Heparin-induced extracorporeal LDL-precipitation(HELP):Mode of action, safety and clinical experience(3)

Safety and practicability of the HELP procedure Compared to standard plasma exchange, HELP-apheresis is much better tolerated, primarily be-cause heparin rather than citrate is used for anti-coagulation and therefore problems due to hypercalce-mia do not play a role.……

Heparin-induced extracorporeal LDL- precipitation(HELP): Mode of action, safety and clinical experience(2)

Rheological effects of acute and chronic HELP - treatment The acute reduction of rheologically relevant plasma constituents such as fibrinogen and largesized lipoproteins significantly improves whole blood and plasma viscosity by a decrease of approximately 15%, erythrocyte aggregability decreases by 53% whereas erythrocyte filtrability improves by 10% (figure 3).……

Molecular Epidemiology of Nontuberculous Mycobacteria in a German CF Center and Clinical Course of NTM Positive Patients

Goal of this study was to analyse the clinical course of cystic fibrosis (CF) patients with nontuberculous mycobacteria (NTM) in their respiratory secretions and to investigate the molecular epidemiology of the most prevalent NTM species by multilocus sequence analysis (MLSA). The respiratory specimen and the clinical parameters forced expiratory volume in one second (FEV1), body-mass-index (BMI), erythrocyte sedimentation rate (ESR) 1 h and immunoglobulin G (IgG) of 357 CF patients, 0 - 52.4 years, mean FEV1 2009 81.5% pred were analysed between 1998 and 2010. In 13 patients NTM were detected. 12 of 13 patients carried M. abscessus, for one patient the NTM species was not characterized. 4 patients carried a second NTM species (M. avium, M. chelonae (2x), M. intracellulare). 6 patients exhibited a significant decline in FEV1, however changes in BMI, IgG and ESR were discordant. Molecular genotyping of M. abscessus isolates revealed a unique MLSA pattern in 6 patients. 2 patients harboured identical strains, and one patient a closely related strain. Whether the presence of identical strains is attributed to the acquisition of NTM clones from common environmental sources or to patient-to-patient transmission cannot be definitely clarified. Although cross-in- fection of the three patients with identical/closely related strains in the present cohort is highly unlikely, we recommend strict hygiene measures for all CF patients harbouring NTM.

Exogenous sphingomyelinase causes impaired intestinal epithelial barrier function

AIM: To test the hypothesis that hydrolysis of sphingomyelin to ceramide changes the composition of tight junctions (TJs) with increasing permeability of the intestinal epithelium. METHODS: Monolayers of Caco-2 cells were used as an in vitro model for the intestinal barrier. Permeability was determined by quantifi cation of transepithelialflux and transepithelial resistance. Sphingolipid-rich membrane microdomains were isolated by a discontinuous sucrose gradient and characterized by Western-blot. Lipid content of microdomains was analysed by tandem mass spectrometry. Ceramide was subcellularly localized by immunofluorescent staining.RESULTS: Exogenous sphingomyelinase increased transepithelial permeability and decreased transepithelial resistance at concentrations as low as 0.01 U/mL. Lipid analysis showed rapid accumulation of ceramide in the membrane fractions containing occludin and claudin-4, representing TJs. In these fractions we observed a concomitant decrease of sphingomyelin and cholesterol with increasing concentrations of ceramide. Immunofluorescent staining confirmed clustering of ceramide at the sites of cell-cell contacts. Neutralization of surface ceramide prevented the permeability-increase induced by platelet activating factor. CONCLUSION: Our findings indicate that changes in lipid composition of TJs impair epithelial barrier functions. Generation of ceramide by sphingomyelinases might contribute to disturbed barrier function seen in diseases such as inflammatory, infectious, toxic or radiogenic bowel disease.

