Open-label, multicentre, dose-escalating phase II clinical trial on the safety and efficacy of tadekinig alfa (IL-18BP) in adult-onset Still's disease.

OBJECTIVES: Adult-onset Still's disease (AOSD) is a rare systemic autoinflammatory disease; its management is largely empirical. This is the first clinical study to determine if interleukin (IL)-18 inhibition, using the recombinant human IL-18 binding protein, tadekinig alfa, is a therapeutic option in AOSD. METHODS: In this phase II, open-label study, patients were ≥18 years with active AOSD plus fever or C reactive protein (CRP) levels ≥10 mg/L despite treatment with prednisone and/or conventional synthetic disease-modifying antirheumatic drugs (DMARDs). Previous biological DMARD treatment was permitted. Patients received tadekinig alfa 80 mg or 160 mg subcutaneously three times per week for 12 weeks; those receiving 80 mg not achieving early predicted response criteria (reduction of ≥50% CRP values from baseline and fever resolution) were up-titrated to 160 mg for a further 12 weeks. The primary endpoint was the occurrence of adverse events (AEs) throughout the study. RESULTS: Ten patients were assigned to receive 80 mg tadekinig alfa and 13 patients to the 160 mg dose. One hundred and fifty-five treatment-emerging AEs were recorded, and 47 were considered related to the study drug. Most AEs were mild and resolved after drug discontinuation. Three serious AEs occurred, one possibly related to treatment (toxic optic neuropathy). At week 3, 5 of 10 patients receiving 80 mg and 6 of 12 patients receiving 160 mg achieved the predefined response criteria. CONCLUSIONS: Our results indicate that tadekinig alfa appears to have a favourable safety profile and is associated with early signs of efficacy in patients with AOSD. TRIAL REGISTRATION NUMBER: NCT02398435.

Interleukin-18 diagnostically distinguishes and pathogenically promotes human and murine macrophage activation syndrome.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperferritinemic systemic inflammatory disorders. Although profound cytotoxic impairment causes familial HLH (fHLH), the mechanisms driving non-fHLH and MAS are largely unknown. MAS occurs in patients with suspected rheumatic disease, but the mechanistic basis for its distinction is unclear. Recently, a syndrome of recurrent MAS with infantile enterocolitis caused by NLRC4 inflammasome hyperactivity highlighted the potential importance of interleukin-18 (IL-18). We tested this association in hyperferritinemic and autoinflammatory patients and found a dramatic correlation of MAS risk with chronic (sometimes lifelong) elevation of mature IL-18, particularly with IL-18 unbound by IL-18 binding protein, or free IL-18. In a mouse engineered to carry a disease-causing germ line NLRC4T337S mutation, we observed inflammasome-dependent, chronic IL-18 elevation. Surprisingly, this NLRC4T337S-induced systemic IL-18 elevation derived entirely from intestinal epithelia. NLRC4T337S intestines were histologically normal but showed increased epithelial turnover and upregulation of interferon-γ-induced genes. Assessing cellular and tissue expression, classical inflammasome components such as Il1b, Nlrp3, and Mefv predominated in neutrophils, whereas Nlrc4 and Il18 were distinctly epithelial. Demonstrating the importance of free IL-18, Il18 transgenic mice exhibited free IL-18 elevation and more severe experimental MAS. NLRC4T337S mice, whose free IL-18 levels were normal, did not. Thus, we describe a unique connection between MAS risk and chronic IL-18, identify epithelial inflammasome hyperactivity as a potential source, and demonstrate the pathogenicity of free IL-18. These data suggest an IL-18-driven pathway, complementary to the cytotoxic impairment of fHLH, with potential as a distinguishing biomarker and therapeutic target in MAS.

Elevated serum levels of free interleukin-18 in adult-onset Still's disease.

