Safety and immunogenicity of the M72/AS01 candidate tuberculosis vaccine in HIV-infected adults on combination antiretroviral therapy: a phase I/II, randomized trial.

OBJECTIVE: Tuberculosis (TB) is highly prevalent among HIV-infected people, including those receiving combination antiretroviral therapy (cART), necessitating a well tolerated and efficacious TB vaccine for these populations. We evaluated the safety and immunogenicity of the candidate TB vaccine M72/AS01 in adults with well controlled HIV infection on cART. DESIGN: A randomized, observer-blind, controlled trial (NCT00707967). METHODS: HIV-infected adults on cART in Switzerland were randomized 3 : 1 : 1 to receive two doses, 1 month apart, of M72/AS01, AS01 or 0.9% physiological saline (N = 22, N = 8 and N = 7, respectively) and were followed up to 6 months postdose 2 (D210). Individuals with CD4⁺ cell counts below 200 cells/μl were excluded. Adverse events (AEs) including HIV-specific and laboratory safety parameters were recorded. Cell-mediated (ICS) and humoral (ELISA) responses were evaluated before vaccination, 1 month after each dose (D30, D60) and D210. RESULTS: Thirty-seven individuals [interquartile range (IQR) CD4⁺ cell counts at screening: 438-872 cells/μl; undetectable HIV-1 viremia] were enrolled; 73% of individuals reported previous BCG vaccination, 97.3% tested negative for the QuantiFERON-TB assay. For M72/AS01 recipients, no vaccine-related serious AEs or cART-regimen adjustments were recorded, and there were no clinically relevant effects on laboratory safety parameters, HIV-1 viral loads or CD4⁺ cell counts. M72/AS01 was immunogenic, inducing persistent and polyfunctional M72-specific CD4⁺ T-cell responses [medians 0.70% (IQR 0.37-1.07) at D60] and 0.42% (0.24-0.61) at D210, predominantly CD40L⁺IL-2⁺TNF-α⁺, CD40L⁺IL-2⁺ and CD40L⁺IL-2⁺TNF-α⁺IFN-γ⁺]. All M72/AS01 vaccines were seropositive for anti-M72 IgG after second vaccination until study end. CONCLUSION: M72/AS01 was clinically well tolerated and immunogenic in this population, supporting further clinical evaluation in HIV-infected individuals in TB-endemic settings.

Cell-matrix interactions in Schistosomal portal fibrosis: a dynamic event

In recent years, one of the most significant progress in the understanding of liver diseases was the demonstration that liver fibrosis is a dynamic process resulting from a balance between synthesis and degradation of several matrix components, collagen in particular. Thus, fibrosis has been found to be a very early event during liver diseases, be it of toxic, viral or parasitic origin, and to be spontaneously reversible, either partially or totally. In liver fibrosis cell matrix interactions are dependent on the existence of the many factors (sometimes acting in combination) which produce the same events at the cellular and molecular levels. These events are: (i) the recruitment of fiber-producing cells, (ii) their proliferation, (iii) the secretion of matrix constituents of the extracellular matrix, and (iv) the remodeling and degradation of the newly formed matrix. All these events represent, at least in principle, a target for a therapeutic intervention aimed at influencing the experimentally induced hepatic fibrosis. In this context, hepatosplenic schistosomiasis is of particular interest, being an immune cell-mediated granulomatous disease and a model of liver fibrosis allowing extensive studies in human and animals as well as providing original in vitro models.

Microbiota abnormalities in inflammatory airway diseases - Potential for therapy.

