Melchor Alvarez-Mon

Statins Inhibit HIV-1 infection by down-regulating rho activity

Human immunodeficiency virus (HIV)-1 infectivity requires actin-dependent clustering of host lipid raft–associated receptors, a process that might be linked to Rho guanosine triphosphatase (GTPase) activation. Rho GTPase activity can be negatively regulated by statins, a family of drugs used to treat hypercholesterolemia in man. Statins mediate inhibition of Rho GTPases by impeding prenylation of small G proteins through blockade of 3-hydroxy-3-methylglutaryl coenzyme A reductase. We show that statins decreased viral load and increased CD4+ cell counts in acute infection models and in chronically HIV-1–infected patients. Viral entry and exit was reduced in statin-treated cells, and inhibition was blocked by the addition of l-mevalonate or of geranylgeranylpyrophosphate, but not by cholesterol. Cell treatment with a geranylgeranyl transferase inhibitor, but not a farnesyl transferase inhibitor, specifically inhibited entry of HIV-1–pseudotyped viruses. Statins blocked Rho-A activation induced by HIV-1 binding to target cells, and expression of the dominant negative mutant RhoN19 inhibited HIV-1 envelope fusion with target cell membranes, reducing cell infection rates. We suggest that statins have direct anti–HIV-1 effects by targeting Rho.

HCV‐associated thrombocytopenia: clinical characteristics and platelet response after recombinant α2b‐interferon therapy

Hepatitis C virus (HCV) has been proposed as a possible causative agent of chronic thrombocytopenia. We investigated HCV infection in a series of 51 unselected Spanish patients with chronic acquired thrombocytopenia. Anti‐HCV and HCV viraemia were detected in 13/51 (22·5%) of cases; this prevalence was particularly significant when compared with HCV seropositivity in age‐matched controls (0·4%). Anti‐HCV‐positive patients, four men and nine women with a median age of 59·3 years (range 36–72), had a mean platelet count of 55·8 × 109/l (range 12–96). Only one of our HCV‐positive thrombocytopenic patients had hypersplenism. Platelet‐associated IgG (PAIgG) was negative, as measured by immunofluorescent flow cytometric analysis in 11/13 HCV‐positive thrombocytopenic patients. Thus, thrombocytopenia in our HCV‐positive patients appeared to be non‐autoimmune mediated. In six patients, a trial of recombinant α2b‐interferon (IFN‐α) given at a dose of 3 MU three times per week for 6–24 months gave a durable (> 1 year) and significant increase in platelet count in all six patients. The maximum increase occurred after 6 months of IFN‐α therapy. In conclusion, the ability of IFN‐α to increase platelet counts in HCV‐positive thrombocytopenic patients supports mechanisms involving a direct role for HCV inhibiting platelet production.

Serum lipopolysaccharide-binding protein prediction of severe bacterial infection in cirrhotic patients with ascites

Serum lipopolysaccharide-binding protein is increased in a subset of non-infected ascitic cirrhotic patients, a finding previously related to bacterial passage from the gut to the circulation without overt infection. We prospectively analysed the risk factors associated with a first episode of severe bacterial infection in 84 ascitic cirrhotics, followed up for a median of 46 weeks. The cumulative probability of such infection in patients with raised and normal lipopolysaccharide-binding protein was 32.4% and 8.0% (p=0.004), respectively. Increased lipopolysaccharide-binding protein was the only factor independently associated with severe bacterial infection in a multivariate analysis (relative risk 4.49, 95% CI 1.42-14.1). Monitoring of serum lipopolysaccharide-binding protein could, therefore, help to target cirrhotic patients with ascites for antibiotic prophylaxis.

Factors mediating the hemodynamic effects of tumor necrosis factor-α in portal hypertensive rats

Nitric oxide, prostacyclin, and glucagon have been implicated in promoting the hyperdynamic circulatory state of portal hypertension. Recent evidence also indicates that increased tumor necrosis factor-α (TNF-α) production is involved in the pathogenesis of this hemodynamic abnormality. This study was aimed at investigating in rats with portal vein stenosis (PVS) the effects on splanchnic hemodynamics of blocking circulating TNF-α and the factors mediating the vascular action of this cytokine in this setting. Anti-TNF-α polyclonal antibodies or placebo was injected into rats ( n = 96) before and 4 days after PVS (short-term inhibition) and at 24 h and 4, 7, 10 days after PVS (long-term inhibition). Short-term TNF-α inhibition reduced portal venous inflow and cardiac index and increased splanchnic and systemic resistance. Portal pressure was unchanged, but portal-systemic shunting was decreased. After long-term TNF-α inhibition, portal venous inflow and portal pressure were unchanged, but arterial pressure and systemic resistance rose significantly. Anti-TNF-α PVS rats exhibited lower increments of systemic resistance after N ω-nitro-l-arginine methyl ester and indomethacin administration and lower serum levels of TNF-α, nitrates-nitrites, and 6-keto-PGF1α, both over the short and the long term. Serum glucagon levels rose after long-term inhibition. In conclusion, the specific role played by TNF-α in the development of the hyperdynamic state of portal hypertension appears to be mainly mediated through an increased release of nitric oxide and prostacyclin. Maintenance of the splanchnic hyperemia after long-term TNF-α inhibition could be due to a compensatory release of glucagon.

