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Background and Purpose Klotho is a membrane‐bound or soluble protein, originally identified as an age‐suppressing factor and regulator of mineral metabolism. Klotho deficiency is associated with the development of renal disease, but its role in cardiac function in the context of uraemic cardiomyopathy is unknown. Experimental Approach We explored the effects of Klotho on cardiac Ca2+ cycling. We analysed Ca2+ handling in adult cardiomyocytes from Klotho‐deficient (kl/kl) mice and from a murine model of 5/6 nephrectomy (Nfx). We also studied the effect of exogenous Klotho supplementation, by chronic recombinant Klotho treatment, or endogenous Klotho overexpression, using transgenic mice overexpressing Klotho (Tg‐Kl), on uraemic cardiomyopathy. Hearts from Nfx mice were used to study Ca2+ sensitivity of ryanodine receptors and their phosphorylation state. Key Results Cardiomyocytes from kl/kl mice showed decreased amplitude of intracellular Ca2+ transients and cellular shortening together with an increase in pro‐arrhythmic Ca2+ events compared with cells from wild‐type mice. Cardiomyocytes from Nfx mice exhibited the same impairment in Ca2+ cycling as kl/kl mice. Changes in Nfx cardiomyocytes were explained by higher sensitivity of ryanodine receptors to Ca2+ and their increased phosphorylation at the calmodulin kinase type II and protein kinase A sites. Ca2+ mishandling in Nfx‐treated mice was fully prevented by chronic recombinant Klotho administration or transgenic Klotho overexpression. Conclusions and Implications Klotho emerges as an attractive therapeutic tool to improve cardiac Ca2+ mishandling observed in uraemic cardiomyopathy. Strategies that improve Klotho availability are good candidates to protect the heart from functional cardiac alterations in renal disease. Sin financiación 8.740 JCR (2020) Q1, 12/276 Pharmacology & Pharmacy 2.432 SJR (2020) Q1, 16/314 Pharmacology No data IDR 2020 UEM

We report the case of a Caucasian Spanish origin female who showed severe psychomotor developmental delay, hypotonia, strabismus, epilepsy, short stature, and poor verbal language development. Brain magnetic resonance imaging scans showed thickened corpus callosum, cortical malformations, and dilated and abnormal configuration of the lateral ventricles without hydrocephalus. Whole-exome sequence uncovered a de novo variant in the microtubule associated serine/threonine kinase 1 gene (MAST1; NM_014975.3:c.1565G>A:p.(Gly522Glu)) that encodes for the MAST1. Only 12 patients have been identified worldwide with 10 different variants in this gene: six patients with mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations; two patients with microcephaly and cerebellar hypoplasia; two patients with autism, one patient with diplegia, and one patient with microcephaly and dysmorphism. Our patient shows a new phenotypic subtype defined by mega-corpus-callosum syndrome with cortical malformations without cerebellar hypoplasia. In conclusion, our data expand the phenotypic spectrum associated to MAST1 gene variants.

Our objective is to assess the characteristics of respiratory syncytial virus (RSV) infection in adult patients and to establish differences with influenza viruses. Fifty-four patients diagnosed with RSV and 198 with influenza were prospectively included. Compared with influenza, empirical antimicrobial therapy was more frequent in patients diagnosed with RSV, whereas antibiotic withdrawal at the time of diagnosis confirmation was lower (OR, 0.12; CI, 95% 0.01-0.90; P = 0.040). RSV-positive patients were more likely to need hospital readmission (OR, 3.00; CI, 95% 0.98-9.09; P = 0.053). The role of RSV infection in adults is often overlooked, leading to inappropriate use of antibiotics and a probable increase in nosocomial RSV transmission.

We report the clinical and genetic findings in a 15-year-old Spanish boy presenting prenatal and postnatal growth retardation, reduced subcutaneous adipose tissue, premature skin wrinkling, sparse hair, short distal phalanges with small nails, umbilical hernia, wide anterior fontanel, and normal cognitive and motor development. Exome sequencing uncovered a heterozygous mutation in SLC25A24 (NM_013386: c.650G>A: p.R217H) that encodes for the calcium-binding mitochondrial carrier protein SCaMC-1. This gain-of-function variant has been previously associated with Fontaine syndrome and Gorlin-Chaudhry-Moss syndrome, two entities that show overlapping features, and have been recently subsumed under the name Fontaine progeroid syndrome (FPS; MIM: 612289) in OMIM. Here, we describe the first male patient with genetically confirmed FPS who survives at least until adolescence.

