Anti-nuclear antibodies in patients with breast cancer.

To study the prevalence of anti-nuclear antibodies (ANA) in breast cancer patients and its association with tumour characteristics. Ninety-one patients with breast mass detected by image studies and assigned to conduct diagnostic biopsy and eventual surgical treatment were studied for demographical, tumour data and presence of ANA. Serum of positive ANA patients was screened for the extractable nuclear antigen (ENA) profile. As comparison, 91 healthy individuals matched for age and from the same geographical area were included. In this sample 72 of 91 (79·1%) had malignant lesions (83% ductal infiltrative carcinoma). ANA was positive in 44·4% of patients with malignant tumour and in 15·7% of those with benign lesions (malignant versus benign with P = 0·03). Controls had ANA positivity in 5·4%, and when compared with tumour samples showed P < 0·0001. The most common immunofluorescence pattern was a fine dense speckled pattern. In the ANA-positive patients with malignant lesions, seven had positivity for ENA profile (three for anti-RNP and anti-Sm, one for just anti-RNP, two for anti-Ro and anti-La e two for just anti-La). It was not possible to associate ANA positivity with tumour histological characteristics or staging or with patient's age. A negative association of ANA with hormonal (oestrogen or oestrogen plus progesterone) receptor status was found (P = 0·01). In this sample, there was a high prevalence of ANA positivity in breast cancer patients with a negative association with the presence of hormonal receptors. More studies are needed to understand the real value of this finding.

Dynamic flux of microvesicles modulate parasite-host cell interaction of Trypanosoma cruzi in eukaryotic cells.

Extracellular vesicles released from pathogens may alter host cell functions. We previously demonstrated the involvement of host cell-derived microvesicles (MVs) during early interaction between Trypanosoma cruzi metacyclic trypomastigote (META) stage and THP-1 cells. Here, we aim to understand the contribution of different parasite stages and their extracellular vesicles in the interaction with host cells. First, we observed that infective host cell-derived trypomastigote (tissue culture-derived trypomastigote [TCT]), META, and noninfective epimastigote (EPI) stages were able to induce different levels of MV release from THP-1 cells; however, only META and TCT could increase host cell invasion. Fluorescence resonance energy transfer microscopy revealed that THP-1-derived MVs can fuse with parasite-derived MVs. Furthermore, MVs derived from the TCT-THP-1 interaction showed a higher fusogenic capacity than those from META- or EPI-THP-1 interaction. However, a higher presence of proteins from META (25%) than TCT (12%) or EPI (5%) was observed in MVs from parasite-THP-1 interaction, as determined by proteomics. Finally, sera from patients with chronic Chagas disease at the indeterminate or cardiac phase differentially recognized antigens in THP-1-derived MVs resulting only from interaction with infective stages. The understanding of intracellular trafficking and the effect of MVs modulating the immune system may provide important clues about Chagas disease pathophysiology.

Mannose-binding lectin serum levels in patients with systemic lupus erythematosus: association with thrombocytopaenia and seizure.

The complement system contributes to the pathogenesis of systemic lupus erythematosus (SLE). Mannose-binding lectin (MBL) is a key molecule of the lectin pathway of complement and seems to be related to the clinical manifestations of this disease. We evaluated the serum levels of MBL and its relationship with disease onset and clinical findings in SLE patients. Serum samples were analysed in 195 patients and 145 healthy controls from southern Brazil. Patients with high MBL levels (above 2000 ng/ml) showed a significant increase in the frequency of thrombocytopaenia ( p = 0.007; OR = 2.71; 95% CI = 1.32-5.55); and seizures ( p = 0.034; OR = 2.61; 95% CI = 1.07-6.37). A positive correlation between disease activity and MBL levels (>2000 ng/ml; p = 0.031, rho = 0.279) as well as of MBL concentration with accumulated organ damage ( p = 0.021; rho = 0.232) was observed. Our results suggest a role for MBL in the development of clinical manifestations such as thrombocytopaenia and seizures in SLE patients. These findings corroborate the participation of the lectin pathway of complement in the pathophysiologic mechanisms underlying clinical manifestations of SLE.

Complement Factor H as a potential atherogenic marker in chronic Chagas' disease.