Metabolic inflammation as an instigator of fibrosis during nonalcoholic fatty liver disease

Non-alcoholic fatty liver disease(NAFLD)is characterized by excessive storage of fatty acids in the form of triglycerides in hepatocytes.It is most prevalent in western countries and includes a wide range of clinical and histopathological findings,namely from simple steatosis to steatohepatitis and fibrosis,which may lead to cirrhosis and hepatocellular cancer.The key event for the transition from steatosis to fibrosis is the activation of quiescent hepatic stellate cells(qHSC)and their differentiation to myofibroblasts.Pattern recognition receptors(PRRs),expressed by a plethora of immune cells,serve as essential components of the innate immune system whose function is to stimulate phagocytosis and mediate inflammation upon binding to them of various molecules released from damaged,apoptotic and necrotic cells.The activation of PRRs on hepatocytes,Kupffer cells,the resident macrophages of the liver,and other immune cells results in the production of proinflammatory cytokines and chemokines,as well as profibrotic factors in the liver microenvironment leading to qHSC activation and subsequent fibrogenesis.Thus,elucidation of the inflammatory pathways associated with the pathogenesis and progression of NAFLD may lead to a better understanding of its pathophysiology and new therapeutic approaches.

Different effects of a CD14 gene polymorphism on disease outcome in patients with alcoholic liver disease and chronic hepatitis C infection

AIM: Clinical and experimental data suggest that gut-derived endotoxins are an important pathogenic factors for progression of chronic liver disease. Recently, a C-T (-159)polymorphism in the promoter region of the CD14 gene was detected and found to confer increased CD14 expression and to be associated with advanced alcoholic liver damage. Here, we investigated this polymorphism in patients with less advanced alcoholic liver disease (ALD)and chronic hepatitis C virus (HCV) infection.METHODS: CD14 genotyping was performed by PCR-RFLP analysis in (a) 121 HCV patients, (b) 62 patients with alcohol-associated cirrhosis (Alc-Ci), (c) 118 individuals with heavy alcohol abuse without evidence of advanced liver damage (Alc-w/o Ci), and (d) 247 healthy controls.Furthermore, serum levels of soluble CD14 (sCD14) and transaminases were determined.RESULTS: The TT genotype was significantly more frequent in Alc-Ci compared to Alc-w/o Ci or controls (40.3% vs 23.7% or 24.0%, respectively). In Alc-w/o Ci,serum levels of transaminases did not differ significantly between patients with different CD14 genotypes. In HCV patients, TT-homozygotes had significantly higher sCD14 levels and sCD14 serum levels were significantly higher in patients with advanced fibrosis or cirrhosis. However,no association was found between CD14 genotypes and histological staging or grading.CONCLUSION: Considering serum transaminases as surrogate markers for alcoholic liver damage, the CD14 polymorphism seems to exhibit different effects during the course of ALD. Differences in genotype distribution between cirrhotic HCV patients and alcoholics and the known functional impact of this polymorphism on CD14 expression levels further indicate differences in the pathophysiological role of CD14 and CD14-mediated lipopolysaccharides signal transduction with regard to the stage as well as the type of the underlying liver disease.

Tumor-and osteoclast-derived NRP2 in prostate cancer bone metastases

Understanding the role of neuropilin 2(NRP2)in prostate cancer cells as well as in the bone microenvironment is pivotal in the development of an effective targeted therapy for the treatment of prostate cancer bone metastasis.We observed a significant upregulation of NRP2 in prostate cancer cells metastasized to bone.Here,we report that targeting NRP2 in cancer cells can enhance taxane-based chemotherapy with a better therapeutic outcome in bone metastasis,implicating NRP2 as a promising therapeutic target.Since,osteoclasts present in the tumor microenvironment express NRP2,we have investigated the potential effect of targeting NRP2 in osteoclasts.Our results revealed NRP2 negatively regulates osteoclast differentiation and function in the presence of prostate cancer cells that promotes mixed bone lesions.Our study further delineated the molecular mechanisms by which NRP2 regulates osteoclast function.Interestingly,depletion of NRP2 in osteoclasts in vivo showed a decrease in the overall prostate tumor burden in the bone.These results therefore indicate that targeting NRP2 in prostate cancer cells as well as in the osteoclastic compartment can be beneficial in the treatment of prostate cancer bone metastasis.