Increasingly the development of novel therapeutic strategies is taking into consideration the contribution of the intestinal microbiota to health and disease. Dysbiosis of the microbial communities colonizing the human intestinal tract has been described for a variety of chronic diseases, such as inflammatory bowel disease, obesity and asthma. In particular, reduction of several so-called probiotic species including Lactobacilli and Bifidobacteria that are generally considered to be beneficial, as well as an outgrowth of potentially pathogenic bacteria is often reported. Thus a tempting therapeutic approach is to shape the constituents of the microbiota in an attempt to restore the microbial balance towards the growth of 'health-promoting' bacterial species. A twist to this scenario is the recent discovery that the respiratory tract also harbors a microbiota under steady-state conditions. Investigators have shown that the microbial composition of the airway flora is different between healthy lungs and those with chronic lung diseases, such as asthma, chronic obstructive pulmonary disease as well as cystic fibrosis. This is an emerging field, and thus far there is very limited data showing a direct contribution of the airway microbiota to the onset and progression of disease. However, should future studies provide such evidence, the airway microbiota might soon join the intestinal microbiota as a target for therapeutic intervention. In this review, we highlight the major advances that have been made describing the microbiota in chronic lung disease and discuss current and future approaches concerning manipulation of the microbiota for the treatment and prevention of disease.

The human CFTR protein expressed in CHO cells activates aquaporin-3 in a cAMP-dependent pathway: study by digital holographic microscopy.

The transmembrane water movements during cellular processes and their relationship to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) protein activity through a cAMP-stimulated aqueous pore, or be dependent on aquaporin. Here, we used digital holographic microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel, CFTR, measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTRinh172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR (F508del-CFTR), which mimics the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTR-defective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators, genistein and MPB91, failed to trigger water movements. Finally, using a specific small-interfering RNA against the endogenous aquaporin AQP3, the water transport accompanying CFTR activity decreased. We conclude that water fluxes accompanying CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in the CFTR protein.

Risk of Bronchiolitis Obliterans Syndrome Is Twice as High in Cyclosporine Treated Patients in Comparison to Tacrolimus 3 Years after Lung Transplantation: Results of a Prospective Randomized International Trial of 248 Patients

Purpose: In this prospective randomized study efficacy and safety of two immunosuppressive regimens (Tac, MMF, Steroids vs. CsA, MMF, Steroids) after Lung Transplantation were compared. Primary objective was the incidence of bronchiolitis obliterans syndrome (BOS). Secondary objectives were incidence of acute rejection and infection, survival and adverse events. 248 patients with a complete 3 year follow-up were included in the analysis. Methods and Materials: Patients were randomized to treatment group A: Tac (0.01-0.03 mg/kg/d iv-0.05-0.3 mg/kg/d po) or B: CsA (1-3 mg/kg/d iv-2-8 mg/kg/d po). MMF dose was1-4 mg/d in both groups. No induction therapy was given. Patients were stratified for cystic fibrosis. Intention to treat analysis was performed in patients who were switched to a different immunosuppressive regimen. Results: 3 of 123 Tac patients and 41 of 125 CsA patients were switched to another immunosuppressive regimen and were analyzed as intention to treat. Three year follow-up data of the complete patient cohort were included in this final analysis. Groups showed no difference in demographic data. Kaplan Meier analysis revealed significantly less BOS in Tac treated patients (p=0.033, log rank test, pooled over strata). Cox regression showed a twice as high risk for BOS in the CsA group (factor 2.003). Incidence of acute rejection was 67.5% (Tac) and 75.2% (CsA) (p=0.583). One- and 3-year-survival-rates were not different (85.4% Tac vs. 88.8% CsA, and 80.5% Tac vs. 83.2% CsA, p=n.s.). Incidence of infections and renal failure was similar (p=n.s.). Conclusions: Tac significantly reduced the risk for BOS after 3 years in this intention to treat analysis. Both regimens have a good immunosuppressive potential and offer a similar safety profile with excellent one and three year survival rates. Acute rejection rates were similar in both groups. Incidence of infections and renal failure showed no difference.

Ten years of lung transplantation in Switzerland: results of the Swiss Lung Transplant Registry.