We aimed to investigate the association between plasma levels of complement Factor H (FH) with cardiac involvement, inflammatory and cardiometabolic parameters in patients with chronic Chagas' disease (CD). FH plasmatic levels were determined in 80 chronic CD patients. Glycaemic index, lipidogram (high-density lipoprotein cholesterol HDL-C, low-density lipoprotein cholesterol LDL-C, triglycerides and total cholesterol) and Ultrasensitive C-Reactive Protein (uCRP) values were obtained from medical records. Height, weight, body mass index (BMI) blood pressure and left ventricular ejection fraction (LVEF) were obtained from echocardiography examinations. Comparisons between chronic CD clinical forms were performed using Mann-Whitney test and correlation Spearman's test. FH levels were correlated positively with triglycerides (P = .001, r = .39), LDL-C (P = .009, r = .3), cholesterol (P = .02, r = .28), uCRP (P = .029, r = .31) and BMI (P = .008, r = .34); and negatively with HDL-C (P = .03, r = -.25) levels. Dyslipidemic patients showed higher FH levels compared to normolipidemic, although no difference for FH levels was observed between chronic CD clinical forms. Alternative pathway of complement may be a link between immune response and metabolic disorders, with important immunoregulatory role in chronic CD.

Modulating Effects of Arabinogalactans from Plant Gum Exudates on Human Complement System.

Gum arabic and cashew nut tree gum exudate polysaccharide (CNTG) are plant polysaccharides composed of galactose and arabinose known as arabinogalactans (AGs). Although these fractions are used in food and pharmaceutical industry, cases of allergic reactions were described in clinical reports. As AGs were reported as modulators of the classical (CP) and alternative pathways (AP) of complement system (CS), in the present work, we investigate whether gum arabic and CNTG have an effect on both CS pathways. The complement fixation tests were performed with (CP-30 and AP-30) and without pre-incubation (CP-0 and AP-0). For CP-30, CNTG and gum arabic (833 µg/ml) showed a reduction of 28.0% (P = 0.000174) and 48.5% (P = 0.000143), respectively, on CP-induced haemolysis. However, no effect was observed for CP-0 in the CP-induced haemolysis. For AP-30, both CNTG and gum arabic (833 µg/ml) showed 87% reduction on the CP-induced haemolysis, with IC50 values of 100 and 7 µg/ml, respectively. For AP-0, a reduction of 11.3% for gum arabic and no effect for the CNTG on the CP-induced haemolysis were observed. These results suggested that gum arabic and CNTG could be acting as activators of the CS. Thus, this effect on the CS, especially on the AP, which accounts for up to 80-90% of total CS activation, indicates that both fractions may be harmful because of their potential pro-inflammatory action. Considering that CS activation induces inflammatory response, further studies confirming this immunomodulatory effect of these fractions are required to insure their safe use.

BF*F allotype of the alternative pathway of complement: A marker of protection against the development of antiphospholipid antibodies in patients with systemic lupus erythematosus.

BACKGROUND: B factor (BF) from the alternative complement pathway seems to participate in the pathophysiology of systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). OBJECTIVE: To study the allotypic variability of BF in SLE and their associations with clinical and autoantibodies profile. METHODS: BF allotypes were determined by high-voltage agarose gel electrophoresis, under constant cooling, followed by immunofixation with anti-human BF antibody, in 188 SLE patients and 103 controls. Clinical and serological data were obtained from medical examination and records. RESULTS: No significant differences of BF variants between patients and controls were found, neither in relation to epidemiologic or clinical manifestations. Associations of phenotype BF SS07 and allotype BF*S07 were found with anticardiolipin IgM (aCl-IgM) antibodies (p = 0.014 and p = 0.009 respectively), but not with aCl-IgG, lupus anticoagulant (LA), anti ß2GPI or clinical APS. A significant decrease in BF*F allotype (p = 0.043) and BF SF phenotype (p = 0.018) was detected in patients with anti-phospholipid antibodies as a whole (aCl-IgG, aCl-IgM, LA and anti ß2GPI). CONCLUSIONS: There is a link between phenotype BF SS07 and allotype BF*S07 with aCl-IgM in SLE patients; BF*F allotype could be considered a marker of protection against the development of antiphospholipid antibodies in these patients.

Association of complement factor B allotypes and serum biomarkers in rheumatoid arthritis patients and their relatives.

