Identification and characterization of a hepatic IL-13-producing ILC3-like population potentially involved in liver fibrosis.

BACKGROUND AND AIMS: Human innate lymphoid cells (ILCs) are critically involved in the modulation of homeostatic and inflammatory processes in various tissues. However, only little is known about the composition of the intrahepatic ILC pool and its potential role in chronic liver disease. Here, we performed a detailed characterization of intrahepatic ILCs in both healthy and fibrotic livers. APPROACH AND RESULTS: A total of 50 livers (nonfibrotic = 22, and fibrotic = 29) were analyzed and compared with colon and tonsil tissue (each N = 14) and peripheral blood (N = 32). Human intrahepatic ILCs were characterized ex vivo and on stimulation using flow cytometry and single-cell RNA sequencing. ILC differentiation and plasticity were analyzed by both bulk and clonal expansion experiments. Finally, the effects of ILC-derived cytokines on primary human HSteCs were studied. Unexpectedly, we found that an "unconventional" ILC3-like cell represented the major IL-13-producing liver ILC subset. IL-13 + ILC3-like cells were specifically enriched in the human liver, and increased frequencies of this cell type were found in fibrotic livers. ILC3-derived IL-13 production induced upregulation of proinflammatory genes in HSteCs, indicating a potential role in the regulation of hepatic fibrogenesis. Finally, we identified KLRG1-expressing ILC precursors as the potential progenitor of hepatic IL-13 + ILC3-like cells. CONCLUSIONS: We identified a formerly undescribed subset of IL-13-producing ILC3-like cells that is enriched in the human liver and may be involved in the modulation of chronic liver disease.

Single-cell RNA sequencing identifies a population of human liver-type ILC1s.

Group 1 innate lymphoid cells (ILCs) comprise a heterogeneous family of cytotoxic natural killer (NK) cells and ILC1s. We identify a population of "liver-type" ILC1s with transcriptional, phenotypic, and functional features distinct from those of conventional and liver-resident NK cells as well as from other previously described human ILC1 subsets. LT-ILC1s are CD49a+CD94+CD200R1+, express the transcription factor T-BET, and do not express the activating receptor NKp80 or the transcription factor EOMES. Similar to NK cells, liver-type ILC1s produce IFN-γ, TNF-α, and GM-CSF; however, liver-type ILC1s also produce IL-2 and lack perforin and granzyme-B. Liver-type ILC1s are expanded in cirrhotic liver tissues, and they can be produced from blood-derived ILC precursors in vitro in the presence of TGF-ß1 and liver sinusoidal endothelial cells. Cells with similar signature and function can also be found in tonsil and intestinal tissues. Collectively, our study identifies and classifies a population of human cross-tissue ILC1s.

Fecal microbiota transfer for refractory intestinal graft-versus-host disease - Experience from two German tertiary centers.

RATIONALE: Steroid refractory graft-vs-host disease (sr-GvHD) represents a challenging complication after allogeneic hematopoietic cell transplantation (allo-HCT). Intestinal microbiota (IM) diversity and dysbiosis were identified as influencing factors for the development of acute GvHD. Fecal microbiota transfer (FMT) is hypothesized to restore IM dysbiosis, but there is limited knowledge about the significance of FMT in the treatment of sr-GvHD. OBJECTIVES: We studied the effects of FMT on sr-GvHD in allo-HCT patients from two German tertiary clinical centers (n = 11 patients; period: March 2017 until July 2019). To assess safety and clinical efficacy, we analyzed clinical data pre- and post-FMT (day -14 to +30 relative to FMT). Moreover, IM were analyzed in donor samples and in a subset of patients pre- and post-FMT by 16S rRNA sequencing. RESULTS: Post-FMT, we observed no intervention-associated, systemic inflammatory responses and only minor side effects (5/11 patients: abdominal pain and transformation of peristalsis-each 3/11 and vomiting-1/11). Stool frequencies and volumes were significantly reduced [pre- vs post-FMT (d14): P < .05, respectively] as well as clear attenuation regarding both grading and staging of sr-GvHD was present upon FMT. Moreover, IM analyses revealed an increase of alpha diversity as well as a compositional shifts toward the donor post-FMT. CONCLUSIONS: In our study, we observed positive effects on sr-GVHD after FMT without the occurrence of major adverse events. Although these findings are in line with published data on beneficial effects of FMT in sr-GvHD, further randomized clinical studies are urgently needed to better define the clinical validity including mode of action.