Background and Purpose: Immunomodulatory tetracyclines are well-characterised drugs with a pharmacological potential beyond their antibiotic properties. Particulaarly, minocycline and doxycycicline have shown beneficial effects in experimental colitis, although pro-inflammatory actions have also been described in macrophages. Therefore, we aimed to characterise the mechanism behind their effect in acute intestinal inflammation. Experimental Approach: A comparative pharmacological study was first used to elucidate teh most relevant actions of immunomodulatory tetracyclines: doxycycline, minocycline, tigecycline and other antibiotic or immunomodulatory drugs were assessed in bone-marrow derived macrophages and in DSS-induced mouse colitis, where different barrier markers, inflammatory mediators, microRNAs, TLRs, and the gut microbiota composition were evaluated. Then, the sequential immune events that mediate the intestinal anti-inflammatory effect of minocycline in DSS-colitis were characterised. Key Results: We have identified a novel immunomodulatory activity of tetracyclines, potentiating the innate immune response and leading to an enhanced resolution of inflammation. This is also the first report describing the intestinal anti-inflammatory effect of tigecycline. A minor therapeutic benefit seems to derive from their antibiotic properties. Conversely, immunomodulatory tetracyclines potentiate macrophage cytokine release in vitro and, while improving mucosal recovery in colitic mice, they up-regulate Ccl2, miR-142, miR-375 and Tlr4. In particular, minocycline initially enhances IL-1β, IL-6, IL-22, GM-CSF and IL-4 colonic production and monocyte recruitment to the intestine, subsequently increasing Ly6C−MHCII+ macrophages, Tregs and type-2 intestinal immune responses. Conclusion and Implications: Immunomodulatory tetracyclines potentiate protective immune pathways leading to mucosal healing and resolution, representing a promising drug reposition strategy for the treatment of intestinal inflammation.

We report clinical and biochemical finding from three unrelated patients presenting ONCE (Optic Neuropathy, Cardiomyopathy and Encephalopathy with lactic acidosis and combined oxidative phosphorylation deficiency) syndrome. Whole-exome sequencing (WES) of the three patients and the healthy sister of one of them was used to identify the carry gene. Clinical and biochemical findings were used to filter variants, and molecular, in silico and genetic studies were performed to characterize the candidate variants. Mitochondrial DNA (mtDNA) defects involving mutations, deletions or depletion were discarded, whereas WES uncovered a double homozygous mutation in the MTO1 gene (NM_001123226:c.1510C>T, p.R504C, and c.1669G>A, p.V557M) in two of the patients and the homozygous mutation p.R504C in the other. Therefore, our data confirm p.R504C as pathogenic mutation responsible of ONCE syndrome, and p.V557M as a rare polymorphic variant.

Capsicum annuum L. cv Senise is a sweet pepper containing health promoting compounds, that could be modified by ripening and drying. This study focuses on finding the peppers with the best antioxidant properties, which are evaluated on an experimental model of obesity METHODS AND RESULTS: : Phytochemical profile and antioxidant activity were evaluated on several peppers obtained from the same cultivar at different ripening stages. Red sweet peppers showed the highest content in polyphenols, β-carotene, lycopene and capsinoids, and demonstrated the best antioxidant activity in vitro. Mice fed a high fat diet (HFD) were orally treated with an extract from these peppers (CAE) (1, 10 and 25 mg/kg/day). It promoted weight loss and improved plasma markers related to glucose and lipid metabolisms. CAE also ameliorated obesity-associated systemic inflammation reducing the expression of pro-inflammatory cytokines in adipose and hepatic tissues and improving the expression of different markers involved in the gut epithelial barrier function. These effects were associated with a modulation of the intestinal microbiome, which appears altered CONCLUSIONS: The extract could be considered a new potential approach for the treatment of obesity, complementary to dietary restrictions. This article is protected by copyright. All rights reserved.

Scope Obesity is characterized by a dysfunction in the adipose tissue and an inflammatory subclinical state leading to insulin resistance and increased risk of cardiovascular diseases. It is also associated with intestinal dysbiosis that contributes to inflammation development. Lippia citriodora (LCE) contains high levels of polyphenolpropanoids and has shown promising results in obesity. The aim of this study is to investigate a well-characterized extract of LCE in a model of metabolic syndrome in mice, focusing on its effects on metabolic tissues, endothelial dysfunction, and microbiome. Methods Mice are fed a high fat diet (HFD) for six weeks and treated daily with LCE (1, 10, and 25 mg kg-1 ). Glucose and lipid metabolism is investigated. The inflammatory state in the metabolic tissues and the intestinal microbiota composition are characterized, as well as the endothelium-dependent vasodilator response to acetylcholine. Results LCE reduces fat accumulation and improves plasma glycemic and lipid profiles, as well as the inflammatory process and vascular dysfunction. Moreover, LCE lessens intestinal dysbiosis, as it reduces the Firmicutes/Bacteroidetes ratio and increases Akkermansia abundance in comparison with untreated HFD mice. Conclusion The antiobesity therapeutic properties of LCE are most probably mediated by the synergic effects of its bioactive compounds.

