Phenotype, treatment practice and outcome in the cobalamin-dependent remethylation disorders and MTHFR deficiency: Data from the E-HOD registry.

AIM: To explore the clinical presentation, course, treatment and impact of early treatment in patients with remethylation disorders from the European Network and Registry for Homocystinurias and Methylation Defects (E-HOD) international web-based registry. RESULTS: This review comprises 238 patients (cobalamin C defect n = 161; methylenetetrahydrofolate reductase deficiency n = 50; cobalamin G defect n = 11; cobalamin E defect n = 10; cobalamin D defect n = 5; and cobalamin J defect n = 1) from 47 centres for whom the E-HOD registry includes, as a minimum, data on medical history and enrolment visit. The duration of observation was 127 patient years. In 181 clinically diagnosed patients, the median age at presentation was 30 days (range 1 day to 42 years) and the median age at diagnosis was 3.7 months (range 3 days to 56 years). Seventy-five percent of pre-clinically diagnosed patients with cobalamin C disease became symptomatic within the first 15 days of life. Total homocysteine (tHcy), amino acids and urinary methylmalonic acid (MMA) were the most frequently assessed disease markers; confirmatory diagnostics were mainly molecular genetic studies. Remethylation disorders are multisystem diseases dominated by neurological and eye disease and failure to thrive. In this cohort, mortality, thromboembolic, psychiatric and renal disease were rarer than reported elsewhere. Early treatment correlates with lower overall morbidity but is less effective in preventing eye disease and cognitive impairment. The wide variation in treatment hampers the evaluation of particular therapeutic modalities. CONCLUSION: Treatment improves the clinical course of remethylation disorders and reduces morbidity, especially if started early, but neurocognitive and eye symptoms are less responsive. Current treatment is highly variable. This study has the inevitable limitations of a retrospective, registry-based design.

Cynomolgus monkeys (Macaca fascicularis) experimentally infected with B19V and hepatitis A virus: no evidence of the co-infection as a cause of acute liver failure.

This study was conducted to analyse the course and the outcome of the liver disease in the co-infected animals in order to evaluate a possible synergic effect of human parvovirus B19 (B19V) and hepatitis A virus (HAV) co-infection. Nine adult cynomolgus monkeys were inoculated with serum obtained from a fatal case of B19V infection and/or a faecal suspension of acute HAV. The presence of specific antibodies to HAV and B19V, liver enzyme levels, viraemia, haematological changes, and necroinflammatory liver lesions were used for monitoring the infections. Seroconversion was confirmed in all infected groups. A similar pattern of B19V infection to human disease was observed, which was characterised by high and persistent viraemia in association with reticulocytopenia and mild to moderate anaemia during the period of investigation (59 days). Additionally, the intranuclear inclusion bodies were observed in pro-erythroblast cell from an infected cynomolgus and B19V Ag in hepatocytes. The erythroid hypoplasia and decrease in lymphocyte counts were more evident in the co-infected group. The present results demonstrated, for the first time, the susceptibility of cynomolgus to B19V infection, but it did not show a worsening of liver histopathology in the co-infected group.

Hepatitis A and E seroprevalence and associated risk factors: a community-based cross-sectional survey in rural Amazonia.

BACKGROUND: Hepatitis A virus (HAV) and hepatitis E virus (HEV) are both transmitted by the faecal-oral route, and represent common causes of acute hepatitis in developing countries. The endemicity of HAV infection has shifted from high to moderate in Brazil. Human cases of HEV infection seem to be rare, although the virus has been detected in swine livestock and effluents of slaughterhouses. This study was to determine the epidemiology of hepatitis A and E in one of the largest agricultural settlements in the Amazon Basin of Brazil. METHODS: Serum samples collected from 397 individuals aged between 5 and 90 years during a population-based cross-sectional survey were tested for anti-HAV and anti-HEV antibodies. Associated risk factors and spatial clustering of HAV and HEV seropositivity were also analyzed. RESULTS: The overall rate of HAV seropositivity was 82.9% (95% confidence interval (CI), 79.2-86.6%). Multilevel logistic regression analysis identified increasing age (in years; odds ratio (OR), 1.097; 95% CI, 1.050-1.147; P < 0.001) and crowding (OR, 1.603; 95% CI, 1.054-2.440; P = 0.028) as significant risk factors for HAV seropositivity. Anti-HEV IgG was detected in 50/388 settlers (12.9%, 95% CI, 9.5-16.2%). Anti-HEV IgM was detected in 7/43 (16.3%) anti-IgG positive samples, and 4 of them had a confirmed result by immunoblot. Increasing age was the only significant determinant of HEV seropositivity (OR, 1.033; 95% CI, 1.016-1.050; P < 0.001). No significant spatial clustering of HAV and HEV seropositivity was detected in the area. CONCLUSIONS: Both HAV and HEV are endemic, with differing rates of infection in children and adults in this rural setting of the Brazilian Amazon. Anti-HEV prevalence was considerably higher than those previously reported in Brazil. The detection of HEV- specific IgM antibodies in four asymptomatic individuals is highly suggestive of the circulation of HEV in this rural population.