A genetic variant in toll-like receptor 5 is linked to chemokine levels and hepatocellular carcinoma in steatohepatitis.

BACKGROUND & AIMS: Bacterial translocation drives liver disease progression. We investigated whether functional genetic variants in toll-like receptor 5 (TLR5), the receptor for bacterial flagellin, affect the risk for hepatocellular carcinoma (HCC). METHODS: Healthy controls (n = 212), patients with alcohol abuse without liver disease (n = 382), and patients from a discovery cohort of alcohol-associated cirrhosis (n = 372 including 79 HCC cases), a validation cohort of alcohol-associated cirrhosis (n = 355 including 132 HCC cases), and a cohort of cirrhosis due to nonalcoholic steatohepatitis (NASH) (n = 145 including 62 HCC cases) were genotyped for the TLR5 rs5744174 and rs5744168 polymorphisms. Chemokine levels were measured by ELISA in patients' sera and supernatants of flagellin-stimulated healthy monocytes. RESULTS: Frequency of the TLR5 rs5744174 TT genotype was similar in healthy controls (33%), controls with alcohol abuse (34%), and patients with alcohol-associated cirrhosis in the discovery (28%), validation (33%), and NASH cohort (31%). The TT genotype was enriched in patients with versus without HCC in the discovery, validation, and NASH cohort (41% vs 25%; 39% vs 29%; 40% vs 24%; p < .05 each). This genotype remained a risk factor for HCC (OR = 1.9; p = .01) after multivariate correction for age, gender, diabetes, and carriage of the PNPLA3 148M variant. Interleukin-8 induction in monocytes from healthy controls and serum levels of interleukin-8 and CXCL1 from cirrhotic patients with the TT genotype were significantly increased versus C allele carriers. CONCLUSION: The TLR5 rs5744174 polymorphism, affecting immune response to flagellin, is linked to occurrence of HCC in cirrhosis caused by steatohepatitis.

Clinical effectiveness of bidirectional fecal microbiota transfer in the treatment of recurrent Clostridioides difficile infections.

BACKGROUND: Fecal microbiota transfer (FMT) has become a standard of care in the prevention of multiple recurrent Clostridioides difficile (rCDI) infection. AIM: While primary cure rates range from 70-80% following a single treatment using monodirectional approaches, cure rates of combination treatment remain largely unknown. METHODS: In a retrospective case-control study, outcomes following simultaneous bidirectional FMT (bFMT) with combined endoscopic application into the upper and lower gastrointestinal tract, compared to standard routes of application (endoscopy via upper or lower gastrointestinal tract and oral capsules; abbreviated UGIT, LGIT and CAP) on day 30 and 90 after FMT were assessed. Statistical matching partners were identified using number of recurrences (<3; ≥3), age and gender. RESULTS: Primary cure rates at D30 and D90 for bFMT were 100% (p=.001). The matched control groups showed cure rates of 81.3% for LGIT (p=.010), 62.5% for UGIT (p=.000) and 78.1% for CAP (p=.005) on D30 and 81.3% for LGIT (p=.010), 59.4% for UGIT (p=.000) and 71.9% for CAP (p=.001) on D90. CONCLUSION: In our analysis, bFMT on the same day significantly increased primary cure rate at D30 and D90. These data require prospective confirmation but suggest that route of application may play a significant role in optimizing patient outcomes. ClinicalTrials.gov no: NCT02681068.

Renal replacement therapy as bridging-to-recovery in refractory hepatorenal syndrome.