Legume-rhizobium interactions have been widely studied and characterized, and the disaccharide trehalose has been commonly detected during this symbiotic interaction. It has been proposed that trehalose content in nodules during this symbiotic interaction might be regulated by trehalase. In the present study, we assessed the role of trehalose accumulation by down-regulating trehalase in the nodules of common bean plants. We performed gene expression analysis for trehalase (PvTRE1) during nodule development. PvTRE1 was knocked down by RNA interference (RNAi) in transgenic nodules of the common bean. PvTRE1 expression in nodulated roots is mainly restricted to nodules. Down-regulation of PvTRE1 led to increased trehalose content (78%) and bacteroid number (almost one order of magnitude). In addition, nodule biomass, nitrogenase activity, and GOGAT transcript accumulation were significantly enhanced too. The trehalose accumulation, triggered by PvTRE1 down-regulation, led to a positive impact on the legume-rhizobium symbiotic interaction. This could contribute to the agronomical enhancement of symbiotic nitrogen fixation.

BACKGROUND AND PURPOSE Dersalazine sodium (DS) is a new chemical entity formed by combining, through an azo bond, a potent platelet activating factor (PAF) antagonist (UR-12715) with 5-aminosalicylic acid (5-ASA). DS has been demonstrated to have anti-inflammatory effects on trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats and recently in UC patients in phase II PoC. There is Increasing evidence that Th17 cells have an important role in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to further characterize the anti-inflammatory effects of DS. EXPERIMENTAL APPROACH Effect of DS (10 or 30 mg·kg−1 b.i.d.) on TNBS-induced colitis in rats was studied after 2 and 7 days with special focus on inflammatory mediators. Additionally, its anti-inflammatory properties were analysed in two different models of dextran sodium sulphate (DSS)-induced colitis, BALB/c and C57BL/6 mice, the latter being dependent on IL-17. KEY RESULTS DS, when administered for 7 days, showed intestinal anti-inflammatory effects in TNBS-induced colitis; these effects were observed both macroscopically and through the profile of inflammatory mediators (TNF, IL-1β, IL-6 and IL-17). Although the 2 day treatment with DS did not induce intestinal anti-inflammatory effects, it was sufficient to reduce the enhanced IL-17 expression. DS showed beneficial effects on DSS-induced colitis in C57BL/6 mice and reduced colonic pro-inflammatory cytokines IL-1β, IL-6 and IL-17. In contrast, it did not exert intestinal anti-inflammatory effects on DSS-induced colitis in BALB/c mice. CONCLUSIONS AND IMPLICATIONS DS exerts intestinal anti-inflammatory activity in different rodent models of colitis through down-regulation of IL-17 expression.

Scope Propyl-propane thiosulfonate (PTSO) is a component isolated from garlic (Allium sativum) with antioxidant, anti-inflammatory, immunomodulatory, and antimicrobial properties. In consequence, PTSO can be a potential candidate for the treatment of inflammatory bowel diseases. Methods and results The anti-inflammatory effects of PTSO are studied in two mice models of colitis: 2,4-dinitrobenzene sulfonic acid (DNBS) (PTSO doses: 0.01-10 mg kg-1 ) and dextran sodium sulfate (DSS) (PTSO doses: 0.01-0.1 mg kg-1 ). The immunomodulatory effects of PTSO (0.1-25 µm) are also shown in vitro in Caco-2 and THP-1 cells, reducing the production of pro-inflammatory mediators and downregulating mitogen-activated protein kinases (MAPKs) signaling pathways. This compound displays beneficial effects in both models of mouse colitis by reducing the expression of different pro-inflammatory mediators and improving the intestinal epithelial barrier integrity. Moreover, PTSO ameliorates the altered gut microbiota composition observed in DSS colitic mice. Conclusion PTSO exerts intestinal anti-inflammatory activity in experimental colitis in mice. This anti-inflammatory activity can be associated with the immunomodulatory properties of PTSO through the regulation of the activity of cells involved in the inflammatory response. Furthermore, PTSO is able to restore the intestinal epithelial barrier function and to ameliorate the intestinal microbiota homeostasis, thus supporting its future development in human IBD.

Eur J Clin Invest 2010; 40 (6): 511–517 Abstract Background Iron is essential for mammalian metabolism and its cellular concentration is controlled by regulating its acquisition and storage. Haemochromatosis is a condition involving iron overload that is characterised by increased duodenal iron absorption and a progressive accumulation of iron in vital organs. Hepcidin is the main hormone that regulates iron homoestasis and it is secreted by the liver. Materials and methods We have studied how extended hepcidin administration affects the iron load status, plasma and tissue iron concentration, erythropoiesis and the expression of proteins involved on iron homeostasis in haemochromatotic (Hfe−/−) and wild-type mice. Results Hepcidin reverted the high plasma iron concentrations in Hfe−/− mice to normal values. The high concentration of hepatic iron was not altered in the liver of these Hfe−/− mice. Hepcidin administration did not disturb erythropoiesis in either Hfe−/− or wild-type mice and likewise, hepcidin did not modify the expression of any protein analysed in the liver, duodenum or spleen of Hfe−/− and wild-type mice. These data confirm that hepcidin administration diminishes plasma iron concentrations. Conclusion Treatment with sustained doses of hepcidin diminishes plasma iron concentrations in Hfe−/− mice.

