Arsenic Trioxide Triggers Apoptosis of Metastatic Oral Squamous Cells Carcinoma with Concomitant Downregulation of GLI1 in Hedgehog Signaling.

Given the lack of advances in Oral Squamous Cell Carcinoma (OSCC) therapy in recent years, pharmacological strategies to block OSCC-related signaling pathways have gained prominence. The present study aimed to evaluate the therapeutic potential of Arsenic Trioxide (ATO) concerning its antitumoral effects and the inhibition of the Hedgehog (HH) pathway in OSCC. Initially, ATO cytotoxicity was assessed in a panel of cell lines. Cell viability, cell cycle, death patterns, and cell morphology were analyzed, as well as the effect of ATO on the expression of HH pathway components. After the cytotoxic assay, HSC3 cells were chosen for all in vitro assays. ATO increased apoptotic cell death and nuclear fragmentation in the sub-G1 cell cycle phase and promoted changes in cell morphology. In addition, the reduced expression of GLI1 indicated that ATO inhibits HH activity. The present study provides evidence of ATO as an effective cytotoxic drug for oral cancer treatment in vitro.

Performance of Chimeric Trypanosoma cruzi Antigens in Serological Screening for Chagas Disease in Blood Banks.

Chagas disease (CD) is among the top 10 causes of inability to blood donation. Blood donation centers screen for anti-Trypanosoma cruzi antibodies using highly sensitive immunoenzymatic (ELISA) or chemiluminescent methods, which can lead to false positive results. Since positive samples cannot be used, to avoid the loss of valuable blood donations, it is necessary to improve specificity without reducing the sensitivity of the tests used for blood screening. For this purpose, our group has developed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) that have been evaluated in phase I and II studies with high performance and low cross-reactivity rates. The study included a panel of 5,014 serum samples collected from volunteer blood donors at the Hematology and Hemotherapy Foundation of the State of Bahia (Brazil). They were subjected to the detection of anti-T. cruzi antibodies, using all four IBMP antigens individually and latent class analysis (LCA) as a reference test, since there is no gold standard test for this purpose. Considering the sample size analyzed, LCA classified 4,993 (99.6%) samples as T. cruzi-negative and 21 (0.42%) as T. cruzi-positive. Sensitivity values ranged from 85.71% for IBMP-8.1 and 90.48% for IBMP-8.2-95.24% for IBMP-8.3 and 100% for IBMP-8.4, while specificity ranged from 99.98% for IBMP-8.3 and IBMP-8.4-100% for IBMP-8.1 and IBMP-8.2. Accuracy values ranged from 99.4 to 99.98%. The pretest probability for the molecules was 0.42, whereas the positive posttest probability ranged from 95.24 to 99.95% and the negative posttest probability ranged from 0.00001 to 0.0006% for all antigens. The higher odds ratio diagnosis was found for IBMP-8.4, which has been shown to be a safe single antigen for serological screening of CD in blood samples. The use of chimeric IBMP antigens is an alternative to reduce the number of bags discarded due to false-positive results. These molecules have high diagnostic performance and were shown to be suitable for use in screening CD in blood banks, isolated (IBMP-8.4) or in combination; and their use in blood banks could significantly reduce unnecessary disposal of blood bags or the risk of T. cruzi transmission.

Genetic Polymorphisms in Patients With Systemic Lupus Erythematosus and Jaccoud Arthropathy: A Pilot Study.

INTRODUCTION: Jaccoud arthropathy (JA) is a nonerosive and deforming arthropathy experienced frequently by patients with systemic lupus erythematosus (SLE). Although genetic polymorphisms are associated with SLE development, the association between genetic polymorphisms and JA has not been studied to date. The main objective of this study was to evaluate an association between HLA, STAT4, IRF5, and BLK polymorphisms and the presence of JA in Brazilian individuals with SLE. METHODS: Patients were selected from a cohort of individuals with SLE followed at 2 rheumatology reference centers in Salvador, Bahia, Brazil. The JA diagnosis was based on clinical and radiological criteria. The participants were genotyped for rs9271100, rs7574865, rs10488631, and rs13277113 polymorphisms in the HLA, STAT4, IRF5, and BLK genes, respectively, using real-time polymerase chain reaction. The presence of JA was correlated with allele frequencies, and clinical and laboratory data. RESULTS: One hundred forty-four individuals with SLE (38 with JA and 106 with SLE without JA) were studied. The mean age of the patients was 45 ± 12 years; the majority were women and had brown skin. Patients with JA had a longer disease duration than patients without JA. Serositis and neuropsychiatric manifestations were more frequent in the JA population. The A allele of rs13277113 in the BLK gene was associated with the presence of JA. CONCLUSIONS: The rs13277113 polymorphism in the BLK gene was found to be a possible genetic risk for JA development. However, further studies in larger populations should be performed to confirm this finding.

