Hepatitis B virus and hepatitis delta virus genotypes in outbreaks of fulminant hepatitis (Labrea black fever) in the western Brazilian Amazon region

The genotypes of hepatitis B (HBV) and delta (HDV) viruses circulating among fulminant hepatitis cases from the western Amazon Basin of Brazil were characterized in this study. HBV and HDV isolates were obtained from liver samples from 14 patients who developed fulminant hepatitis and died during 1978-1989. HBV DNA and HDV RNA were detected in all samples. Phylogenetic analyses of HDV sequences showed that they all clustered with previously characterized sequences of HDV genotype 3 (HDV-3). HBV genotypes F, A and D were found in 50.0, 28.6 and 21.4% of cases, respectively. These results confirm the predominance of HDV-3 in South America and its association with the severe form of hepatitis, and the finding of the co-infection of HDV-3 with different genotypes of HBV suggests that the association between HDV-3 and HBV-F is not necessarily causally related to a more severe clinical course of infection.

Identification of FAM46D as a novel cancer/testis antigen using EST data and serological analysis

Cancer/testis Antigens (CTAs) are immunogenic proteins with a restricted expression pattern in normal tissues and aberrant expression in different types of tumors being considered promising candidates for immunotherapy. We used the alignment between EST sequences and the human genome sequence to identify novel CT genes. By examining the EST tissue composition of known CT clusters we defined parameters for the selection of 1184 EST clusters corresponding to putative CT genes. The expression pattern of 70 CT gene candidates was evaluated by RT-PCR in 21 normal tissues, 17 tumor cell lines and 160 primary tumors. We were able to identify 4 CT genes expressed in different types of tumors. The presence of antibodies against the protein encoded by 1 of these 4 CT genes (FAM46D) was exclusively detected in plasma samples from cancer patients. Due to its restricted expression pattern and immunogenicity FAM46D represents a novel target for cancer immunotherapy. (c) 2009 Elsevier Inc. All rights reserved.

Association of the UGT1A1-53(TA)(n) polymorphism with L-thyroxine doses required for thyrotropin suppression in patients with differentiated thyroid cancer

There is a considerable interindividual variation in L-thyroxine [ 3,5,3`,5`-tetraiodo-l-thyronine (T(4))] dose required for thyrotropin (thyroid-stimulating hormone) suppression in patients with differentiated thyroid cancer. To investigate whether uridine diphosphate-glucuronosyl transferase 1A1 (UGT1A1)-mediated T(4) glucuronidation in liver affects T(4) dose, we genotyped 101 patients for the common UGT1A1-53(TA)(n) polymorphism and compared T(4) doses among patients having zero (5/6 and 6/6 genotypes), one (6/7 genotype), or two (7/7 and 7/8 genotypes) copies of the low-expression (TA) 7 and (TA) 8 alleles. A significant trend for decreasing T(4) dose with increasing number of copies of (TA)(7) and (TA)(8) (P = 0.037) and significant difference in T(4) dose across the UGT1A1-53(TA)(n) genotypes (P = 0.048) were observed, despite considerable overlap of T(4) doses among different genotypes. These results are consistent with reduced T(4) glucuronidation in patients with low-expression (TA) 7 and (TA) 8 alleles and provide the first evidence for association between UGT1A1-53(TA)(n) and T(4)-dose requirement for thyroid-stimulating hormone suppression in a natural clinical setting. Pharmacogenetics and Genomics 21: 341-343 (C) 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins. Pharmacogenetics and Genomics 2011, 21: 341-343

Evaluation of the genotoxicity of treated urban sludge in the Tradescantia micronucleus assay

The sludge produced in sewage treatment plants can contain toxic substances. Among these, the genotoxic substances are of great concern. The present paper aimed at evaluating the genotoxicity of treated sludge samples collected at four different sewage treatment plants (STP) located in the State of Sao Paulo Brazil, using the Trad-MN assay. Another objective of the study was to compare the responses of the Clone #4430 with the Tradescantia pallida. Sludge samples mixed with reference soil in concentrations of 10, 25 and 50% (v/v) were tested in experiments with 3 months exposure in the field. Negative and positive controls (arsenic trioxide) were also tested with both plants. In Clone #4430 two sludge samples induced genotoxicity while in T pallida three were positive, although no clear dose-response were observed for both plants. Results with the negative and positive controls suggest that T pallida presented similar results when compared to the Clone #4430. The protocol using plants chronically exposed to sludge mixed with soil seems to be a promising tool to assess the genotoxicity of sludge although time consuming. (C) 2008 Elsevier B.V. All rights reserved.

