José M. González-Navajas

Bacterial DNA in patients with cirrhosis and noninfected ascites mimics the soluble immune response established in patients with spontaneous bacterial peritonitis

Bacterial infections and severity of associated inflammatory reaction influence prognosis in patients with advanced cirrhosis. We compared the innate immune response to bacterial DNA (bactDNA) translocation with that caused by viable bacteria translocation in patients with spontaneous bacterial peritonitis and the relationship between the cytokine response and serum levels of bactDNA. The bactDNA translocation was investigated in 226 patients with cirrhosis and noninfected ascites, 22 patients with spontaneous bacterial peritonitis, and 10 patients with ascites receiving continuous norfloxacin. Serum and ascitic fluid tumor necrosis factor α, interferon‐γ, interleukin‐12, and nitric oxide metabolites were measured via enzyme‐linked immunosorbent assay. Bacterial genomic identifications were made via amplification and sequencing of the 16S ribosomal RNA gene and digital quantization with DNA Lab‐on‐chips. The bactDNA was present in 77 noninfected patients (34%) and in all cases of spontaneous bacterial peritonitis, even in those with culture‐negative ascitic fluid. No patient receiving norfloxacin showed bactDNA translocation. Levels of all cytokines were similar in patients with bactDNA translocation or spontaneous bacterial peritonitis and significantly higher than in patients without bactDNA or in those receiving norfloxacin. Serum bactDNA concentration paralleled levels of all cytokines and nitric oxide in a series of patients with bactDNA translocation or spontaneous bacterial peritonitis followed during 72 hours. Antibiotic treatment in the series of patients with spontaneous bacterial peritonitis did not abrogate bactDNA translocation in the short term. Conclusion: bactDNA translocation‐associated cytokine response is indistinguishable from that in patients with spontaneous bacterial peritonitis and is dependent on bactDNA concentration. Norfloxacin abrogates bactDNA translocation and cytokine response. (HEPATOLOGY 2008;47:978–985.)

Serum and ascitic fluid bacterial DNA: A new independent prognostic factor in noninfected patients with cirrhosis

We tested the hypothesis that the presence of bacterial DNA (bactDNA) in ascitic fluid and serum is associated with decreased survival in patients with cirrhosis. In a prospective, multicenter study, we analyzed the clinical evolution of 156 patients with cirrhosis and ascites (first or recurrence) with lower than 250 polymorphonuclear cells (PMN)/μL, negative ascites bacteriological culture, and absence of other bacterial infections being admitted for evaluation of large‐volume paracentesis, according to the presence of bactDNA at admission. Survival, causes of death, and successive hospital admissions were determined during a 12‐month follow‐up period. BactDNA was detected in 48 patients. The most prevalent identified bactDNA corresponded to Escherichia coli (n = 32/48 patients, 66.6%). Patients were followed for 12 months after inclusion and in this period 34 patients died: 16 of 108 (15%) bactDNA negative versus 18 of 48 (38%) bactDNA positive (P = 0.003). The most frequent cause of death was acute‐on‐chronic liver failure in both groups (7/16 and 9/18 in patients without or with bactDNA, respectively), although more prevalent in the first month of follow‐up in patients with presence of bactDNA (0 versus 4/7). When considering patients with model for end‐stage liver disease (MELD) score less than 15, mortality was significantly higher in those with presence of bactDNA. Spontaneous bacterial peritonitis developed similarly in patients with or without bactDNA at admission. Conclusion: The presence of bactDNA in a patient with cirrhosis during an ascitic episode is an indicator of poor prognosis. This fact may be related to the development of acute‐on‐chronic liver failure at short term and does not predict the development of spontaneous bacterial peritonitis. (HEPATOLOGY 2008;48:1924‐1931.)