A 50-year-old female patient with cirrhosis due to alcoholic steatohepatitis was referred to our department because of recurrent hepatorenal syndrome (HRS) and hepatic hydrothorax. Clinically, severe anasarca was the leading problem. In contrast to previous episodes, HRS did not respond to standard treatment including terlipressin.Given the severe, refractory hyperhydration, we finally initiated renal replacement therapy (RRT). Subsequently, RRT was performed without severe side effects for more than 100 days. In the meantime, liver function remarkably improved, most probably due to the prolonged abstinence from alcohol. Finally, RRT could be stopped. Since then, our patient has remained in good clinical condition for more than 6 months, with well-compensated Child-Pugh stage A cirrhosis and only mild chronic kidney disease stage III.In conclusion, this case highlights that RRT may be considered in individual cases as bridging therapy in refractory HRS until the liver regenerates due to the absence of damaging mechanisms.

The impact of technical and clinical factors on fecal microbiota transfer outcomes for the treatment of recurrent Clostridioides difficile infections in Germany.

Introduction: Fecal microbiota transfer (FMT) is highly effective in the treatment and prevention of recurrent Clostridioides difficile infection (rCDI) with cure rates of about 80% after a single treatment. Nevertheless, the reasons for failure in the remaining 20% remain largely elusive. The aim of the present study was to investigate different potential clinical predictors of response to FMT in Germany. Methods: Information was extracted from the MicroTrans Registry (NCT02681068), a retrospective observational multicenter study, collecting data from patients undergoing FMT for recurrent or refractory CDI in Germany. We performed binary logistic regression with the following covariates: age, gender, ribotype 027, Eastern Co-operative Oncology Group score, immunosuppression, preparation for FMT by use of proton pump inhibitor, antimotility agents and bowel lavage, previous recurrences, severity of CDI, antibiotic induction treatment, fresh or frozen FMT preparation, and route of application. Results: Treatment response was achieved in 191/240 evaluable cases (79.6%) at day 30 (D30) post FMT and 78.1% at day 90 (D90) post FMT. Assessment of clinical predictors for FMT failure by forward and confirmatory backward-stepwise regression analysis yielded higher age as an independent predictor of FMT failure (p = 0.001; OR 1.060; 95%CI 1.025-1.097). Conclusion: FMT in Germany is associated with high cure rates at D30 and D90. No specific pre-treatment, preparation or application strategy had an impact on FMT success. Only higher age was identified as an independent risk factor for treatment failure. Based on these and external findings, future studies should focus on the assessment of microbiota and microbiota-associated metabolites as factors determining FMT success.

Relative Ascites Polymorphonuclear Cell Count Indicates Bacterascites and Risk of Spontaneous Bacterial Peritonitis.

BACKGROUND AND AIMS: Absolute polymorphonuclear (PMN) counts in ascites define spontaneous bacterial peritonitis (SBP), a severe form of bacterial infection in liver cirrhosis. Bacterascites, another form of ascites infection, can progress to SBP or may resolve spontaneously but is not reflected by absolute PMN counts. We investigated whether the relative ascites PMN count (the absolute PMN count divided by the absolute leukocyte count) provides additional information to detect bacterascites or predict SBP. METHODS: Hospitalized patients with liver cirrhosis requiring paracentesis were stratified with respect to a diagnosis of bacterascites and SBP with a prospective follow-up for 1 year. Diagnostic power of relative PMN counts in ascites was evaluated by receiver operating characteristics curves. RESULTS: At inclusion, we observed 28/269 (10%) and 43/269 (16%) episodes of BA and SBP, respectively. Unlike absolute PMN counts, relative PMN counts in ascites were significantly elevated in bacterascites (p = 0.001). During follow-up, 16 and 30 further episodes of BA and SBP were detected, respectively. Relative PMN counts increased significantly once patients developed BA (p = 0.001). At a threshold of 0.20 for the relative PMN count, sensitivity, specificity, positive and negative predictive values for bacterascites which required antibiotic treatment were 83, 75, 26 and 98%, respectively (p < 0.001). Furthermore, a relative PMN count in ascites ≥0.13 and MELD score >17 was independent factors associated with occurrence of SBP during follow-up. CONCLUSION: The relative PMN count is a cheap immunological marker linked to bacterascites and future SBP, which may help to stratify patients according to their risk of infection.