The aim of the study was to investigate the allotypic variability of complement factor B (BF) in patients and relatives with rheumatoid arthritis (RA) and its association with serological biomarkers and clinical features of the disease. BF allotypes were determined by high-voltage agarose gel electrophoresis in serum samples of 180 patients with RA, 198 relatives and 98 controls from Southern Brazil. Anticyclic citrullinated peptide (anti-CCP), antimutated citrullinated vimentin (anti-MCV) and IgA-rheumatoid factor (RF) were determined by ELISA and IgM-RF by latex agglutination in all samples. No significant differences were found in the allotypic variants of BF between patients with RA, relatives and controls, nor associations with gender and age of RA onset. BF*S07 allotype was significantly associated with extra-articular manifestations (EAMs; Secondary Sjögren Syndrome, pneumonitis, rheumatoid nodules) in patients with RA (P = 0.02; OR = 6.62). Patients with phenotype BF F had lower positivity for anti-MCV biomarker (P = 0.02; OR = 0.22) and those with allotype BF*S had higher prevalence of this autoantibody (P = 0.02; OR = 3.77). An increased frequency of RF-IgA was detected in relatives of patients with RA with BF FS07 phenotype (P = 0.02; OR = 7.78). Complement BF variability did not influence the development of RA in the studied patients, but BF variants may act as markers of disease prognosis, such as development of EAMs, corroborating with the role of the alternative pathway in the pathogenesis of RA.

Analysis of polymorphic sites in the promoter of the nitric oxide synthase 2 gene in Brazilian patients with leprosy.

Leprosy is one of the most neglected infectious tropical diseases of the skin and the nerves caused by the intracellular pathogen Mycobacterium leprae. The inducible NOS isoform encoded by NOS2A plays a vital role in host defence against bacterial infections. The functional promoter polymorphisms in NOS2A are associated with various autoimmune and infectious diseases. We investigated the association of NOS2A variants with progression of leprosy in a Brazilian cohort including 221 clinically classified patients and 103 unrelated healthy controls. We observed a novel variant ss528838018A/G in the promoter region at position -6558. The other functional variants were observed with low frequency of minor allele (<0.005). NOS2A promoter variant (-954G/C) was not observed in Brazilian populations, and the new observed promoter variant (ss528838018A/G) as well as other promoter variants were not associated with any clinical forms of leprosy in the Brazilian populations.

Spectrum of autoantibodies for gastrointestinal autoimmune diseases in systemic lupus erythematosus patients.

BACKGROUND: Organ-specific autoimmune diseases may appear in patients with systemic lupus erythematosus (SLE). Gastrointestinal symptoms are well documented in SLE and may be similar to those related to autoimmune gastrointestinal diseases. OBJECTIVE: Our aim was to search for gastrointestinal organ-specific autoantibodies in 194 patients with systemic lupus and 103 healthy controls from Southern Brazil. Methods Anti-endomysium antibodies (IgA-EmA), anti-gastric parietal cells (GPC) antibodies, anti-smooth muscle antibodies (ASMA), anti-mitochondrial antibodies (AMA) and anti-LKM-1 (liver-kidney microsomal) were searched for using indirect immunofluorescence in the sera of patients and controls. RESULTS: The total positivity of antibodies in SLE patients was 14.4% (28/194) and differed significantly from healthy individuals (0.97%; p<0.001). IgA-EmA was more common in lupus patients than in controls (11/194; p=0.009), and one of these patients had dermatitis herpetiformis. Clinical association revealed that IgA-EmA was more common in SLE patients with discoid lesions. The frequency of anti-GPC (p=0.10), ASMA (p=0.16) and AMA (p=0.55) did not differ significantly between groups. No patient presented LKM-1 autoantibodies. One patient presenting anti-GPC was diagnosed with atrophic gastritis and pernicious anemia. CONCLUSION: Only IgA-EmA was significantly associated with lupus and with the presence of discoid lesions. Until now, no obvious association with celiac disease has been found.

Ficolin 2 (FCN2) functional polymorphisms and the risk of rheumatic fever and rheumatic heart disease.