The detection of bacterial DNA in blood of rats with CCl4-induced cirrhosis with ascites represents episodes of bacterial translocation†

Bacterial DNA (bactDNA) is present in blood and ascitic fluid (AF) in a third of patients with cirrhosis and ascites, but whether this phenomenon represents episodes of bacterial translocation (BT), strictly considered when culture of mesenteric lymph nodes (MLNs) are positive, remains unknown. This study assessed the relationship between bactDNA detection in biological fluids and MLNs and went on to investigate the local and systemic inflammatory status according to its presence. Cirrhosis was induced in rats by ingestion of CCL4. A subgroup of five animals with cirrhosis received norfloxacin (5 mg/kg/day) for 7 days. MLNs and ascitic and pleural fluids were collected at laparotomy and cultured; samples were collected for identification of bactDNA and measurement of tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), and nitric oxide (NO). BactDNA was detected in MLNs in 12 of 19 animals (63.1%), corresponding in seven cases to culture‐positive MLNs, and in five to culture‐negative MLNs. BactDNA was detected in biological fluids in 11 of 19 animals (57.9%), and in all cases the same bacteria spp. detected in samples was present in MLNs. BactDNA was not detected in any biological sample from animals receiving norfloxacin. Tumor necrosis factor alpha (TNF‐α), IL‐6, and NO were similar in culture‐positive and culture‐negative/bactDNA‐positive samples, and significantly higher than those observed in animals with culture‐negative/bactDNA‐negative MLNs, animals with cirrhosis that were receiving norfloxacin, and controls. In conclusion, the presence of bactDNA in biological fluids in rats with cirrhosis constitutes a marker of BT, and it is associated with a marked inflammatory response, independent of the result of the culture. (HEPATOLOGY 2006.)

Genetic susceptibility to increased bacterial translocation influences the response to biological therapy in patients with Crohn's disease

Objective The aetiology of Crohns disease (CD) has been related to nucleotide-binding oligomerisation domain containing 2 (NOD2) and ATG16L1 gene variants. The observation of bacterial DNA translocation in patients with CD led us to hypothesise that this process may be facilitated in patients with NOD2/ATG16L1-variant genotypes, affecting the efficacy of anti-tumour necrosis factor (TNF) therapies. Design 179 patients with Crohns disease were included. CD-related NOD2 and ATG16L1 variants were genotyped. Phagocytic and bactericidal activities were evaluated in blood neutrophils. Bacterial DNA, TNFα, IFNγ, IL-12p40, free serum infliximab/adalimumab levels and antidrug antibodies were measured. Results Bacterial DNA was found in 44% of patients with active disease versus 23% of patients with remitting disease (p=0.01). A NOD2-variant or ATG16L1-variant genotype was associated with bacterial DNA presence (OR 4.8; 95% CI 1.1 to 13.2; p=0.001; and OR 2.4; 95% CI 1.4 to 4.7; p=0.01, respectively). This OR was 12.6 (95% CI 4.2 to 37.8; p=0.001) for patients with a double-variant genotype. Bacterial DNA was associated with disease activity (OR 2.6; 95% CI 1.3 to 5.4; p=0.005). Single and double-gene variants were not associated with disease activity (p=0.19). Patients with a NOD2-variant genotype showed decreased phagocytic and bactericidal activities in blood neutrophils, increased TNFα levels in response to bacterial DNA and decreased trough levels of free anti-TNFα. The proportion of patients on an intensified biological therapy was significantly higher in the NOD2-variant groups. Conclusions Our results characterise a subgroup of patients with CD who may require a more aggressive therapy to reduce the extent of inflammation and the risk of relapse.

Bacterial DNA Induces the Complement System Activation in Serum and Ascitic Fluid from Patients with Advanced Cirrhosis

Translocation of intestinal bacteria to ascitic fluid is, probably, the first step in the development of spontaneous bacterial peritonitis in patients with cirrhosis. Proteins of the complement system are soluble mediators implicated in the host immune response to bacterial infections and its activation has been traditionally considered to be an endotoxin-induced phenomenon. The aim of this study was to compare the modulation of these proteins in response to the presence of bacterial DNA and/or endotoxin in patients with advanced cirrhosis and ascites in different clinical conditions. Groups I and II consisted of patients without/with bacterial DNA. Group III included patients with spontaneous bacterial peritonitis and Group IV with patients receiving norfloxacin as secondary long-term prophylaxis of spontaneous bacterial peritonitis. Serum and ascitic fluid levels of endotoxin and truncated residues of the complement system were measured by ELISA. The complement system is triggered in response to bacterial DNA, as evidenced by significantly increased levels of C3b, membrane attack complex, and C5a in patients from Groups II and III compared with patients without bacterial DNA (Group I) and those receiving norfloxacin (Group IV). Gram classification did not further differentiate the immune response between patients within groups II and III, even though endotoxin levels were, as expected, significantly higher in patients with bacterial DNA from gram-negative microorganisms. The complement protein activation observed in patients with bacterial DNA in blood and ascitic fluid is indistinguishable from that observed in patients with spontaneous bacterial peritonitis and may occur in an endotoxin-independent manner.