Lung transplantation has evolved from an experimental procedure to a viable therapeutic option in many countries. In Switzerland, the first lung transplant was performed in November 1992, more than ten years after the first successful procedure world-wide. Thenceforward, a prospective national lung transplant registry was established, principally to enable quality control. The data of all patients transplanted in the two Swiss Lung Transplant centres Zurich University Hospital and Centre de Romandie (Geneva-Lausanne) were analysed. In 10 years 242 lung transplants have been performed. Underlying lung diseases were cystic fibrosis including bronchiectasis (32%), emphysema (32%), parenchymal disorders (19%), pulmonary hypertension (11%) and lymphangioleiomyomatosis (3%). There were only 3% redo procedures. The 1, 5 and 9 year survival rates were 77% (95% CI 72-82), 64% (95% CI 57-71) and 56% (95% CI 45-67), respectively. The 5 year survival rate of patients transplanted since 1998 was 72% (95% CI 64-80). Multivariate Cox regression analysis revealed that survival was significantly better in this group compared to those transplanted before 1998 (HR 0.44, 0.26-0.75). Patients aged 60 years and older (HR 5.67, 95% CI 2.50-12.89) and those with pulmonary hypertension (HR 2.01, 95% CI 1.10-3.65) had a significantly worse prognosis The most frequent causes of death were infections (29%), bronchiolitis obliterans syndrome (25%) and multiple organ failure (14%). The 10-year Swiss experience of lung transplantation compares favourably with the international data. The best results are obtained in cystic fibrosis, pulmonary emphysema and parenchymal disorders.

Intestinal fibrovascular nodules caused by Schistosoma mansoni infection in Calomys callosus Rengger, 1830 (Rodentia: Cricetidae): a model of concomitant fibrosis and angiogenesis

Human schistosomiasis develops extensive and dense fibrosis in portal space, together with congested new blood vessels. This study demonstrates that Calomys callosus infected with Schistosoma mansoni also develops fibrovascular lesions, which are found in intestinal subserosa. Animals were percutaneously infected with 70 cercariae and necropsied at 42, 45, 55, 80, 90 and 160 days after infection. Intestinal sections were stained for brightfield, polarization microscopy, confocal laser scanning, transmission and scanning electron microscopies. Immunohistological analysis was also performed and some nodules were aseptically collected for cell culture. Numerous intestinal nodules, appearing from 55 up to 160 days after infection, were localized at the interface between external muscular layer and intestinal serosa, consisting of fibrovascular tissue forming a shell about central granuloma(s). Intranodular new vessels were derived from the vasculature of the external vascular layer and were positive for laminin, chondroitin-sulfate, smooth muscle alpha-actin and FVIII-RA. Fibroblastic cells and extracellular matrix components (collagens I, III and VI, fibronectin and tenascin) comprised the stroma. Intermixed with the fibroblasts and vessels there were variable number of eosinophils, macrophages and haemorrhagic foci. In conclusion, the nodules constitute an excellent and accessible model to study fibrogenesis and angiogenesis, dependent on S. mansoni eggs. The fibrogenic activity is fibroblastic and not myofibroblastic-dependent. The angiogenesis is so prominent that causes haemorrhagic ascites.

Tacrolimus and cyclosporine have differential effects on the risk of development of bronchiolitis obliterans syndrome: results of a prospective, randomized international trial in lung transplantation.

BACKGROUND: Chronic lung allograft dysfunction, which manifests as bronchiolitis obliterans syndrome (BOS), is recognized as the primary cause of morbidity and mortality after lung transplantation. In this study we assessed the efficacy and safety of two de novo immunosuppression protocols to prevent BOS. METHODS: Our study approach was a multicenter, prospective, randomized (1:1) open-label superiority investigation of de novo tacrolimus vs cyclosporine, with both study arms given mycophenolate mofetil and prednisolone after lung transplantation. Cytolytic induction therapy was not employed. Patients were stratified at entry for cystic fibrosis. Primary outcome was incidence of BOS 3 years after transplant (intention-to-treat analysis). Secondary outcomes were survival and incidence of acute rejection, infection and other adverse events. RESULTS: Group demographic data were well matched: 110 of 124 tacrolimus vs 74 of 125 cyclosporine patients were treated per protocol (p < 0.01 by chi-square test). Cumulative incidence of BOS Grade ≥1 at 3 years was 11.6% (tacrolimus) vs 21.3% (cyclosporine) (cumulative incidence curves, p = 0.037 by Gray's test, pooled over strata). Univariate proportional sub-distribution hazards regression confirmed cyclosporine as a risk for BOS (HR 1.97, 95% CI 1.04 to 3.77, p = 0.039). Three-year cumulative incidence of acute rejection was 67.4% (tacrolimus) vs 74.9% (cyclosporine) (p = 0.118 by Gray's test). One- and 3-year survival rates were 84.6% and 78.7% (tacrolimus) vs 88.6% and 82.8% (cyclosporine) (p = 0.382 by log-rank test). Cumulative infection rates were similar (p = 0.91), but there was a trend toward new-onset renal failure with tacrolimus (p = 0.09). CONCLUSIONS: Compared with cyclosporine, de novo tacrolimus use was found to be associated with a significantly reduced risk for BOS Grade ≥1 at 3 years despite a similar rate of acute rejection. However, no survival advantage was detected.

