Salivary and serum cortisol levels, salivary alpha-amylase and unstimulated whole saliva flow rate in pregnant and non-pregnant

PURPOSE: To compare salivary and serum cortisol levels, salivary alpha-amylase (sAA), and unstimulated whole saliva (UWS) flow rate in pregnant and non-pregnant women. METHOD: A longitudinal study was conducted at a health promotion center of a university hospital. Nine pregnant and 12 non-pregnant women participated in the study. Serum and UWS were collected and analyzed every trimester and twice a month during the menstrual cycle. The salivary and serum cortisol levels were determined by chemiluminescence assay and the sAA was processed in an automated biochemistry analyzer. RESULTS: Significant differences between the pregnant and non-pregnant groups were found in median [interquartile range] levels of serum cortisol (23.8 µL/dL [19.4-29.4] versus 12.3 [9.6-16.8], p<0.001) and sAA (56.7 U/L [30.9-82.2] versus 31.8 [18.1-53.2], p<0.001). Differences in salivary and serum cortisol (µL/dL) and sAA levels in the follicular versus luteal phase were observed (p<0.001). Median UWS flow rates were similar in pregnant (0.26 [0.15-0.30] mL/min) and non-pregnant subjects (0.23 [0.20-0.32] mL/min). Significant correlations were found between salivary and serum cortisol (p=0.02) and between salivary cortisol and sAA (p=0.01). CONCLUSIONS: Serum cortisol and sAA levels are increased during pregnancy. During the luteal phase of the ovarian cycle, salivary cortisol levels increase, whereas serum cortisol and sAA levels decline.

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.

alpha-Tocopherol enhances tumour growth inhibition by cis-dichlorodiammine platinum (II)

Present studies indicate that alpha-tocopherol enhances the efficacy of cisplatin as demonstrated by inoculation of Dalton's lymphoma cells incubated with either cisplatin (5 or 10 µg/ml) alone or cisplatin + alpha-tocopherol (25 or 50 µg/ml) into C3H/He mice. Tumour cells (3 x 10(6) cells/mouse) incubated with cisplatin grow slowly in syngeneic mice as indicated by the late appearance of tumour. However, mice failed to develop tumour when inoculated with tumour cells incubated with cisplatin + alpha-tocopherol. When the animals were challenged with tumour cells (3 x 10(6) cells/mouse) on the 15th day after the initial inoculation, 30-50% survived more than 60 days, with 10% tumour-free survivors being observed in some groups. Antitumour activity was higher in mice receiving lymphoma cells (3 x 10(6) cells/mouse) preincubated with cisplatin + alpha-tocopherol compared to cisplatin alone. Tumour-bearing mice receiving cisplatin in combination with different concentrations of alpha-tocopherol exhibited significantly higher (P<0.001) intratumour platinum content (123-306%) but without any change in the kidney platinum content as compared to those receiving cisplatin (5 or 10 µg/ml) alone. Enhancement of cisplatin-induced tumour growth inhibition is probably due to the modulation of tumour cell membrane permeability by alpha-tocopherol. alpha-Tocopherol might increase the influx of cisplatin into tumour cells, causing the DNA repair machinery to be less efficient due to increased efficiency of adduct formation in the DNA molecule. This effect of alpha-tocopherol can render cisplatin more effective as an antitumour agent.

alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

Seven unrelated patients with hemoglobin (Hb) H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha)20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, aaNcoI, alphaIcalpha and alphaTSaudialpha) was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha)20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(aa)T]. Among the 27 patients with structural alterations, 15 (of Italian descent) had Hb Hasharon (alpha47Asp->His) associated with the -alpha3.7 deletion, 4 (of Italian descent) were heterozygous for Hb J-Rovigo (alpha53Ala->Asp), 4 (3 Blacks and 1 Caucasian) were heterozygous for Hb Stanleyville-II (alpha78Asn->Lys) associated with the alpha+-thalassemia, 1 (Black) was heterozygous for Hb G-Pest (alpha74Asp->Asn), 1 (Caucasian) was heterozygous for Hb Kurosaki (alpha7Lys->Glu), 1 (Caucasian) was heterozygous for Hb Westmead (alpha122His->Gln), and 1 (Caucasian) was the carrier of a novel silent variant (Hb Campinas, alpha26Ala->Val). Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population.