Overweight/obese persons usually have an inadequate vitamin D status, a situation commonly made worse by an inadequate intake of this vitamin. For this reason, the aim of this study was to analyze dietetic and anthropometric differences in a group of young, overweight/obese Spanish women with respect to their vitamin D status. The study subjects were 66 white Spanish women (aged 20-35 years) with a BMI of 24-35 kg/m(2). Dietetic, anthropometric, and biochemical data were collected. Women were divided into two groups depending on their serum vitamin D concentrations: LD (women with90 nmol/l 25(OH)D) and HD (women withor=90 nmol/l 25(OH)D). The intakes of vitamin D, calcium, and supplements were similar in both groups. The body weight, BMI, and waist circumference of the HD subjects were smaller than those recorded for the LD subjects (68.6 +/- 4.2 kg, 26.0 +/- 1.3 kg/m(2), and 79.4 +/- 3.4 cm compared to 76.2 +/- 9.8, 28.6 +/- 3.2 kg/m(2), and 86.2 +/- 9.3 cm, respectively; P0.05). The hip circumference and the waist/hip ratio were similar in both groups. A BMI of27.7 kg/m(2) (P50) was associated with serum vitamin D concentrations ofor=90 nmol/l (odds ratio = 0.1313; confidence interval: 0.0149-1.1599; P0.05). Overweight/obese women are at higher risk of vitamin D deficiency, largely due to excess adiposity rather than inadequate intake.

SUMMARY Background Hepatitis C virus replicates by the synthesis of an antigenomic HCV-RNA. As the end point of anti-viral therapy is to decrease viral replication, the amount of antigenomic HCV-RNA could influence the response. Aim To study if amounts of genomic and antigenomic HCV-RNA in the baseline liver biopsy are predictive factors of response to anti-viral therapy. Methods Eighty-eight patients with chronic HCV infection (anti-HIV-negative) treated with pegyltaed-interferon-α2b plus ribavirin for 12 months were included. Intrahepatic genomic and antigenomic HCV-RNA concentrations were determined by real-time polymerase chain reaction and percentage of infected hepatocytes by in situ hybridization. Results Of the 88 patients, 31% were responders while 69% were not. Median of antigenomic HCV-RNA in liver of responders and non-responders was 120 000 copies/μg RNA (range: 10 000–775 000) vs. 150 000 copies/μg RNA (range: 100–3 200 000; P = 0.38). Median of genomic HCV-RNA in liver of responders was 1 250 000 copies/μg RNA (range: 5000–9 000 000) and in non-responders 3 180 000 copies/μg RNA (range: 4600–18 000 000; P = 0.0191). Predictive factors of response in the logistic regression were: intrahepatic amount of genomic HCV-RNA, percentage of infected hepatocytes and previous therapy. Conclusion Response to 12 months of therapy with pegylated interferon-α2b plus ribavirin depends on the amount of genomic HCV-RNA in the pre-treatment liver biopsy.

We have recently described the presence of occult hepatitis C virus (HCV) infection (HCV-RNA in liver in the absence of anti-HCV and serum HCV-RNA) in patients with persistently abnormal liver function tests of unknown aetiology. The aim of this study was to compare the characteristics of patients with occult HCV infection vs those of patients with chronic hepatitis C. We compared clinical features of 68 patients with occult HCV infection and 69 untreated chronic HCV patients (anti-HCV and serum HCV-RNA positive), matched for age, gender, duration of abnormal liver function tests and body mass index. Aspartate aminotransferase and alanine aminotransferase were higher (P < 0.001) in chronic HCV, but cholesterol and triglycerides were significantly higher in patients with occult HCV infection (P < 0.001 and P = 0.002). Chronic HCV patients had higher gamma-globulin (P = 0.005), alpha-foetoprotein (P < 0.001) and iron (P < 0.001) levels. Percentage of patients with necroinflammatory activity and fibrosis was higher (P < 0.001) in chronic HCV than in occult HCV infection. Mean percentage of infected hepatocytes was higher (P = 0.001) in chronic HCV (10.1%) than in occult HCV infection (5.3%). This occult HCV infection is a milder disease than chronic HCV, and this could be related to the significantly lower number of infected hepatocytes observed in occult HCV.