Vasopressin-stimulated CFTR Cl- currents are increased in the renal collecting duct cells of a mouse model of Liddle's syndrome.

Liddle's syndrome is a genetic form of hypertension linked to Na(+) retention caused by activating mutations in the COOH terminus of the beta or gamma subunit of the epithelial sodium channel (ENaC). In this study, we used the short-circuit current (I(sc)) method to investigate the effects of deamino-8-d-arginine vasopressin (dDAVP) on Na(+) and Cl(-) fluxes in primary cultures of cortical collecting ducts (CCDs) microdissected from the kidneys of mice with Liddle's syndrome carrying a stop codon mutation, corresponding to the beta-ENaC R(566) stop mutation (L) found in the original pedigree. Compared to wild-type (+/+) CCD cells, untreated L/+ and L/L CCD cells exhibited 2.7- and 4.2-fold increases, respectively, in amiloride-sensitive (Ams) I(sc), reflecting ENaC-dependent Na(+) absorption. Short-term incubation with dDAVP caused a rapid and significant increase (approximately 2-fold) in Ams I(sc) in +/+, but not in L/+ or L/L CCD cells. In sharp contrast, dDAVP induced a greater increase in 5-nitro-2-(3-phenylpropamino)benzoate (NPPB)-inhibited apical Cl(-) currents in amiloride-treated L/L and L/+ cells than in their +/+ counterparts. I(sc) recordings performed under apical ion substituted conditions revealed that the dDAVP-stimulated apical secretion of Cl(-), which was absent in cultured CCDs lacking CFTR, was 1.8-fold greater in L/+ and 3.7-fold greater in L/L CCD cells than in their +/+ CCD counterparts. After the basal membrane had been permeabilized with nystatin and a basal-to-apical Cl(-) gradient had been imposed, dDAVP also stimulated larger Cl(-) currents across L/L and L/+ CCD layers than +/+ CCD layers. These findings demonstrate that vasopressin stimulates greater apical CFTR Cl(-) conductance in the renal CCD cells of mice with Liddle's syndrome than in wild-type mice. This effect could contribute to the enhanced NaCl reabsorption observed in the distal nephron of patients with Liddle's syndrome.

Neutrophil transepithelial migration: role of toll-like receptors in mucosal inflammation

The symptomatic phases of many inflammatory diseases are characterized by migration of large numbers of neutrophils (PMN) across a polarized epithelium and accumulation within a lumen. For example, acute PMN influx is common in diseases of the gastrointestinal system (ulcerative colitis, Crohn's disease, bacterial enterocolitis, gastritis), hepatobiliary system (cholangitis, acute cholecystitis), respiratory tract (bronchial pneumonia, bronchitis, cystic fibrosis, bronchiectasis), and urinary tract (pyelonephritis, cystitis). Despite these observations, the molecular basis of leukocyte interactions with epithelial cells is incompletely understood. In vitro models of PMN transepithelial migration typically use N-formylated bacterial peptides such as fMLP in isolation to drive human PMNs across epithelial monolayers. However, other microbial products such as lipopolysaccharide (LPS) are major constituents of the intestinal lumen and have potent effects on the immune system. In the absence of LPS, we have shown that transepithelial migration requires sequential adhesive interactions between the PMN beta2 integrin CD11b/CD18 and JAM protein family members. Other epithelial ligands appear to be abundantly represented as fucosylated proteoglycans. Further studies indicate that the rate of PMN migration across mucosal surfaces can be regulated by the ubiquitously expressed transmembrane protein CD47 and microbial-derived factors, although many of the details remain unclear. Current data suggests that Toll-like receptors (TLR), which recognize specific pathogen-associated molecular patterns (PAMPs), are differentially expressed on both leukocytes and mucosal epithelial cells while serving to modulate leukocyte-epithelial interactions. Exposure of epithelial TLRs to microbial ligands has been shown to result in transcriptional upregulation of inflammatory mediators whereas ligation of leukocyte TLRs modulate specific antimicrobial responses. A better understanding of these events will hopefully provide new insights into the mechanisms of epithelial responses to microorganisms and ideas for therapies aimed at inhibiting the deleterious consequences of mucosal inflammation.

