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Background and AimsChronic hepatitis C virus (HCV) infection can be cured with direct-acting antiviral agents (DAA). However, not all sequelae of chronic hepatitis C appear to be completely reversible after sustained virologic response (SVR). Recently, chronic viral infections have been shown to be associated with biological age acceleration defined by the epigenetic clock. The aim of this study was to investigate whether chronic HCV infection is associated with epigenetic changes and biological age acceleration and whether this is reversible after SVR.MethodsWe included 54 well-characterized patients with chronic hepatitis C at three time points: DAA treatment initiation, end of treatment, and long-term follow-up (median 96 weeks after end of treatment). Genome-wide DNA methylation status from peripheral blood mononuclear cells (PBMC) was generated and used to calculate epigenetic age acceleration (EAA) using Horvath’s clock.ResultsHCV patients had an overall significant EAA of 3.12 years at baseline compared with -2.61 years in the age-matched reference group (pConclusionsOur data contribute to the understanding of the biological impact of HCV elimination after DAA and demonstrate that HCV elimination can lead to “reverse inflammaging”. In addition, we provide new conceptual ideas for the use of biological age as a potential biomarker for HCV sequelae after SVR.Lay SummaryChronic hepatitis C virus infection is now curable with direct acting antiviral agents (DAA), but are concomitant and sequelae also fully reversible after cure? Recent data demonstrate that chronic viral infections lead to an increase in biological age as measured by epigenetic DNA methylation status. Using a unique cohort of hepatitis C patients with and without cirrhosis as well as progression to HCC, we demonstrated that these epigenetic changes and concomitant increase in biological age are also observed in chronic HCV infection. Our data further suggest that this effect seems to be partially reversible in the long-term course after sustained virological response (SVR) by DAA therapy and that biological regeneration occurs. In this regard, the recovery effect appears to be dependent on disease course and was significantly lower in patients with progression to HCC. This suggests the use of biological age based on epigenetic state as a potential biomarker for HCV sequelae.Graphical abstractHighlights-Patients with chronic hepatitis C have accelerated epigenetic age compared with healthy controls.-DAA treatment and HCV elimination partially reverse the accelerated epigenetic age in the long-term follow-up.-Patients who developed hepatocellular carcinoma after HCV elimination did not show reversal of accelerated epigenetic aging during the follow-up.

Soluble immune markers (SIMs) are associated with the risk of virological relapse in HBeAg‐negative patients with chronic hepatitis B after discontinuation of nucleos(t)ide analogue therapy. Machine learning identified a combination of five SIMs (IL‐2, MIG, RANTES, SCF, and TRAIL), which are highly predictive for virological relapse (AUC 0.89; CI: 0.5‐1.1) and can be validated in an independent cohort (AUC 0.63; CI: 0.1‐0.99)., Treatment with nucleos(t)ide analogues (NAs) may be stopped after 1‐3 years of hepatitis B virus DNA suppression in hepatitis B e antigen (HBeAg)‐negative patients according to Asian Pacific Association for the Study of Liver and European Association for the Study of Liver guidelines. However, virological relapse (VR) occurs in most patients. We aimed to analyze soluble immune markers (SIMs) and use machine learning to identify SIM combinations as predictor for early VR after NA discontinuation. A validation cohort was used to verify the predictive power of the SIM combination. In a post hoc analysis of a prospective, multicenter therapeutic vaccination trial (ABX‐203, NCT02249988), hepatitis B surface antigen, hepatitis B core antigen, and 47 SIMs were repeatedly determined before NA was stopped. Forty‐three HBeAg‐negative patients were included. To detect the highest predictive constellation of host and viral markers, a supervised machine learning approach was used. Data were validated in a different cohort of 49 patients treated with entecavir. VR (hepatitis B virus DNA ≥ 2,000 IU/mL) occurred in 27 patients. The predictive value for VR of single SIMs at the time of NA stop was best for interleukin (IL)‐2, IL‐17, and regulated on activation, normal T cell expressed and secreted (RANTES/CCL5) with a maximum area under the curve of 0.65. Hepatitis B core antigen had a higher predictive power than hepatitis B surface antigen but lower than the SIMs. A supervised machine‐learning algorithm allowed a remarkable improvement of early relapse prediction in patients treated with entecavir. The combination of IL‐2, monokine induced by interferon γ (MIG)/chemokine (C‐C motif) ligand 9 (CCL9), RANTES/CCL5, stem cell factor (SCF), and TNF‐related apoptosis‐inducing ligand (TRAIL) was reliable in predicting VR (0.89; 95% confidence interval: 0.5‐1.0) and showed viable results in the validation cohort (0.63; 0.1‐0.99). Host immune markers such as SIMs appear to be underestimated in guiding treatment cessation in HBeAg‐negative patients. Machine learning can help find predictive SIM patterns that allow a precise identification of patients particularly suitable for NA cessation.