The efficiency of concentration methods used to detect enteric viruses in anaerobically digested sludge.

The presence of enteric viruses in biosolids can be underestimated due to the inefficient methods (mainly molecular methods) used to recover the viruses from these matrices. Therefore, the goal of this study was to evaluate the different methods used to recover adenoviruses (AdV), rotavirus species A (RVA), norovirus genogroup II (NoV GII) and the hepatitis A virus (HAV) from biosolid samples at a large urban wastewater treatment plant in Brazil after they had been treated by mesophilic anaerobic digestion. Quantitative polymerase chain reaction (PCR) was used for spiking experiments to compare the detection limits of feasible methods, such as beef extract elution and ultracentrifugation. Tests were performed to detect the inhibition levels and the bacteriophage PP7 was used as an internal control. The results showed that the inhibitors affected the efficiency of the PCR reaction and that beef extract elution is a suitable method for detecting enteric viruses, mainly AdV from biosolid samples. All of the viral groups were detected in the biosolid samples: AdV (90%), RVA, NoV GII (45%) and HAV (18%), indicating the viruses' resistance to the anaerobic treatment process. This is the first study in Brazil to detect the presence of RVA, AdV, NoV GII and HAV in anaerobically digested sludge, highlighting the importance of adequate waste management.

Evaluation of n-butyl cyanoacrylate adhesive in rat subcutaneous tissue.

BACKGROUND: Tissue adhesives have been widely used for wound closure, especially in children, because they are painless, fast, and easy to use and result in minimal scarring. OBJECTIVE: To analyze the biocompatibility of an adhesive based on n-butyl-cyanoacrylate in the subcutaneous tissue of rats. MATERIALS AND METHODS: Two surgical sites were prepared (approximately 3 cm apart): one on the left side of the animal and the other on the right side); polyethylene tubes were implanted in each surgical site. The tube on the left was filled with n-butyl-cyanoacrylate (treated group) and the tube on the right side was unfilled (control group). After 7, 30, and 120 days, the animals were killed, and the specimens were processed for histologic analysis. RESULTS: No significant inflammatory reaction occurred in the treated group, showing results similar to the control group. CONCLUSION: This adhesive based on n-butyl-cyanoacrylate is biocompatible in the subcutaneous tissue of rats.

Detection of enteric viruses in recreational waters of an urban lagoon in the city of Rio de Janeiro, Brazil.

The aim of this study was to determine the occurrences of the group A rotavirus (RVA), norovirus (NoV) and human adenovirus (HAdV) in the surface waters of an urban lagoon (Rodrigo de Freitas Lagoon) in the city of Rio de Janeiro, Brazil. During one year of surveillance, water samples were obtained from the lagoon and other interconnected ecosystems (river and beach). The samples were concentrated using an adsorption-elution method with a negatively charged membrane and tested by qualitative and quantitative polymerase chain reaction assays. RVA was the most prevalent virus detected (24.3%) with a viral load ranging from 3.0 x 10¹-5.6 x 10(4) genome copies/L, followed by NoV (18.8%) and HAdV (16.7%). Considering water samples suitable for bathing, according to Escherichia coli criterion (< 2,000 most probable number/100 mL), viruses were detected in 50% (57/114) of them. Physicochemical parameters were also measured and showed possible correlations between turbidity and RVA presence and between pH and NoV presence. These data demonstrate the importance of considering viral parameters to ensure water quality and the utilisation of these parameters as additional tools for the characterisation of environmental contamination.

Declining prevalence of hepatitis A virus antibodies among children from low socioeconomic groups reinforces the need for the implementation of hepatitis A vaccination in Brazil.