Ficolins are pattern-recognition proteins involved in innate immunity, which upon binding to their specific pathogen-associated molecular patterns on the microbial surfaces trigger the immune response either by binding to collectin cellular receptors or by initiating the complement lectin pathway. In humans, three ficolin genes have been identified, which encode ficolin-1 (M-ficolin), ficolin-2 (L-ficolin) and ficolin-3 (H-ficolin or Hakata antigen). Ficolin-2 was shown to bind to lipoteichoic acid, a cell wall constituent in all Gram-positive bacteria such as Streptococcus pyogenes, which is the aetiological agent of rheumatic fever (RF) and its most severe sequelae, chronic rheumatic heart disease (CRHD). Here we investigated polymorphisms in the promoter region of the FCN2 gene (at positions -986/-602 and +4) in 122 patients with RF and CRHD and in 210 healthy subjects from the same geographic region and socioeconomic background. The haplotype -986/-602/-4 G/G/A, which is related to low levels of L-ficolin, was observed more frequently in the CRHD group when compared to the healthy subjects [99/162, 61.1% versus 211/420, 50.2%, odds ratio (OR) 1.6, confidence interval (CI) 95% 1.1-2.3, P = 0.021]. The haplotype -986/-602/-4 A/G/A was observed more frequently in the healthy group when compared to the affected (RF plus CRHD) subjects (31/420, 7.4% versus 6/244, 2.5%, OR 3.2, CI 95% 0.13-0.77, P = 0.008). Based on those findings, one can conclude that polymorphisms associated with low levels of L-ficolin level may predispose an individual to recurrent and/or more severe streptococcal infection.

Mannan-binding lectin MBL2 gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy.

Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan-binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2 polymorphism in 102 Euro-Brazilian patients with moderate and severe chronic hepatitis C, paired for gender and age with 102 HCV seronegative healthy individuals. Six common single nucleotide polymorphisms in the MBL2 gene, three in the promoter (H/L, X/Y and P/Q) and three in exon 1 (A, the wild-type, and B, C or D also known as O) were evaluated using real-time polymerase chain reaction with fluorescent hybridization probes. The concentration of MBL in plasma was measured by enzyme-linked immunosorbent assay. The frequency of the YA/YO genotype was significantly higher in the HCV patients compared with the controls (P = 0.022). On the other hand, the genotypes associated with low levels of MBL (XA/XA, XA/YO and YO/YO) were decreased significantly in the patients with severe fibrosis (stage F4), when compared with the patients with moderate fibrosis (stage F2) (P = 0.04) and to the control group (P = 0.011). Furthermore, MBL2 genotypes containing X or O mutations were found to be associated with non-responsiveness to pginterferon and ribavirin treatment (P = 0.023). MBL2 polymorphisms may therefore be associated not only with the development of chronic hepatitis C, but also with its clinical evolution and response to treatment.

Monoquaternary ammonium derivatives inhibit growth of protozoan parasites.

The phospholipid metabolism of Plasmodium falciparum-infected erythrocytes has been shown to be an effective pharmacological target for novel chemotherapy. Thirty-seven monoquaternary ammonium derivatives analogous to choline were screened for their potential antiprotozoal activity against P. falciparum and Leishmania braziliensis. Twenty-three compounds inhibited chloroquine resistant and sensitive P. falciparum strains with inhibitory concentrations ranging from 0.001 microM to 47 microM. Among the inhibitors were six compounds with nanomolar activity containing at least one ethyl group in the polar head and a hydrophobic alkyl chain with 10 to 14 methylene groups. Four compounds also exhibited in vitro antileishmanial properties in the micromolar range.

Circulating levels of mannan-binding lectin (MBL) and MBL-associated serine protease 2 in endemic pemphigus foliaceus.

Endemic pemphigus foliaceus (EPF) is an autoimmune disease, which occurs in Brazil and other regions of South America. Mannose-binding lectin (MBL) and MBL-associated serine protease (MASP-2) play a key role in innate immunity, and its deficiency has been related to increased susceptibility to infection and autoimmune diseases. MBL and MASP-2 serum levels were measured in 114 patients with EPF and in 100 healthy individuals in Brazil. MBL and MASP-2 levels were measured by sandwich assays (time-resolved immunofluorimetic assay) using monoclonal antibodies. No difference was observed in the MBL level in patients with EPF compared with controls [mean +/- SEM 1230.07 +/- 132.18 ng/mL (median 789.0 ng/mL) vs. 1036.98 +/- 117.99 ng/mL (median 559.5 ng/mL), P = 0.32]. Non-significant lower MASP-2 levels were observed in EPF [274.34 +/- 15.66 ng/mL (median 239.5 ng/mL ) vs. 304.72 +/- 15.28 ng/mL [median 261.0 ng/mL ), P = 0.06]. MBL deficiency (< 10 ng/mL) or MASP-2 deficiency (< 100 ng/mL) did not differ significantly between patients and controls. These data indicate that MBL and MASP-2 deficiency are not associated with susceptibility to EPF.

