Nucleos(t)ide analogue treatment reduces apoptotic activity in patients with chronic hepatitis B.

Background: Reduction of necroinflammatory activity is a major goal of antiviral therapy of patients with chronic hepatitis B. Serum ALT does not detect all forms of cell death.Objectives: To analyze dynamics of novel serum cell death markers for apoptosis and necrosis in association with virologic response to nucleos(t)ide (Nuc) analogue treatment.Study design: Quantification of the M30-apoptosis neoepitope and the cytokeratin-18 (M65-necrosis) serum levels before and during treatment of patients with chronic hepatitis B with Nuc (n = 26).Results: Before treatment, M30-apoptotic activity was significantly correlated with M65-necrosis and fibrosis but not with serum ALT. During therapy with Nucs, cell death parameters M30-apoptosis, M65-necrosis, and ALT declined in association with virologic response. The most frequent cell death pattern was simultaneous decline of ALT and M30-apoptosis which occurred more frequently in patients with HBs-Antigen decline than in patients with HBs-Antigen increase during treatment (87.5% vs. 40.0%; p = 0.024). ALT decline in association with increase of M30 apoptosis was frequent in patients with HBs-Antigen increase during treatment (36.3%) but was not observed in patients with HBs-Antigen decline during treatment.Conclusion: Decline of cell death parameters in association with decline of HBV-DNA and HBs-Antigen indicates a reduction in overall cell death activity during Nuc treatment supporting the concept that response to Nuc therapy reduces necroinflammatory activity and progression of liver disease. (C) 2011 Elsevier B. V. All rights reserved.

Recirculating CD4 memory T cells mount rapid secondary responses without major contributions from follicular CD4 effectors and B cells.

For weeks after primary immunization with thymus-dependent antigens the responding lymph nodes contain effector CD4 T cells in T zones and germinal centers as well as recirculating memory T cells. Conversely, remote nodes, not exposed to antigen, only receive recirculating memory cells. We assessed whether lymph nodes with follicular effector CD4 T cells in addition to recirculating memory CD4 T cells mount a more rapid secondary response than nodes that only contain recirculating memory cells. Also, the extent to which T cell frequency governs accelerated CD4 T cell recall responses was tested. For this, secondary antibody responses to a superantigen, where the frequency of responding T cells is not increased at the time of challenge, were compared with those to conventional protein antigens. With both types of antigens similar accelerated responses were elicited in the node draining the site of primary immunization and in the contralateral node, not previously exposed to antigen. Thus recirculating memory cells are fully capable of mounting accelerated secondary responses, without the assistance of CD4 effector T cells, and accelerated memory responses are not solely dependent on higher T cell frequencies. Accelerated memory CD4 T cell responses were also seen in B cell-deficient mice.

Quantification of brain glycogen concentration and turnover through localized 13C NMR of both the C1 and C6 resonances.

We have recently shown that at isotopic steady state (13)C NMR can provide a direct measurement of glycogen concentration changes, but that the turnover of glycogen was not accessible with this protocol. The aim of the present study was to design, implement and apply a novel dual-tracer infusion protocol to simultaneously measure glycogen concentration and turnover. After reaching isotopic steady state for glycogen C1 using [1-(13)C] glucose administration, [1,6-(13)C(2)] glucose was infused such that isotopic steady state was maintained at the C1 position, but the C6 position reflected (13)C label incorporation. To overcome the large chemical shift displacement error between the C1 and C6 resonances of glycogen, we implemented 2D gradient based localization using the Fourier series window approach, in conjunction with time-domain analysis of the resulting FIDs using jMRUI. The glycogen concentration of 5.1 +/- 1.6 mM measured from the C1 position was in excellent agreement with concomitant biochemical determinations. Glycogen turnover measured from the rate of label incorporation into the C6 position of glycogen in the alpha-chloralose anesthetized rat was 0.7 micromol/g/h.

