Effect of berberine associated with photodynamic therapy in cell lines.

Cervical cancer is a serious worldwide health problem. In view of the potentially harmful effects of current conventional therapies, photodynamic therapy may be an option as it is a minimally invasive therapy and can promote selective cytotoxic activity for neoplastic cells in the target tissue., Berberine (BBR) as an isolated molecule is a natural compound that has antineoplastic properties and potential action as a photosensitizer agent. The purpose of this study was to evaluate the use of berberine as a photosensitizer in photodynamic therapy (PDT) protocols and observe the effects produced by this association in cervical carcinoma cells and in immortalized keratinocytes. Incubation with 2.5 µM berberine promoted less than 10 % of cellular death in both cell lines studied. In addition, by fluorescence microscopy, we demonstrated that berberine was internalized by the cells, and after a period of 48 h, it was still present in the intracellular environment preferentially localized in the cytoplasm. After photodynamic therapy using berberine as a photosensitizer and visible light activation at 447 (±10) nm, we observed a phototoxic effect, which resulted in 19.84 % cell viability for Caski cells and 47.22 % cell viability for HaCaT. Treatment with berberine associated with photodynamic therapy promoted an increase in the production of reactive species of oxygen (ROS) and caspase-3 activity, indicating a preferential cell death mechanism by caspase-dependent apoptosis. Therefore, we demonstrated that berberine is an efficient photosensitizer and that its association with photodynamic therapy may be a potential anticancer treatment strategy for cervical cancer.

Adverse birth outcomes associated with Zika virus exposure during pregnancy in São José do Rio Preto, Brazil.

OBJECTIVES: We aimed to report the first 54 cases of pregnant women infected by Zika virus (ZIKV) and their virologic and clinical outcomes, as well as their newborns' outcomes, in 2016, after the emergence of ZIKV in dengue-endemic areas of São Paulo, Brazil. METHODS: This descriptive study was performed from February to October 2016 on 54 quantitative real-time PCR ZIKV-positive pregnant women identified by the public health authority of São José do Rio Preto, São Paulo, Brazil. The women were followed and had clinical and epidemiologic data collected before and after birth. Adverse outcomes in newborns were analysed and reported. Urine or blood samples from newborns were collected to identify ZIKV infection by reverse transcription PCR (RT-PCR). RESULTS: A total of 216 acute Zika-suspected pregnant women were identified, and 54 had the diagnosis confirmed by RT-PCR. None of the 54 women miscarried. Among the 54 newborns, 15 exhibited adverse outcomes at birth. The highest number of ZIKV infections occurred during the second and third trimesters. No cases of microcephaly were reported, though a broad clinical spectrum of outcomes, including lenticulostriate vasculopathy, subependymal cysts, and auditory and ophthalmologic disorders, were identified. ZIKV RNA was detected in 18 of 51 newborns tested and in eight of 15 newborns with adverse outcomes. CONCLUSIONS: Although other studies have associated many newborn outcomes to ZIKV infection during pregnancy, these same adverse outcomes were rare or nonexistent in this study. The clinical presentation the newborns we studied was mild compared to other reports, suggesting that there is significant heterogeneity in congenital Zika infection.

Natural compounds isolated from Brazilian plants are potent inhibitors of hepatitis C virus replication in vitro.

Compounds extracted from plants can provide an alternative approach to new therapies. They present characteristics such as high chemical diversity, lower cost of production and milder or inexistent side effects compared with conventional treatment. The Brazilian flora represents a vast, largely untapped, resource of potential antiviral compounds. In this study, we investigate the antiviral effects of a panel of natural compounds isolated from Brazilian plants species on hepatitis C virus (HCV) genome replication. To do this we used firefly luciferase-based HCV sub-genomic replicons of genotypes 2a (JFH-1), 1b and 3a and the compounds were assessed for their effects on both HCV replication and cellular toxicity. Initial screening of compounds was performed using the maximum non-toxic concentration and 4 compounds that exhibited a useful therapeutic index (favourable ratio of cytotoxicity to antiviral potency) were selected for extra analysis. The compounds APS (EC50=2.3µM), a natural alkaloid isolated from Maytrenus ilicifolia, and the lignans 3(∗)43 (EC50=4.0µM), 3(∗)20 (EC50=8.2µM) and 5(∗)362 (EC50=38.9µM) from Peperomia blanda dramatically inhibited HCV replication as judged by reductions in luciferase activity and HCV protein expression in both the subgenomic and infectious systems. We further show that these compounds are active against a daclatasvir resistance mutant subgenomic replicon. Consistent with inhibition of genome replication, production of infectious JFH-1 virus was significantly reduced by all 4 compounds. These data are the first description of Brazilian natural compounds possessing anti-HCV activity and further analyses are being performed in order to investigate the mode of action of those compounds.