Translocation of bacterial DNA from Gram-positive microorganisms is associated with a species-specific inflammatory response in serum and ascitic fluid of patients with cirrhosis

Translocation of bacterial‐DNA in patients with cirrhosis and ascites triggers an innate immune response. Identification of characteristics to which this response is sensitive is relevant from a clinical standpoint. The aim of this study has been to determine if the proinflammatory immune response established in vivo in cirrhotic patients with ascites as a consequence of bacterial‐DNA translocation is related to the identified bacterial species and their frequency of cytosine‐guanosine content in serum and ascitic fluid. Patients with advanced cirrhosis and ascites were included in the study and distributed into groups I and II according to the absence or presence of bacterial‐DNA translocation, respectively. Serum and ascitic fluid levels of proinflammatory cytokines after normalization of bacterial‐DNA concentration and the activated form of nuclear factor‐kappa B in ascitic fluid pellets were measured by enzyme‐linked immunosorbent assay techniques. Translocation of bacterial‐DNA with higher cytosine‐guanosine content induced the highest cytokine response, which was higher than that in patients without bacterial‐DNA translocation. The activated form of nuclear factor‐kappa B in ascitic fluid pellets of patients with bacterial‐DNA translocation was greater in patients with higher bacterial‐DNA cytosine‐guanosine content, whereas the amount of total nuclear factor‐kappa B remained unaltered. Bacterial‐DNA translocation induces a marked immune reaction in vivo in patients with advanced cirrhosis and ascites which is related, among other factors, to the bacterial‐DNA cytosine‐guanosine content. Therefore, the hosts immune response to bacterial‐DNA translocation constitutes a species‐specific phenomenon.

Protective effect of Bifidobacterium pseudocatenulatum CECT7765 against induced bacterial antigen translocation in experimental cirrhosis.

Intervention in the gut ecosystem is considered as a potential strategy to treat liver diseases and their complications. We have evaluated the effects of Bifidobacterium pseudocatenulatum CECT7765 on bacterial translocation and the liver status in experimental cirrhosis.

Absent in melanoma 2 triggers a heightened inflammasome response in ascitic fluid macrophages of patients with cirrhosis.

BACKGROUND & AIMS Inflammation is a common event in the pathogenesis of liver cirrhosis. The inflammasome pathway has acquired significant relevance in the pathogenesis of inflammation, but its role in the inflammatory response in patients with decompensated cirrhosis remains unexplored. METHODS We performed a prospective study in which 44 patients with decompensated cirrhosis and 12 healthy volunteers were included. We isolated macrophages from blood and ascitic fluid and assessed the expression and activation of the inflammasome, its response to priming by bacterial products, and its association with the degree of liver disease. RESULTS Macrophages from sterile ascitic fluids showed constitutive activation of caspase-1 and a marked increase in the expression of IL-1β, IL-18, and absent in melanoma 2 (AIM2) when compared to blood macrophages. Pre-stimulation of blood-derived macrophages from cirrhotic patients with bacterial DNA increased the expression of AIM2 and induced a higher AIM2-mediated inflammasome response than priming with other bacterial products such as lipopolysaccharide. By contrast, activation of the AIM2 inflammasome did not require a priming signal in ascitic fluid-derived macrophages, demonstrating the preactivated state of the inflammasome in these cells. Last, higher IL-1β and IL-18 production by ascitic fluid macrophages correlated with a more advanced Child-Pugh score. CONCLUSIONS The inflammasome is highly activated in the ascitic fluid of cirrhotic patients, which may explain the exacerbated inflammatory response observed in these patients under non-infected conditions. Clinically, activation of the inflammasome is associated with a higher degree of liver disease.

