Dysregulated autoantibodies targeting vaso- and immunoregulatory receptors in Post COVID Syndrome correlate with symptom severity.

Most patients with Post COVID Syndrome (PCS) present with a plethora of symptoms without clear evidence of organ dysfunction. A subset of them fulfills diagnostic criteria of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Symptom severity of ME/CFS correlates with natural regulatory autoantibody (AAB) levels targeting several G-protein coupled receptors (GPCR). In this exploratory study, we analyzed serum AAB levels against vaso- and immunoregulatory receptors, mostly GPCRs, in 80 PCS patients following mild-to-moderate COVID-19, with 40 of them fulfilling diagnostic criteria of ME/CFS. Healthy seronegative (n=38) and asymptomatic post COVID-19 controls (n=40) were also included in the study as control groups. We found lower levels for various AABs in PCS compared to at least one control group, accompanied by alterations in the correlations among AABs. Classification using random forest indicated AABs targeting ADRB2, STAB1, and ADRA2A as the strongest classifiers (AABs stratifying patients according to disease outcomes) of post COVID-19 outcomes. Several AABs correlated with symptom severity in PCS groups. Remarkably, severity of fatigue and vasomotor symptoms were associated with ADRB2 AAB levels in PCS/ME/CFS patients. Our study identified dysregulation of AAB against various receptors involved in the autonomous nervous system (ANS), vaso-, and immunoregulation and their correlation with symptom severity, pointing to their role in the pathogenesis of PCS.

Serum of Post-COVID-19 Syndrome Patients with or without ME/CFS Differentially Affects Endothelial Cell Function In Vitro.

A proportion of COVID-19 reconvalescent patients develop post-COVID-19 syndrome (PCS) including a subgroup fulfilling diagnostic criteria of Myalgic encephalomyelitis/Chronic Fatigue Syndrome (PCS/CFS). Recently, endothelial dysfunction (ED) has been demonstrated in these patients, but the mechanisms remain elusive. Therefore, we investigated the effects of patients' sera on endothelia cells (ECs) in vitro. PCS (n = 17), PCS/CFS (n = 13), and healthy controls (HC, n = 14) were screened for serum anti-endothelial cell autoantibodies (AECAs) and dysregulated cytokines. Serum-treated ECs were analysed for the induction of activation markers and the release of small molecules by flow cytometry. Moreover, the angiogenic potential of sera was measured in a tube formation assay. While only marginal differences between patient groups were observed for serum cytokines, AECA binding to ECs was significantly increased in PCS/CFS patients. Surprisingly, PCS and PCS/CFS sera reduced surface levels of several EC activation markers. PCS sera enhanced the release of molecules associated with vascular remodelling and significantly promoted angiogenesis in vitro compared to the PCS/CFS and HC groups. Additionally, sera from both patient cohorts induced the release of molecules involved in inhibition of nitric oxide-mediated endothelial relaxation. Overall, PCS and PCS/CFS patients' sera differed in their AECA content and their functional effects on ECs, i.e., secretion profiles and angiogenic potential. We hypothesise a pro-angiogenic effect of PCS sera as a compensatory mechanism to ED which is absent in PCS/CFS patients.

Endothelial dysfunction and altered endothelial biomarkers in patients with post-COVID-19 syndrome and chronic fatigue syndrome (ME/CFS).

BACKGROUND: Fatigue, exertion intolerance and post-exertional malaise are among the most frequent symptoms of Post-COVID Syndrome (PCS), with a subset of patients fulfilling criteria for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). As SARS-CoV-2 infects endothelial cells, causing endotheliitis and damaging the endothelium, we investigated endothelial dysfunction (ED) and endothelial biomarkers in patients with PCS. METHODS: We studied the endothelial function in 30 PCS patients with persistent fatigue and exertion intolerance as well as in 15 age- and sex matched seronegative healthy controls (HCs). 14 patients fulfilled the diagnostic criteria for ME/CFS. The other patients were considered to have PCS. Peripheral endothelial function was assessed by the reactive hyperaemia index (RHI) using peripheral arterial tonometry (PAT) in patients and HCs. In a larger cohort of patients and HCs, including post-COVID reconvalescents (PCHCs), Endothelin-1 (ET-1), Angiopoietin-2 (Ang-2), Endocan (ESM-1), IL-8, Angiotensin-Converting Enzyme (ACE) and ACE2 were analysed as endothelial biomarkers. RESULTS: Five of the 14 post-COVID ME/CFS patients and five of the 16 PCS patients showed ED defined by a diminished RHI (< 1.67), but none of HCs exhibited this finding. A paradoxical positive correlation of RHI with age, blood pressure and BMI was found in PCS but not ME/CFS patients. The ET-1 concentration was significantly elevated in both ME/CFS and PCS patients compared to HCs and PCHCs. The serum Ang-2 concentration was lower in both PCS patients and PCHCs compared to HCs. CONCLUSION: A subset of PCS patients display evidence for ED shown by a diminished RHI and altered endothelial biomarkers. Different associations of the RHI with clinical parameters as well as varying biomarker profiles may suggest distinct pathomechanisms among patient subgroups.