Age-related seroprevalence studies that have been conducted in Brazil have indicated a transition from a high to a medium endemicity of hepatitis A virus (HAV) infection in the population. However, most of these studies have focused on urban populations that experience lower incidence rates of HAV infection. In the current study, the prevalence of anti-HAV antibodies was investigated in children with a low socioeconomic status (SES) that live on the periphery of three capital cities in Brazil. A total of 1,162 dried blood spot samples were collected from individuals whose ages ranged from one-18 years and tested for anti-HAV antibodies. A large number of children under five years old (74.1-90%) were identified to be susceptible to HAV infection. The anti-HAV antibody prevalence reached ≥ 50% among those that were 10-14 years of age or older. The anti-HAV prevalence rates observed were characteristics of regions with intermediate level of hepatitis A endemicity. These data indicated that a large proportion of children with a low SES that live at the periphery of urban cities might be at risk of contracting an HAV infection. The hepatitis A vaccine that is currently offered in Brazil is only available for high-risk groups or at private clinics and is unaffordable for individuals with a lower SES. The results from this study suggest that the hepatitis A vaccine should be included in the Brazilian National Program for Immunisation.

Characterization and in vitro evaluation of bacterial cellulose membranes functionalized with osteogenic growth peptide for bone tissue engineering.

The aim of this study was to characterize the physicochemical properties of bacterial cellulose (BC) membranes functionalized with osteogenic growth peptide (OGP) and its C-terminal pentapeptide OGP[10-14], and to evaluate in vitro osteoinductive potential in early osteogenesis, besides, to evaluate cytotoxic, genotoxic and/or mutagenic effects. Peptide incorporation into the BC membranes did not change the morphology of BC nanofibers and BC crystallinity pattern. The characterization was complemented by Raman scattering, swelling ratio and mechanical tests. In vitro assays demonstrated no cytotoxic, genotoxic or mutagenic effects for any of the studied BC membranes. Culture with osteogenic cells revealed no difference in cell morphology among all the membranes tested. Cell viability/proliferation, total protein content, alkaline phosphatase activity and mineralization assays indicated that BC-OGP membranes enabled the highest development of the osteoblastic phenotype in vitro. In conclusion, the negative results of cytotoxicity, genotoxicity and mutagenicity indicated that all the membranes can be employed for medical supplies, mainly in bone tissue engineering/regeneration, due to their osteoinductive properties.

Exposure to multiple subgenotypes of hepatitis A virus during an outbreak using matched serum and saliva specimens.

Matched serum and saliva samples were collected simultaneously from 124 subjects exposed during a hepatitis A virus (HAV) outbreak at a daycare center in Rio de Janeiro, Brazil. All samples were tested for IgM and total anti-HAV antibodies by enzyme immunoassay (EIA). HAV was detected by nested PCR in serum, saliva, and water samples employing primers for the VP1/2A region of the viral RNA; all positive products were then sequenced. The viral load of the matched samples was determined by real-time PCR using the TaqMan system. HAV-RNA was identified by nested PCR in 37.7% of the saliva samples, 29% of the serum samples, and one drinking water sample. The mean HAV viral load was similar in the serum and saliva specimens (10(3) copies/ml). HAV genotypes IA and IB were detected in both specimen types, and the water sample isolate was classified as genotype IB, indicating the existence of more than one source of infection at the daycare center. In six infected patients, a different HAV subgenotype was found in their serum than in their saliva, and this unusual pattern of mixed HAV infection was investigated further by molecular cloning followed by nucleotide sequencing. All clones derived from the saliva samples belonged to subgenotype IB and shared 96.5-100% identity. However, clones derived from their corresponding serum sample belonged to subgenotype IA and shared 90.5-100% identity. This study showed the important role that non-invasive saliva samples can play in the molecular epidemiological analysis of a hepatitis A outbreak.

Suturable elastomeric tubular grafts with patterned porosity for rapid vascularization of 3D constructs.

