Culture of mouse peritoneal macrophages with mouse serum induces lipid bodies that associate with the parasitophorous vacuole and decrease their microbicidal capacity against Toxoplasma gondii

Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.

Serum intact parathyroid hormone levels in cats with chronic kidney disease

Chronic kidney disease (CKD) is frequently observed in cats and it is characterized as a multisystemic illness, caused by several underlying metabolic changes, and secondary renal hyperparathyroidism (SRHPT) is relatively common; usually it is associated with the progression of renal disease and poor prognosis. This study aimed at determining the frequency of SRHPT, and discussing possible mechanisms that could contribute to the development of SRHPT in cats at different stages of CKD through the evaluation of calcium and phosphorus metabolism, as well as acid-base status. Forty owned cats with CKD were included and divided into three groups, according to the stages of the disease, classified according to the International Renal Interest Society (IRIS) as Stage II (n=12), Stage III (n=22) and Stage IV (n=6). Control group was composed of 21 clinically healthy cats. Increased serum intact parathyroid hormone (iPTH) concentrations were observed in most CKD cats in all stages, and mainly in Stage IV, which hyperphosphatemia and ionized hypocalcemia were detected and associated to the cause for the development of SRHPT. In Stages II and III, however, ionized hypercalcemia was noticed suggesting that the development of SRHPT might be associated with other factors, and metabolic acidosis could be involved to the increase of serum ionized calcium. Therefore, causes for the development of SRHPT seem to be multifactorial and they must be further investigated, mainly in the early stages of CKD in cats, as hyperphosphatemia and ionized hypocalcemia could not be the only factors involved.

The effect of methotrexate and azathioprine on the serum levels of IgA-a1-antitrypsin complex in juvenile chronic arthritis

In the present study we investigated the influence of methotrexate (MTX) and azathioprine (AZA) on the serum levels of the IgA-a1-antitrypsin (IgA-AT) complex in patients with the systemic form of juvenile chronic arthritis (JCA). Fifty-six JCA patients (22 treated with MTX, 18 treated with AZA, and 16 not treated with any immunosuppressive agent) were enrolled in the study. MTX dosage ranged from 0.3 to 0.5 mg kg-1 week-1, while AZA was given daily at an average dose of 1 mg/kg. MTX was given for 13 months (SD = 7 months) whereas AZA for 11 months (SD = 6 months). The average value of the complex was higher in JCA patients than in both control groups (0.74 ± 0.73 U vs 0.37 ± 0.13 U (control children), P<0.001 and vs 0.23 ± 0.12 U (control adults), P<0.001). Values exceeding the normal range were found in twenty-two JCA patients (39.4%). Serum IgA-AT level was lowest in the MTX group compared to AZA and non-treated patients (0.56 ± 0.24 U, 0.76 ± 0.43 U, 0.95 ± 0.52 U, respectively, P<0.05). IgA values exceeding normal levels for age were found in 14% of the patients. A correlation between the levels of the IgA-AT complex and C-reactive protein (r = 0.43, P<0.01), a1-acid-glycoprotein (r = 0.45, P<0.01), a1-antichymotrypsin (r = 0.52, P<0.01), a1-antitrypsin (r = 0.40, P<0.01) and IgA (r = 0.56, P<0.01) was established

Lipid peroxidation, antioxidant enzymes and glutathione levels in human erythrocytes exposed to colloidal iron hydroxide in vitro

The free form of the iron ion is one of the strongest oxidizing agents in the cellular environment. The effect of iron at different concentrations (0, 1, 5, 10, 50, and 100 µM Fe3+) on the normal human red blood cell (RBC) antioxidant system was evaluated in vitro by measuring total (GSH) and oxidized (GSSG) glutathione levels, and superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and reductase (GSH-Rd) activities. Membrane lipid peroxidation was assessed by measuring thiobarbituric acid reactive substance (TBARS). The RBC were incubated with colloidal iron hydroxide and phosphate-buffered saline, pH 7.45, at 37oC, for 60 min. For each assay, the results for the control group were: a) GSH = 3.52 ± 0.27 µM/g Hb; b) GSSG = 0.17 ± 0.03 µM/g Hb; c) GSH-Px = 19.60 ± 1.96 IU/g Hb; d) GSH-Rd = 3.13 ± 0.17 IU/g Hb; e) catalase = 394.9 ± 22.8 IU/g Hb; f) SOD = 5981 ± 375 IU/g Hb. The addition of 1 to 100 µM Fe3+ had no effect on the parameters analyzed. No change in TBARS levels was detected at any of the iron concentrations studied. Oxidative stress, measured by GSH kinetics over time, occurs when the RBC are incubated with colloidal iron hydroxide at concentrations higher than 10 µM of Fe3+. Overall, these results show that the intact human RBC is prone to oxidative stress when exposed to Fe3+ and that the RBC has a potent antioxidant system that can minimize the potential damage caused by acute exposure to a colloidal iron hydroxide in vitro.