Summary Background Occult hepatitis C virus infection is defined by the presence of hepatitis C virus-RNA in liver but with undetectable anti-hepatitis C virus and serum viral RNA. Aim To study the response to anti-viral therapy in occult hepatitis C virus infection to assess the pathogenic effect of occult hepatitis C virus. Methods Ten patients with occult hepatitis C virus infection were treated with pegylated-interferon plus ribavirin for 24 weeks and were followed-up 24 weeks after therapy. All patients had abnormal alanine aminotransferase, hepatitis C virus-RNA positive in peripheral blood mononuclear cells and liver necroinflammation. Results At the end of treatment and follow-up, the percentage of patients with normal alanine aminotransferase was 80% (95% CI: 48–96%) and 60% (95% CI: 31–84%) respectively, and hepatitis C virus-RNA in peripheral blood mononuclear cells was negative in 80% (95% CI: 48–96%) and 70% (95% CI: 40–90%) cases. At the end of follow-up sustained response was observed in 30% (95% CI: 11–61%) of cases. Five patients underwent a second liver biopsy. In all cases, liver hepatitis C virus-RNA persisted, although hepatitis C virus-RNA load was significantly lower (3.2 × 104 ± 5.1 × 104 copies/μg RNA) than in the basal biopsy (2.4 × 105 ± 3.8 × 105 copies/μg RNA); (P = 0.043). Necroinflammation and fibrosis decreased in three cases. Conclusion The biochemical, virological and histological response to therapy achieved in patients with occult hepatitis C virus infection demonstrates the pathologic effects of occult hepatitis C virus.

Hepatitis C virus (HCV) RNA persistence in the liver has been described even after apparent resolution of HCV infection. Because T-cell reactivity plays a role in recovery from HCV infection, virus-specific T-cell responses were investigated in apparently recovered individuals in whom hepatic HCV RNA persistence was documented: 15 sustained virological responders to interferon (IFN)-treatment and 9 asymptomatic aviremic anti-HCV carriers. HCV-specific CD4(+) T-cell proliferative responses were detected significantly more often in apparently recovered individuals (sustained virological responders: 60%; asymptomatic anti-HCV carriers: 66%) compared with 50 chronic hepatitis C patients (28%; P < 0.05). However, T-cell frequencies and numbers tended to decline over time and the number of HCV proteins targeted by CD4(+) T-cell proliferative responses was limited. Interestingly, liver viral load correlated inversely with virus-specific immune responses. Thus, CD4(+) T-cell responders showed significantly lower hepatic HCV RNA levels (P < 0.05). HCV-specific IFN-gamma-secreting CD4(+) T-cells were not detected in all the apparently recovered patients although they were found significantly more often compared with chronic hepatitis C patients (P < 0.05). Also, HCV NS3-specific CD8(+) T-cells were detected in 11 HLA-A2-positive apparently recovered individuals (8 sustained virological responders and 3 asymptomatic anti-HCV carriers); T-cell frequencies tended to be greater in those patients who had lower hepatic viral levels. In conclusion, HCV-specific T-cells are detectable in apparently recovered individuals in whom HCV RNA can persist in the liver indicating that HCV replication may be prolonged in the face of an insufficient or inadequate virus-specific CD4(+) and CD8(+) T-cell response.

The mechanical and thermodynamic properties of poly(acrylic acid-co-methyl methacrylate) hydrogels with varying crosslinker N,N′-methylenebisacrylamide (NMBA) content are reported. A higher NMBA content generally led to a stronger and harder gel with lower water content. Swelling capacity decreased as the NMBA concentration increased between 0.5% and 2%, remaining constant beyond this range. The temperature changes of the partial molar Gibbs free energy of dilution and enthalpic and entropic contributions were examined. The thermodynamic parameters showed that swelling was an unfavorable and endothermic process. The freezing and nonfreezing water in the hydrogel was determined by differential scanning calorimetry (DSC). Freezing water content decreased with increasing crosslinker (NMBA) content, whereas the ratio of nonfreezing water to total water content increased with NMBA content because of the promoting of hydrophobic interactions in the hydrogels. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 4016–4022, 2006

Hydrogels whose diameters range in the nanosize scale were synthesized by precipitation copolymerization of 4-nitrophenol acrylate (NPA) with methacrylamide (MeAM) and N-isopropylacrylamide (NIPAM). The polymerization reaction process was followed by attenuated total reflectance Fourier-transform infrared spectrometry (ATR-FTIR) and the conversion was studied by UV-vis spectrometry. Poly(NPA-co-MeAM) and poly(NPA-co-NIPAM) microgels were characterized by proton nuclear magnetic resonance (1H-NMR) spectrometry. The thermal properties were studied by both thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) analysis. Transmission electron microscopy (TEM) of these microgels showed that the copolymer is located in spherical particles with a moderate polydispersity. The average particle diameter and the particle size distribution were measured in acetone by quasi-elastic light scattering (QELS), which indicated a mean diameter close to 50 nm. Copyright © 2005 Society of Chemical Industry