Vascularization is considered to be one of the key challenges in engineering functional 3D tissues. Engineering suturable vascular grafts containing pores with diameter of several tens of microns in tissue engineered constructs may provide an instantaneous blood perfusion through the grafts improving cell infiltration and thus, allowing rapid vascularization and vascular branching. The aim of this work was to develop suturable tubular scaffolds to be integrated in biofabricated constructs, enabling the direct connection of the biofabricated construct with the host blood stream, providing an immediate blood flow inside the construct. Here, tubular grafts with customizable shapes (tubes, Y-shape capillaries) and controlled diameter ranging from several hundreds of microns to few mm are fabricated based on poly(glycerol sebacate) (PGS)/poly(vinyl alcohol) (PVA) electrospun scaffolds. Furthermore, a network of pore channels of diameter in the order of 100µm was machined by laser femtosecond ablation in the tube wall. Both non-machined and laser machined tubular scaffolds elongated more than 100% of their original size have shown suture retention, being 5.85 and 3.96 N mm-2respectively. To demonstrate the potential of application, the laser machined porous grafts were embedded in gelatin methacryloyl (GelMA) hydrogels, resulting in elastomeric porous tubular graft/GelMA 3D constructs. These constructs were then co-seeded with osteoblast-like cells (MG-63) at the external side of the graft and human umbilical vein endothelial cells inside, forming a bone osteon model. The laser machined pore network allowed an immediate endothelial cell flow towards the osteoblasts enabling the osteoblasts and endothelial cells to interact and form 3D structures. This rapid vascularization approach could be applied, not only for bone tissue regeneration, but also for a variety of tissues and organs.

Experimental hepatitis A virus (HAV) infection in cynomolgus monkeys (Macaca fascicularis): evidence of active extrahepatic site of HAV replication.

This work studied the replication sites of hepatitis A virus (HAV) in cynomolgus monkeys (Macaca fascicularis) after intravenous inoculation. The cynomolgus monkeys were inoculated with the Brazilian hepatitis A virus strain (HAF-203). Monkeys were euthanized on days 15, 30, 45 and 60 postinoculation (pi). Liver samples, submandibular salivary gland, mesenteric lymph node and tonsils were removed for virological and pathological evaluation. Immunofluorescence analyses on liver and salivary gland sections using confocal laser scanning microscopy revealed the presence of HAV antigen (HAV Ag). The presence of HAV genome was monitored by real-time PCR. The HAV RNA was detected at 7 days postinoculation (dpi), concomitantly in serum, saliva and faeces. The highest HAV viral load was observed in faeces at 15 dpi (10(5) copies/ml), followed by serum viral load of 10(4) copies/ml at 20 dpi and saliva viral load of 10(3 )copies/ml at 7 dpi. The animals showed first histological and biochemical signs of hepatitis at 15 dpi. The HAV antigen (Ag) was present from day 7 until day 60 pi in the liver and salivary glands. The HAV replicative intermediate was also detected in the liver (4.5 x 10(4) copies/mg), salivary glands (1.9 x 10(3) copies/mg), tonsils (4.2 x 10(1) copies/mg) and lymph nodes (3.4 x 10(1) copies/mg). Our data demonstrated that the salivary gland as an extrahepatic site of early HAV replication could create a potential risk of saliva transmitted infection. In addition, the cynomolgus monkey was confirmed as a suitable model to study the pathogenesis of HAV human infection.

Evidences of HEV genotype 3 persistence and reactivity in liver parenchyma from experimentally infected cynomolgus monkeys (Macaca fascicularis).

Hepatitis E virus genotype 3 (HEV-3) is an emerging zoonotic pathogen, responsible for sporadic cases of acute hepatitis E worldwide. Primate models have proven to be an essential tool for the study of HEV pathogenesis. Here we describe the outcomes of HEV infection in Macaca fascicularis (cynomolgus) inoculated experimentally with genotype 3. Eight adult cynomolgus macaques were inoculated intravenously with HEV-3 viral particles isolated from swine and human samples. Liver, spleen, duodenum, gallbladder and bile were sequential assessed up to the end-point of this study, 67 days post-inoculation (dpi). Our previously published findings showed that biochemical parameters return gradually to baseline levels at 55 dpi, whereas anti-HEV IgM and HEV RNA become undetectable in the serum and feces of all animals, indicating a non-viremic phase of recovery. Nevertheless, at a later stage during convalescence (67 dpi), the presence of HEV-3 RNA and antigen persist in central organs, even after peripheral viral clearance. Our results show that two cynomolgus inoculated with swine HEV-3 (animals I3 and O1) presented persistence of HEV RNA low titers in liver, gallbladder and bile. At this same stage of infection, HEV antigen (HEV Ag) could be detected in all infected animals, predominantly in non-reactive Kupffer cells (CD68+iNOS-) and sinusoidal lining cells. Simultaneously, CD4+, CD3+CD4+, and CD3+CD8+ immune cells were identified in hepatic sinusoids and small inflammatory clusters of lobular mononuclear cells, at the end-point of this study. Inability of HEV clearance in humans can result in chronic hepatitis, liver cirrhosis, with subsequent liver failure requiring transplantation. The results of our study support the persistence of HEV-3 during convalescence at 67 dpi, with active immune response in NHP. We alert to the inherent risk of viral transmission through liver transplantation, even in the absence of clinical and biochemical signs of acute infection. Thus, besides checking conventional serological markers of HEV infection, we strongly recommend HEV-3 RNA and antigen detection in liver explants as public health measure to prevent donor-recipient transmission and spread of hepatitis E.