Deregulated expression of superoxide dismutase-2 correlates with different stages of cervical neoplasia

Objective: Superoxide dismutase-2 (SOD2) is considered one of the most important antioxidant enzymes that regulate cellular redox state in normal and tumorigenic cells. Overexpression of this enzyme may be involved in carcinogenesis, particularly in lung, gastric, colorectal and breast cancer. Methods: In the present study, we have evaluated SOD2 protein levels by immunohistochemistry (IHC) in 331 cervical histological samples including 31 low-grade cervical intraepithelial neoplasia (LSIL), 51 high-grade cervical intraepithelial neoplasia (HSIL), 197 squamous cervical carcinomas (SCC) and 52 cervical adenocarcinomas (ADENO). Results: We observed that SOD2 staining increases with cervical disease severity. Intense SOD2 staining was found in 13% of LSIL, 25.5% of HSIL and 40% of SCC. Moreover, 65.4% of ADENO exhibited intense SOD2 staining. Conclusions: Differences in the expression of SOD2 could potentially be used as a biomarker for the characterization of different stages of cervical disease.

Effects of anti-TNF therapy on glucose metabolism in patients with ankylosing spondylitis, psoriatic arthritis or juvenile idiopathic arthritis

The objective of this study was to evaluate the influence of anti-tumor necrosis factor (anti-TNF) in juvenile idiopathic arthritis (DA), ankylosing spondylitis (AS) or psoriatic arthritis (PsA). Sixty-two patients were investigated: 7 DA; 37 AS; and 18 PsA. Caucasian race accounted for 79% and 29% were female. Mean age was 40.4 +/- 12.6years. None of the patients had a history of diabetes, and none had used oral hypoglycemic agents or insulin. Treatment was with adalimumab, infliximab and etanercept. Glucose, inflammatory markers and prednisone dose were assessed at baseline, as well as after three and six months of treatment. The mean erythrocyte sedimentation rate was significantly lower at three months and six months than at baseline (13.7 +/- 18.0 and 18 +/- 22.5 vs. 27.9 +/- 23.4 mm; p = 0.001). At baseline, three months and six months, we found the following: mean C-reactive protein levels were comparable (22.1 +/- 22.7, 14.5 +/- 30.7 and 16.0 +/- 23.8 mg/L, respectively; p = 0.26); mean glucose levels remained unchanged (90.8 +/- 22.2 mg/dl, 89.5 +/- 14.6 mg/dl and 89.8 +/- 13.6 mg/dl, respectively; p = 0.91); and mean prednisone doses were low and stable (3.9 +/- 4.9 mg/day, 3.7 +/- 4.8 mg/day and 2.6 +/- 4.0 mg/day, respectively; p = 0.23). During the first six months of treatment, anti-TNF therapy does not seem to influence glucose metabolism in JIA, AS or PsA. (C) 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

HLA-DR in Brazilian patients with polyarteritis nodosa (PAN) and microscopic polyangiitis (MPA)