Acetylcholine-induced vasodilation and reactive hyperemia are not affected by acute cyclo-oxygenase inhibition in human skin

Résumé Il est actuellement reconnu que l'endothélium vasculaire joue un rôle primordial dans la genèse des maladies cardiovasculaires, notamment l'artériosclérose. Dès lors, il est important de pouvoir investiguer la fonction endothéliale en clinique. Pour ce faire, il est particulièrement simple d'examiner la microcirculation cutanée, car celle-ci est très simplement accessible, de manière non-invasive, par fluxmétrie laser-Doppler. Pratiquement, on mesure l'augmentation du flux sanguin dermique en réponse à des stimuli connus pour agir via l'endothélium vasculaire. Les stimuli endothélium-dépendants les plus courants sont l'interruption temporaire du flux sanguin qui est suivie d'une hyperémie réactive, et l'administration transcutanée d'acétylcholine (Ach) par iontophorèse. La iontophorèse consiste à obtenir le transfert d' une substance ionisée, telle l'Ach, par l'application d'un courant électrique de polarité appropriée. L'objectif du présent travail était de déterminer le rôle des prostaglandines dans ces réponse vasodilatatrices dépendante de l'endothélium, rôle actuellement peu clair. 23 jeunes hommes volontaires non fumeurs et en bonne santé habituelle ont été examinés lors de deux visites séparées par 1 à 3 semaines. Lors de chaque visite, l'hyperémie réactive et la réponse vasodilatatrice à l'Ach ont été déterminées dans la peau de l'avant bras après administration soit d'un placebo, soit d'un inhibiteur de la cyclooxygénase (COX, enzyme qui contrôle la synthèse des prostaglandines). Chez certains sujets, l'inhibiteur était de l'acétylsalicylate de lysine (900 mg par voie intraveineuse). Chez d'autres sujets, il s'agissait d'indométhacine. (75 mg par voie orale). Comme la stimulation nociceptive liée au courant iontophorétique peut influencer la réponse à l'Ach, celle-ci a été déterminée en présence et en l'absence d'anesthésie de surface (crème de lidocaine). La réponse à l'Ach a été obtenue pour 4 doses différentes de cet agent (exprimées sous la forme de la densité de charge iontophorétique appliquée : 0.28, 1.4, 7, et 14 millicoulombs par cm2 de peau exposée). Le flux sanguin dermique était mesuré par imagerie laser-Doppler, une variante de la fluxmétrie laser-Doppler classique permettant l'exploration d'une surface de peau de taille arbitraire. Quelle que soit la condition testée, nous n'avons jamais observé la moindre influence de l'inhibition de la COX sur l'hyperémie réactive, ni sur la réponse à l'Ach. Cette dernière était augmentée significativement par l'anesthésie cutanée, que les sujets aient reçu ou non de l'acétylsalicylate de lysine ou de l'indométhacine . Par exemple, la réponses moyenne (±SD) à la plus haute dose d'Ach (testée sur 6 sujets, et exprimée en unités de perfusion, comme il est d'usage en fluxmétrie laser-Doppler ) était la suivante : en l'absence d'anesthésie : acétylsalicylate de lysine 339 ± 105, placebo 344 ± 68 ; avec l'anesthésie : acétylsalicylate de lysine 453 ± 76 , placebo 452 ± 65 (p * 0.001 pour les effets de l'anesthésie). En conclusion, nos résultats infirment une contribution des prostaglandines à l'hyperémie réactive ou à la vasodilatation induite par l'acétylcholine dans la microcirculation cutanée. Dans ce lit vasculaire, l'anesthésie locale accroît la vasodilatation induite par l'acétylcholine par un mécanisme indépendant des prostaglandines.

Maturation of marginal zone and follicular B cells requires B cell activating factor of the tumor necrosis factor family and is independent of B cell maturation antigen.