Papel da proteína Mx1 na resposta a interferons e no processo neoplásico / Role of Mx1 protein in the response to interferons and neoplastic process

A proteína Mx1 é codificada por um gene induzido por interferon e compartilha a organização de seus domínios, a capacidade de homo-oligomerização e associação com membranas com as grandes dinaminas GTPases. A proteína Mx1 está envolvida na resposta contra um grande número de vírus de RNA, como aqueles pertencentes à família Buniavírus e o vírus influenza. Curiosamente, o gene MX1 foi encontrado como silenciado por metilação em diversos processos neoplásicos, incluindo carcinomas de cabeça e pescoço de células escamosas. Neste cenário, o silenciamento gênico de MX1 está associado à imortalização de uma série de linhagens celulares neoplásicas. Assim, Mx1 se destaca como uma das principais proteínas envolvidas nas respostas imunes induzidas por interferon e também desempenha um importante papel no controle do ciclo celular. Aqui discutimos os aspectos funcionais da proteína Mx1 abordando sua atividade antiviral, organização estrutural, envolvimento com neoplasias e, principalmente, os aspectos funcionais obtidos pela determinação de seus parceiros celulares.

The Mx1 protein is encoded by an interferon-induced gene and shares domain organization, homo-oligomerization capacity and membrane association with the large dynamin-like GTPases. The Mx1 protein is involved in the response to a large number of RNA viruses, such as the bunyavirus family and the influenza virus. Interestingly, it has also been found as a methylation-silenced gene in several types of neoplasm, including head and neck squamous cell carcinoma. In this scenario, MX1 gene silencing is associated with immortalization in several neoplastic cell lines. Thus, Mx1 stands out as one of the key proteins involved in interferon-induced immune response and also plays an important role in cell cycle control. Here we discuss some of the functions of the Mx1 protein, including its antiviral activity, protein folding and involvement in neoplasia, as well as those revealed by investigating its cellular partners.

Expression of TERT in precancerous gastric lesions compared to gastric cancer

The objective of this study was to determine the levels of TERT mRNA and TERT protein expression in stomach precancerous lesions such as intestinal metaplasia (IM) and gastric ulcer (GU) and compare them to gastric cancer (GC). Real-time PCR was performed to detect TERT mRNA expression levels in 35 biopsies of IM, 30 of GU, and 22 of GC and their respective normal mucosas. TERT protein was detected by immunohistochemistry in 68 samples, 34 of IM, 23 of GU, and 11 of GC. Increased TERT mRNA expression levels were observed in a significant number of cases, i.e., 46 percent of IM, 50 percent of GU, and 79 percent of GC. The relative mean level of TERT mRNA after normalization with the β-actin reference gene and comparison with the respective adjacent normal mucosa was slightly increased in the IM and GU groups, 2.008 ± 2.605 and 2.730 ± 4.120, respectively, but high TERT mRNA expression was observed in the GC group (17.271 ± 33.852). However, there were no statistically significant differences between the three groups. TERT protein-positive immunostaining was observed in 38 percent of IM, 39 percent of GU, and 55 percent of GC. No association of TERT mRNA and protein expression with Helicobacter pylori infection or other clinicopathological variables was demonstrable, except for the incomplete type vs the complete type of IM. This study confirms previous data of the high expression of both TERT mRNA and protein in gastric cancer and also demonstrates this type of changed expression in IM and GU, thus suggesting that TERT expression may be deregulated in precursor lesions that participate in the early stages of gastric carcinogenesis.

Expression of TERT in precancerous gastric lesions compared to gastric cancer.