The aim of this study was to evaluate the frequency and clinical associations of HLA-DR alleles in Brazilian Caucasian patients with polyarteritis nodosa (PAN) or microscopic polyangiitis (MPA). We evaluated 29 Caucasian patients with vasculitis classified as PAN or MPA according to the American College of Rheumatology (ACR) 1990 Criteria, Chapel Hill Consensus Conference (CHCC) nomenclature for vasculitis and EULAR recommendations for conducting clinical studies in systemic vasculitis. HLA-DR alleles were typed using polymerase chain reaction-amplified DNA, hybridized with sequence-specific low resolution primers. DNA obtained from 59 Caucasian healthy blood donors were used as control. In order to evaluate if a specific HLA may have influence on the clinical profile of those diseases, we also divided the patients according to Birmingham vasculitis score (BVAS) and Five-Factors Score (FFS) at the time of diagnosis. Increased frequency of HLA-DRB1*16 (p = 0.023) and DRB4*01 (p = 0.048) was found in patients with higher disease activity at the time of diagnosis (BVAS >= 22). Patients with less severe disease (FFS = 0) had a higher frequency of HLA-DRB1*03 (p = 0.011). Patients with gastrointestinal tract involvement had significantly increased frequency of HLA-DRB1*11 or B1*12 (p = 0.046), B1*13 (p = 0.021) and B3 (p = 0.008). In contrast, patients with renal disease, had higher frequency of DRB1*15 or DRB1*16 (p = 0.035) and B5 (p = 0.035). In the subgroup of patients with MPA, increased frequency of HLA-DRB1*15 was found in patients with BVAS >= 22 (p = 0.038) and FFS >= 1 (p = 0.039) suggesting that this allele is associated with more aggressive disease. Antineutrophil cytoplasmic antibodies (ANCA) negative MPA patients had significantly increased frequency of HLA-DRB1*11 or DRB1*12 when compared to ANCA positive patients (p = 0.023). Our results suggest that HLA-DR alleles may influence PAN and MPA clinical expression and outcome and that in MPA they participate in the mechanisms involved in the development to ANCA.

Association of EGFR c.2073A > T polymorphism with decreased risk of diffusely infiltrating astrocytoma in a Brazilian case-control study

Epidermal growth factor receptor (EGFR) gene overexpression has been implicated in the development of many types of tumors, including glioblastomas, the most frequent diffusely infiltrating astrocytomas. However, little is known about the influence of the polymorphisms of EGFR on EGFR production and/or activity, possibly modulating the susceptibility to astrocytomas. This study aimed to examine the association of two EGFR promoter polymorphisms (c.-191C > A and c.-216G > T) and the c.2073A > T polymorphism located in exon 16 with susceptibility to astrocytomas, EGFR gene expression and survival in a case-control study of 193 astrocytoma patients and 200 cancer-free controls. We found that the variant TT genotype of the EGFR c.2073A > T polymorphism was associated with a significantly decreased risk of astrocytoma when compared with the AA genotype [sex- and age-adjusted odds ratio 0.51, 95% confidence interval 0.26-0.98]. No association of the two promoter EGFR polymorphisms (or combinations of these polymorphisms) and risk of astrocytomas, EGFR expression or survival was found. Our findings suggest that modulation of the EGFR c.2073A > T polymorphism could play a role in future therapeutic approaches to astrocytoma. (Int J Biol Markers 2008; 23: 140-6)

Trans-resveratrol inhibits early blood vessel formation (Vasculogenesis) without impairment of embryonic growth

Resveratrol is a stilbene compound found in grapes and other sources. In this study we examined the effects of trans-resveratrol (4.38-438 mu M/implant) in the vasculogenesis of yolk-sac membranes and its capacity to improve chick embryo growth. High concentrations of the stilbene (43.8-438 mu M) significantly inhibited early vessel formation, decreasing the percentage vitelline vessels of 3.5-day embryos by 50% compared to the control. In addition, basic fibroblast growth factor-stimulated vasculogenesis (140% of vessels as compared to control) was partially reversed by t-resveratrol (35% of inhibition) and treatments with cyclooxygenase inhibitors (acetylsalicylic acid and indomethacin) as well a protein-kinase C (PKC) activator (phorbol-12,13-dibutyrate) decreased the vessel number to 60%, 50%, and 44%, respectively. Treatments with t-resveratrol (4.38-43.8 mu M/implant) significantly increased the body length of embryos incubated in vitro uncoupled from any impairment in the body shape or detectable embryotoxic effect. We suggest that the antivasculogenic activity and the enhancement in embryonic growth promoted by non acute treatments with t-resveratrol were, at least in part, due to PKC inhibition. We suggest that t-resveratrol can be usable not only as a reliable functional nutriment, but also is useful for the development of prophylactic and/or therapeutic agents for treatment of angiogenic-degenerative diseases.