Compartment-specific distribution of human intestinal innate lymphoid cells is altered in HIV patients under effective therapy.

Innate lymphocyte cells (ILCs), a novel family of innate immune cells are considered to function as key orchestrators of immune defences at mucosal surfaces and to be crucial for maintaining an intact intestinal barrier. Accordingly, first data suggest depletion of ILCs to be involved in human immunodeficiency virus (HIV)-associated damage of the intestinal mucosa and subsequent microbial translocation. However, although ILCs are preferentially localized at mucosal surfaces, only little is known regarding distribution and function of ILCs in the human gastrointestinal tract. Here, we show that in HIV(-) individuals composition and functional capacity of intestinal ILCs is compartment-specific with group 1 ILCs representing the major fraction in the upper gastrointestinal (GI) tract, whereas ILC3 are the predominant population in ileum and colon, respectively. In addition, we present first data indicating that local cytokine concentrations, especially that of IL-7, might modulate composition of gut ILCs. Distribution of intestinal ILCs was significantly altered in HIV patients, who displayed decreased frequency of total ILCs in ileum and colon owing to reduced numbers of both CD127(+)ILC1 and ILC3. Of note, frequency of colonic ILC3 was inversely correlated with serum levels of I-FABP and sCD14, surrogate markers for loss of gut barrier integrity and microbial translocation, respectively. Both expression of the IL-7 receptor CD127 on ILCs as well as mucosal IL-7 mRNA levels were decreased in HIV(+) patients, especially in those parts of the GI tract with reduced ILC frequencies, suggesting that impaired IL-7 responses of ILCs might contribute to incomplete reconstitution of ILCs under effective anti-retroviral therapy. This is the first report comparing distribution and function of ILCs along the intestinal mucosa of the entire human gastrointestinal tract in HIV(+) and HIV(-) individuals.

Alterations of the NK cell pool in HIV/HCV co-infection.

BACKGROUND: A relevant proportion of human immunodeficiency virus (HIV) infected patients is co-infected with the hepatitis C virus (HCV). HCV co-infection in HIV-positive patients is associated with faster progression of liver disease in comparison to HCV mono-infection. Natural killer (NK) cells critically modulate the natural course of HCV infection. Both HIV and HCV mono-infection are associated with alterations of the NK cell pool. However, little data is available concerning phenotype and function of NK cells in HIV/HCV co-infection. METHODS: A total of 34 HIV/HCV co-infected, 35 HIV and 39 HCV mono-infected patients and 43 healthy control persons were enrolled into this study. All HIV-positive patients were under effective antiretroviral therapy. NK cell phenotype, IFN-γ production and degranulation were studied by flow cytometry. RESULTS: NK cell frequency in HIV/HCV co-infection was significantly lower than in healthy individuals but did not differ from HIV and HCV mono-infection. HIV/HCV co-infection was associated with significantly decreased expression of the maturation/differentiation markers CD27/62L/127 on NK cells but increased expression of CD57 compared to healthy controls. Of note, expression also differed significantly from HCV mono-infection but was similar to HIV mono-infection, suggesting a pronounced impact of HIV on these alterations. Similar findings were made with regard to the NK cell receptors NKG2A/C and NKp30. More importantly, NK cells in co-infection displayed a highly impaired functional activity with significantly lower IFN-γ production and degranulation than in healthy donors as well as HIV and HCV mono-infection, suggesting a synergistic effect of both viruses. CONCLUSIONS: Our data indicate that HIV/HCV co-infection is associated with significant alterations of the NK cell pool, which might be involved in the rapid progression of liver disease in co-infected patients and which mainly reflect alterations observed in HIV mono-infection.

Antibiotic resistance in healthcare-related and nosocomial spontaneous bacterial peritonitis.