The objective of this study was to determine the levels of TERT mRNA and TERT protein expression in stomach precancerous lesions such as intestinal metaplasia (IM) and gastric ulcer (GU) and compare them to gastric cancer (GC). Real-time PCR was performed to detect TERT mRNA expression levels in 35 biopsies of IM, 30 of GU, and 22 of GC and their respective normal mucosas. TERT protein was detected by immunohistochemistry in 68 samples, 34 of IM, 23 of GU, and 11 of GC. Increased TERT mRNA expression levels were observed in a significant number of cases, i.e., 46% of IM, 50% of GU, and 79% of GC. The relative mean level of TERT mRNA after normalization with the ß-actin reference gene and comparison with the respective adjacent normal mucosa was slightly increased in the IM and GU groups, 2.008 ± 2.605 and 2.730 ± 4.120, respectively, but high TERT mRNA expression was observed in the GC group (17.271 ± 33.852). However, there were no statistically significant differences between the three groups. TERT protein-positive immunostaining was observed in 38% of IM, 39% of GU, and 55% of GC. No association of TERT mRNA and protein expression with Helicobacter pylori infection or other clinicopathological variables was demonstrable, except for the incomplete type vs the complete type of IM. This study confirms previous data of the high expression of both TERT mRNA and protein in gastric cancer and also demonstrates this type of changed expression in IM and GU, thus suggesting that TERT expression may be deregulated in precursor lesions that participate in the early stages of gastric carcinogenesis.

Detection of bovine Herpesvirus type 5 in formalin-fixed, paraffin-embedded bovine brain by PCR: a useful adjunct to conventional tissue-based diagnostic test of bovine encephalitis.

The aim of this study was to evaluate the application of PCR technique for the detection of BoHV-5 in routinely formalin-fixed, paraffin-embedded brain tissues in 20 naturally infected calves affected by fatal meningoencephalitis. Brains were divided into two halves, one kept fresh for virus isolation and PCR assay, targeting the glycoprotein C gene from BoHV-5 genome. The other half brain, corresponding to posterior cortex region, was submitted to formalin fixation and embedded into paraffin blocks for microscopic evaluation and total DNA isolation. Most of the slides showed severe multifocal non-supurative encephalitis with neuronal degeneration, neurophagia, and no acidophilic intranuclear inclusions could be found in neurons and glial. The 20 fresh samples were confirmed, by virus isolation and PCR assay, as having the BoHV-5 virus and, respective glicoprotein C sequence, while 15 of 20 formalin-fixed, paraffin-embedded samples were considered positive for the same analysis. The results revealed the first description of PCR efficiency, applied to formalin-fixed, paraffin-embedded brain collected from naturally infected calves, improving the detection of BoHV-5 from archival samples in South America.

HFE gene mutations in Brazilian thalassemic patients

Hereditary hemochromatosis is a disorder of iron metabolism characterized by increased iron intake and progressive storage and is related to mutations in the HFE gene. Interactions between thalassemia and hemochromatosis may further increase iron overload. The ethnic background of the Brazilian population is heterogeneous and studies analyzing the simultaneous presence of HFE and thalassemia-related mutations have not been carried out. The aim of this study was to evaluate the prevalence of the H63D, S65C and C282Y mutations in the HFE gene among 102 individuals with alpha-thalassemia and 168 beta-thalassemia heterozygotes and to compare them with 173 control individuals without hemoglobinopathies. The allelic frequencies found in these three groups were 0.98, 2.38, and 0.29 percent for the C282Y mutation, 13.72, 13.70, and 9.54 percent for the H63D mutation, and 0, 0.60, and 0.87 percent for the S65C mutation, respectively. The chi-square test for multiple independent individuals indicated a significant difference among groups for the C282Y mutation, which was shown to be significant between the beta-thalassemia heterozygote and the control group by the Fisher exact test (P value = 0.009). The higher frequency of inheritance of the C282Y mutation in the HFE gene among beta-thalassemic patients may contribute to worsen the clinical picture of these individuals. In view of the characteristics of the Brazilian population, the present results emphasize the need to screen for HFE mutations in beta-thalassemia carriers.

Spatial expression of two anti-inflammatory mediators, annexin 1 and galectin-1, in nasal polyposis.

BACKGROUND: There is renewed interest in the role played by specific counter-regulatory mechanisms to control the inflammatory host response, poorly investigated in human pathology. Here, we monitored the expression of two anti-inflammatory mediators, annexin 1 and galectin-1, and assessed their potential link to glucocorticoids' (GCs) effective control of nasal polyposis (NP). METHODS: Total patterns of mRNA and protein expression were analysed by quantitative real-time PCR (qPCR) and Western blotting analyses, whereas ultrastructural immunocytochemistry was used for spatial localization and quantification of each mediator, focusing on mast cells, eosinophils and epithelial cells. RESULTS: Up-regulation of the annexin 1 gene, and down-regulation of galectin-1 gene, was detected in polypoid tissue compared with nasal mucosa. Patient treatment with betamethasone augmented galectin-1 protein expression in polyps. At the cellular level, control mast cells and eosinophils displayed higher annexin 1 expression, whereas marked galectin-1 immunolabelling was detected in the granule matrix of mast cells. Cells of glandular duct epithelium also displayed expression of both annexin 1 and galectin-1, augmented after treatment. CONCLUSION: Mast cells and epithelial cells appeared to be pivotal cell types involved in the expression of both annexin 1 and galectin-1. It is possible that annexin 1 and galectin-1 could be functionally associated with a specific mechanism in NP and that GC exert at least part of their beneficial effects on the airway mucosa by up-regulating, in a specific cell target fashion, these anti-inflammatory agonists.