B cells undergo a complex series of maturation and selection steps in the bone marrow and spleen during differentiation into mature immune effector cells. The tumor necrosis factor (TNF) family member B cell activating factor of the TNF family (BAFF) (BLyS/TALL-1) plays an important role in B cell homeostasis. BAFF and its close homologue a proliferation-inducing ligand (APRIL) have both been shown to interact with at least two receptors, B cell maturation antigen (BCMA) and transmembrane activator and cyclophilin ligand interactor (TACI), however their relative contribution in transducing BAFF signals in vivo remains unclear. To functionally inactivate both BAFF and APRIL, mice transgenic for a soluble form of TACI were generated. They display a developmental block of B cell maturation in the periphery, leading to a severe depletion of marginal zone and follicular B2 B cells, but not of peritoneal B1 B cells. In contrast, mice transgenic for a soluble form of BCMA, which binds APRIL, have no detectable B cell phenotype. This demonstrates a crucial role for BAFF in B cell maturation and strongly suggests that it signals via a BCMA-independent pathway and in an APRIL-dispensable way.

When "enough" is not enough: new perspectives on optimal methadone maintenance dose.

Some methadone maintenance treatment (MMT) programs prescribe inadequate daily methadone doses. Patients complain of withdrawal symptoms and continue illicit opioid use, yet practitioners are reluctant to increase doses above certain arbitrary thresholds. Serum methadone levels (SMLs) may guide practitioners dosing decisions, especially for those patients who have low SMLs despite higher methadone doses. Such variation is due in part to the complexities of methadone metabolism. The medication itself is a racemic (50:50) mixture of 2 enantiomers: an active "R" form and an essentially inactive "S" form. Methadone is metabolized primarily in the liver, by up to five cytochrome P450 isoforms, and individual differences in enzyme activity help explain wide ranges of active R-enantiomer concentrations in patients given identical doses of racemic methadone. Most clinical research studies have used methadone doses of less than 100 mg/day [d] and have not reported corresponding SMLs. New research suggests that doses ranging from 120 mg/d to more than 700 mg/d, with correspondingly higher SMLs, may be optimal for many patients. Each patient presents a unique clinical challenge, and there is no way of prescribing a single best methadone dose to achieve a specific blood level as a "gold standard" for all patients. Clinical signs and patient-reported symptoms of abstinence syndrome, and continuing illicit opioid use, are effective indicators of dose inadequacy. There does not appear to be a maximum daily dose limit when determining what is adequately "enough" methadone in MMT.

Decrease in the prevalence of hepatitis B and a low prevalence of hepatitis C virus infections in the general population of the Seychelles.

A serological survey of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections was carried out on a random sex- and age-stratified sample of 1006 individuals aged 25-64 years in the Seychelles islands. Anti-HBc and anti-HCV antibodies were detected using commercially available enzyme-linked immunosorbent assays (ELISA), followed by a Western blot assay in the case of a positive result for anti-HCV. The age-adjusted seroprevalence of anti-HBc antibodies was 8.0% (95% CI: 6.5-9.9%) and the percentage prevalence among males/females increased from 7.0/3.1 to 19.1/13.4 in the age groups 25-34 to 55-64 years, respectively. Two men and three women were positive for anti-HCV antibodies, with an age-adjusted seroprevalence of 0.34% (95% CI: 0.1-0.8%). Two out of these five subjects who were positive for anti-HCV also had anti-HBc antibodies. The seroprevalence of anti-HBc was significantly higher in unskilled workers, persons with low education, and heavy drinkers. The age-specific seroprevalence of anti-HBc in this population-based survey, which was conducted in 1994, was approximately three times lower than in a previous patient-based survey carried out in 1979. Although there are methodological differences between the two surveys, it is likely that the substantial decrease in anti-HBc prevalence during the last 15 years may be due to significant socioeconomic development and the systematic screening of blood donors since 1981. Because hepatitis C virus infections are serious and the cost of treatment is high, the fact that the prevalence of anti-HCV antibodies is at present low should not be an argument for not screening blood donors for anti-HCV and eliminating those who are positive.