Low specificity of point-of-care circulating cathodic antigen (POCCCA) diagnostic test in a non-endemic area for schistosomiasis mansoni in Brazil.

A point-of-care test for detecting schistosome circulating cathodic antigen in urine (POCCCA) has been proposed for mapping infection and defining prevalence thresholds for mass drug administration (MDA). However, there is increasing evidence that POCCCA may yield false-positive results, which requires rigorous specificity evaluation in non-endemic areas. POCCCA was applied in an area known to be free from infection and devoid of any condition for schistosomiasis transmission as part of a multicentre study to evaluate the performance of POCCCA in Brazil's low or potentially endemic settings. Besides POCCCA detection in urine, a search for eggs in stool was performed by Kato-Katz (KK) and Helmintex (HTX) methods. One-hundred-and-seventy-four participants returned urine samples, 140 of which delivered stool samples. All these were HTX-negative for Schistosoma mansoni, and all 118 tested with KK were negative for both S. mansoni and soil-transmitted helminths. POCCCA results from freshly collected urine yielded a specificity of 62.1% (95% CI: 53.6% - 70.2%), taking trace outcomes as positive according to the manufacturer's instructions. Retesting urine from the 140 HTX-negatives after one-year storage at -20 °C with two new POCCCA batches simultaneously yielded significantly different specificities (34.3%; 95%CI: 26.5% - 42.8% and 75.0%; 95% CI: 67.0% - 81.9%). These two batches had a weak agreement (Cohen's kappa: 0.56; 95%CI: 0.44-0.68) among the 174 urine samples retested. At present, POCCCA cannot be recommended either as a cut-off point for MDA or a reliable diagnostic tool for treatment of the infection carriers (selective chemotherapy) in low endemic areas and at final stages of transmission interruption. Manufacturers should be required to optimize production standardization and to assure quality and reproducibility of the test. Extended rigorous performance evaluations by different users from different regions are needed before POCCCA is widely recommended.

Inhibition of CAL27 Oral Squamous Carcinoma Cell by Targeting Hedgehog Pathway With Vismodegib or Itraconazole.

Oral Squamous Cell Carcinoma (OSCC) presents an important challenge for the health systems worldwide. Thus, unraveling the biological mechanisms involved in OSCC pathogenesis is essential to the discovery of new drugs with anticancer potential. The Hedgehog (HH) pathway has shown promising results as a therapeutic target both in vitro and in vivo. This study aimed to investigate the effects of vismodegib and itraconazole on the expression of Hedgehog (HH) genes (PTCH1, SMO, and GLI1), cell cycle and cell death in OSCC cells. Alamar Blue assay was used to assess the cytotoxicity of vismodegib and itraconazole in a panel of oral cancer cell lines, including CAL27. The expression of HH signaling components after treatment with vismodegib and itraconazole, at concentrations of 25 or 50 µg/ml was evaluated by qPCR. Cell cycle and apoptosis were evaluated by flow cytometry after 72 h treatment with 50 µg/ml of vismodegib or itraconazole. HH signaling was activated in OSCC cell lines CAL27, SCC4, SCC9, and HSC3. Vismodegib and itraconazole significantly reduced CAL27 cell viability after 48 h of treatment. Gene expression of PTCH1, SMO, and GLI1 decreased in response to 24 h of treatment with vismodegib or itraconazole. Furthermore, CAL27 cells exhibited alterations in morphology, cell size, and cellular granularity. An increase in the DNA fragmentation was observed after treatment and both inhibitors induced apoptosis after 72 h. In conclusion, SMO inhibitors vismodegib and itraconazole demonstrably reduced the expression of HH genes in CAL27 OSCC cell line. In addition, treatment with vismodegib and itraconazole reduced cellular viability and altered the morphology of CAL27 cells, and also induced apoptosis.