Several in vitro studies have shown that HIV-1 can infect CD4 negative epithelial cells of different origin including normal human oral keratinocytes, but whether this infection of mucosal epithelial cells occurs in vivo is still unclear. In this report, the presence and cell types infected by HIV-1 in paraffin embedded oral mucosa biopsies from 17 anti-HIV-1 positive patients have been examined by in situ hybridization and immunohistochemistry. As controls, oral mucosa biopsies from eight patients without HIV-1 infection markers were also analyzed. The results showed that 8 out of the 17 anti-HIV-1 positive patients had HIV-1 RNA detectable in plasma. Positive hybridization signals were observed in the mucosa biopsies from 14 of the 17 anti-HIV-1 patients (82.3%). The mean percentage of cells showing HIV-1 RNA was 2.64% +/- 1.77% (range: 1% to 5.5%). No differences in the mean percentage of HIV-1 infected cells were found between patients with and without HIV-1 RNA in plasma (3.01% +/- 1.57% vs. 3.4% +/- 1.27% respectively), or between untreated patients and patients under antiretroviral therapy (2.83% +/- 1.63% vs. 3.42% +/- 1.29% respectively). Immunohistochemical detection of S-100 antigen, cytokeratin and CD4 showed that hybridization signals appeared in cytokeratin positive cells and CD4 positive cells but not in S-100 positive cells. In conclusion, this study has demonstrated that HIV-1 infects and replicates in oral mucosa epithelial cells in vivo and that these cells could represent a reservoir of the virus that may escape to the currently used antiretroviral therapy.

1 Quercitrin, 3-rhamnosylquercetin, is a bioflavonoid with antioxidant properties, which exerts antiinflammatory activity in experimental colitis. In the present study, different in vivo experiments were performed in order to evaluate the mechanisms of action involved in this effect, with special attention to its effects on proinflammatory mediators, including nitric oxide (NO). 2 Experimental colitis was induced in female Wistar rats by incorporation of dextran sodium sulfate (DSS) in drinking water. Oral treatment of quercitrin (1 or 5 mg kg � 1 day � 1 ) to colitic rats ameliorated the evolution of the inflammatory process induced when administered in a preventative dosing protocol. When quercitrin (1 mg kg � 1 day � 1 ) was administered on established colitis, it facilitated the recovery of the inflamed mucosa. 3 The beneficial effects exerted by quercitrin were evidenced both histologically and biochemically, and were associated with an improvement in the colonic oxidative status, altered as a consequence of the colonic insult induced by DSS. In addition, a reduction of colonic NO synthase activity was observed, probably related to a decreased expression in the inducible form of the enzyme via downregulation in the colonic activity of the nuclear factor-kB. 4 Immunohistochemical studies showed that quercitrin treatment reduced macrophage and granulocyte infiltration in the inflamed tissue. British Journal of Pharmacology (2004) 143, 908–918. doi:10.1038/sj.bjp.0705941

The swelling capacity and release rate of two homologous drugs, theophylline and aminophylline, from acrylic acid/n-alkyl methacrylate hydrogels have been studied. The maximum equilibrium swelling increases as the molar fraction of acrylic acid or the chain length of the methacrylate in the hydrogels increases. Water diffusion to the hydrogels is non-Fickian. Both drugs are released from the fully swollen hydrogels according to Fick's law. However, the drug release from xerogels deviates from Fick's law, especially for aminophylline. As expected because of its larger size, aminophylline diffuses more slowly than theophylline under similar experimental conditions. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 2756–2765, 2004

An experimental research of the absorption properties of metal ions onto synthetic hydrogel obtained by solution polymerization of acrylic acid and itaconic acid in presence of N,N′-methylenebisacrylamide as crosslinking agent was carried out. The swelling behavior in aqueous salt solutions was studied as a function of divalent cation concentration (Cu2+, Zn2+, Ni2+, Co2+, Cd2+, Pb2+, Hg2+) in the external solution ranging from 10−5 to 1M, at 25°C. The ability of these hydrogels to bind cations was measured at different pH values and metal ion concentrations. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 530–536, 2003

Two series of hydrogels of poly[(acrylic acid)-co-(itaconic acid)] have been preparted by copolymerization in solution using tetrafunctional N,N-methylenebisacrylamide (NMBA) as cross-linker. The resulting polymer was swollen in water at 298 K to yield homogenous transparent hydrogels. These hydrogels were characterized in terms of swelling and compression-strain measurcements. The influence of the comonomer composition and concentration of cross-linking agent on volumetric swelling and the mechanical properties of these hydrogels were investigated. Inefficient cross-linking is indicated by the small values of ν 1 relative to the theoretical cross-linking density

Although occult hepatitis B virus (HBV) infection (HBV-DNA in serum in the absence of hepatitis B surface antigen [HBsAg]) is common in chronic hepatitis C, its characteristics are not well known. In this work, the presence of HBV-DNA (by polymerase chain reaction; PCR) and its distribution (by in situ hybridization) in liver biopsies and peripheral blood mononuclear cells (PBMCs) from 32 patients with chronic hepatitis C and occult HBV infection and in 20 HBsAg chronic carriers were determined. The results showed that serum HBV-DNA levels were statistically lower (P = 0.001) in patients with occult HBV infection than in HBsAg chronic carriers. The HBV infection pattern in liver cells was identical between patients with occult HBV infection and those with chronic hepatitis B. However, the mean percentage of HBV-infected hepatocytes was significantly lower (P = 0.001) in patients with occult HBV infection (5 +/- 4.44%) than in HBsAg chronic carriers (17.99 +/- 11.58%). All patients with chronic hepatitis B have HBV-DNA in their PBMCs while this occurred in 50% of the cases with occult HBV infection. In conclusion, patients with occult HBV infection have a low number of HBV-infected hepatocytes and this fact could explain the lack of HBsAg detection and low viremia levels found in these cases.