Ectodysplasin A2 receptor-NF-κB-inducing kinase axis: a new player in muscle wasting to cancer cachexia

Cancer cachexia is a complex syndrome characterized by weight loss,muscle atrophy,and metabolic dysregulation,impeding cancer treatment and reducing patients’quality of life.Cachexia can be seen as end-stage systemic manifestation common to various chronic diseases.Distinct triggers like aging,COPD,HIV,and cancer result in unresolved metabolic or inflammatory stress,leading to a maladaptive state.

Neutrophils are shaped by the tumor microenvironment:novel possibilities for targeting neutrophils in cancer

In a recent paper published in Science,Ng et al.provide seminal insights into the process through which neutrophils are reprogrammed by the tumor environment,leading to a convergent transition from different neutrophil states towards a unique end-stage differentiated pro-tumor phenotype.1 These findings have major translational implications as they provide potential neutrophil-related targets for tumor immunotherapy.

The deubiquitinase OTUB1 augments NF-κB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13

Dendritic cells(DCs)are indispensable for defense against pathogens but may also contribute to immunopathology.Activation of DCs upon the sensing of pathogens by Toll-like receptors(TLRs)is largely mediated by pattern recognition receptor/nuclear factor-κB(NF-κB)signaling and depends on the appropriate ubiquitination of the respective signaling molecules.However,the ubiquitinating and deubiquitinating enzymes involved and their interactions are only incompletely understood.Here,we reveal that the deubiquitinase OTU domain,ubiquitin aldehyde binding 1(OTUB1)is upregulated in DCs upon murine Toxoplasma gondii infection and lipopolysaccharide challenge.Stimulation of DCs with the TLR11/12 ligand T.gondii profilin and the TLR4 ligand lipopolysaccharide induced an increase in NF-κB activation in OTUB1-competent cells,resulting in elevated interleukin-6(IL-6),IL-12,and tumor necrosis factor(TNF)production,which was also observed upon the specific stimulation of TLR2,TLR3,TLR7,and TLR9.Mechanistically,OTUB1 promoted NF-κB activity in DCs by K48-linked deubiquitination and stabilization of the E2-conjugating enzyme UBC13,resulting in increased K63-linked ubiquitination of IRAK1(IL-1 receptor-associated kinase 1)and TRAF6(TNF receptor-associated factor 6).Consequently,DC-specific deletion of OTUB1 impaired the production of cytokines,in particular IL-12,by DCs over the first 2 days of T.gondii infection,resulting in the diminished production of protective interferon-γ(IFN-γ)by natural killer cells,impaired control of parasite replication,and,finally,death from chronic T.encephalitis,all of which could be prevented by low-dose IL-12 treatment in the first 3 days of infection.In contrast,impaired OTUB1-deficient DC activation and cytokine production by OTUB1-deficient DCs protected mice from lipopolysaccharide-induced immunopathology.Collectively,these findings identify OTUB1 as a potent novel regulator of DCs during infectious and inflammatory diseases.

Trilineage Sequencing Reveals Complex TCRβTranscriptomes in Neutrophils and Monocytes Alongside T Cells

Recent findings indicate the presence of T cell receptor(TCR)-based combinatorial immune receptors beyond T cells in neutrophils and monocytes/macrophages.In this study,using a semiquantitative trilineage immune repertoire sequencing approach as well as under rigorous bioinformatic conditions,we identify highly complex TCRβtranscriptomes in human circulating monocytes and neutrophils that separately encode repertoire diversities one and two orders of magnitude smaller than that of T cells.Intraindividual transcriptomic analyses reveal that neutrophils,monocytes,and T cells express distinct TCRβrepertoires with less than 0.1%overall trilineage repertoire sharing.Interindividual comparison shows that in all three leukocyte lineages,the vast majority of the expressed TCRβvariants are private.We also find that differentiation of monocytes into macrophages induces dramatic individual-specific repertoire shifts,revealing a surprising degree of immune repertoire plasticity in the monocyte lineage.These results uncover the remarkable complexity of the two phagocyte-based flexible immune systems which until now has been hidden in the shadow of T cells.