Serum antigliadin antibody levels as a screening criterion before jejunal biopsy indication for celiac disease in a developing country

The objective of the present study was to determine the efficacy of detection of antigliadin immunoglobulins G and A (IgG and IgA) for the diagnosis of celiac disease in a developing country, since other enteropathies might alter the levels of these antibodies. Three groups were studied: 22 patients with celiac disease (mean age: 30.6 months), 61 patients with other enteropathies (mean age: 43.3 months), and 46 patients without enteropathies (mean age: 96.9 months). Antigliadin IgG and IgA ELISA showed sensitivity of 90.9 and 95.5%, respectively. With the hypothetical values of prevalence ranging from 1:500 to 1:2000 liveborns, the positive predictive value varied from 8.5 to 2.3% for IgG and from 4.8 to 1.1% for IgA. Considering the patients without enteropathies, specificity was 97.8 and 95.7% for IgG and IgA, respectively. In patients with other enteropathies, specificity was 82.0 and 84.1%, respectively. When patients with and without other enteropathies were considered as a whole, specificity was 88.8 and 91.6%, respectively. The specificity of positive IgG or IgA was 93.5% in children without enteropathies and 78.7% in the presence of other enteropathies. The negative predictive value for hypothetical prevalences varying from 1:500 to 1:2000 liveborns was 99.9%. Thus, even in developing countries where the prevalence of non-celiac enteropathies is high, the determination of serum antigliadin antibody levels is a useful screening test prior to the jejunal biopsy in the investigation of intestinal malabsorption.

Positive correlation of serum leptin with estradiol levels in patients with polycystic ovary syndrome

Patients with polycystic ovary syndrome (PCOS) usually are obese, insulin resistant and hyperinsulinemic. The known association between leptin, obesity andinsulin action suggests that leptin may have a role in PCOS but this has only been addressed peripherally. This study was designed to assess the relationship between serum leptin and the anthropometric, metabolic and endocrine variables of obese (body mass index, BMI ³30 kg/m²) and non-obese (BMI <30 kg/m²) PCOS patients. Twenty-eight PCOS patients and 24 control women subdivided into obese and non-obese groups were evaluated. Leptin, androgens, lipids, gonadotrophins and insulin-glucose response to the oral glucose tolerance test were measured by radioimmunoassay in all participants. The assays were done all in one time. The areas under the insulin curve (AUC-I) and the glycemia curve were calculated to identify patients with insulin resistance. Mean leptin levels were not significantly higher in patients with PCOS compared to the control group (21.2 ± 10.2 vs 27.3 ± 12.4 ng/ml). Leptin levels were found to be significantly higher in the obese subgroups both in patients with PCOS (26.9 ± 9.3 vs 14.1 ± 7.0 ng/ml) and in the control group (37.3 ± 15.5 vs 12.9 ± 5.8 ng/ml). The leptin of the PCOS group was correlated with BMI (r = 0.74; P < 0.0001) and estradiol (r = 0.48; P < 0.008) and tended to be correlated with the AUC-I (r = 0.36; P = 0.05). Of the parameters which showed a correlation with leptin in PCOS, only estradiol and probably insulinemia (AUC-I) did not show a significant correlation with BMI, suggesting that the other parameters were correlated with leptin due to their correlation with BMI. Estradiol correlated with leptin in PCOS patients regardless of their weight.

Elevated levels of erythrocyte-conjugated dienes indicate increased lipid peroxidation in schistosomiasis mansoni patients