OBJECTIVE: IL-18 is a pro-inflammatory cytokine of the IL-1 family that is naturally inhibited by IL-18 binding protein (IL-18BP). High levels of IL-18 have been described in the serum of adult-onset Still's disease (AOSD) patients, but only total IL-18 levels (including inactive IL-18 bound to IL-18BP) have been measured. With a specific immunoassay, we aimed to measure free IL-18 serum levels in AOSD patients and other rheumatic diseases. METHODS: An ELISA was developed to measure free IL-18. Its sensitivity and specificity were tested by spiking recombinant IL-18 or IL-18BP in serum and PBS supplemented with 5% BSA. The binding affinity of IL-18 to IL-18BP was calculated by titration experiments using the ELISA and by Biacore analysis. Sera of 37 AOSD patients and 138 controls (40 healthy controls, 30 RA, 29 SLE, 21 AS and 18 PsA) were assayed for free IL-18, IL-18BP, total IL-18 and other cytokines. Correlations were performed between free IL-18 and markers of disease activity in AOSD patients. RESULTS: Free IL-18 serum levels were significantly higher in AOSD patients (median 8.89 pg/ml) than in healthy and disease controls (1.37 pg/ml; P < 0.01). Free IL-18 serum levels correlated with AOSD activity. The affinity of IL-18 to IL-18BP was found to be much higher than previously described, with a dissociation constant ranging from 30 to 50 pM. CONCLUSION: Free IL-18 levels are specifically elevated in AOSD compared with other inflammatory diseases, suggesting that IL-18 represents a potential target for the treatment of AOSD.

Investigation of effect of nutritional drink on chemotherapy-induced mucosal injury and tumor growth in an established animal model.

Chemotherapy-induced mucositis represents a significant burden to quality of life and healthcare costs, and may be improved through enhanced nutritional status. We first determined the safety of two nutritional drinks (plus placebo), and then potential gut protection in tumor-bearing rats in a model of methotrexate-induced mucositis. In study 1, animals were fed one of two test diets (or placebo or control chow pellets) for a total of 60 days and were monitored daily. All diets were found to be safe to administer. In study 2, after seven days of receiving diets, a Dark Agouti Mammary Adenocarcinoma (DAMA) was transplanted subcutaneously. Ten days after starting diets, animals had 2 mg/kg intramuscular methotrexate administered on two consecutive days; after this time, all animals were given soaked chow. Animals were monitored daily for changes in bodyweight, tumor burden and general health. Animals were killed 10, 12 and 16 days after initially starting diets, and tissues were collected at necropsy. In study 1, animals receiving diets had gained 0.8% and 10.8% of their starting bodyweight after 60 days, placebo animals 4.4%, and animals fed on standard chow had gained 15.1%. In study 2, there was no significant influence of test diet on bodyweight, organ weight, tumor burden or biochemical parameters. Only animals treated with MTX exhibited diarrhea, although animals receiving Diet A and Diet C showed a non-significant increase in incidence of diarrhea. Administration of these nutritional drinks did not improve symptoms of mucositis.

Falls and risk factors for falls in community-dwelling adults with dementia (NutriAlz trial).

To estimate the number of fallers and risk factors for falls in a cohort with dementia, we did a secondary analysis of a cluster-randomized controlled trial (NutriAlz) in 11 outpatient and day care centers in Catalonia (Spain) including 626 community-dwelling patients with dementia, followed for 12 months. Participants' characteristics were assessed at baseline, at 6 and 12 months [fall in the earlier 6 mo, anthropometric data, comorbidities, Mini-Mental State Examination, Clinical Dementia Rating, Basic Activities of Daily Living (BADL), Instrumental Activities of Daily Living, Neuropsychiatric Inventory Questionnaire, Zarit Caregiver Burden Interview and Mini-Nutritional Assessment]. Multivariate logistic regression models and generalized linear models were used to explore risk factors for falls and changes in health and function. Two hundred twenty-three participants fell during the 12 months follow-up (35.62%). Risk factors identified for falls were age (odds ratio (OR)=1.03, 95% confidence interval (CI), 1.00-1.05), BADL (OR=1.18, 95% CI, 1.05-1.32), and earlier fall (OR=2.30, 95% CI, 1.57-3.35). Fallers had worse health than nonfallers, and their dependence increased significantly more in BADL during the study, compared with nonfallers. Dependence in BADL is a risk factor and a consequence of falls; interventions aimed at preventing falls in dementia patients could promote autonomy in BADL and slow its decline.