ß-Lapachone and its iodine derivatives cause cell cycle arrest at G2/M phase and reactive oxygen species-mediated apoptosis in human oral squamous cell carcinoma cells.

ß-Lapachone is a natural naphthoquinone originally obtained from the bark of the purple Ipe (Tabebuia avellanedae Lor, Bignoniaceae) and its therapeutic potential in human cancer cells has been evaluated in several studies. In this study, we examined the effects of ß-lapachone and its 3-iodine derivatives (3-I-α-lapachone and 3-I-ß-lapachone) on cell proliferation, cell death, and cancer-related gene expression in human oral squamous cell carcinoma cells. ß-Lapachone and its 3-iodine derivatives showed potent cytotoxicity against different types of human cancer cell lines. Indeed, treatment with these compounds induced cell cycle arrest at G2/M phase, followed by internucleosomal DNA fragmentation, and caused significant increases in phosphatidylserine externalization, caspase-8 and -9 activation, mitochondrial membrane depolarization, reactive oxygen species (ROS) production, and apoptotic cell death morphology. The apoptosis induced by the compounds was prevented by pretreatment with a pan-caspase inhibitor (Z-VAD-FMK) and an antioxidant (N-acetyl-l-cysteine). In vivo, ß-lapachone and its 3-iodine derivatives significantly reduced tumor burden and did not alter any of the biochemical, hematological, or histological parameters of the animals. Overall, ß-lapachone and its 3-iodine derivatives showed promising cytotoxic activity due to their ability to induce cell cycle arrest at G2/M phase and promote caspase- and ROS-mediated apoptosis. In addition, ß-lapachone and its 3-iodine derivatives were able to suppress tumor growth in vivo, indicating that these compounds may be new antitumor drug candidates.

Dissemination of the ST-103 clonal complex serogroup C meningococci in Salvador, Brazil.

Invasive meningococcal disease (IMD) is a major public health problem worldwide. An epidemic of serogroup C (NmC) IMD occurred in 2010 in the city of Salvador. In this study, we describe the antigenic and genetic characterization of meningococcal isolates collected from meningitis cases in Salvador from 2001 to 2012. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed for the analysis of IMD isolates. A total of 733 cases were identified, and the serogroup was determined for 391 (53.0%) of these. Most cases were caused by NmC (53%) or B (47%). The most prevalent strains were B:4,7:P1.19,15 (32.9%; 129/391) and C:23:P1.14-6 (28.6%; 112/391). Based on PFGE/MLST analysis, 71.3% (77/108 PFGE-tested isolates) clustered as two clones of sequence type ST-3779 and ST-3780, both belonging to the ST-103 clonal complex. ST-3779 has been detected in Salvador since 1996 and together with ST-3780 became predominant after 2005. There was a predominance of C:23:P1.14-6, ST-3779/3780 in Salvador during the period of 2007-2012, establishing a major clonal lineage, which remained in the community for a long time; this has serious implications for public health, particularly in terms of prevention and control strategies of IMD.

Genotyping of HBV and tracking of resistance mutations in treatment-naïve patients with chronic hepatitis B.

BACKGROUND AND AIMS: Resistance mutation analogs to nucleos(t)ides have been described in treatment-naïve patients with chronic hepatitis B (CHB), with clinical implications. The aim of this study was to investigate primary resistance mutations and genotypes circulating in patients naïve to chronic hepatitis B, in the Northern and Northeastern regions of Brazil. METHODS: We conducted a study of resistance mutations and genotypic characterization of hepatitis B virus (HBV) in 189 treatment-naïve patients chronically infected with HBV. RESULTS: Drug resistance-associated mutations located in the RT domain of the P gene (rtHBV) were found in 6% of the treatment-naïve patients from the Northeastern Region. The mutations were rtA194T, rtL180M + rtM204V, rtS202I, rtM204I, and rtA181S. No patient in the Northern Region had the resistance mutation. In the gene S region, the frequency of vaccine escape mutations was 2.4% in the Northeastern Region and 8.6% in the Northern Region. CONCLUSION: This information before the start of treatment may contribute to clinical decision making, reducing treatment failure and the risk of progression to cirrhosis and hepatocellular carcinoma for CHB.