Expression of Mycobacterium leprae HSP65 in tobacco and its effectiveness as an oral treatment in adjuvant-induced arthritis

Transgenic plants are able to express molecules with antigenic properties. In recent years, this has led the pharmaceutical industry to use plants as alternative systems for the production of recombinant proteins. Plant-produced recominant proteins can have important applications in therapeutics, such as in the treatment of rheumatoid arthritis (RA). In this study, the mycobacterial HSP65 protein expressed in tobacco plants was found to be effective as a treatment for adjuvant-induced arthritis (AIA). We cloned the hsp65 gene from Mycobacterium leprae into plasmid pCAMBIA 2301 under the control of the double 35S promoter from cauliflower mosaic virus. Agrobacterium tumefaciens bearing the pChsp65 plasmid was used to transform tobacco plants. Incorporation of the hsp65 gene was confirmed by PCR, reverse transcription-PCR, histochemistry, and western blot analyses in several transgenic lines of tobacco plants. Oral treatment of AIA rats with the HSP65 protein allowed them to recover body weight and joint inflammation was reduced. Our results suggest a synergistic effect between the HSP65 expressed protein and metabolites presents in tobacco plants.

A subunit vaccine based on biodegradable microspheres carrying rHsp65 protein and KLK protects BALB/c mice against tuberculosis infection

Of the hundreds of new tuberculosis ( TB) vaccine candidates some have therapeutic value in addition to their prophylactic properties. This is the case for the DNA vaccine encoding heat-shock protein 65 (DNAhsp65) from Mycobacterium leprae. However, there are concerns about the use of DNA vaccines in certain populations such as newborns and pregnant women. Thus, the optimization of vaccination strategies that circumvent this limitation is a priority. This study evaluated the efficacy of a single dose subunit vaccine based on recombinant Hsp65 protein against infection with M. tuberculosis H37Rv. The Hsp65 protein in this study was either associated or not with immunostimulants, and was encapsulated in biodegradable PLGA microspheres. Our results demonstrate that the protein was entrapped in microspheres of adequate diameter to be engulfed by phagocytes. Mice vaccinated with a single dose of Hsp65-microspheres or Hsp65 + CpG-microspheres developed both humoral and cellular-specific immune responses. However, they did not protect mice against challenge with M. tuberculosis. By contrast, Hsp65+KLK-microspheres induced specific immune responses that reduced bacilli loads and minimized lung parenchyma damage. These data suggest that a subunit vaccine based on recombinant protein Hsp65 is feasible.

Protective efficacy of different strategies employing Mycobacterium leprae heat-shock protein 65 against tuberculosis

Background: Tuberculosis is a major threat to human health. The high disease burden remains unaffected and the appearance of extremely drug-resistant strains in different parts of the world argues in favor of the urgent need for a new effective vaccine. One of the promising candidates is heat-shock protein 65 when used as a genetic vaccine (DNAhsp65). Nonetheless, there are substantial data indicating that BCG, the only available anti-TB vaccine for clinical use, provides other important beneficial effects in immunized infants. Methods: We compared the protective efficacy of BCG and Hsp65 antigens in mice using different strategies: i) BCG, single dose subcutaneously; ii) naked DNAhsp65, four doses, intramuscularly; iii) liposomes containing DNAhsp65, single dose, intranasally; iv) microspheres containing DNAhsp65 or rHsp65, single dose, intramuscularly; and v) prime-boost with subcutaneous BCG and intramuscular DNAhsp65. Results: All the immunization protocols were able to protect mice against infection, with special benefits provided by DNAhsp65 in liposomes and prime-boost strategies. Conclusion: Among the immunization protocols tested, liposomes containing DNAhsp65 represent the most promising strategy for the development of a new anti-TB vaccine.