Despite an available vaccine and efficient treatment for hepatitis B virus (HBV) infection, chronic HBV infection still remains a major global threat, and one of the top 20 causes of human mortality worldwide. One of the major challenges in controlling HBV infection is the high number of undiagnosed chronic carriers and the lack of access to prophylaxis and treatment in several parts of the world. We discuss relevant barriers that need to be overcome to achieve global control of HBV infection and make eradication possible. Most important, vaccination must be scaled-up to lower the risk of vertical transmission and decrease the number of new infections, and comprehensive screening programs must be linked to care to obtain a better rate of diagnosis and treatment. This can probably only be achieved if sustainable funding is available. We therefore emphasize the importance of making the management of viral hepatitis a global health priority.

Using an in vitro model for the differentiation of human blood-derived monocytes into dendritic cells (DCs), we have undertaken an investigation of epigenetic changes that take place at CD14 and CD209 (DC-SIGN) genes that encode cell surface antigens that are crucial for the function of monocytes and DCs, respectively. Upon differentiation the cell surface expression of CD14 is lost, whilst CD209 expression is increased. These reciprocal changes are associated with the loss of epigenetic markers of "activation" at the CD14 locus, but the acquisition of the same at the CD209 locus. There is little change in "repressive" histone marks and CpG methylation at the CD14 locus. By contrast there are changes in both the "repressive" histone marks and CpG methylation at the CD209 locus. In particular, two CpG dinucleotides, designated CpG2 and CpG3, show a marked demethylation at the CD209 promoter upon differentiation. These data provide insight into the epigenetic demands that are necessary to effect the reciprocal changes in expression of the CD14 and CD209 genes upon terminal differentiation of monocytes into DCs. For repression of the active CD14 gene the loss of "activation" histone modifications is likely necessary and sufficient for silencing. By contrast the activation of the silent CD209 gene appears to require an acquisition of "active" histone modifications and concomitant loss of both "repressive" histone marks and CpG methylation.

To cite this article: Debarry J, Hanuszkiewicz A, Stein K, Holst O, Heine H. The allergy-protective properties of Acinetobacter lwoffii F78 are imparted by its lipopolysaccharide. Allergy 2010; 65: 690–697. Abstract Background: An increasing number of epidemiological studies show that exposure to farming environment during early childhood strongly influences the development of allergic reactions later in life (‘hygiene hypothesis’). Also, it had been shown that certain bacteria from this environment may have allergy-protective properties. In the present study, we further characterized one of these bacteria, namely Acinetobacter lwoffii F78, with regard to the bacteria-induced signaling and possible mechanisms of allergy protection. Methods: The impact of A. lwoffii F78 on human monocyte-derived dendritic cells especially with respect to their THelper cell polarization capacity was investigated by ELISA and real-time PCR experiments as well as confocal microscopy. The responsible molecule for these effects was further characterized and identified using blocking experiments. Results: It was shown that A. lwoffii F78 induced a TH1-polarizing program in human dendritic cells which led to TH1 differentiation. In addition, a positive influence on the TBet/GATA3 level could be detected. Blocking experiments revealed that the lipopolysaccharide (LPS) of A. lwoffii F78 was the responsible molecule promoting these effects. Conclusion: We found evidence that the allergy-protecting effects of A. lwoffii F78 are because of the activation of a TH1-polarizing program in human dendritic cells, and that the LPS of A. lwoffii F78 is responsible for these beneficial effects.