Induction of the alternative NF-κB pathway by lymphotoxin αβ (LTαβ) relies on internalization of LTβ receptor.

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-κB pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin β receptor (LTβR) as a prototype, we showed that activation of the alternative, but not the classical, NF-κB pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LTβR essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, but clathrin-independent, internalization of LTβR appeared to be required for the activation of the alternative, but not the classical, NF-κB pathway. In vivo, ligand-induced internalization of LTβR in mesenteric lymph node stromal cells correlated with induction of alternative NF-κB target genes. Thus, our data shed light on LTβR cellular trafficking as a process required for specific biological functions of NF-κB.

Myosin motors and not actin comets are mediators of the actin-based Golgi-to-endoplasmic reticulum protein transport

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2AA). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2AA mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2AA. Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport.

When genetic load does not correlate with phenotypic spectrum: lessons from the GnRH receptor (GNRHR).

CONTEXT: A broad spectrum of GnRH-deficient phenotypes has been identified in individuals with both mono- and biallelic GNRHR mutations. OBJECTIVE: The objective of the study was to determine the correlation between the severity of the reproductive phenotype(s) and the number and functional severity of rare sequence variants in GNRHR. SUBJECTS: Eight hundred sixty-three probands with different forms of GnRH deficiency, 46 family members and 422 controls were screened for GNRHR mutations. The 70 subjects (32 patients and 38 family members) harboring mutations were divided into four groups (G1-G4) based on the functional severity of the mutations (complete or partial loss of function) and the number of affected alleles (monoallelic or biallelic) with mutations, and these classes were mapped on their clinical phenotypes. RESULTS: The prevalence of heterozygous rare sequence variants in GNRHR was significantly higher in probands vs. controls (P < 0.01). Among the G1-G3 groups (homozygous subjects with successively decreasing severity and number of mutations), the hypogonadotropic phenotype related to their genetic load. In contrast, subjects in G4, with only monoallelic mutations, demonstrated a greater diversity of clinical phenotypes. CONCLUSIONS: In patients with GnRH deficiency and biallelic mutations in GNRHR, genetic burden defined by severity and dose is associated with clinical phenotype. In contrast, for patients with monoallelic GNRHR mutations this correlation does not hold. Taken together, these data indicate that as-yet-unidentified genetic and/or environmental factors may combine with singly mutated GNRHR alleles to produce reproductive phenotypes.

Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) but not PPARalpha serves as a plasma free fatty acid sensor in liver.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is an important transcription factor in liver that can be activated physiologically by fasting or pharmacologically by using high-affinity synthetic agonists. Here we initially set out to elucidate the similarities in gene induction between Wy14643 and fasting. Numerous genes were commonly regulated in liver between the two treatments, including many classical PPARalpha target genes, such as Aldh3a2 and Cpt2. Remarkably, several genes induced by Wy14643 were upregulated by fasting independently of PPARalpha, including Lpin2 and St3gal5, suggesting involvement of another transcription factor. Using chromatin immunoprecipitation, Lpin2 and St3gal5 were shown to be direct targets of PPARbeta/delta during fasting, whereas Aldh3a2 and Cpt2 were exclusive targets of PPARalpha. Binding of PPARbeta/delta to the Lpin2 and St3gal5 genes followed the plasma free fatty acid (FFA) concentration, consistent with activation of PPARbeta/delta by plasma FFAs. Subsequent experiments using transgenic and knockout mice for Angptl4, a potent stimulant of adipose tissue lipolysis, confirmed the stimulatory effect of plasma FFAs on Lpin2 and St3gal5 expression levels via PPARbeta/delta. In contrast, the data did not support activation of PPARalpha by plasma FFAs. The results identify Lpin2 and St3gal5 as novel PPARbeta/delta target genes and show that upregulation of gene expression by PPARbeta/delta is sensitive to plasma FFA levels. In contrast, this is not the case for PPARalpha, revealing a novel mechanism for functional differentiation between PPARs.