BACKGROUND: Spontaneous bacterial peritonitis (SBP) can be life threatening in patients with liver cirrhosis. In contrast to community-acquired SBP, no standard treatment has been established for healthcare-related and nosocomial SBP. MATERIALS AND METHODS: We prospectively collected healthcare-related and nosocomial SBP cases from March 2012 till February 2016 at the Department of Internal Medicine I of the University of Bonn and analysed the prevalence of antibiotic resistance among the isolated bacteria. SBP was diagnosed according to international guidelines. Ciprofloxacin, ceftriaxone and meropenem were used as reference substance for resistance to quinolones, third-generation cephalosporins and carbapenems, respectively. RESULTS: Ninety-two SBP episodes in 86 patients were identified: 63 episodes (69%) were nosocomial. Escherichia coli, Klebsiella species, enterococci and streptococci were most frequently isolated. Frequencies of these microorganisms were comparable for healthcare-related and nosocomial SBP (14% vs. 11%, 14% vs. 8%, 14% vs. 5% and 10% vs. 6%, respectively). In general, antibiotic resistance was higher in isolates from nosocomial than from healthcare-related SBP (50% vs. 18% for quinolones, 30% vs. 11% for piperacillin-tazobactam; P > 0·05), but comparable concerning third-generation cephalosporins (30% vs. 33%). All microorganisms were sensitive to carbapenems apart from nosocomial infections with Enterococcus faecium (n = 3) and Candida albicans (n = 1) due to intrinsic resistance or lack of microbiological efficacy, respectively. No multidrug-resistant microorganisms were detected. Resistance to initial antibiotic treatment affected 30-day survival negatively (18% vs. 68%; P = 0·002). CONCLUSION: Resistance to initial antibiotic treatment was associated with increased mortality. With resistance to cephalosporins being frequent, piperacillin-tazobactam or carbapenems might be preferred as treatment of SBP.

Fecal Microbiota Transplant in Patients With Recurrent Clostridium Difficile Infection.

BACKGROUND: The clinical effectiveness of fecal microbiota transplant (FMT) for the treatment of recurrent Clostridium difficile infections (rCDI) has been demonstrated in randomized controlled trials. To assess the current status of FMT in Germany with respect to active centers, local standards, clinical effectiveness and safety, the MicroTrans Registry (NCT02681068) was established. METHODS: In a long-term retrospective multicenter observational study by the German Clinical Microbiome Study Group (GCMSG), primary and secondary cure on day 30 and 90, as well as occurrence of treatment-related adverse events were assessed. In addition to patient demographic data, we provide an overview of the FMT procedures and techniques used at different centers. RESULTS: Overall, 133 eligible patients from 33 centers were included, of which 64.7% were female (n = 86). The mean age was 75 years (interquartile range: 59.5-81.5). Administration via the duodenal route (n = 59; 44.4%) was the most frequently applied option, followed by colonic (n = 55; 41.1%), capsule (n = 13; 9.8%), and gastric administration (n = 4; 3.0%). Primary cure on day 30 and 90 was achieved in 84.2% (n = 101/120) and 78.3% (n = 72/92) of patients, respectively. Including re-treatment, secondary response was achieved in 87.5% (d 30; n = 105/120) and 85.9% (d 90; n = 79/92), respectively. Treatment- elated adverse events were documented in 16 patients (12.0%). CONCLUSION: FMT is a safe and effective treatment option for rCDI. However, FMT is currently available only in few centers in Germany, and treatment options vary from one center to another.

IL-28B Genetic Variants Determine the Extent of Monocyte-Induced Activation of NK Cells in Hepatitis C.