Over 800 million people worldwide are infected with hepatitis viruses, human immunodeficiency virus (HIV), and malaria, resulting in more than 5 million deaths annually. Here we discuss the potential and challenges of humanized mouse models for developing effective and affordable therapies and vaccines, which are desperately needed to combat these diseases.

Regulators of G-protein signalling accelerate the GTPase activity of G(alpha) subunits, driving G proteins in their inactive GDP-bound form. This property defines them as GTPase activating proteins. Here the effect of different Toll-like receptor agonists on RGS1 and RGS2 expression in murine bone marrow-derived macrophages and J774 cells was analysed. After stimulation with TLR2/1 or TLR2/6 lipopeptide ligands and the TLR4/MD2 ligand lipopolysaccharide, microarray analyses show only modulation of RGS1 and RGS2 among all the regulators of G-protein signalling tested. Real-time PCR confirmed modulation of RGS1 and RGS2. In contrast to RGS2, which was always downregulated, RGS1 mRNA was upregulated during the first 30 min after stimulation, followed by downregulation. Similar results were also found in the murine macrophage cell line J774. The ligand for intracellular TLR9 modulates RGS1 and RGS2 in a similar manner. However, the TLR3 ligand poly(I:C) permanently upregulates RGS1 and RGS2 expression indicating a different modulation by the MyD88- and TRIF-signalling pathway. This was confirmed using MyD88(-/-) and TRIF(-/-) bone marrow-derived macrophages. Modulation of RGS1 and RGS2 by Toll-like receptor ligands plays an important role during inflammatory and immunological reactions after bacterial and viral infection.

The capsular polysaccharide from Acinetobacter lwoffii F78 was isolated and purified, and its structure was elucidated by chemical analyses, NMR spectroscopy, and mass spectrometry. The presence of a capsule on this bacterium was confirmed by transmission electron microscopy experiments, and the utilization of the antilipid A monoclonal antibody proved the nonendotoxin origin of the isolated material. The structure represents a novel nonbranched aminopolysaccharide with high heterogeneity of amino groups substituents: →3)-α-L-FucNAc-(1→3)-β-D-QuiNR14NR2-(1→4)-β-L-GlcNR33NR4A-(1→, where R1 = 3-hydroxybutyric acid (3-HBA) or N-acetylated alanine (AlaNAc), R2 = AlaNAc or 3-HBA, R3 = acetyl or 3-HBA, and R4 = 3-HBA or acetyl.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Chemical analyses, NMR spectroscopy, and mass spectrometry were used to elucidate the structure of the rough lipopolysaccharide (LPS) isolated from Acinetobacter lwoffii F78. As a prominent feature, the core region of this LPS contained the disaccharide alpha-Kdo-(2-->8)-alpha-Kdo (Kdo=3-deoxy-d-D-manno-oct-2-ulopyranosonic acid), which so far has been identified only in chlamydial LPS. In serological investigations, the anti-chlamydial LPS monoclonal antibody S25-2, which is specific for the epitope alpha-Kdo-(2-->8)-alpha-Kdo, reacted with A. lwoffii F78 LPS. Thus, an LPS was identified outside Chlamydiaceae that contains a Chlamydia-specific LPS epitope in its core region.