Role of interleukin 10 in norfloxacin prevention of luminal free endotoxin translocation in mice with cirrhosis

BACKGROUND & AIMS Bacterial endotoxin is present in patients with advanced cirrhosis and can induce an immunogenic response without an overt infection. Norfloxacin is a gram-negative bactericidal drug able to maintain low endotoxin levels and stimulate IL-10 production. We aimed at investigating the role of IL-10 in decreasing endotoxin absorption in cirrhotic mice treated with norfloxacin. METHODS Cirrhosis was induced by carbon tetrachloride or bile duct ligation in wild type and IL10-deficient mice with or without norfloxacin prior to an intragastrical administration of E. coli, K. pneumonia or E. faecalis. Spontaneous and induced bacterial translocation, free endotoxin and cytokine levels were evaluated in mesenteric lymph nodes. Intestinal permeability was followed by fluorimetry and barrier integrity markers were measured in disrupted intestinal samples. The inflammatory-modulating mechanism was characterized in purified intestinal mononuclear cells. RESULTS Norfloxacin reduced spontaneous and induced MLN positive-cultures in wild type and IL-10-deficient animals. However, reduction of free endotoxin levels was associated with norfloxacin in wild type but not in IL-10-deficient mice. Wild type but not IL-10-deficient mice treated with norfloxacin significantly normalized intestinal permeability and improved gut barrier integrity markers. The toll-like receptor 4-mediated pro-inflammatory milieu was modulated by norfloxacin in a concentration-dependent manner in cultured intestinal mononuclear cells of wild type mice but not of IL-10-deficient mice. The restoration of IL-10 levels in IL-10-deficient animals reactivated the norfloxacin effect on inflammatory-modulation, gut barrier permeability, and luminal endotoxin absorption. CONCLUSION Norfloxacin not only reduces gram-negative intestinal flora but also participates in an IL-10-driven modulation of gut barrier permeability, thus reducing luminal free endotoxin absorption in experimental cirrhosis.

Gut Bacterial DNA Translocation is an Independent Risk Factor of Flare at Short Term in Patients With Crohn's Disease.

OBJECTIVES:We aimed at evaluating bacterial DNA (bactDNA) presence in blood of Crohn’s disease (CD) patients in remission as an independent risk factor of flare at 6 months.METHODS:This is a prospective, multicenter study on CD patients with Crohn’s disease activity index (CDAI)<150. The primary end point was time-to-relapse as evaluated by CDAI>150 in the following 6 months. BactDNA in blood, the nucleotide-binding oligomerization domain containing 2 (NOD2) genotype, and serum cytokine levels were determined at baseline.RESULTS:A total of 288 patients were included. BactDNA was detected in 98 patients (34.0%). A variant-NOD2 genotype was identified in 114 patients (39.6%). Forty patients (14%) relapsed during follow-up. Multivariate survival analysis identified bactDNA as an independent risk factor of flare (hazard ratio (HR) 8.75 (4.02–19.06) 95% confidence interval (CI)). Hospitalization, surgery, switch of treatment, initiation and escalation of anti-tumor necrosis factor (TNF) therapy, steroids initiation, and increased fecal calprotectin levels at 6 months were associated with bactDNA at baseline. A logistic regression analysis showed bactDNA as an independent and significant predictive factor of hospitalization (odds ratio (OR) 11.9 (3.4–42.3); P<0.001), steroids startup (OR 8.5 (2.7–27.1); P<0.001), and switch of treatment (OR 3.5 (1.6–7.7); P=0.002) at 6 months. No relationship was observed between bactDNA and mucosal lesions in patients with colonoscopy at admission. Serum pro-inflammatory cytokines were significantly increased in patients with bactDNA or a variant-NOD2 genotype. The combination of both factors induced decreased anti-TNF-α levels and a higher percentage of patients on intensified anti-TNF therapy.CONCLUSIONS:BactDNA is an independent risk factor of relapse at 6 months in CD patients. BactDNA is also independently associated with an increased risk of hospitalization, switch of treatment, and steroids initiation.