Further evidence of inflammation in chronic rheumatic valve disease (CRVD): high levels of advanced oxidation protein products (AOPP) and high sensitive C-reactive protein (hs-CRP).

Chronic rheumatic valve disease (CRVD) is a late sequel of Rheumatic Fever (RF) which appears in approximately 30% of RF patients, leading to valve injury. Advanced Oxidation Protein Products (AOPP) and high sensitive C-Reactive Protein (hs-CRP) plasma levels were measured in patients with CRVD in order to evaluate the presence of oxidative stress and systemic inflammation. A total of 90 patients (70 female, 20 male, mean age 46.01 +/- 11.72 years, range 24-69 years) with CRVD, who have or have not undergone valve replacement due to rheumatic etiology, and 46 healthy subjects (27 female, 19 male, mean age 41.89 +/- 9.02 years range 28-60 years) were studied. Levels of AOPP were measured by the determination of optical density (OD) at 340 nm under acidic conditions and hs-CRP by enhanced immunonephelometric assays. Significantly elevated levels of AOPP and hs-CRP were observed in CRVD patients when compared to the controls (AOPP 212.62 +/- 34.14 umol/l vs. 126.97 +/- 27.74 umol/l p < 0.00006 and for hs-CRP 5.40 +/- 1.98 mg/l vs. 2.66 +/- 1.36 mg/l p < 0.05). In addition, high levels of AOPP were associated to the presence of prosthetic valve and time after surgery (p < 0.0008 and p < 0.005, respectively). No correlation was observed between the levels of AOPP and hs-CRP with age, sex and degree of mitral valve stenosis. No correlation was found between AOPP and hs-CRP plasma values. These results suggest the involvement of oxidative stress and systemic inflammation in the pathogenesis of CRVD.

Prevalence of Mycobacterium avium and Mycobacterium tuberculosis in blood cultures of Brazilian AIDS patients after introduction of highly active retroviral therapy.

The use of highly active antiretroviral therapy (HAART) for the treatment of HIV infection has been associated with a marked reduction in the incidence of most opportunistic infections. From April 2001 to February 2002, 80 blood samples from patients who were suspected to have disseminated mycobacterial infection, presenting fever and (preferably) a CD4 T cell count < 100.0 cell/mL were investigated. Twelve (15%) of the 80 blood cultures were positive for mycobacteria, with Mycobacterium avium being identified in 7 (8.8%) samples and M. tuberculosis in 5 (6.2%). The TCD4+ count at the time of M. avium bacteremia ranged from 7 cells/microL (average of 48.5 cell/microL), while in M. tuberculosis bacteremia it ranged from 50.0 cells/microL (average of 80.0 cell/microL). The prevalence of M. avium bacteremia in our study follows the expected decline in opportunistic infections observed after the introduction of HAART; however, mycobacteremia by M. tuberculosis still indicates a high prevalence of tuberculosis infection in AIDS patients.

SC5b-9 is the most sensitive marker in assessing disease activity in Brazilian SLE patients.

This study investigated whether increased plasma levels of terminal complement complex (SC5b-9) or split products correlate with disease activity and clinical manifestations in Brazilian systemic lupus erythematosus (SLE) patients. Comparisons with conventional measurements of complement and other inflammatory markers were also performed. Plasma levels of SC5b-9, C3a desArg, C1rs-C1Inhibitor, C3b(Bb)P, C3, C4, erythrocyte sedimentation rate (ESR) and mucoproteins (MP) were measured in 41 patients with SLE of different disease activity: 10 patients with none, 15 patients with mild, and 16 patients with moderate or severe activity. All parameters, with the exception of C3 and C3b(Bb)P, showed a statistically significant correlation with disease activity. Plasma levels of SC5b-9, C3a desArg, C4, CH50, ESR and MP revealed significant differences between the groups of patients without activity and those with moderate or severe disease. Although none of the variables were able to discriminate between patients without and those with mild activity, SC5b-9, C3a desArg, C4, ESR and mucoproteins showed significant differences between the patients with mild and those with moderate or severe disease. Among all the variables, SC5b-9 levels showed the most significant results and correlated well with the severity of the disease (p < 0.0005). Our data suggest that elevated levels of complement activation products, particularly of SC5b-9 are more sensitive markers in assessing disease activity than conventional laboratory diagnosis. Modern complement diagnosis is therefore recommended for monitoring disease progress in SLE patients.