Schistosoma mansoni causes liver disease by inducing granulomatous inflammation. This favors formation of reactive oxygen species, including superoxide ions, hydrogen peroxide and hydroxyl radicals all of which may induce lipid peroxidation. We have evaluated lipid peroxidation in 18 patients with hepatosplenic schistosomiasis mansoni previously treated with oxamniquine followed by splenectomy, ligature of the left gastric vein and auto-implantation of spleen tissue, by measuring levels of erythrocyte-conjugated dienes and plasma malondialdehyde (MDA). Age-matched, healthy individuals (N = 18) formed the control group. Erythrocyte-conjugated dienes were extracted with dichloromethane/methanol and quantified by UV spectrophotometry, while plasma MDA was measured by reaction with thiobarbituric acid. Patient erythrocytes contained two times more conjugated dienes than control cells (584.5 ± 67.8 vs 271.7 ± 20.1 µmol/l, P < 0.001), whereas the increase in plasma MDA concentration (about 10%) was not statistically significant. These elevated conjugated dienes in patients infected by S. mansoni suggest increased lipid peroxidation in cell membranes, although this was not evident when a common marker of oxidative stress, plasma MDA, was measured. Nevertheless, these two markers of lipid peroxidation, circulating MDA and erythrocyte-conjugated dienes, correlated significantly in both patient (r = 0.62; P < 0.01) and control (r = 0.57; P < 0.05) groups. Our data show that patients with schistosomiasis have abnormal lipid peroxidation, with elevated erythrocyte-conjugated dienes implying dysfunctional cell membranes, and also imply that this may be attenuated by the redox capacity of antioxidant agents, which prevent accumulation of plasma MDA.

Correlation between serum E-selectin levels and panoramic nailfold capillaroscopy in systemic sclerosis

E-selectin is expressed by the activated endothelium and its plasma levels are increased in patients with systemic sclerosis. Eighteen patients fulfilling the American Rheumatism Association criteria for systemic sclerosis, 15 females and 3 males, 42-70 years old, 9 with diffuse and 9 with limited forms, were sequentially recruited for this study. Serum E-selectin levels were determined by commercially available ELISA and their association with nailfold capillaroscopic abnormalities was investigated. Nailfold capillaries were analyzed by 16X magnification wide-field capillaroscopy. Two parameters on capillaroscopy were used to correlate to serum E-selectin: deletion and ectasia. Data were analyzed statistically by the Student t-test and Spearman correlation. Two-tailed P values below 0.05 were considered significant. E-selectin range was 38 to 200 ng/ml (80 ± 39.94). There was a correlation between serum E-selectin levels and the deletion capillaroscopic score (r = 0.50, P < 0.035). This correlation was even stronger within the first 48 months of diagnosis (r = 0.63, P < 0.048). On the other hand, no association was observed between selectin and ectasia. Patients with diffuse disease presented higher serum E-selectin levels than patients with limited disease, although the difference was not statistically significant (96.44 ± 48.04 vs 63.56 ± 21.77 ng/dl; P = 0.08). The present study is the first showing a correlation between soluble serum E-selectin levels and alterations in capillaroscopy. The stronger correlation of deletion score in capillaroscopy in early disease suggests that serum E-selectin levels might be a useful biochemical marker of disease activity in systemic sclerosis.

Association between dental-oral health in young adults and salivary glutathione, lipid peroxidation and sialic acid levels and carbonic anhydrase activity

The aim of the present study was to evaluate the relationship between salivary oxidative stress and dental-oral health. Healthy young adults, matched for gender and age, with (N = 21, 10 men, mean age: 20.3 ± 1 years) and without (N = 16, 8 men, mean age: 21.2 ± 1.8 years) caries were included in this study. The World Health Organization (WHO) caries diagnostic criteria were used for determining the decayed, missing, filled teeth (DMFT) index. The oral hygiene and gingival status were assessed using the simplified oral hygiene index and gingival index, respectively. Unstimulated salivary total protein, glutathione (GSH), lipid peroxidation and total sialic acid levels, carbonic anhydrase activity, and salivary buffering capacity were determined by standard methods. Furthermore, salivary pH was measured with pH paper and salivary flow rate was calculated. Simplified oral hygiene index and gingival index were not significantly different between groups but DMFT scores were significant (P < 0.01). Only, GSH values were significantly different (P < 0.05) between groups (2.2 and 1.6 mg/g protein in young adults without caries and with caries, respectively). There was a significant negative correlation between DMFT and GSH (r = -0.391; P < 0.05; Pearson's correlation coefficient). Our results suggest that there is an association between caries history and salivary GSH levels.