A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes.

The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a limiting step in sodium reabsorption across distal airway epithelium and controlling mucociliary clearance. ENaC is activated by serine proteases secreted in the extracellular milieu. In cystic fibrosis lungs, high concentrations of secreted neutrophil elastase (NE) are observed. hNE could activate ENaC and contribute to further decreased mucociliary clearance. The aims of this study were (i) to test the ability of an engineered human neutrophil elastase inhibitor (EPI-hNE4) to specifically inhibit the elastase activation of ENaC-mediated amiloride-sensitive currents (I(Na)) and (ii) to examine the effect of elastase on cell surface expression of ENaC and its cleavage pattern (exogenous proteolysis). Oocytes were exposed to hNE (10-100 microg/ml) and/or trypsin (10 microg/ml) for 2-5 min in the presence or absence of EPI-hNE4 (0.7 microm). hNE activated I(Na) 3.6-fold (p < 0.001) relative to non-treated hENaC-injected oocytes. EPI-hNE4 fully inhibited hNE-activated I(Na) but had no effect on trypsin- or prostasin-activated I(Na). The co-activation of I(Na) by hNE and trypsin was not additive. Biotinylation experiments revealed that cell surface gamma ENaC (but not alpha or beta ENaC) exposed to hNE for 2 min was cleaved (as a 67-kDa fragment) and correlated with increased I(Na). The elastase-induced exogenous proteolysis pattern is distinct from the endogenous proteolysis pattern induced upon preferential assembly, suggesting a causal relationship between gamma ENaC cleavage and ENaC activation, taking place at the plasma membrane.

Correlation of biological serum markers with the degree of hepatic fibrosis and necroinflammatory activity in hepatitis C and schistosomiasis patients

Liver biopsy is the gold-standard method to stage fibrosis; however, it is an invasive procedure and is potentially dangerous. The main objective of this study was to evaluate biological markers, such as cytokines IL-13, IFN-γ, TNF-α and TGF-β, platelets, bilirubins (Bil), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), total proteins, γ-glutamil transferase (γ-GT) and alkaline phosphatase (AP), that could be used to predict the severity of hepatic fibrosis in schistosomiasis and hepatitis C (HC) as isolated diseases or co-infections. The following patient groups were selected: HC (n = 39), HC/hepatosplenic schistosomiasis (HSS) (n = 19), HSS (n = 22) and a control group (n = 13). ANOVA and ROC curves were used for statistical analysis. P < 0.05 was considered significant. With HC patients we showed that TNF-α (p = 0.020) and AP (p = 0.005) could differentiate mild and severe fibrosis. With regard to necroinflammatory activity, AST (p = 0.002), γ-GT (p = 0.034) and AP (p = 0.001) were the best markers to differentiate mild and severe activity. In HC + HSS patients, total Bil (p = 0.008) was capable of differentiating between mild and severe fibrosis. In conclusion, our study was able to suggest biological markers that are non-invasive candidates to evaluate fibrosis and necroinflammatory activity in HC and HC + HSS.

Airway surface liquid volume regulation determines different airway phenotypes in liddle compared with betaENaC-overexpressing mice.