Complement activation in Brazilian patients with preeclampsia.

The aim of this study was to evaluate the complement activation in Brazilian patients with preeclampsia and to correlate it with the severity and clinical outcome of the disease. Plasma levels of C3d, SC5b-9, C3 and C4 were measured in 16 patients with preeclampsia and in 17 normotensive pregnant women. Ten patients developed severe and six mild disease. C3 and C4 levels were determined by turbidimetry using polyclonal specific antisera. C3d concentrations were evaluated through double-decker rocket immunoelectrophoresis and SC5b-9 was assayed by a double-antibody enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against a neoantigen expressed in the formed complex. The mean levels of all variables were significantly higher in the preeclamptic group (before the delivery) when compared to the normal pregnancies. The complex SC5b-9 followed by C3d showed the most significant results for those comparisons (p < or = 0.00001). The levels of all parameters in the preeclampsia patients decreased significantly after the delivery. Again, the complex SC5b-9 and C3d showed the most significant results (p < or = 0.0004). None of the studied variables showed statistically significant differences regarding the severity of preeclampsia. These results confirm the activation of complement in preeclampsia, suggesting that this activation is related to the disease manifestation. Our findings further emphasize the involvement of complement activation in the pathological manifestations of preeclampsia.

Utilização de planejamento fatorial para a determinação da capacidade antioxidante e doseamento de flavonoides totais em Verbena minutiflora Briq. ex Moldenke (gervai) / The use of factorial design for determining the antioxidant capacity and dosing the total flavonoids in Verbena minutiflora Briq. Ex Moldenke (Gervai)

RESUMO Verbena minutiflora Briq. ex Moldenke (gervai) tem seu uso medicinal relatado popularmente para tratamento de doenças hepáticas, diarreia e outros problemas de saúde. Entretanto, pouco se conhece a respeito de seus componentes químicos e estudos que comprovem suas propriedades medicinais são escassos. O objetivo desse estudo foi avaliar a composição química dos extratos aquosos e etanólicos de flores de V. minutiflora e otimizar processos de obtenção de extratos com maiores capacidades antioxidantes e maiores concentrações de flavonoides. O método de extração foi desenhado por planejamento fatorial, onde as variáveis para a determinação da capacidade antioxidante foram: pH, extração líquida, método e tempo de extração. Para a determinação de flavonoides totais as variáveis avaliadas por planejamento fatorial foram: concentração de hexametilenotetramina, tipo de ácido, volume de ácido e tempo de aquecimento. Os resultados das análises químicas dos extratos mostraram: aminogrupos, taninos e ácidos fixos (extrato aquoso) aminogrupos, flavonoides, triterpenos, esteroides, alcaloides e cumarinas (extrado hidroetanólico). Os resultados dos planejamentos fatoriais mostraram que o melhor método de extração para a capacidade antioxidante foi o que usou vórtex, por 35 min, com água:etanol 50:50, com pH1, obtendo 0,1899± 5,8.10-3 mmol expressos em ácido ascórbico g-1 nos extratos de V. minutiflora. Enquanto, para as dosagens de flavonoides totais as variáveis significantes foram: tipo de ácido e volume de ácido. A melhor extração obtida foi: 6,748. 10-2± 2,085 10-3% expressos em quercetina. Os resultados mostraram que o planejamento fatorial é uma importante ferramenta para a otimização de extração de componentes químicos em produtos naturais.