LHX6 is a sensitive methylation marker in head and neck carcinomas.

Head and neck cancer remains a morbid and often fatal disease and at the present time few effective molecular markers have been identified. The purpose of the present work was to identify new molecular markers for head and neck squamous cell carcinoma (HNSCC). We applied methylation-sensitive arbitrarily primed PCR (MS/AP-PCR) to isolate sequences differentially methylated in HNSCC. The most frequently hypermethylated fragment we found maps close to a cytosine guanine dinucleotide (CpG) island on chromosome 9q33.2, and hypermethylation of this CpG island was associated with transcriptional silencing of an alternative transcript of the LHX6 gene. Using combined bisulfite restriction analysis (COBRA), hypermethylation of this fragment was detected in 13 of 14 (92.8%) HNSCC cell lines studied and 21 of 32 (65.6%) primary tumors, whereas little or no methylation was seen in 10 normal oral mucosa samples. We extended this investigation to other cancer cell lines and methylation was found in those derived from colon, breast, leukemia and lung, and methylation was also found in 12/14 primary colon tumors. These findings suggest that differentially methylated (DIME)-6 hypermethylation is a good cancer marker in HNSCC as well as in other kinds of neoplasias and confirm the importance of searching for markers of epigenetic dysregulation in cancer.

Human papillomavirus DNA and p53 status in penile carcinomas.

Our study aimed at evaluating the presence of human papillomavirus (HPV) DNA in a series of 84 paraffin-embedded (PET) penile carcinomas. We have also investigated the presence of p53 mutations in these tumors by immunohistochemistry (IHC), single-stranded conformational polymorphism (SSCP) and DNA sequencing. Tissues were submitted to amplification of a 268 bp fragment from the beta-globin gene and a fragment of the E6 gene of HPV types 6, 11, 16 and 18. Twenty samples (18 fixed in Bouin's solution and 2 in buffered formalin) were found inadequate and were excluded from the analysis. In the remaining 64 tumors, HPV DNA was found in 26% of the samples. The prevalence of HPV in fresh samples of the same tumors was 56%. The most prevalent type was HPV 16 in both fresh samples and PET. Isotopic in situ hybridization was performed in all PET samples, but only 2 cases were positive, 1 for HPV 16 and 1 for HPV 18. Immunohistochemistry with anti-p53 pAb 1801 antibody showed a positive nuclear reaction over more than 5% of tumor cells in 26% of the cases. SSCP of exons 5-8 of the p53 gene was performed on 9 HPV-positive and 12 HPV-negative specimens. Abnormal mobility was found in 26% of the tumors, of which 2 were HPV-positive and 5 HPV-negative. Point mutations were detected in p53 exons 6 (1 case), 7 (1 case) and 8 (5 cases), showing that high-risk type HPVs and mutated p53 may coexist in these tumors. Our data indicate that a subset of penile carcinomas are etiologically related to HPV and that an overlapping subset may arise from mutational events in the p53 gene.

Cytological diagnosis of serous effusions in a cancer hospital in Brazil.

The performance of cytological diagnosis in serous effusions was evaluated through a historical large series study which was carried out at Department of Surgical Pathology of Hospital A.C. Camargo. Files from the period of 1966 to 1990 were reviewed. Out of 4297 serous effusions samples (from 3379 patients), 2520 were pleural, 1763 ascitic and 14 pericardiac, with cytological report of malignancy in 917, 688 and 4 cases, respectively. Cytological diagnoses were confirmed true or false after correlation with the final diagnosis of the patients (clinical follow-up and/or histological evaluation). The results observed were 1982 false-negative, 21 false-positive, 1588 true-positive and 468 true-negative cases. Suspicious (161 or 3.75%) and inconclusive (77 or 1.79%) cases were not considered for statistical evaluation. Sensitivity, specificity, efficiency, as well as positive and negative predictive values were 44.5%, 95.7%, 50.1%, 98.7% and 20%, respectively. These values in pleural and ascitic effusions, separately showed similar performance, which was not observed in pericardiac samples due to small casistics. These data show low sensitivity and negative predictive values which contrast with the high specificity and positive predictive values that can be partially explained by the methodological limitations and invasive features of neoplasias.