Cerebral venous sinus thrombosis after ChAdOx1 nCov- 19 vaccination with a misleading first cerebral MRI scan

SUMMARY Vaccine-induced immune thrombotic thrombocytopenia(VITT)and cerebral venous sinus thrombosis(CVST)have been recently described as rare complications following vaccination against SARS-CoV-2 with vector vaccines.We report a case of a young woman who presented with VITT and cerebral CVST 7 days following vaccination with ChAdOx1 nCov-19(AstraZeneca).While the initial MRI was considered void of pathological findings,MRI 3 days later revealed extensive CVST of the transversal and sigmoidal sinus with intracerebral haemorrhage.Diagnostic tests including a platelet-factor-4-induced platelet activation assay confirmed the diagnosis of VITT.Treatment with intravenous immunoglobulins and argatroban resulted in a normalisation of platelet counts and remission of CVST.

Neutrophil extracellular traps contribute to the development of hepatocellular carcinoma in NASH by promoting Treg differentiation

Non-alcoholic fatty liver disease(NAFLD)is an umbrella term,describing a range of chronic liver disorders associated with fat deposition in hepatocytes,defined as steatosis,and it is mostly emerged in the presence of obesity,hyperlipidemia and insulin resistance.NAFLD has been classified as the most frequent liver condition worldwide with more than 10%of NAFLD patients progressing to the inflammatory and fibrotic disorder of non-alcoholic steatohepatitis(NASH),which in turn can lead to end stage liver disease including hepatocellular carcinoma(HCC);the most frequent malignant liver tumor(1).During HCC development the immune system interacts with the tumour microenvironment and it can stimulate its growth by maintaining a chronic inflammatory milieu and the production of factors that favor tumor growth,survival,and angiogenesis.Similar phenomena may also take place and have significant implications in the transition from NASH to HCC(2).Although,it is well established that chronic inflammation within the hepatic microenvironment is of great significance in HCC development,the exact mechanisms leading to the transition from NASH to HCC are yet to be elucidated.

PBX/Knotted 1 homeobox-2(PKNOX2)is a novel regulator of myocardial fibrosis

Much effort has been made to uncover the cellular heterogeneities of human hearts by single-nucleus RNA sequencing.However,the cardiac transcriptional regulation networks have not been systematically described because of the limitations in detecting transcription factors.In this study,we optimized a pipeline for isolating nuclei and conducting single-nucleus RNA sequencing targeted to detect a higher number of cell signal genes and an optimal number of transcription factors.With this unbiased protocol,we characterized the cellular composition of healthy human hearts and investigated the transcriptional regulation networks involved in determining the cellular identities and functions of the main cardiac cell subtypes.Particularly in fibroblasts,a novel regulator,PKNOX2,was identified as being associated with physiological fibroblast activation in healthy hearts.To validate the roles of these transcription factors in maintaining homeostasis,we used single-nucleus RNA-sequencing analysis of transplanted failing hearts focusing on fibroblast remodelling.The trajectory analysis suggested that PKNOX2 was abnormally decreased from fibroblast activation to pathological myofibroblast formation.Both gain-and loss-of-function in vitro experiments demonstrated the inhibitory role of PKNOX2 in pathological fibrosis remodelling.Moreover,fibroblast-specific overexpression and knockout of PKNOX2 in a heart failure mouse model induced by transverse aortic constriction surgery significantly improved and aggravated myocardial fibrosis,respectively.In summary,this study established a high-quality pipeline for single-nucleus RNA-sequencing analysis of heart muscle.With this optimized protocol,we described the transcriptional regulation networks of the main cardiac cell subtypes and identified PKNOX2 as a novel regulator in suppressing fibrosis and a potential therapeutic target for future translational studies.