Studies in cystic fibrosis patients and mice overexpressing the epithelial Na(+) channel beta-subunit (betaENaC-Tg) suggest that raised airway Na(+) transport and airway surface liquid (ASL) depletion are central to the pathogenesis of cystic fibrosis lung disease. However, patients or mice with Liddle gain-of-function betaENaC mutations exhibit hypertension but no lung disease. To investigate this apparent paradox, we compared the airway phenotype (nasal versus tracheal) of Liddle with CFTR-null, betaENaC-Tg, and double mutant mice. In mouse nasal epithelium, the region that functionally mimics human airways, high levels of CFTR expression inhibited Liddle epithelial Nat channel (ENaC) hyperfunction. Conversely, in mouse trachea, low levels of CFTR failed to suppress Liddle ENaC hyperfunction. Indeed, Na(+) transport measured in Ussing chambers ("flooded" conditions) was raised in both Liddle and betaENaC-Tg mice. Because enhanced Na(+) transport did not correlate with lung disease in these mutant mice, measurements in tracheal cultures under physiologic "thin film" conditions and in vivo were performed. Regulation of ASL volume and ENaC-mediated Na(+) absorption were intact in Liddle but defective in betaENaC-Tg mice. We conclude that the capacity to regulate Na(+) transport and ASL volume, not absolute Na(+) transport rates in Ussing chambers, is the key physiologic function protecting airways from dehydration-induced lung disease.

Low transformation growth factor-β1 production and collagen synthesis correlate with the lack of hepatic periportal fibrosis development in undernourished mice infected with Schistosoma mansoni

Undernourished mice infected (UI) submitted to low and long-lasting infections by Schistosoma mansoni are unable to develop the hepatic periportal fibrosis that is equivalent to Symmers’ fibrosis in humans. In this report, the effects of the host’s nutritional status on parasite (worm load, egg viability and maturation) and host (growth curves, biology, collagen synthesis and characteristics of the immunological response) were studied and these are considered as interdependent factors influencing the amount and distribution of fibrous tissue in hepatic periovular granulomas and portal spaces. The nutritional status of the host influenced the low body weight and low parasite burden detected in UI mice as well as the number, viability and maturation of released eggs. The reduced oviposition and increased number of degenerated or dead eggs were associated with low protein synthesis detected in deficient hosts, which likely induced the observed decrease in transformation growth factor (TGF)-β1 and liver collagen. Despite the reduced number of mature eggs in UI mice, the activation of TGF-β1 and hepatic stellate cells occurred regardless of the unviability of most miracidia, due to stimulation by fibrogenic proteins and eggshell glycoproteins. However, changes in the repair mechanisms influenced by the nutritional status in deficient animals may account for the decreased liver collagen detected in the present study.

A comparison of the spatial dependence of body mass index among adults and children in a Swiss general population.

BACKGROUND: Body mass index (BMI) may cluster in space among adults and be spatially dependent. Whether BMI clusters among children and how age-specific BMI clusters are related remains unknown. We aimed to identify and compare the spatial dependence of BMI in adults and children in a Swiss general population, taking into account the area's income level. METHODS: Geo-referenced data from the Bus Santé study (adults, n=6663) and Geneva School Health Service (children, n=3601) were used. We implemented global (Moran's I) and local (local indicators of spatial association (LISA)) indices of spatial autocorrelation to investigate the spatial dependence of BMI in adults (35-74 years) and children (6-7 years). Weight and height were measured using standardized procedures. Five spatial autocorrelation classes (LISA clusters) were defined including the high-high BMI class (high BMI participant's BMI value correlated with high BMI-neighbors' mean BMI values). The spatial distributions of clusters were compared between adults and children with and without adjustment for area's income level. RESULTS: In both adults and children, BMI was clearly not distributed at random across the State of Geneva. Both adults' and children's BMIs were associated with the mean BMI of their neighborhood. We found that the clusters of higher BMI in adults and children are located in close, yet different, areas of the state. Significant clusters of high versus low BMIs were clearly identified in both adults and children. Area's income level was associated with children's BMI clusters. CONCLUSIONS: BMI clusters show a specific spatial dependence in adults and children from the general population. Using a fine-scale spatial analytic approach, we identified life course-specific clusters that could guide tailored interventions.