Active Crohn's disease patients show a distinctive expansion of circulating memory CD4+CD45RO+CD28null T cells.

In a previous study we found an expansion of circulating memory (CD45RO+) CD4+ T cells in patients with Crohns disease (CD). The aim of this work was to investigate the phenotypic and functional characteristics of this T-cell subset in CD. We analyzed in peripheral blood CD4+CD45RO+ T cells from CD patients the expression of surface markers associated to immune activation, costimulation, and apoptosis. In sorted CD4+CD45RO+ T cells apoptosis was quantified by fluorescent annexin V binding. Healthy subjects and patients with ulcerative colitis and acute bacterial enterocolitis served as control groups. An increased percentage of memory CD4+CD45RO+ T cells lacking the expression of costimulatory receptor CD28 was detected in patients with active CD when compared to the other groups evaluated. This expanded CD4+CD45RO+CD28null T-cell subset expressed mostly the effector-cell marker CD57+. Both CD28 downregulation and CD57 expression correlated to CDAI and surrogate markers of disease activity. These phenotypic changes observed on CD4+CD45RO+ T cells from active CD returned to values similar to healthy controls after clinical remission. Moreover, this memory CD28νll T-cell subset might express more intracytoplasmic TNF and IFN-γ than their CD28+ counterpart. Significantly lower frequencies of memory CD4+CD45RO+ T cells expressing CD95 apoptosis receptor were found in patients with active CD. Moreover, sorted CD4+CD45RO+and CD4+CD45RO+ CD28null T cells from patients with active CD exhibited a lower apoptotic rate than that found in healthy controls and inactive CD patients. According to our data, circulating T lymphocytes from active CD patients show distinctive phenotypic and functional changes, characterized by an expansion of memory CD4+CD45RO+CD28null T cells expressing effector-associated cell surface molecules and displaying enhanced resistance to apoptosis.

Cytokine levels after open and laparoscopic cholecystectomy.

A prospective study of serum cytokine levels was performed in patients randomly assigned to undergo either laparoscopic cholecystectomy (LC) or open cholecystectomy (OC). The kinetics of serum interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), and cortisol were studied in both groups of patients. Cytokine and cortisol levels were measured in serum samples from patients who underwent either LC (n = 14) or OC (n = 14) using enzyme-linked immunosorbent assay and radioimmunoassay, respectively. Serum samples were obtained 24 h before surgery and 24 h and 7 days after surgery. IL-6 levels differed significantly (p < 0.05) between the LC and OC groups. IL-1 beta, IL-10, TNF-alpha and cortisol levels showed no significant differences (p > 0.05). Kinetic studies of IL-6, IL-1 beta and TNF-alpha levels revealed them to behave similarly, 24 h after surgery the levels of these cytokines were higher than those 24 h before surgery. These levels normalized by 7 days after surgery. Cytokine concentrations were always higher in the OC group than in the LC group. IL-1 beta and IL-10 levels were the most stable in both groups, though cortisol levels were also fairly stable.

Two different maturational stages of natural killer lymphocytes in human newborn infants

The objective of this study was to assess the basis for the diminished natural killer (NK) lymphocyte activity of neonates. We found either severely reduced (63% of 68 neonates) or normal (similar to healthy adult) levels of NK activity. The percentages of cord blood mononuclear cells from the two groups of infants that expressed CD16, a differentiation antigen found in NK cells, were similar and within the range found in peripheral blood mononuclear cells of adults. However, infants with low NK activity had reduced numbers of cells in the CD16+56+ subpopulation, whereas the number of these effector cells present in cord blood mononuclear cells from infants with normal NK activity was within the range found in adults. Recombinant interleukin-2, but not recombinant interferon-gamma, normalized the low NK activity of infants in a dose- and time-dependent manner. Analysis of the pattern of target cell susceptibility to lysis, together with the CD16+CD3- phenotype of the precursor and effector lymphocytes, demonstrated that the induced cytotoxicity was mediated by NK cells. In contrast, NK cells from infants with normal cytotoxic levels exhibited a functional response to interleukin-2 and interferon-gamma similar to that of adults. Our results indicate that NK cells in human neonates go through two different maturational stages.

Regulation of natural killer cytotoxicity by 1,25-dihydroxyvitamin D3.