Comparison between serum and saliva for the detection of hepatitis A virus RNA.

Due to the ease of collection, oral fluid is being investigated as an alternative to serum for diagnostic and epidemiological purposes. However, for prospective studies involving hepatitis A virus (HAV) RNA detection, a standard methodology must be developed. In the present study, nested RT-PCR and real-time PCR were optimized and evaluated for HAV detection and quantification, using oral fluid from healthy volunteers (n=20) and paired serum/oral fluid samples from individuals involved in a hepatitis A outbreak (n=78). Using nested RT-PCR, HAV RNA was detected in 50% of oral fluid and in 42% of serum samples from acute cases, as well as in 12% of all samples from cases without IgM and total anti-HAV. Using real-time PCR, HAV RNA was detected in 61% of oral fluid and in 71% of serum samples from acute cases, as well as in 17 and 12%, respectively, from patients without HAV markers. Mean viral loads were 1.7+/-3.24 x 10(3)copies/ml in oral fluid and 2.8+/-6.46 x 10(3)copies/ml in serum. Although nested RT-PCR and real-time PCR both detected HAV RNA in oral fluid, real-time PCR was more sensitive. Oral fluid sample testing could be used as a noninvasive method of detecting HAV RNA during HAV outbreaks.

Biopolymer-based membranes associated with osteogenic growth peptide for guided bone regeneration.

Barrier membranes for guided bone regeneration (GBR) mainly promote mechanical maintenance of bone defect space and induce osteopromotion. Additionally, biopolymer-based membranes may provide greater bioactivity and biocompatibility due to their similarity to extracellular matrix (ECM). In this study, biopolymers-based membranes from bacterial cellulose (BC) and collagen (COL) associated with osteogenic growth peptide (OGP(10-14)) were evaluated to determine in vitro osteoinductive potential in early osteogenesis; moreover, histological study was performed to evaluate the BC-COL OGP(10-14) membranes on bone healing after GBR in noncritical defects in rat femur. The results showed that the BC-COL and BC-COL OGP(10-14) membranes promoted cell proliferation and alkaline phosphatase activity in osteoblastic cell cultures. However, ECM mineralization was similar between cultures grown on BC OGP(10-14) and BC-COL OGP(10-14) membranes. In vivo results showed that all the membranes tested, including the peptide-free BC membrane, promoted better bone regeneration than control group. Furthermore, the BC-COL OGP(10-14) membranes induced higher radiographic density in the repaired bone than the other groups at 1, 4 and 16 weeks. Histomorphometric analyses revealed that the BC-COL OGP(10-14) induced higher percentage of bone tissue in the repaired area at 2 and 4 weeks than others membranes. In general, these biopolymer-based membranes might be potential candidates for bone regeneration applications.

Development of coverage and its evaluation in the treatment of chronic wounds.

OBJECTIVES: To describe the development of the bacterial cellulose coating with anti-inflammatory Ibuprofen (BC/Ibu) and to evaluate the cicatrization process with its use in patients with chronic wounds of venous and diabetic etiology. METHODS: Longitudinal descriptive study. The cellulose membrane, cultivated with bacteria Gluconacetobacter xylinus and with incorporation of Ibuprofen, was used in the treatment of patients with chronic wounds in public health services in a Brazilian municipality. The ideal coverage characteristics were evaluated through physical, chemical and cell proliferation tests. RESULTS: The sample consisted of 14 patients (10 women and 4 men), 8 with venous ulcer, 5 with diabetic foot and one with mixed wound. There was reduction of area and pain in 9 lesions; total healing of 3 wounds; and debridement of the devitalized tissue in 5 wounds with increased area. The use of the membrane was important in the reduction of pain, exudation and ease in the accomplishment of the curative. CONCLUSIONS: BC/Ibu favored the cicatrization process of patients with chronic vasculogenic wounds.