Tolerance, safety, and effect on the faecal microbiota of an enteral formula supplemented with pre- and probiotics in critically ill children.

OBJECTIVES: The aim of this study was to demonstrate the tolerance and safety of an enteral formula containing prebiotics/probiotics, and its effect on the faecal microbiota in critically ill children. SUBJECTS AND METHODS: Ninety-four patients between 1 and 3 years old under mechanical ventilation requiring enteral feeding were randomised to receive either a test formula containing a synbiotic blend (composed of 2 probiotic strains [Lactobacillus paracasei NCC 2461 and Bifidobacterium longum NCC 3001], fructooligosaccharides [FOS], inulin, and Acacia gum), or a control formula. Patients remained in the intensive care unit for 7 days and were examined at day 14. Tolerance was assessed by overall caloric intake and time to reach caloric goal. Safety was assessed by abdominal distention, vomiting, and stool frequency. Microbiota was analysed by culture- and molecular-based methods. RESULTS: Overall caloric intake and time to reach caloric goal were similar between groups (noninferiority was shown). Abdominal distention, vomiting, and stool frequency were not affected by the supplementation with pre- and probiotics. Faecal bifidobacteria were higher in the test group at the end of the study. A similar trend was observed for total lactobacilli. L paracasei NCC 2461 and B longum NCC 3001 were detected in 80.4% and 17% of the test group patients, respectively. Enterobacteria levels remained unchanged during hospitalisation in the control group but diminished in the test group. CONCLUSIONS: The enteral formula supplemented with synbiotics was well tolerated by children in intensive care units; it was safe and produced an increase in faecal bacterial groups of previously reported beneficial effects.

Intestinal Host-Microbe Interactions under Physiological and Pathological Conditions.

The intestinal mucosa is unique in that it can be tolerant to the resident, symbiotic microbiota but remaining, at the same time, responsive to and able to fight pathogens. The close interaction between host-symbiotic microbiota at the mucosal level poses important challenges since microbial breaches through the gut barrier can result in the breakdown of gut homeostasis. In this paper, hosts-integrated components that help to preserve intestinal homeostasis including barrier and immune function are discussed. In addition global alterations of the microbiota that can play a role in the initiation of an exaggerated inflammatory response through an abnormal signaling of the innate and adaptive immune response are briefly described.

Potential role of the intestinal microbiota of the mother in neonatal immune education.

Mucosal dendritic cells are at the heart of decision-making processes that dictate immune reactivity to intestinal microbes. They ensure tolerance to commensal bacteria and a vigorous immune response to pathogens. It has recently been demonstrated that the former involves a limited migration of bacterially loaded dendritic cells from the Peyer's patches to the mesenteric lymph nodes. During lactation, cells from gut-associated lymphoid tissue travel to the breast via the lymphatics and peripheral blood. Here, we show that human peripheral blood mononuclear cells and breast milk cells contain bacteria and their genetic material during lactation. Furthermore, we show an increased bacterial translocation from the mouse gut during pregnancy and lactation and the presence of bacterially loaded dendritic cells in lactating breast tissue. Our observations show bacterial translocation as a unique physiological event, which is increased during pregnancy and lactation. They suggest endogenous transport of intestinally derived bacterial components within dendritic cells destined for the lactating mammary gland. They also suggest neonatal immune imprinting by milk cells containing commensal-associated molecular patterns.

The inflammatory status of the elderly: the intestinal contribution.