The steroid hormone 1 alpha, 25-dihydroxyvitamin D3, calcitriol, is crucial in calcium homeostasis. Calcium plays a central role in T, B, and NK cell functions, and calcitriol is a known inhibitor of T cell proliferation and immunoglobulin production. We have analyzed here the immunoregulatory effects of calcitriol on NK cell function. We show that calcitriol specifically specifically inhibits, in a time- and dose-dependent fashion, the generation of cytotoxic activity from cultured CD16+ peripheral blood NK cells. It also suppresses, at similar molar concentrations (1-10 nM), interleukin 2 (IL-2) production by PHA-activated peripheral blood lymphocytes. Calcitriol does not interfere with the cytotoxic function of NK cells, whether fresh or generated in vitro, placing the inhibition at the level of NK cell activation. Interestingly enough, exogenous IL-2 can completely reverse the suppressive effect. These findings suggest that modulation of NK cell activation by control of the internal level of IL-2 may reflect an additional paracrine calcitriol-dependent circuit with immunoregulatory consequences.

Obeticholic acid reduces bacterial translocation and inhibits intestinal inflammation in cirrhotic rats.

BACKGROUND & AIMS In advanced cirrhosis, gut bacterial translocation is the consequence of intestinal barrier disruption and leads to bacterial infection. Bile acid abnormalities in cirrhosis could play a role in the integrity of the intestinal barrier and the control of microbiota, mainly through the farnesoid X receptor. We investigated the long-term effects of the farnesoid X receptor agonist, obeticholic acid, on gut bacterial translocation, intestinal microbiota composition, barrier integrity and inflammation in rats with CCl4-induced cirrhosis with ascites. METHODS Cirrhotic rats received a 2-week course of obeticholic acid or vehicle starting once ascites developed. We then determined: bacterial translocation by mesenteric lymph node culture, ileum expression of antimicrobial peptides and tight junction proteins by qPCR, fecal albumin loss, enteric bacterial load and microbiota composition by qPCR and pyrosequencing of ileum mucosa-attached contents, and intestinal inflammation by cytometry of the inflammatory infiltrate. RESULTS Obeticholic acid reduced bacterial translocation from 78.3% to 33.3% (p<0.01) and upregulated the expression of the farnesoid X receptor-associated gene small heterodimer partner. Treatment improved ileum expression of antimicrobial peptides, angiogenin-1 and alpha-5-defensin, tight junction proteins zonulin-1 and occludin, and reduced fecal albumin loss and liver fibrosis. Enteric bacterial load normalized, and the distinctive mucosal microbiota of cirrhosis was reduced. Gut immune cell infiltration was reduced and inflammatory cytokine and Toll-like receptor 4 expression normalized. CONCLUSIONS In ascitic cirrhotic rats, obeticholic acid reduces gut bacterial translocation via several complementary mechanisms at the intestinal level. This agent could be used as an alternative to antibiotics to prevent bacterial infection in cirrhosis.

Clinical relevance of the severe abnormalities of the T cell compartment in septic shock patients

IntroductionGiven the pivotal role of T lymphocytes in the immune system, patients with septic shock may show T cell abnormalities. We have characterised the T cell compartment in septic shock and assess its clinical implications.MethodsT lymphocytes from the peripheral blood of 52 patients with septic shock and 36 healthy control subjects were analysed on admission to the intensive care unit, baseline, and 3, 7, 14 and 28 days later. T cell phenotypes (CD3+CD4+/CD3+CD8+, CD45RA+/CD45RO+, CD62L+/CD28+) were assessed by quantitative flow cytometry.ResultsCD3+, CD3+CD4+ and CD3+CD8+ lymphocyte counts were significantly lower in patients with septic shock than control subjects. In surviving patients, CD3+CD4+ lymphocytes had normalised after 14 days, yet CD3+CD8+ numbers were still low. Non effector CD45RA+CD45RO- subsets of CD3+CD4+ and CD3+CD8+ were persistently low during patient follow up. CD3+CD8+CD28+ and CD3+CD8+CD62L+ were reduced in patients versus controls and survivors versus nonsurvivors in the first three days. A prediction receptor operative curve revealed that for the CD3+CD8+CD28+ subset, a cutoff of 136 cells/ml showed 70% sensitivity and 100% specificity for predicting death and the area under the curve was 0.84 at admission. Corresponding values for CD3+CD8+CD62L+ were 141 cells/ml, 60% sensitivity, 100% specificity and an area under the curve of 0.75.ConclusionsA severe redistribution of T lymphocyte subsets is found in septic shock patients. A different kinetic pattern of T cell subset involvement is observed in surviving and nonsurviving patients, with lower numbers of circulating CD3+CD8+CD28+ and CD3+CD8+CD62L+ associated with a better disease outcome.