Hepatitis C virus (HCV) replicates in salivary glands of chronic hepatitis C patients and is released into the saliva, suggesting that HCV may replicate in other exocrine glands. The presence of positive and negative HCV RNA strands was demonstrated by in situ hybridization, and of HCV core protein by immunohistochemistry, in sweat glands and keratinocytes in healthy skin biopsies from 15 patients with chronic hepatitis C and 10 anti-HCV negative patients with chronic liver disease. Positive and negative HCV RNA strands were detected in 9.6 +/- 5.2% and 4.2 +/- 3.8%, respectively, of the epithelial cells of eccrine sweat glands. Core protein was detected in 6.0 +/- 3.93% of these cells. HCV RNA resistant to RNase digestion (encapsidated HCV RNA) was detected in 10/10 sweat samples from HCV-infected patients. Positive and negative HCV RNA strands were detected in 6.7 +/- 2.97% and 3.0 +/- 3.08% of the keratinocytes, respectively. HCV core protein was found in 4.5 +/- 2.76% of these cells. No HCV RNA or HCV core protein was detected in the skin biopsies from the 10 anti-HCV negative patients. In conclusion, HCV replicates in eccrine sweat glands cells and keratinocytes in healthy skin and is released into the sweat.

Background: Morin, a bioflavonoid with antioxidant properties, shows intestinal anti-inflammatory activity in the acute phase of the trinitrobenzenesulphonic acid model of rat colitis. Aim: To assess the anti-inflammatory activity of morin in the chronic stages of trinitrobenzenesulphonic acid-induced rat colitis. Methods: Rats were rendered colitic by a single colonic instillation of 30 mg of the hapten trinitrobenzenesulphonic acid dissolved in 0.25 mL of 50% ethanol. A group of colitic animals was given morin orally at doses of 25 mg/kg daily. Animals were sacrificed every week for 4 weeks. Colonic damage was evaluated macroscopically and microscopically. Different biochemical markers of colonic inflammation were also assayed, including myeloperoxidase activity, leukotriene B4 and interleukin-1β synthesis, glutathione and malonyldialdehyde levels and nitric oxide synthase activity. Results: The administration of morin facilitated tissue recovery during the 4 weeks following colonic insult with trinitrobenzenesulphonic acid, as demonstrated macroscopically and microscopically, as well as biochemically by a reduction in myeloperoxidase activity. The intestinal anti-inflammatory effect of morin was accompanied by a significant reduction in colonic leukotriene B4 and interleukin-1β levels, improvement in colonic oxidative stress and inhibition of colonic nitric oxide synthase activity. Conclusions: Morin exerts a beneficial anti-inflammatory effect in the chronic phase of trinitrobenzenesulphonic acid-induced rat colitis through the down-regulation of some of the mediators involved in the intestinal inflammatory response, including free radicals, cytokines, leukotriene B4 and nitric oxide.

Epidemiological studies have demonstrated a correlation between oral lichen planus and different liver diseases. The new virus termed TT virus (TTV) is highly prevalent in patients with chronic hepatitis of different etiology and it may be speculated that TT virus may be involved in the pathogenesis of oral lichen planus. This study examined the presence of TT virus DNA in serum by PCR and in oral mucosa biopsies by in situ hybridization from 20 patients with oral lichen planus (13 with chronic hepatitis and seven without liver disease). Serum and oral mucosa biopsies from six patients all with chronic hepatitis with leukoplakia were also studied as controls. TT virus DNA was positive in the serum of 17/20 (85%) of the patients with oral lichen planus and in all the controls. TT virus DNA hybridization signals were detected in mucosa biopsies from all the patients with TT virus DNA in serum but in none of the three cases without this marker. The percentage of positive cells ranged from 1.6-80%. No differences were found in the percentage of positive cells between TT virus positive patients with and without oral lichen planus and there was no relationship between the number of positive cells and the intensity of the inflammatory infiltrate. In conclusion, TT virus infects oral epithelial cells but the results do not support a role for TT virus in causing oral lichen planus.