Heterogenic colonization patterns by Leptospira interrogans in Rattus norvegicus from urban slums.

We evaluated the renal colonization by Leptospira interrogans in Rattus norvegicus (rats), as it is the major natural reservoir of urban leptospirosis. We caught 72 R. norvegicus, out of which 32 were found to be positive for L. interrogans by immunofluorescence assay. From these rats, we selected 17 and divided them into six groups based on the mass-age/sex. We performed the immunohistochemistry test against L. interrogans in the kidney sections of the rats and systematically counted the colonized tubules (CTs) in 20 fields. The proportion of positive fields varied from 5% to 95%. The number of CTs in 20 fields varied from 0.5 to 85.5. These differences were not related to age or sex of the animals. The characterization of leptospiral colonization patterns in the natural reservoirs is important to better understand the host-pathogen interactions in leptospirosis.

Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor.

BACKGROUND: Leptospirosis is a zoonose that is increasingly endemic in built-up areas, especially where there are communities living in precarious housing with poor or non-existent sanitation infrastructure. Leptospirosis can kill, for its symptoms are easily confused with those of other diseases. As such, a rapid diagnosis is required so it can be treated effectively. A test for leptospirosis diagnosis using Leptospira Immunoglobulin-like (Lig) proteins is currently at final validation at Fiocruz. RESULTS: In this work, the process for expression of LigB (131-645aa) in E. coli BL21 (DE3)Star™/pAE was evaluated. No significant difference was found for the experiments at two different pre-induction temperatures (28 °C and 37 °C). Then, the strain was cultivated at 37 °C until IPTG addition, followed by induction at 28°C, thereby reducing the overall process time. Under this condition, expression was assessed using central composite design for two variables: cell growth at which LigB (131-645aa) was induced (absorbance at 600 nm between 0.75 and 2.0) and inducer concentration (0.1 mM to 1 mM IPTG). Both variables influenced cell growth and protein expression. Induction at the final exponential growth phase in shaking flasks with Abs(ind) = 2.0 yielded higher cell concentrations and LigB (131-645aa) productivities. IPTG concentration had a negative effect and could be ten-fold lower than the concentration commonly used in molecular biology (1 mM), while keeping expression at similar levels and inducing less damage to cell growth. The expression of LigB (131-645aa) was associated with cell growth. The induction at the end of the exponential phase using 0.1 mM IPTG at 28 °C for 4 h was also performed in microbioreactors, reaching higher cell densities and 970 mg/L protein. LigB (131-645aa) was purified by nickel affinity chromatography with 91% homogeneity. CONCLUSIONS: It was possible to assess the effects and interactions of the induction variables on the expression of soluble LigB (131-645aa) using experimental design, with a view to improving process productivity and reducing the production costs of a rapid test for leptospirosis diagnosis.

Immunomodulatory treatment with thalidomide in experimental leptospirosis in Golden Syrian hamsters (Mesocricetus auratus).

BACKGROUND: The benefit of antibiotics in leptospirosis is limited when treatment is started four days after symptoms appear, and new adjuvant therapeutic options are urgently needed. METHODS: Hamsters (Mesocricetus auratus) were infected by Leptospira interrogans strain L1-130, and groups were assigned based on no treatment (NONE), thalidomide only (TAL), ampicillin only (AMP) or both (AMP-TAL). Treatment was started two days after the onset of symptoms (experiment 1) and immediately after detection of the first death (experiment 2). RESULTS: Experiment 1: all hamsters from the groups AMP and AMP-TAL survived (n=8), while all hamsters from groups NONE (n=6) and TAL (n=8) died. The AMP and the AMP-TAL groups showed no renal or liver pathology and absent or very low leptospiral burden in target organs. Experiment 2: lethal outcome was observed in 6/6 hamsters in the NONE group, 8/8 in the TAL group, and 6/8 in both the AMP and AMP-TAL groups. Thalidomide showed no survival benefit when compared to hamsters treated with ampicillin alone. The TAL, AMP and AMP-TAL groups had very low tissue leptospiral counts. CONCLUSION: Thalidomide had minimal impact on survival in the late treatment of leptospirosis hamster model.