Nanocellulose-collagen-apatite composite associated with osteogenic growth peptide for bone regeneration.

Despite advances in the field of biomaterials for bone repair/regeneration, some challenges for developing an ideal bone substitute need to be overcome. Herein, this study synthesized and evaluated in vitro a nanocomposite based on bacterial cellulose (BC), collagen (COL), apatite (Ap) and osteogenic growth peptide (OGP) or its C-terminal pentapeptide [OGP(10-14)] for bone regeneration purposes. The BC-COL nanocomposites were successfully obtained by carbodiimide-mediated coupling as demonstrated by spectroscopy analysis. SEM, FTIR and 31P NMR analyses revealed that in situ synthesis to apatite was an effective route for obtaining of bone-like apatite. The OGP-containing (BC-COL)-Ap stimulated the early development of the osteoblastic phenotype. Additionally, the association among collagen, apatite, and OGP peptides enhanced cell growth compared with OGP-containing BC-Ap. Furthermore, none of the nanocomposites showed cytotoxic, genotoxic or mutagenic effects. These promising results suggest that the (BC-COL)-Ap associated with OGP peptides might be considered a potential candidate for bone tissue engineering applications.

Cynomolgus monkeys are successfully and persistently infected with hepatitis E virus genotype 3 (HEV-3) after long-term immunosuppressive therapy.

Epidemiological studies found that hepatitis E virus genotype 3 (HEV-3) infection was associated with chronic hepatitis and cirrhosis in immunocompromised patients. Our study aimed to investigate the relationship between the host immunosuppressive status and the occurrence of HEV-related chronic hepatitis. Here we describe a successful experimental study, using cynomolgus monkeys previously treated with tacrolimus, a potent calcineurin inhibitor immunosuppressant, and infected with a Brazilian HEV-3 strain isolated from naturally infected pigs. HEV infected monkeys were followed up during 160 days post infection (dpi) by clinical signs; virological, biochemical and haematological parameters; and liver histopathology. The tacrolimus blood levels were monitored throughout the experiment. Immunosuppression was confirmed by clinical and laboratorial findings, such as: moderate weight loss, alopecia, and herpes virus opportunistic infection. In this study, chronic HEV infection was characterized by the mild increase of liver enzymes serum levels; persistent RNA viremia and viral faecal shedding; and liver histopathology. Three out of four immunosuppressed monkeys showed recurrent HEV RNA detection in liver samples, evident hepatocellular ballooning degeneration, mild to severe macro and microvesicular steatosis (zone 1), scattered hepatocellular apoptosis, and lobular focal inflammation. At 69 dpi, liver biopsies of all infected monkeys revealed evident ballooning degeneration (zone 3), discrete hepatocellular apoptosis, and at most mild portal and intra-acinar focal inflammation. At 160 dpi, the three chronically HEV infected monkeys showed microscopic features (piecemeal necrosis) corresponding to chronic hepatitis in absence of fibrosis and cirrhosis in liver parenchyma. Within 4-months follow up, the tacrolimus-immunosuppressed cynomolgus monkeys infected with a Brazilian swine HEV-3 strain exhibited more severe hepatic lesions progressing to chronic hepatitis without liver fibrosis, similarly as shown in tacrolimus-immunosuppressed solid organ transplant (SOT) recipients. The cause-effect relationship between HEV infection and tacrolimus treatment was confirmed in this experiment.

Evaluation of methods used to concentrate and detect hepatitis A virus in water samples.

Two adsorption-elution concentration methods, both involving negatively charged membranes, were evaluated in order to monitor hepatitis A virus (HAV) contamination in tap, river, mineral and coastal water samples: elution with urea-arginine phosphate buffer/reconcentration with magnesium chloride (method 1); and sodium hydroxide elution/reconcentration with a commercial concentrator (method 2). Nested (qualitative) reverse transcriptase PCR (RT-PCR) and real-time (quantitative) RT-PCR were used to detect and quantify HAV RNA in concentrated water samples. For concentrating HAV, method 1 was found to be the most suitable for tap water and method 2 most suitable for mineral water. HAV inoculated experimentally was detected in river water samples by both methods and in coastal water samples by neither method. The detection limits were 6 x 10(9) g equiv./ml for qualitative PCR and 60 g equiv./ml for quantitative PCR. In a field application study, HAV was detected in 20% of river and tap water samples but not in coastal or mineral water samples. River water samples contained subgenotype IA, and tap water samples contained subgenotype IB. It is concluded that, although influencing qualitative PCR, the concentration method does not affect quantitative PCR, which could therefore be used for all types of water samples. Both techniques are recommended for detecting HAV in environmental water samples.

