Felix Goeser

Functional implications of microbial and viral gut metagenome changes in early stage L-DOPA-naïve Parkinson’s disease patients

BackgroundParkinson’s disease (PD) presently is conceptualized as a protein aggregation disease in which pathology involves both the enteric and the central nervous system, possibly spreading from one to another via the vagus nerves. As gastrointestinal dysfunction often precedes or parallels motor symptoms, the enteric system with its vast diversity of microorganisms may be involved in PD pathogenesis. Alterations in the enteric microbial taxonomic level of L-DOPA-naïve PD patients might also serve as a biomarker.MethodsWe performed metagenomic shotgun analyses and compared the fecal microbiomes of 31 early stage, L-DOPA-naïve PD patients to 28 age-matched controls.ResultsWe found increased Verrucomicrobiaceae (Akkermansia muciniphila) and unclassified Firmicutes, whereas Prevotellaceae (Prevotella copri) and Erysipelotrichaceae (Eubacterium biforme) were markedly lowered in PD samples. The observed differences could reliably separate PD from control with a ROC-AUC of 0.84. Functional analyses of the metagenomes revealed differences in microbiota metabolism in PD involving the ẞ-glucuronate and tryptophan metabolism. While the abundances of prophages and plasmids did not differ between PD and controls, total virus abundance was decreased in PD participants. Based on our analyses, the intake of either a MAO inhibitor, amantadine, or a dopamine agonist (which in summary relates to 90% of PD patients) had no overall influence on taxa abundance or microbial functions.ConclusionsOur data revealed differences of colonic microbiota and of microbiota metabolism between PD patients and controls at an unprecedented detail not achievable through 16S sequencing. The findings point to a yet unappreciated aspect of PD, possibly involving the intestinal barrier function and immune function in PD patients. The influence of the parkinsonian medication should be further investigated in the future in larger cohorts.

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

Abstract 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.

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.

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

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.

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.

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-&ggr;) 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-&ggr; 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-&ggr; production was significantly impaired in HIV infection. Accordingly, we found low frequency of IFN-&ggr;(+) 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-&ggr; 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.

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.

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.

Dysregulierte Interaktion zwischen CD4+ T Zellen und NK Zellen in der HIV/HCV Koinfektion

Hintergrund: Die HCV/HIV Koinfektion ist im Vergleich zur HCV Monoinfektion mit einer schnelleren Progression einer Lebererkrankung assoziiert. Naturliche Killer (NK) Zellen haben auf die Viruseliminierung bei Hepatitis C einen enormen Einfluss. Bei der Aktivierung von NK Zellen sind nach bisheriger Erkenntnis CD4+ T Zellen involviert. Allerdings ist eine HIV Infektion insbesondere durch dysfunktionelle CD4+ T Zellen gekennzeichnet. Aus diesem Grunde vermuten wir bei der Immunpathogense der HIV/HCV Koinfektion eine gestorte Interaktion zwischen CD4+ T Zellen und NK Zellen. Material undMethoden: In diese Studie wurden 40 HIV Patienten, davon 21 HIV RNA(-) Patienten mit HAART und 19 HIV RNA(+) Patienten ohne Therapie, und 20 gesunde HIV(-)/HCV(-)Probanden eingeschlossen. Die IL2-Produktion CD4+ T Zellen wurde nach CD3/CD28 Stimulation mittels Durchflusszytometrie getestet. Die anti-HCV Aktivitat von PBMC wurde mit Hilfe des HCV-Replikon-Systems untersucht. Die AnaIyse der IFN-γ Produktion isolierter NK Zellen und der anti-HCV Aktivitat erfolgte nach Koinkubation mit HCV-Replikonzellen in Kokultur mit bzw. ohne aktivierten CD4+ T Zellen. Ergebnisse: Die Hemmung der HCV Replikation war bei PBMC von HIV Infizierten signifikant niedriger als bei gesunden Probanden (p < 0.001). Passend dazu war bei HIV Infizierten die IFN-γ Produktion der NK Zellen signifikant gehemmt (p < 0.001). Interessanterweise korrelierte bei Therapie-naiven HIV RNA(+) Patienten die CD4+ T Zellzahl positiv mit der anti-HCV Aktivitat und IFN-γ Produktion von NK Zellen, allerdings nicht bei HIV RNA(-) Patienten unter HAART (p < 0.5). Zudem war die viramische HIV Infektion im Vergleich zu gesunden Kontrollen und HIV RNA(-) Patienten mit einer verminderten IL2 Sezernierung von CD4+ T Zellen assoziiert. In Ubereinstimmung mit bisher von uns veroffentlichten Arbeiten induzieren aktivierte CD4+ T Zellen effektiv die IFN-γ Produktion von gesunden NK Zellen. Allerdings gab es zwischen CD4+ T Zellen von HIV Patienten und Gesunden keine Unterschiede in der Stimulierbarkeit der NK Zellen gesunder Probanden. Uberraschenderweise liesen sich NK Zellen von viramischen HIV Patienten weder durch autologe CD4+ T Zellen noch von gesunden Kontrollen stimulieren. Das lasst einen intrinsischen Defekt der NK Zellen vermuten. Diese dysregulierte Funktion wurde auch bei NK Zellen von therapierten HIV RNA(-) Patienten beobachtet. Diskussion: Unsere Daten deuten auf eine gestorte Interaktion zwischen CD4+ T Zellen und NK Zellen bei der HIV Infektion hin. Dies hat einen negativen Einfluss auf die anti-HCV Aktivitat von NK Zellen und fuhrt moglicherweise zur schnelleren Progression einer HCV assoziierten Lebererkrankung in der HIV Koinfektion. Korrespondierender Autor: Kramer, Benjamin E-Mail:m.benjamin.kraemer@gmail.com

P485 THE RATIO OF ASCITES CALPROTECTIN TO TOTAL PROTEIN IS A DIAGNOSTIC AND PROGNOSTIC MARKER FOR SPONTANEOUS BACTERIAL PERITONITIS IN LIVER CIRRHOSIS

inclusion. The median follow up time was 283 days (range 1– 1382 days). M30 levels differed between Child Pugh stages and correlated with the MELD score (model of end stage liver disease). When patients were grouped according to M30 levels in individuals with low ( 3000U/l) M30 levels, M30 could differentiate overall survival of patients according to the log-rank test (P < 0.001). Furthermore, M30 level was an independent risk factor for mortality in addition to age, gender, Child Pugh stage and the MELD score in a multivariate Cox regression model. Conclusions: The M30 serum level correlates with the stage of cirrhosis and is predictive for survival in patients with liver cirrhosis.