Ionic imbalance and lack of effect of adjuvant treatment with methylene blue in the hamster model of leptospirosis.

Leptospirosis in humans usually involves hypokalaemia and hypomagnesaemia and the putative mechanism underlying such ionic imbalances may be related to nitric oxide (NO) production. We previously demonstrated the correlation between serum levels of NO and the severity of renal disease in patients with severe leptospirosis. Methylene blue inhibits soluble guanylyl cyclase (downstream of the action of any NO synthase isoforms) and was recently reported to have beneficial effects on clinical and experimental sepsis. We investigated the occurrence of serum ionic changes in experimental leptospirosis at various time points (4, 8, 16 and 28 days) in a hamster model. We also determined the effect of methylene blue treatment when administered as an adjuvant therapy, combined with late initiation of standard antibiotic (ampicillin) treatment. Hypokalaemia was not reproduced in this model: all of the groups developed increased levels of serum potassium (K). Furthermore, hypermagnesaemia, rather than magnesium (Mg) depletion, was observed in this hamster model of acute infection. These findings may be associated with an accelerated progression to acute renal failure. Adjuvant treatment with methylene blue had no effect on survival or serum Mg and K levels during acute-phase leptospirosis in hamsters.

Surveillance for leptospirosis in the Americas, 1996-2005: a review of data from ministries of health.

OBJECTIVE: To characterize current leptospirosis reporting practices in the Americas. METHODS: Information was collected from the official websites of national ministries of health from the Americas region and two international organizations; personal communications; and three international morbidity databases. For all sources other than the morbidity databases, the review was limited to official reports citing clinically suspected and laboratory confirmed leptospirosis cases or deaths during the period 1996-2005. RESULTS: A total of 73 out of 1 644 reports met the selection criteria and were included in the analysis. Published leptospirosis data were available from half of the countries/sovereign territories (24 out of 48), and 18 of them had mandatory notification policies for leptospirosis. The sum of the median number of leptospirosis cases notified annually by the 24 countries/territories was 4 713.5, but just three countries (Brazil, Costa Rica, and Cuba) accounted for 83.1% (3 9cas20 es) of the notifications. Eight (16.7%) countries reported deaths due to leptospirosis. The sum of the median number of deaths reported annually for the eight countries was 380, but 349 (91.8%) were reported by Brazil. CONCLUSIONS: Notification practices in the Americas for leptospirosis are limited. Therefore, the numbers of cases and deaths reported are not representative for the region. The lack of leptospirosis data for many countries/territories may reflect weaknesses in certain aspects of national surveillance systems, including mandatory reporting policies, clinical laboratory infrastructure for performing case confirmation, and capacity to collect reported cases. Improved surveillance of leptospirosis cases and deaths in the Americas is needed to allow monitoring of regional epidemiological patterns and to estimate the burden of this important disease.

Inverse association between Lancefield group G Streptococcus colonization and sore throat in slum and nonslum settings in Brazil.

Group G Streptococcus has been implicated as a causative agent of pharyngitis in outbreak situations, but its role in endemic disease remains elusive. We found an unexpected inverse association of Streptococcus dysgalactiae subsp. equisimilis colonization and sore throat in a study of 2,194 children of 3 to 15 years of age in Salvador, Brazil.

[Detection of genotype 4 of the hepatitis C virus in Salvador, BA]. / Detecção do genótipo 4 do vírus da hepatite C em Salvador, BA.

The first detected case of genotype 4 of the hepatitis C virus (HCV) in Salvador, Bahia, is described. RT-PCR tests were used to detect HCV-RNA, and LIPA was used for genotyping. Genotype 4 responds poorly to treatment, and it is therefore important to actively search for people who have been in contact with it.