Central cellular signaling pathways involved with the regulation of lipid metabolism in the liver: a review

The liver is primarilyresponsible for energy homeostasis and the regulation of lipid, carbohydrate and protein metabolism. Lipid metabolism consists of distributing lipids to peripheral tissues or ensuring their return to the liver to be reprocessed. Additionally, cellular metabolism isregulated by several molecules in different signaling pathways. Lipid homeostasis in the liver is mainly regulated by AKT, AMPK, SREBP, PPAR, and JNK. The PI3K/AKT/mTOR signaling pathway results in the biosynthesis of macromolecules and regulates lipogenesis and the expression of lipogenic genes. AMPK is an energy sensor that regulates metabolism and is activated when stored ATP is depleted, and it is responsible for the suppression of several key lipogenic factors in the liver related to cholesterol and fatty acid synthesis. SREBPs control lipogenic geneexpressionandcholesterol metabolism and actin the nutritional regulation of fatty acids and triglycerides. The continued activation of SREBPs is associated with cellular stress, inflammation and ultimately steatosis. PPARs are intrinsically important regulators of lipid metabolism. These genes are essential tovarious metabolic processes, especially lipid and glucose homeostasis, and can play a role in cell differentiation. JNK signaling is related to insulin resistance and its activation results in decreased mitochondrial activity and fat accumulation. Therefore, the study of cell signaling pathways related to lipid metabolism and liver function may help to identify abnormalities and develop strategies to manage and regulate metabolic disorders and resulting complications.

Longitudinal Study of Hepatitis A Infection by Saliva Sampling: The Kinetics of HAV Markers in Saliva Revealed the Application of Saliva Tests for Hepatitis A Study.

Despite the increasing numbers of studies investigating hepatitis A diagnostic through saliva, the frequency and the pattern of hepatitis A virus (HAV) markers in this fluid still remains unknown. To address this issue, we carried on a longitudinal study to examine the kinetics of HAV markers in saliva, in comparison with serum samples. The present study followed-up ten patients with acute hepatitis A infection during 180 days post diagnosis (dpd). Total anti-HAV was detected in paired serum and saliva samples until the end of the follow-up, showing a peak titer at 90th. However, total anti-HAV level was higher in serum than in saliva samples. This HAV marker showed a probability of 100% to be detected in both serum and saliva during 180 dpd. The IgM anti-HAV could be detected in saliva up to 150 dpd, showing the highest frequency at 30th, when it was detected in all individuals. During the first month of HAV infection, this acute HAV marker showed a detection probability of 100% in paired samples. The detection of IgM anti-HAV in saliva was not dependent on its level in serum, HAV-RNA detection and/or viral load, since no association was found between IgM anti-HAV positivity in saliva and any of these parameter (p>0.05). Most of the patients (80%) were found to contain HAV-RNA in saliva, mainly at early acute phase (30th day). However, it was possible to demonstrate the HAV RNA presence in paired samples for more than 90 days, even after seroconversion. No significant relationship was observed between salivary HAV-RNA positivity and serum viral load, demonstrating that serum viral load is not predictive of HAV-RNA detection in saliva. Similar viral load was seen in paired samples (on average 104 copies/mL). These data demonstrate that the best diagnostic coverage can be achieved by salivary anti-HAV antibodies and HAV-RNA tests during 30-90 dpd. The long detection and high probability of specific-HAV antibodies positivity in saliva samples make the assessment of salivary antibodies a useful tool for diagnosis and epidemiological studies. The high frequency of HAV-RNA in saliva and the probability of detection of about 50%, during the first 30 dpd, demonstrate that saliva is also useful for molecular investigation of hepatitis A cases, mainly during the early course of infection. Therefore, the collection of saliva may provide a simple, cheap and non-invasive means of diagnosis, epidemiological surveys and monitoring of hepatitis A infection purposes.