High levels of serum mannose-binding lectin are associated with the severity of clinical signs of leptospirosis

The clinical heterogeneity observed in leptospirosis may be associated with host factors or bacteria virulence. Human serum mannose-binding lectin (MBL) recognizes many pathogens, and low levels of this lectin are associated with susceptibility to infection. MBL is also implicated in the modulation of the inflammatory process. We determined the levels of serum MBL during leptospirosis infection. A double-antibody sandwich ELISA was used to detect the immunoreactive serum MBL. The ELISA plates were coated with monoclonal antibody to MBL and bound MBL or recombinant human MBL were detected by rabbit anti-human MBL serum. HRPO-conjugated goat anti-rabbit antibody was used for detection of the reaction. Two groups of patients seen at referral hospitals in Recife, PE, Brazil, were divided according to the year of infection, 2001 (N = 61) or 2002 (N = 57) and compared in terms of disease severity and levels of serum MBL. A group of healthy volunteers (N = 97) matched by age, gender, and ethnic background was used as control. Patients infected in 2001 had more severe outcomes than those infected in 2002, including jaundice, hemorrhage, respiratory alteration, and renal complication (P = 0.0009; chi-square test). The frequency of patients producing serum MBL >1000 ng/mL was higher in the 2001 group than in the 2002 and control groups (P < 0.01), suggesting an association of MBL level with disease severity. The involvement of MBL and genetic variation of the MBL2 gene should be further evaluated to establish the role of this lectin in the pathogenesis of leptospirosis.

Association between apolipoprotein E genotype, serum lipids, and colorectal cancer in Brazilian individuals

We evaluated genetic variants of apolipoprotein E (APOE HhaI) and their association with serum lipids in colorectal cancer (CRC), together with eating habits and personal history. Eight-seven adults with CRC and 73 controls were studied. APOE*2 (rs7412) and APOE*4 (rs429358) were identified by polymerase chain reaction-restriction fragment length polymorphism. APOE gene polymorphisms were similar in both groups, but the ε4/ε4 genotype (6%) was present only in controls. The patients had reduced levels (mean ± SD) of total cholesterol and low-density lipoprotein cholesterol fraction (180.4 ± 49.5 and 116.1 ± 43.1 mg/dL, respectively) compared to controls (204.2 ± 55.6, P = 0.135 and 134.7 ± 50.8 mg/dL; P = 0.330, respectively) indicating that they were not statistically significant after the Bonferroni correction. The APOE*4 allele was associated with lower levels of total cholesterol, low- and high-density lipoprotein cholesterol fraction and increased levels of very low-density lipoprotein cholesterol fraction and triglycerides only among patients (P = 0.014). There was a positive correlation between the altered lipid profile and increased body mass indexes in both groups (P < 0.010). Moreover, a higher rate of hypertension and overweight was observed in controls (P < 0.002). In conclusion, the presence of the ε4/ε4 genotype only in controls may be due to a protective effect against CRC. Lower lipid profile values among patients, even those on lipid-rich diets associated with the APOE*4 allele, suggest alterations in the lipid synthesis and metabolism pathways in CRC.

Peritoneal and serum interleukin-18 levels are not increased in women with minimum or mild endometriosis

Interleukin-18 (IL-18) is a cytokine that belongs to the IL-1 family. Endometriosis is strongly associated with sub-fertility, and affects about 15% of women of reproductive age. IL-18 may favor the progression of endometriosis. The objective of the present study was to determine the concentration of IL-18 in the serum and peritoneal fluid of infertile women with endometriosis. Forty infertile and 25 fertile women were screened in a teaching hospital. Thirty-four infertile patients with minimal or mild endometriosis and 22 fertile controls were enrolled in the study. The primary outcome was the estimate of IL-18 levels and the secondary outcome was the correlation between serum and peritoneal levels of IL-18. There were no differences between the two groups regarding age, body mass index and levels of peritoneal fluid IL-18 (mean ± SD): 290.85 ± 173.02 pg/mL for infertile women vs 374.21 ± 330.15 pg/mL for controls; or serum IL-18: 391.07 ± 119.71 pg/mL for infertile women vs 373.42 ± 129.11 pg/mL for controls. However, a positive association was found between serum and peritoneal IL-18 levels in patients with endometriosis: r = 0.794, P = 0.0001. All measurements were carried out at the same time by the Human IL-18 Immuno Assay ELISA kit (MBL Co. Ltd., Japan). The present study did not find evidence supporting the hypothesis that IL-18 levels are associated with infertility in women with minimal and mild endometriosis, although a positive correlation was detected in these women between peritoneal and serum levels of IL-18.