TACI, unlike BAFF-R, is solely activated by oligomeric BAFF and APRIL to support survival of activated B cells and plasmablasts.

The cytokine BAFF binds to the receptors TACI, BCMA, and BAFF-R on B cells, whereas APRIL binds to TACI and BCMA only. The signaling properties of soluble trimeric BAFF (BAFF 3-mer) were compared with those of higher-order BAFF oligomers. All forms of BAFF bound BAFF-R and TACI, and elicited BAFF-R-dependent signals in primary B cells. In contrast, signaling through TACI in mature B cells or plasmablasts was only achieved by higher-order BAFF and APRIL oligomers, all of which were also po-tent activators of a multimerization-dependent reporter signaling pathway. These results indicate that, although BAFF-R and TACI can provide B cells with similar signals, only BAFF-R, but not TACI, can respond to soluble BAFF 3-mer, which is the main form of BAFF found in circulation. BAFF 60-mer, an efficient TACI agonist, was also detected in plasma of BAFF transgenic and nontransgenic mice and was more than 100-fold more active than BAFF 3-mer for the activation of multimerization-dependent signals. TACI supported survival of activated B cells and plasmablasts in vitro, providing a rational basis to explain the immunoglobulin deficiency reported in TACI-deficient persons.

High b-value diffusion-weighted imaging: A sensitive method to reveal white matter differences in schizophrenia.

Over the last 10 years, diffusion-weighted imaging (DWI) has become an important tool to investigate white matter (WM) anomalies in schizophrenia. Despite technological improvement and the exponential use of this technique, discrepancies remain and little is known about optimal parameters to apply for diffusion weighting during image acquisition. Specifically, high b-value diffusion-weighted imaging known to be more sensitive to slow diffusion is not widely used, even though subtle myelin alterations as thought to happen in schizophrenia are likely to affect slow-diffusing protons. Schizophrenia patients and healthy controls were scanned with a high b-value (4000s/mm(2)) protocol. Apparent diffusion coefficient (ADC) measures turned out to be very sensitive in detecting differences between schizophrenia patients and healthy volunteers even in a relatively small sample. We speculate that this is related to the sensitivity of high b-value imaging to the slow-diffusing compartment believed to reflect mainly the intra-axonal and myelin bound water pool. We also compared these results to a low b-value imaging experiment performed on the same population in the same scanning session. Even though the acquisition protocols are not strictly comparable, we noticed important differences in sensitivities in the favor of high b-value imaging, warranting further exploration.

Local and global error models to improve uncertainty quantification

In groundwater applications, Monte Carlo methods are employed to model the uncertainty on geological parameters. However, their brute-force application becomes computationally prohibitive for highly detailed geological descriptions, complex physical processes, and a large number of realizations. The Distance Kernel Method (DKM) overcomes this issue by clustering the realizations in a multidimensional space based on the flow responses obtained by means of an approximate (computationally cheaper) model; then, the uncertainty is estimated from the exact responses that are computed only for one representative realization per cluster (the medoid). Usually, DKM is employed to decrease the size of the sample of realizations that are considered to estimate the uncertainty. We propose to use the information from the approximate responses for uncertainty quantification. The subset of exact solutions provided by DKM is then employed to construct an error model and correct the potential bias of the approximate model. Two error models are devised that both employ the difference between approximate and exact medoid solutions, but differ in the way medoid errors are interpolated to correct the whole set of realizations. The Local Error Model rests upon the clustering defined by DKM and can be seen as a natural way to account for intra-cluster variability; the Global Error Model employs a linear interpolation of all medoid errors regardless of the cluster to which the single realization belongs. These error models are evaluated for an idealized pollution problem in which the uncertainty of the breakthrough curve needs to be estimated. For this numerical test case, we demonstrate that the error models improve the uncertainty quantification provided by the DKM algorithm and are effective in correcting the bias of the estimate computed solely from the MsFV results. The framework presented here is not specific to the methods considered and can be applied to other combinations of approximate models and techniques to select a subset of realizations