ABSTRACT Verbena minutiflora Briq. ex Moldenke (gervai) has its popular use reported for liver disorders treatments, diarrhea, and other health problems. However, little is known about its chemical components and studies that proves its medicinal properties are rare. The aim of this study was to evaluate the chemical composition of aqueous and ethanolic extracts from flowers of V. minutiflora and to optimize processes to obtain extracts with higher antioxidant capacity and greater concentration of flavonoids. The methods of extraction were designed by factorial planning, where the variables to determine the antioxidant capacity were: pH; extraction liquid; method and extraction time. To determinate the total flavonoids the variables evaluated by factorial design were: concentration of hexamethylenetetramine; type of acid; volume of acid and warming time. The results of chemical analysis of the extracts showed: amino groups, tannins and fixed acids (aqueous extract) amino groups, flavonoids, triterpenes, steroids, alkaloids and coumarins (hydroalcoholic extract). The factorial designs results showed that the best extraction method for the antioxidant capacity was the one that uses vortex, for 35 min, with water: ethanol 50:50, at pH 1, getting 0,1899 ± 5,8.10-3 mmol expressed in ascorbic acid g-1 in extracts of V. minutiflora . While, for dosages of total flavonoids the significant variables were the type of acid and volume of acid. The best extraction obtained was: 6,748. 10-2± 2,085 10-3% expressed in quercetin. These data showed that the factorial design is an important tool in optimizing the extraction of chemical components in natural products.

Multiplex sequence-specific polymerase chain reaction reveals new MASP2 haplotypes associated with MASP-2 and MAp19 serum levels.

Deficiency of mannan-binding lectin-associated serine protease 2 (MASP-2) has been associated with infections, whereas high levels appear to increase the risk of inflammatory disorders. Nevertheless, MASP2 haplotypes have been poorly investigated. To overcome haplotyping cost and time consumption, we developed multiplex polymerase chain reactions with sequence-specific primers (PCR-SSP) for 8 single nucleotide polymorphisms (SNPs), reducing the number of necessary reactions from 18 to 7. SNPs were distributed from the promoter to the last exon, and a single PCR-SSP was used for p.D120G. We evaluated the phylogenetic relationships and global distribution of 10 identified haplotypes in 338 Danish individuals with known MASP-2 and MAp19 levels and 309 South Brazilians. Four haplotypes were associated with reduced MASP-2 levels in plasma (lower than 200 ng/mL). Simultaneous association with the highest MASP-2 (over 600 ng/mL) and lowest MAp19 levels (lower than 200 ng/mL) was demonstrated with the intron 9 mutation (Kruskal-Wallis p < 0.0001). Cumulative genotype frequencies predict approximately 0.4% severely deficient and 25% overproducing individuals in both populations. Rapid and low-cost screening of patients with multiplex MASP2 PCR-SSP could be used to identify clinical conditions where MASP-2 (or MAp19) levels may be disease modifying, possibly improving disease outcome through early therapeutic and preventive measures.

Mannan-binding lectin plasma levels in leprosy: deficiency confers protection against the lepromatous but not the tuberculoid forms.

Mannan-binding lectin (MBL) is an important component of the first-line defence against infections. Evidence has shown that MBL deficiency, reducing phagocytosis and internalization of intracellular pathogens may protect the host against intracellular infections such as leprosy. In this study, we speculated whether genetically determined low MBL serum levels confer protection against Mycobacterium leprae infection. One hundred and ninety-one patients with leprosy, presenting lepromatous (n = 118), tuberculoid (n = 31), dimorph (n = 30) and indeterminate (n = 12) clinical forms and 110 healthy controls matched with the patients according to sex, age and ethnic background were investigated. MBL concentrations were measured in a double-antibody enzyme immune assay and C-reactive protein (CRP) serum levels by nephelometry. A significant negative association of MBL low values (< 100 ng/ml) was observed with lepromatous patients when comparing with controls and tuberculoid patients [10/118, 8.47%versus 21/110, 19.09%P = 0.03 chi(2) with Yates' correction, odds ratio (OR) 0.39, confidence interval (CI) 0.18-0.88 and 8/31, 25.81%, P = 0.02, OR 0.27, CI 0.09-0.75, respectively]. There was no significant difference in the distribution of MBL levels between patients and controls or among the clinical forms. The concentration of CRP was significantly increased in the patients (P = 0.0002) and in the lepromatous form (P = 0.0001) when compared to controls. A weak positive correlation between MBL and CRP levels was observed in the patients (P = 0.010, R = 0.255). These data suggest a protective role for MBL deficiency against the development of the most severe and multi-bacillary form of leprosy.