N-terminal amino acids of bovine alpha interferons are relevant for the neutralization of their antiviral activity

The structure-function relationship of interferons (IFNs) has been studied by epitope mapping. Epitopes of bovine IFNs, however, are practically unknown, despite their importance in virus infections and in the maternal recognition of pregnancy. It has been shown that recombinant bovine (rBo)IFN-alphaC and rBoIFN-alpha1 differ only in 12 amino acids and that the F12 monoclonal antibody (mAb) binds to a linear sequence of residues 10 to 34. We show here that the antiviral activities of these two IFNs were neutralized by the F12 mAb to different extents using two tests. In residual activity tests the antiviral activity dropped by more than 99% with rBoIFN-alphaC and by 84% with rBoIFN-alpha1. In checkerboard antibody titrations, the F12 mAb titer was 12,000 with rBoIFN-alphaC and only 600 with rBoIFN-alpha1. Since these IFNs differ in their amino acid sequence at positions 11, 16 and 19 of the amino terminus, only these amino acids could account for the different neutralization titers, and they should participate in antibody binding. According to the three-dimensional structure described for human and murine IFNs, these amino acids are located in the alpha helix A; amino acids 16 and 19 of the bovine IFNs would be expected to be exposed and could bind to the antibody directly. The amino acid at position 11 forms a hydrogen bond in human IFNs-alpha and it is possible that, in bovine IFNs-alpha, the F12 mAb, binding near position 11, would disturb this hydrogen bond, resulting in the difference in the extent of neutralization observed.

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.

Influences of alpha-tocopherol on cholesterol metabolism and fatty streak development in apolipoprotein E-deficient mice fed an atherogenic diet

Although the role of oxidized lipoproteins is well known in atherogenesis, the role of vitamin E supplementation is still controversial. There is also little information about cholesterol metabolism (hepatic concentration and fecal excretion) in the new models of atherosclerosis. In the present study, we evaluated the effect of moderate vitamin E supplementation on cholesterol metabolism and atherogenesis in apolipoprotein E (apo E)-deficient mice. Apo E-deficient mice were fed an atherogenic diet containing 40 or 400 mg/kg of alpha-tocopherol acetate for 6 weeks. Total cholesterol in serum and liver and 3-OH-alpha-sterols in feces, and fecal excretion of bile acids were determined and histological analyses of aortic lesion were performed. A vitamin E-rich diet did not affect body weight, food intake or serum cholesterol. Serum and hepatic concentrations of cholesterol as well as sterol concentration in feces were similar in both groups. However, when compared to controls, the alpha-tocopherol-treated mice showed a reduction of about 60% in the atherosclerotic lesions when both the sum of lesion areas and the average of the largest lesion area were considered. These results demonstrate that supplementation of moderate doses of alpha-tocopherol was able to slow atherogenesis in apo E-deficient mice and to reduce atherogenic lipoproteins without modifying the hepatic pool or fecal excretion of cholesterol and bile acids.