Chronic hepatitis C in children is characterized by milder forms of liver damage than those found in adults. Such a difference has been attributed to a low viral load in children that may lead to poor recognition of infected cells by the immune system. One approach that could be used to confirm this hypothesis may be to examine the number of infected hepatocytes in liver biopsies. Paraffin embedded liver biopsies from 21 children and 15 adults with chronic hepatitis C virus (HCV) infection (with a similar duration of the infection) were hybridized in situ and the percentage of infected hepatocytes was correlated with the histological activity index, alanine aminotransferase levels and HCV viraemia levels. Histological activity index and HCV viraemia levels were statistically higher (P

The main site of TT virus (TTV) replication remains unknown. Therefore, we have studied the presence and titres of TTV DNA in paired serum, liver and PBMC samples from 50 patients with liver disease (32 with chronic hepatitis B or C, seven with cryptogenic hepatitis and 11 with nonviral liver disease) were included. TTV DNA was analysed by polymerase chain reaction (PCR) using primers from the open reading frame 1 (ORF 1) and from the untranslated region (UTR) and titres were semiquantified by PCR using an external standard. TTV DNA was detected in 26% of serum, 24% of liver and 14% of PBMC samples with ORF 1 primers. When UTR primers were used, 70% of serum and liver samples and 64% of PBMC were TTV DNA positive. No differences between TTV positive and negative patients were found regarding epidemiological or biochemical parameters. Trypsin treatment and fluorescent in situ hybridization confirm the intracellular location of TTV in PBMC. The mean of TTV DNA titres was statistically higher in liver than in serum or PBMC. TTV titres in serum correlated with those in PBMC but not with those in liver. In conclusion, although the liver seems to be the main site for TTV replication, this virus is also able to infect PBMC.

Although the liver is the main target for hepatitis C virus (HCV) infection, HCV RNA of positive and negative polarity has also been detected in peripheral blood mononuclear cells (PBMCs) by polymerase chain reaction. However, no data have been published on the relationship between the number of HCV-infected PBMCs and serum viremia levels. To address this issue, PBMC samples from 20 patients with chronic hepatitis C were examined by fluorescent “in situ” hybridization. Serum viremia levels and viral load in infected PBMC were measured using the Amplicor Monitor test. HCV was detected in all PBMC samples corresponding to the HCV-positive patients. Fluorescent signals were found mainly in the cytoplasm of the cell. The percentage of positive cells ranged from 0.08% to 4%, with a statistical correlation with the viral load in PBMC (r = 0.69; p = .001) but not with the serum viremia levels (r = 0.23). It was demonstrated that HCV infection of PBMCs is a common feature of HCV chronic carriers. The results suggest that HCV infection of PBMCs does not contribute significantly to HCV viremia. J. Med. Virol. 60:269–274, 2000. © 2000 Wiley-Liss, Inc.

A modification of the process of oil extraction from rice bran is proposed, introducing one or two enzymatic reactions previous to solvent extraction. Although a total aqueous enzymatic extraction process did not result in reasonable oil extraction yields, an interesting alternative results from enzymatic reactions previous to solvent extraction or pressing. A thermal treatment of rice bran is first applied to deactivate lipase, but also to gelatinize starch previous to reaction with α-amylase. This is followed by a saccharifying step with glucoamylase to produce glucose (28 g/100 g of rice bran treated), while the residual paste, 66.7% of the original bran, may be subjected to a proteolytic process for protein extraction or directly treated with the solvent to obtain bran oil. Finally, under the defined extraction conditions using hexane, yields of oil are 5% higher when rice bran has been previously treated with α-amylase.

Fifty percent of healthy hepatitis B surface antigen carriers may have histologically proven chronic hepatitis. Our aim was to study the benefit of interferon-alpha in healthy patients. Twenty-nine hepatitis B surface antigen carriers with normal liver enzymes and with serum hepatitis B virus DNA were randomized into two groups: Group I, 14 patients treated with 9 megaunits of interferon alpha-2a thrice weekly for six months, and Group II, 15 control patients. A liver biopsy was obtained from each patient at study initiation. A second biopsy was available in nine treated patients and six controls. During treatment, a significant increase in alanine amino transferase levels was observed in treated patients as compared with the controls (P < 0.05). After treatment, transaminase levels decreased to normal values. No differences between treated and control patients were observed in clearance of hepatitis B virus markers. A significant increase in the total histological activity index between base line and final liver biopsies was observed in treated patients (P < 0.05). It is concluded that interferon alpha treatment may induce a biochemical and histological activation of liver disease. Accordingly, interferon alpha should not be administered to healthy hepatitis B surface antigen carriers, at least with the schedule used in this work.

Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6-aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide-directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431-Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half-life of the mutant enzyme when stored at pH 8.5 as compared with the wild-type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH-stability profile. (c) 1995 John Wiley & Sons, Inc.

Novel methyl 4,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carboxylates 3a–e have been prepared in a two step procedure from the readily available 2-oxo-1,2,3,4-tetrahydropyridine-5-carboxylates 1a–e by treatment with the Vilsmeier–Haack reagent. Further treatment of the novel o-chloroformyl substituted methyl 1,4-dihydropyridine-5-carboxylates 2a–e with hydrazine affords the corresponding methyl pyrazolo[3,4-b]pyridine-5-carboxylates in good yields. Semiempirical calculations reveal a favoured geometry with a boat conformation in the dihydropyridine system and a planar pyrazole ring.