A common finding in the elderly population is a chronic subclinical inflammatory status that coexists with immune dysfunction. These interconnected processes are of sufficient magnitude to impact health and survival time. In this review we discuss the different signals that may stimulate the inflammatory process in the aging population as well as the molecular and cellular components that can participate in the initiation, the modulation or termination of the said process. A special interest has been devoted to the intestine as a source of signals that can amplify local and systemic inflammation. Sentinel cells in the splanchnic area are normally exposed to more than one stimulus at a given time. In the intestine of the elderly, endogenous molecules produced by the cellular aging process and stress as well as exogenous evolutionarily conserved molecules from bacteria, are integrated into a network of receptors and molecular signalling pathways that result in chronic inflammatory activation. It is thus possible that nutritional interventions which modify the intestinal ecology can diminish the pro-inflammatory effects of the microbiota and thereby reinforce the mucosal barrier or modulate the cellular activation pathways.

Probiotic yogurt in the elderly with intestinal bacterial overgrowth: endotoxaemia and innate immune functions.

A study was conducted in healthy elderly living independently in senior housing to assess the impact of a probiotic yoghurt supplement on small intestinal bacterial overgrowth. Twenty-three participants with positive and thirteen participants with negative hydrogen breath test were studied before and after a period of 4 weeks of probiotic yoghurt administration. Intestinal permeability, plasma endotoxin levels, phagocytic activity of leucocytes, cytokine production by monocytes and free radical response of neutrophils were determined. Intestinal permeability was similar for the two groups and was unaffected by probiotic treatment. Both plasma endotoxin levels and the basal phagocytic activity of leucocytes decreased after yoghurt intake in the two groups. Exposure of monocytes and neutrophils ex vivo led to an increased cytokine response and free radical response, respectively. The normalisation of the various cytokine responses was more apparent in the group with positive breath test. In addition, the plasma levels of lipoplysaccharide binding protein and soluble CD14, lipoplysaccharide pattern recognition receptors and surrogate markers of lipoplysaccharide permeability were diminished by the end of the study. In conclusion, probiotic administration in the elderly normalises the response to endotoxin, and modulates activation markers in blood phagocytes, and therefore may help reduce low-grade chronic inflammation.

Feeding a diet containing a fructooligosaccharide mix can enhance Salmonella vaccine efficacy in mice.

Fructooligosaccharides (FOS) are considered prebiotics because of their ability to promote growth of specific beneficial gut bacteria, such as bifidobacteria. Some studies reported potential immune-modulating properties. The aim of this study was to investigate the effect of FOS:inulin mix on murine response to Salmonella vaccine and evaluate the relevance toward protection against Salmonella infection. Balb/c mice were fed a diet containing 5% FOS:inulin mix or a control diet 1 wk before oral immunization with a suboptimal dose of live attenuated Salmonella typhimurium vaccine. Four weeks after vaccination, mice were infected with LD100 of virulent S. typhimurium. Specific blood Salmonella immunoglobulin G and fecal immunoglobulin A significantly increased in mice fed the diet containing prebiotics compared with control mice 4 wk postimmunization. Peritoneal macrophage phagocytic activity also significantly increased in FOS:inulin-fed mice at 1 wk postimmunization compared with control mice. No detectable effects were observed on the percentage of lymphoid cell subsets in the spleen. However, production of cytokines, interferon-gamma, interleukin-12, and tumor necrosis factor alpha, was numerically increased in spleen cell cultures stimulated with mitogens from FOS:inulin-fed mice 1 and 4 wk postimmunization. Salmonella translocation to lymphoid organs was not affected by feeding FOS:inulin. However, the improved response to Salmonella vaccine was concomitant with an increase in the survival rate of FOS:inulin-fed mice upon challenge with virulent Salmonella. No detectable effects were observed on the composition or the metabolic activity of the microbiota. Overall, the data suggest that a diet supplemented with FOS:inulin mix stimulates mucosal immunity and seems to improve efficacy of an oral vaccine.

The inflammatory status of old age can be nurtured from the intestinal environment.