Alterations in myocardial gene expression associated with experimental Trypanosoma cruzi infection

Chagas disease, characterized by acute myocarditis and chronic cardiomyopathy, is caused by infection with the protozoan parasite Trypanosoma cruzi. We sought to identify genes altered during the development of parasite-induced cardiomyopathy. Microarrays containing 27,400 sequence-verified mouse cDNAs were used to analyze global gene expression changes in the myocardium of a murine model of chagasic cardiomyopathy. Changes in gene expression were determined as the acute stage of infection developed into the chronic stage. This analysis was performed on the hearts of male CD-1 mice infected with trypomastigotes of T. cruzi (Brazil strain). At each interval we compared infected and uninfected mice and confirmed the microarray data with dye reversal. We identified eight distinct categories of mRNAs that were differentially regulated during infection and identified dysregulation of several key genes. These data may provide insight into the pathogenesis of chagasic cardiomyopathy and provide new targets for intervention. (c) 2008 Elsevier Inc. All rights reserved.

Magnetic purification of biotinylated cDNA removes false priming and ensures strand-specificity of RT-PCR for enteroviral RNAs

The detection of replicative intermediate RNAs as markers of active replication of RNA viruses is an essential tool to investigate pathogenesis in acute viral infections, as well as in their long-term sequelae. In this regard, strand-specific PCR has been used widely to distinguish (-) and (+) enteroviral RNAs in pathogenesis studies of diseases such as dilated cardiomyopathy. It has been generally assumed that oligonucleotide-primed reverse transcription of a given RNA generates only the corresponding specific cDNA, thus assuring the specificity of a PCR product amplified from it. Nevertheless, such assumed strand-specificity is a fallacy, because falsely primed cDNAs can be produced by RNA reverse transcription in the absence of exogenously added primers, (cDNA(primer)(-)), and such falsely primed cDNAs are amplifiable by PCR in the same way as the correctly primed cDNAs. Using as a prototype the coxsackievirus B5 (CVB5), a (+) strand RNA virus, it was shown that cDNA(primer)(-) renders the differential detection of viral (-) and (+) RNAs by conventional PCR virtually impossible, due to gross non-specificity. Using in vitro transcribed CVB5 RNAs (+) and (-), it was shown that cDNA(primer)(-) could be removed effectively by magnetic physical separation of correctly primed biotinylated cDNA. Such strategy enabled truly strand-specific detection of RNA (-) and (+), not only for CVB5, but also for other non-polio enteroviruses. These findings indicate that previous conclusions supporting a role for the persistence of actively replicating enterovirus in the pathogenesis of chronic myocarditis should be regarded with strong skepticism and purification of correctly primed cDNA should be used for strand-specific PCR of viral RNA in order to obtain reliable information on this important subject. (C) 2009 Elsevier B.V. All rights reserved.

Stable and high-level production of recombinant Factor IX in human hepatic cell line

Hemophilia B is a genetic disease of the coagulation system that affects one in 30,000 males worldwide. Recombinant human Factor IX (rhFIX) has been used for hemophilia B treatment, but the amount of active protein generated by these systems is inefficient, resulting in a high-cost production of rhFIX. In this study, we developed an alternative for rhFIX production. We used a retrovirus system to obtain two recombinant cell lines. We first tested rhFIX production in the human embryonic kidney 293 cells (293). Next, we tested a hepatic cell line (HepG2) because FIX is primarily expressed in the liver. Our results reveal that intracellular rhFIX expression was more efficient in HepG2/rhFIX (46%) than in 293/rhFIX (21%). The activated partial thromboplastin time test showed that HepG2/rhFIX expressed biologically active rhFIX 1.5 times higher than 293/rhFIX (P = 0.016). Recovery of rhFIX from the HepG2 by reversed-phase chromatography was straightforward. We found that rhFIX has a pharmacokinetic profile similar to that of FIX purified from human plasma when tested in hemophilic B model. HepG2/rhFIX cell line produced the highest levels of rhFIX, representing an efficient in vitro expression system. This work opens up the possibility of significantly reducing the costs of rhFIX production, with implications for expanding hemophilia B treatment in developing countries.