BACKGROUND: Immuno-genetic studies suggest a functional link between NK cells and λ-IFNs. We recently showed that NK cells are negative for the IFN-λ receptor IFN-λR1 and do not respond to IFN-λ, suggesting a rather indirect association between IL-28B genotype and NK cell activity. METHODS: A total of 75 HCV(+) patients and 67 healthy controls were enrolled into this study. IL-28B (rs12979860) and IFNL-4 (rs368234815) genotypes were determined by rtPCR. Total PBMC, monocytes, and NK cells were stimulated with IL-29, the TLR-7/8 agonist R848, or a combination of both. NK cell IFN-γ response was analysed by FACS. IL-12 and IL-18 secretion of monocytes was studied by ELISA. In blocking experiments anti-IL-12/anti-IL-18 were used. RESULTS: Following stimulation of total PBMCs with R848 we found NK cell IFN- γ responses to vary with the IL-28B genotype, with carriers of a T/T genotype displaying the lowest frequency of IFN-γ(+)NK cells. When isolated NK cells were studied no such associations were observed, indicating an indirect association between IL-28B genotype and NK cell activity. Accordingly, we found R848-stimulated monocytes of patients with a T/T genotype to be significantly less effective in triggering NK cell IFN- γ production than monocytes from carriers of a non-T/T genotype. In line with these findings we observed monocytes from T/T patients to secrete significantly lower concentrations of IL-12 than monocytes from non-T/T individuals. CONCLUSIONS: Our data indicate that monocytes from carriers of an IL-28B T/T genotype display a reduced ability to stimulate NK cell activity and, thus, provide a link between IL-28B genotype and NK functions.

HIV mono-infection is associated with an impaired anti-hepatitis C virus activity of natural killer cells.

OBJECTIVE: Hepatitis C virus (HCV) infection in HIV(+) patients is associated with faster liver disease progression compared with HCV mono-infection. HIV-associated immune defects are considered to play an important role in this context. Here, we analyzed the effects of HIV infection on natural killer (NK)-cell-mediated anti-HCV activity. DESIGN: NK cell phenotype and interferon gamma (IFN-γ) production, NK cell-mediated inhibition of HCV replication and CD4 T-cell/NK cell interactions were studied in treatment naive HIV (n = 22), and HIV patients under combined antiretroviral therapy (n = 29), compared with healthy controls (n = 20). METHODS: NK cell-mediated inhibition of HCV replication was analyzed using the HuH7A2HCVreplicon model. IFN-γ production of NK cells as well as interleukin-2 secretion of CD4 T lymphocytes were studied by flow cytometry. RESULTS: Peripheral blood mononuclear cells from HIV(+) patients displayed a significantly impaired anti-HCV activity, irrespective of combined antiretroviral therapy. This could in part be explained by HIV-associated decline in NK cell numbers. In addition, NK cell IFN-γ production was significantly impaired in HIV infection. Accordingly, we found low frequency of IFN-γ(+) NK cells in HIV(+) patients to be associated with ineffective inhibition of HCV replication. Finally, we show that CD4 T-cell-mediated stimulation of NK cell IFN-γ production was dysregulated in HIV infection with an impaired interleukin-2 response of NK cells. CONCLUSION: HIV infection has a strong suppressive effect on anti-HCV activity of NK cells. This may contribute to low spontaneous clearance rate and accelerated progression of HCV-associated liver disease observed in HIV(+) patients.

Hypoxia impairs anti-viral activity of natural killer (NK) cells but has little effect on anti-fibrotic NK cell functions in hepatitis C virus infection.

BACKGROUND & AIMS: Natural killer (NK) cells have been shown to exert anti-viral as well as anti-fibrotic functions in hepatitis C virus (HCV) infection. Previous studies, however, analyzed NK cell functions exclusively under atmospheric oxygen conditions despite the fact that the liver microenvironment is hypoxic. Here, we analyzed the effects of low oxygen tension on anti-viral and anti-fibrotic NK cell activity. METHODS: Peripheral (n=34) and intrahepatic (n=15) NK cells from HCV(+) patients as well as circulating NK cells from healthy donors (n=20) were studied with respect to anti-viral and anti-fibrotic activity via co-culture experiments with HuH7 replicon cells and hepatic stellate cells, respectively. RESULTS: Anti-viral activity of resting NK cells from healthy controls was not affected by hypoxia. However, hypoxia significantly reduced the response of healthy NK cells to cytokine stimulation. In contrast to healthy controls, we observed resting and cytokine activated peripheral NK cells from HCV patients to display a significantly decreased anti-viral activity when cultured at 5% or 1% oxygen, suggesting HCV NK cells to be very sensitive to hypoxia. These findings could be confirmed when intrahepatic NK cells were tested. Finally, we show that anti-fibrotic NK cell activity was not affected by low oxygen tension. CONCLUSIONS: Our results show that anti-viral function of NK cells from HCV(+) patients is critically affected by a hypoxic microenvironment and, therefore, indicate that in order to obtain an accurate understanding of intrahepatic NK cell anti-HCV activity, the laboratory modelling should take into account the liver specific levels of oxygen.