PURPOSE OF REVIEW: Recent studies suggest an association between inflammation status and the presence of chronic disease in the elderly. The review examines publications that address the low level of chronic inflammation and emphasizes how an altered host-microbiota interaction at the gut level could contribute to maintaining a low systemic inflammatory status in the elderly. RECENT FINDINGS: The first population cross-sectional studies with relevant numbers of healthy elderlies show age-related global changes in gut microbiota with a consistent increase in nonpathogenic Gram-negative mainly Enterobacteria and country-specific changes in bifidobacteria. Noninvasive methods have permitted us to detect subclinical intestinal inflammation in the elderly population. Furthermore, few studies report on immune and/or inflammatory response; however, prebiotics, probiotics or synbiotics might improve the inflammatory condition of the elderly. SUMMARY: A better understanding of the mechanisms of host-gut microbiota cross-talk would significantly help in the design of novel nutritional strategies targeting immune reactivity at the mucosal level.

Bacterial imprinting of the neonatal immune system: lessons from maternal cells?

OBJECTIVE: We examined the presence of a natural bacterial inoculum in breast milk and its intracellular transport from the maternal intestine to the breast through the circulation. METHODS: Breast milk and peripheral blood were collected aseptically from healthy donors at various times after delivery, and the presence of viable bacteria was determined through plating. Temporal temperature gradient gel electrophoresis was used to examine the bacterial ribosomal DNA content in milk cells, maternal peripheral blood mononuclear cells, and feces and in corresponding infant feces. Blood from nongravid nonlactating women served as control samples. Bacterial translocation to extraintestinal tissues was also evaluated in virgin, pregnant, and lactating mice. RESULTS: Breast milk contained a low total concentration of microbes of <10(3) colony-forming units per mL. Temporal temperature gradient gel electrophoresis revealed that maternal blood and milk cells contained the genetic material of a greater biodiversity of enteric bacteria. Some bacterial signatures were common to infant feces and to samples of maternal origin. Bacterial translocation from the gut to mesenteric lymph nodes and mammary gland occurred during late pregnancy and lactation in mice. CONCLUSIONS: Bacterial translocation is a unique physiologic event, which is increased during pregnancy and lactation in rodents. Human breast milk cells contain a limited number of viable bacteria but a range of bacterial DNA signatures, as also found in maternal peripheral blood mononuclear cells. Those peripheral blood mononuclear cells showed greater biodiversity than did peripheral blood mononuclear cells from control women. Taken together, our results suggest that intestinally derived bacterial components are transported to the lactating breast within mononuclear cells. We speculate that this programs the neonatal immune system to recognize specific bacterial molecular patterns and to respond appropriately to pathogens and commensal organisms.

Patchy distribution of mucosal lesions in ileal Crohn's disease is not linked to differences in the dominant mucosa-associated bacteria: a study using fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis.

BACKGROUND: The mucosa-associated bacteria (MAB) are suspected of being involved in the pathogenesis of Crohn's disease. We analyzed and compared the MAB in noninflamed and inflamed ileal mucosa of Crohn's disease patients (n = 22). METHODS: Tissue samples from the inflamed ileal mucosa and from the adjacent noninflamed ileal mucosa were taken from surgical resection specimens. The MAB were investigated using fluorescence in situ hybridization with 7 group-specific probes and temporal temperature gradient gel electrophoresis (TTGE). RESULTS: Samples from both noninflamed and inflamed mucosa were obtained from 15 patients. The distribution of the bacterial populations was not different between noninflamed and inflamed mucosa. The Bacteroidetes phylum was dominant and accounted for 29% of MAB (0%-74%) in noninflamed tissues and 32% (0%-70%) in inflamed areas. The gamma Proteobacteria represented 12% (0%-70%) of MAB both in noninflamed and inflamed areas. The Clostridium coccoides group (Firmicutes phylum) represented 15% of MAB in noninflamed tissues versus 7% in inflamed areas. For most of the patients the similarity index between TTGE paired profiles was very high. CONCLUSION: The dominant MAB do not differ between noninflamed and inflamed ileal mucosa in Crohn's disease. This argues against a localized dysbiosis to explain the patchy distribution of mucosal lesions.