Background Children who grow up in a farming environment show lower levels of atopic sensitization, hay fever, and asthma than children of the same age not living in such an environment. A number of investigations provided good evidence that this is due to an early-life contact with cowsheds, farm animals, and/or consumption of products like raw milk. Also, it had been indicated that microorganisms might have an important effect on the development of allergies, and thus the question arose of which farm microbial organisms, their products, or both might induce or influence allergy-protective mechanisms. Objective We sought to gain further insight into the potential allergy-protective properties of microbes isolated from the farming environment. Methods Of a number of bacterial species identified in cowsheds of farms, 2 were selected, isolated, and characterized, namely Acinetobacter lwoffii F78 and Lactococcus lactis G121. The isolates were investigated with regard to their activation of pattern-recognition receptors, the maturation of human monocyte-derived dendritic cells, the upregulation of inflammatory cytokines, the T H 1-polarizing Notch ligand expression, and their influence on the allergic phenotype. Results It is shown that both bacterial isolates were able to reduce allergic reactions in mice, to activate mammalian cells in vitro , and to induce a T H 1-polarizing program in dendritic cells. Conclusion Our data strongly support the hygiene hypothesis, which states that an environment rich in microbiologic structures, such as a farming environment, might protect against the development of allergies. Clinical implications This work provides the first data on a potential application of cowshed bacteria in allergy protection.

Regulators of G-protein signalling accelerate the GTPase activity of G(alpha) subunits, driving G proteins in their inactive GDP-bound form. This property defines them as GTPase activating proteins. Here the effect of different Toll-like receptor agonists on RGS1 and RGS2 expression in murine bone marrow-derived macrophages and J774 cells was analysed. After stimulation with TLR2/1 or TLR2/6 lipopeptide ligands and the TLR4/MD2 ligand lipopolysaccharide, microarray analyses show only modulation of RGS1 and RGS2 among all the regulators of G-protein signalling tested. Real-time PCR confirmed modulation of RGS1 and RGS2. In contrast to RGS2, which was always downregulated, RGS1 mRNA was upregulated during the first 30 min after stimulation, followed by downregulation. Similar results were also found in the murine macrophage cell line J774. The ligand for intracellular TLR9 modulates RGS1 and RGS2 in a similar manner. However, the TLR3 ligand poly(I:C) permanently upregulates RGS1 and RGS2 expression indicating a different modulation by the MyD88- and TRIF-signalling pathway. This was confirmed using MyD88(-/-) and TRIF(-/-) bone marrow-derived macrophages. Modulation of RGS1 and RGS2 by Toll-like receptor ligands plays an important role during inflammatory and immunological reactions after bacterial and viral infection.

TLR are primary triggers of the innate immune system by recognizing various microorganisms through conserved pathogen-associated molecular patterns. TLR2 is the receptor for a functional recognition of bacterial lipopeptides (LP) and is up-regulated during various disorders such as chronic obstructive pulmonary disease and sepsis. This receptor is unique in its ability to form heteromers with TLR1 or TLR6 to mediate intracellular signaling. According to the fatty acid pattern as well as the assembling of the polypeptide tail, LP can signal through TLR2 in a TLR1- or TLR6-dependent manner. There are also di- and triacylated LP, which stimulate TLR1-deficient cells and TLR6-deficient cells. In this study, we investigated whether heterodimerization evolutionarily developed to broaden the ligand spectrum or to induce different immune responses. We analyzed the signal transduction pathways activated through the different TLR2 dimers using the three LP, palmitic acid (Pam)octanoic acid (Oct)2C-(VPGVG)4VPGKG, fibroblast-stimulating LP-1, and Pam2C-SK4. Dominant-negative forms of signaling molecules, immunoblotting of MAPK, as well as microarray analysis indicate that all dimers use the same signaling cascade, leading to an identical pattern of gene activation. We conclude that heterodimerization of TLR2 with TLR1 or TLR6 evolutionarily developed to expand the ligand spectrum to enable the innate immune system to recognize the numerous, different structures of LP present in various pathogens. Thus, although mycoplasma and Gram-positive and Gram-negative bacteria may activate different TLR2 dimers, the development of different signal pathways in response to different LP does not seem to be of vital significance for the innate defense system.