Transdermal nicotine and gastroesophageal reflux.

OBJECTIVES: Studies have shown that cigarette smoking acutely increases gastroesophageal reflux (GER). Patients prescribed transdermal nicotine often complain of pyrosis. The purpose of this study was to ascertain the effect of transdermal nicotine on GER. METHODS: Twenty volunteers (12 smokers and eight nonsmokers) were studied with a 24-h pH/motility apparatus while wearing a placebo patch for 24 h and a nicotine patch for a subsequent 24-h period. Each subject completed the entire 48-h monitoring session. RESULTS: A significant increase in the total acid score (p = 0.005), duration of acid exposure (p = 0.010), and supine duration of acid exposure (p = 0.004) were noted with transdermal nicotine versus placebo. There were no significant differences in esophageal motility after transdermal nicotine. CONCLUSIONS: Whether enhanced supine reflux is related to lower esophageal sphincter dysfunction, increased acid secretion, or defective acid clearance is not clear. Nonetheless, it may be prudent to recommend removal of the transdermal nicotine patch at night in the patient with symptomatic nocturnal GERD.

Alterations in upper gastrointestinal motility in Helicobacter pylori-positive nonulcer dyspepsia.

OBJECTIVE: To study the association between gastrointestinal motility and Helicobacter pylori (HP) among patients with nonulcer dyspepsia (NUD). METHODS: We examined the gastric emptying and orocecal transit times (OCTT) in patients with NUD who were colonized with Helicobacter pylori (n = 27). NUD was defined as dyspeptic symptoms for at least 3 months in the absence of gastrointestinal pathology as seen on endoscopy and ultrasound. Subjects with diabetes mellitus, thyroid disorder, or abdominal surgery except appendectomy were excluded. The HP-negative patients with NUD (n = 38) served as controls. Solid phase gastric emptying was assessed by radionuclide scintigraphy. OCTT was determined by measuring exhaled breath hydrogen upon administration of lactulose. RESULTS: The two groups were similar with respect to age, sex, race, and history of smoking. Gastric emptying (t1/2) was 64.96 +/- 3.61 min in the HP-negative and 61.0 +/- 6.59 in the HP-positive group (p = NS). The OCTT was 130.9 +/- 17.26 minutes in the HP-negative and 84.28 +/- 11.07 in the HP-positive group (p = 0.03). There was no difference in the prevalence of nonhydrogen producers between the two groups. There was no correlation between gastric emptying and OCTT (p > 0.05). CONCLUSIONS: OCTT is faster among HP-positive patients with NUD than among HP-negative patients. However, gastric emptying is similar in the two groups.

Helicobacter pylori is associated with alterations in intestinal gas profile among patients with nonulcer dyspepsia.

The pathogenesis of nonulcer dyspepsia (NUD) is unknown. Gas and postprandial bloating are frequent symptoms. The role of Helicobacter pylori (HP) in the pathogenesis of NUD is controversial. We studied the intestinal gas profile of NUD patients (N = 34) at baseline and after lactulose administration. The prevalence of hydrogen and methane producers was similar among HP+ and HP- patients. Breath H2 concentrations in response to lactulose showed significantly greater rise among HP+ subjects (P < 0.0001). HP positivity was associated with higher total breath excretion for H2 and methane combined (2984 +/- 1038 vs 1776 +/- 521 ppm/hr) compared to HP- subjects (P < 0.05). There was no correlation between peak H2 and methane levels. The role of alterations in intestinal gas in producing symptoms in HP+ patients with NUD needs further investigation.

Oat cell carcinoma of the lung in patients with CLL.

Two malignancies in one patient was a remote possibility in the distant past, but currently more of these cases are being seen. At our institution, several patients with Chronic Lymphocytic Leukemia (CLL) were seen to develop oat cell carcinoma of the lung. The decreased immunity and B-cell dysfunction in CLL probably accounts for this secondary malignancy. We are presenting these cases to alert physicians of combined malignancies in a patient with increased aggressiveness of oat cell carcinoma.