The ratio of calprotectin to total protein as a diagnostic and prognostic marker for spontaneous bacterial peritonitis in patients with liver cirrhosis and ascites.

BACKGROUND: Diagnosis of spontaneous bacterial peritonitis (SBP) is based on a differential ascites leukocyte count which does not provide prognostic information. We performed a pilot study to assess calprotectin in ascites as an alternative diagnostic and prognostic marker. METHODS: We collected ascites from patients with liver cirrhosis from March 2012 to July 2013. Routine clinical and laboratory data of the patients were recorded. Ascites calprotectin levels were determined by ELISA. RESULTS: Overall, we collected 120 ascites samples from 100 patients with liver cirrhosis and from eight patients with malignant peritoneal effusion as disease control. Samples without infection had significantly lower calprotectin levels (median 34 ng/mL, range 5-795) than SBP samples (median 928 ng/mL, range 21-110,480; p<0.001) and malignant effusions (median 401, range 47-2596 ng/mL; p<0.001). In non-infected ascites, calprotectin levels were higher in Child-Pugh stage B versus C (median 57 ng/mL vs. 17 ng/mL; p<0.001) and in alcoholic versus viral cirrhosis (median 37 ng/mL vs. 10 ng/mL; p=0.015). The ratio of ascites calprotectin to total protein was a better marker for SBP than calprotectin alone (AUROC=0.93; p<0.001; sensitivity 93%, specificity 79%; positive predictive value 60%; negative predictive value 97%). In addition, high levels of the ratio to total protein were associated with poor 30-day survival (p=0.042). CONCLUSIONS: The ratio of ascites calprotectin to total protein may be a promising new diagnostic and prognostic marker in patients with liver cirrhosis and SBP and should be evaluated further.

Polo-like kinase 1 is a therapeutic target in high-risk neuroblastoma.

PURPOSE: High-risk neuroblastoma remains a therapeutic challenge for pediatric oncologists. The Polo-like kinase 1 (PLK1) is highly expressed in many human cancers and is a target of the novel small-molecule inhibitor BI 2536, which has shown promising anticancer activity in adult malignancies. Here, we investigated the effect of BI 2536 on neuroblastoma cells in vitro and in vivo to explore PLK1 as a potential target in high-risk neuroblastoma therapy. EXPERIMENTAL DESIGN: PLK1 transcript levels were analyzed by microarrays in 476 primary neuroblastoma specimens, and correlation with prognostic markers and patient outcome was examined. To explore the effect of PLK1 inhibition on neuroblastoma cells, 7 cell lines were treated with BI 2536 and changes in growth properties were determined. Furthermore, nude mice with IMR-32 and SK-N-AS xenografts were treated with BI 2536. RESULTS: PLK1 is highly expressed in unfavorable neuroblastoma and in neuroblastoma cell lines. Expression of PLK1 is associated with unfavorable prognostic markers such as stage 4, age >18 months, MYCN amplification, unfavorable gene expression-based classification, and adverse patient outcome (P < 0.001 each). On treatment with nanomolar doses of BI 2536, all neuroblastoma cell lines analyzed showed significantly reduced proliferation, cell cycle arrest, and cell death. Moreover, BI 2536 abrogated growth of neuroblastoma xenografts in nude mice. CONCLUSIONS: Elevated PLK1 expression is significantly associated with high-risk neuroblastoma and unfavorable patient outcome. Inhibition of PLK1 using BI 2536 exhibits strong antitumor activity on human neuroblastoma cells in vitro and in vivo, opening encouraging new perspectives for the treatment of high-risk neuroblastoma.