Metformin Attenuates ROS via FOXO3 Activation in Immune Cells.

Forkhead box O 3 (FOXO3) is a transcription factor involved in cell metabolism, inflammation and longevity. Here, we investigated if metformin can activate FOXO3 in human immune cells and affects the subsequent level of reactive oxygen/nitrogen species (ROS/RNS) in immune cells. AMP-activated protein kinase (AMPK) and FOXO3 activation were investigated by immunoblot or flow cytometry (FC) analysis, respectively. FOXO3 target gene expression was quantified by real-time PCR. ROS/RNS measurement using dichlorodihydrofluorescein diacetate (DCFH-DA) dye was investigated by FC. The role of the FOXO3 single nucleotide polymorphisms (SNPs) rs12212067, rs2802292 and rs12206094 on ROS/RNS production was studied using allelic discrimination PCR. Metformin induced activation of AMPK (pT172) and FOXO3 (pS413). ROS/RNS level was reduced in immune cells after metformin stimulation accompanied by induction of the FOXO3 targets mitochondrial superoxide dismutase and cytochrome c. Studies in Foxo3 deficient (Foxo3-/- ) mouse splenocytes confirmed that metformin mediates its effects via Foxo3 as it attenuates ROS/RNS in myeloid cells of wildtype (WT) but not of Foxo3-/- mice. Our results suggest that FOXO3 can be activated by metformin leading to reduced ROS/RNS level in immune cells. This may add to the beneficial clinical effects of metformin observed in large cohort studies on longevity, cardiovascular and cancer risk.

Delineating the Association Between Soluble CD26 and Autoantibodies Against G-Protein Coupled Receptors, Immunological and Cardiovascular Parameters Identifies Distinct Patterns in Post-Infectious vs. Non-Infection-Triggered Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Soluble cluster of differentiation 26 (sCD26) has a wide range of enzymatic functions affecting immunological, metabolic and vascular regulation. Diminished sCD26 concentrations have been reported in various autoimmune diseases and also in Myalgic Encephalomyelitis/Chronic fatigue syndrome (ME/CFS). Here we re-evaluate sCD26 as a diagnostic marker and perform a comprehensive correlation analysis of sCD26 concentrations with clinical and paraclinical parameters in ME/CFS patients. Though this study did find significantly lower concentrations of sCD26 only in the female cohort and could not confirm diagnostic suitability, results from correlation analyses provide striking pathomechanistic insights. In patients with infection-triggered onset, the associations of low sCD26 with elevated autoantibodies (AAB) against alpha1 adrenergic (AR) and M3 muscarinic acetylcholine receptors (mAChR) point to a pathomechanism of infection-triggered autoimmune-mediated vascular and immunological dysregulation. sCD26 concentrations in infection-triggered ME/CFS were found to be associated with activated T cells, liver enzymes, creatin kinase (CK) and lactate dehydrogenase (LDH) and inversely with Interleukin-1 beta (IL-1b). Most associations are in line with the known effects of sCD26/DPP-4 inhibition. Remarkably, in non-infection-triggered ME/CFS lower sCD26 in patients with higher heart rate after orthostatic challenge and postural orthostatic tachycardia syndrome (POTS) suggest an association with orthostatic regulation. These findings provide evidence that the key enzyme sCD26 is linked to immunological alterations in infection-triggered ME/CFS and delineate a different pathomechanism in the non-infectious ME/CFS subset.

Autoimmunity-Related Risk Variants in PTPN22 and CTLA4 Are Associated With ME/CFS With Infectious Onset.