Gender differences in serum CK-MB mass levels in healthy Braziliansubjects

The creatine kinase-isoenzyme MB (CK-MB) mass assay is one of the laboratory tests used for the diagnosis of myocardial infarction. It is recommended, however, that reference limits should take gender and race into account. In the present study, we analyzed the plasma CK-MB mass and troponin levels of 244 healthy volunteers without a personal history of coronary artery disease and with no chronic diseases, muscular trauma or hypothyroidism, and not taking statins. The tests were performed with commercial kits, CK-MB mass turbo kit and Troponin I turbo kit, using the Immulite 1000 analyzer from Siemens Healthcare Diagnostic. The values were separated according to gender and showed significant differences by the Mann-Whitney test. Mean (± SD) CK-MB mass values were 2.55 ± 1.09 for women (N = 121; age = 41.20 ± 10.13 years) and 3.49 ± 1.41 ng/mL for men (N = 123; age = 38.16 ± 11.12 years). Gender-specific reference values at the 99th percentile level, according to the Medicalc statistical software, were 5.40 ng/mL for women and 7.13 ng/mL for men. The influence of race was not considered because of the high miscegenation of the Brazilian population. The CK-MB values obtained were higher than the 5.10 mg/mL proposed by the manufacturer of the laboratory kit. Therefore, decision limits should be related to population and gender in order to improve the specificity of this diagnostic tool, avoiding misclassification of patients

Maternal serum progesterone, estradiol and estriol levels in successful dinoprostone-induced labor

Hormone-mediated quiescence involves the maintenance of a decreased inflammatory responsiveness. However, no study has investigated whether labor induction with prostanoids is associated with changes in the levels of maternal serum hormones. The objective of this study was to determine whether labor induction with dinoprostone is associated with changes in maternal serum progesterone, estradiol, and estriol levels. Blood samples were obtained from 81 pregnant women at term. Sixteen patients had vaginal birth after spontaneous labor, 12 required cesarean section after spontaneous labor and 16 underwent elective cesarean. Thirty-seven patients had labor induction with dinoprostone. Eligible patients received a vaginal insert of dinoprostone (10 mg) and were followed until delivery. Serum progesterone (P4), estradiol (E2) and estriol (E3) levels and changes in P4/E2, P4/E3 and E3/E2 ratios were monitored from admission to immediately before birth, and the association of these measures with the resulting clinical classification outcome (route of delivery and induction responsiveness) was assessed. Progesterone levels decreased from admission to birth in patients who underwent successful labor induction with dinoprostone [vaginal and cesarean birth after induced labor: 23% (P < 0.001) and 18% (P < 0.025) decrease, respectively], but not in those whose induction failed (6.4% decrease, P > 0.05). Estriol and estradiol levels, P4/E2, P4/E3 and E3/E2 ratios did not differ between groups. Successful dinoprostone-induced labor was associated with reduced maternal progesterone levels from induction to birth. While a causal relationship between progesterone decrease and effective dinoprostone-induced labor cannot be established, it is tempting to propose that dinoprostone may contribute to progesterone withdrawal and favor labor induction in humans.

Relationship of macrophage migration inhibitory factor levels in PBMCs, lesional skin and serum with disease severity and activity in vitiligo vulgaris

Melanocyte loss in vitiligo vulgaris is believed to be an autoimmune process. Macrophage migration inhibitory factor (MIF) is involved in many autoimmune skin diseases. We determined the possible role of MIF in the pathogenesis of vitiligo vulgaris, and describe the relationship between MIF expressions and disease severity and activity. Serum MIF concentrations and mRNA levels in PBMCs were measured in 44 vitiligo vulgaris patients and 32 normal controls, using ELISA and real-time RT-PCR. Skin biopsies from 15 patients and 6 controls were analyzed by real-time RT-PCR. Values are reported as median (25th-75th percentile). Serum MIF concentrations were significantly increased in patients [35.81 (10.98-43.66) ng/mL] compared to controls [7.69 (6.01-9.03) ng/mL]. MIF mRNA levels were significantly higher in PBMCs from patients [7.17 (3.59-8.87)] than controls [1.67 (1.23-2.42)]. There was also a significant difference in MIF mRNA levels in PBMCs between progressive and stable patients [7.86 (5.85-9.13)vs 4.33 (2.23-8.39)] and in serum MIF concentrations [40.47 (27.71-46.79) vs 26.80 (10.55-36.07) ng/mL]. In addition, the vitiligo area severity index scores of patients correlated positively with changes of both serum MIF concentrations (r = 0.488) and MIF mRNA levels in PBMCs (r = 0.426). MIF mRNA levels were significantly higher in lesional than in normal skin [2.43 (2.13-7.59)vs 1.18 (0.94-1.83)] and in patients in the progressive stage than in the stable stage [7.52 (2.43-8.84)vs 2.13 (1.98-2.64)]. These correlations suggest that MIF participates in the pathogenesis of vitiligo vulgaris and may be useful as an index of disease severity and activity.