Uniform decrease of alpha-global field power induced by intermittent photic stimulation of healthy subjects

Nineteen-channel EEGs were recorded from the scalp surface of 30 healthy subjects (16 males and 14 females, mean age: 34 years, SD: 11.7 years) at rest and under trains of intermittent photic stimulation (IPS) at rates of 5, 10 and 20 Hz. Digitalized data were submitted to spectral analysis with fast fourier transformation providing the basis for the computation of global field power (GFP). For quantification, GFP values in the frequency ranges of 5, 10 and 20 Hz at rest were divided by the corresponding data obtained under IPS. All subjects showed a photic driving effect at each rate of stimulation. GFP data were normally distributed, whereas ratios from photic driving effect data showed no uniform behavior due to high interindividual variability. Suppression of alpha-power after IPS with 10 Hz was observed in about 70% of the volunteers. In contrast, ratios of alpha-power were unequivocal in all subjects: IPS at 20 Hz always led to a suppression of alpha-power. Dividing alpha-GFP with 20-Hz IPS by alpha-GFP at rest (R = alpha-GFP IPS/alpha-GFPrest) thus resulted in ratios lower than 1. We conclude that ratios from GFP data with 20-Hz IPS may provide a suitable paradigm for further investigations.

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.

Immunocytochemical and biochemical detection of alpha-L-fucosidase in Trypanosoma cruzi

The aim of the present study was to demonstrate the presence of alpha-L-fucosidase in Trypanosoma cruzi. Immunocytochemical and biochemical techniques were used to localize and characterize a membrane-associated, neutral-pH-optimum, alpha-L-fucosidase from the parasite. Light and electron microscopy localized the alpha-L-fucosidase specifically on the surface of the parasite and on membranes in the posterior region of the epimastigote stage. Although much less intense, labeling was also detected on the surface of trypomastigotes. At least 50% of the alpha-L-fucosidase activity was associated with epimastigote membrane solubilized with 1 M NaCl or 1% Triton X-100, suggesting that alpha-L-fucosidase is peripherally associated with membranes. The enzyme from epimastigotes had a neutral pH optimum (near 7) but displayed low specific activity when p-nitrophenyl-alpha-L-fucoside was employed as substrate (0.028 U/mg protein for epimastigotes and 0.015 U/mg protein for tissue culture-derived trypomastigotes). Polyacrylamide gel electrophoresis and Western blotting analysis both showed an expected 50-kDa polypeptide which was immunoreactive with anti-alpha-L-fucosidase antibodies.

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.

Interferon-alpha receptor 1 mRNA expression in peripheral blood mononuclear cells is associated with response to interferon-alpha therapy of patients with chronic hepatitis C

Interferon (IFN)-alpha receptor mRNA expression in liver of patients with chronic hepatitis C has been shown to be a response to IFN-alpha therapy. The objective of the present study was to determine whether the expression of mRNA for subunit 1 of the IFN-alpha receptor (IFNAR1) in peripheral blood mononuclear cells (PBMC) is associated with the response to IFN-alpha in patients with chronic hepatitis C. Thirty patients with positive anti-HCV and HCV-RNA, and abnormal levels of alanine aminotransferase in serum were selected and treated with IFN-alpha2b for one year. Those with HBV or HIV infection, or using alcohol were not included. Thirteen discontinued the treatment and were not evaluated. The IFN-alpha response was monitored on the basis of alanine aminotransferase level and positivity for HCV-RNA in serum. IFNAR1-mRNA expression in PBMC was measured by reverse transcription-polymerase chain reaction before and during the first three months of therapy. The results are reported as IFNAR1-mRNA/ß-actin-mRNA ratio (mean ± SD). Before treatment, responder patients had significantly higher IFNAR1-mRNA expression in PBMC (0.67 ± 0.15; N = 5; P < 0.05) compared to non-responders (0.35 ± 0.17; N = 12) and controls (0.30 ± 0.16; N = 9). Moreover, IFNAR1-mRNA levels were significantly reduced after 3 months of treatment in responders, whereas there were no differences in IFNAR1 expression in non-responders during IFN-alpha therapy. Basal IFNAR1-mRNA expression was not correlated with the serum level of alanine and aspartate aminotransferases or the presence of cirrhosis. The present results suggest that IFNAR1-mRNA expression in PBMC is associated with IFN-alpha response to hepatitis C and may be useful for monitoring therapy in patients with chronic hepatitis C.

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.