAM-resistant 1 (SAMR1) mice. Mice were sacrificed, and blood was analyzed by 2-dimensional electrophoresis combined with mass spectrometry. Five protein spots were present in all SAMP8 serum samples, but only appeared in SAMR1 samples at 15 months of age except for spot 3, which also showed a slight expression in SAMR1-12 m sera. Two proteins decreased in the sera from SAMP8-2 m, -6 m, and -12 m mice, and divided into 2 spots each in SAMP8-15 m sera. Thus, the total number of altered spots in SAMP8 sera was 7; of these, 4 were identified as Ig kappa chain V region (M-T413), chain A of an activity suppressing Fab fragment to cytochrome P450 aromatase (32C2_A), alpha-fetoprotein, and apolipoprotein A-II. M-T413 is a monoclonal CD4 antibody, which inhibits T cell proliferation. We found that M-T413 RNA level was significantly enhanced in splenocytes from SAMP8-2 m mice. This agreed with serum M-T413 protein alterations and a strikingly lower blood CD4+ T cell count in SAMP8 mice when compared to the age-matched SAMR1 mice, with the latter negatively correlating with serum M-T413 protein volume. Age-related changes in serum proteins favored an increase in autoantibodies and alpha-fetoprotein and a decrease of apolipoprotein A-II, which occurred in SAMP8 mice at 2 months of age and onwards. These proteins may serve as candidate biomarkers for early aging.