Single nucleotide polymorphisms (SNP) in various genes have been described to be associated with susceptibility to autoimmune disease. In this study, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients and controls were genotyped for five immune gene SNPs in tyrosine phosphatase non-receptor type 22 (PTPN22, rs2476601), cytotoxic T-lymphocyte-associated protein 4 (CTLA4, rs3087243), tumor necrosis factor (TNF, rs1800629 and rs1799724), and interferon regulatory factor 5 (IRF5, rs3807306), which are among the most important risk variants for autoimmune diseases. Analysis of 305 ME/CFS patients and 201 healthy controls showed significant associations of the PTPN22 rs2476601 and CTLA4 rs3087243 autoimmunity-risk alleles with ME/CFS. The associations were only found in ME/CFS patients, who reported an acute onset of disease with an infection (PTPN22 rs2476601: OR 1.63, CI 1.04-2.55, p = 0.016; CTLA4 rs3087243: OR 1.53, CI 1.17-2.03, p = 0.001), but not in ME/CFS patients without infection-triggered onset (PTPN22 rs2476601: OR 1.09, CI 0.56-2.14, p = 0.398; CTLA4 rs3087243: OR 0.89, CI 0.61-1.30, p = 0.268). This finding provides evidence that autoimmunity might play a role in ME/CFS with an infection-triggered onset. Both genes play a key role in regulating B and T cell activation.

The expression signature of very long non-coding RNA in myalgic encephalomyelitis/chronic fatigue syndrome.

BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic debilitating disease with huge social-economic impact. It has been suggested that immune dysregulation, nitrooxidative stress, and metabolic impairment might contribute to disease pathogenesis. However, the etiology of ME/CFS remains largely unclear, and diagnostic/prognostic disease markers are lacking. Several long noncoding RNAs (lncRNA, > 200 bp) have been reported to play roles in immunological diseases or in stress responses. METHODS: In our study, we examined the expression signature of 10 very long lncRNAs (> 5 kb, CR933609, His-RNA, AK124742, GNAS1-AS, EmX2OS, MIAT, TUG1, NEAT1, MALAT1, NTT) in the peripheral blood mononuclear cells of 44 ME/CFS patients. RESULTS: LncRNAs NTT, MIAT and EmX2OS levels were found to be significantly elevated in ME/CFS patients as compared with healthy controls. Furthermore, NTT and EmX2OS levels increased with disease severity. Stimulation of human monocytic cell line THP-1 and glioma cell line KALS1 with H2O2 (oxidative stress) and poly (I:C) (double strand RNA, representing viral activation) increased the expression levels of NTT and MIAT. CONCLUSIONS: Our study revealed a ME/CFS-associated very long lncRNA expression signature, which might reflect the regulatory response in ME/CFS patients to oxidative stress, chronic viral infection and hypoxemia. Further investigations need to be done to uncover the functions and potential diagnostic value of these lncRNAs in ME/CFS.

Immunoadsorption to remove ß2 adrenergic receptor antibodies in Chronic Fatigue Syndrome CFS/ME.

INTRODUCTION: Infection-triggered disease onset, chronic immune activation and autonomic dysregulation in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) point to an autoimmune disease directed against neurotransmitter receptors. We had observed elevated autoantibodies against ß2 adrenergic receptors, and muscarinic 3 and 4 acetylcholine receptors in a subset of patients. Immunoadsorption (IA) was shown to be effective in removing autoantibodies and improve outcome in various autoimmune diseases. METHODS: 10 patients with post-infectious CFS/ME and elevated ß2 autoantibodies were treated with IA with an IgG-binding column for 5 days. We assessed severity of symptoms as outcome parameter by disease specific scores. Antibodies were determined by ELISA and B cell phenotype by flow cytometry. RESULTS: IgG levels dropped to median 0.73 g/l (normal 7-16 g/l) after the 4th cycle of IA, while IgA and IgM levels remained unchanged. Similarly, elevated ß2 IgG antibodies rapidly decreased during IA in 9 of 10 patients. Also 6 months later ß2 autoantibodies were significantly lower compared to pretreatment. Frequency of memory B cells significantly decreased and frequency of plasma cells increased after the 4th IA cycle. A rapid improvement of symptoms was reported by 7 patients during the IA. 3 of these patients had long lasting moderate to marked improvement for 6-12+ months, 2 patients had short improvement only and 2 patients improved for several months following initial worsening. CONCLUSIONS: IA can remove autoantibodies against ß2 adrenergic receptor and lead to clinical improvement. B cell phenotyping provides evidence for an effect of IA on memory B cell development. Data from our pilot trial warrants further studies in CFS/ME.

The forkhead transcription factor Foxo3 negatively regulates natural killer cell function and viral clearance in myocarditis.

Aims: Foxo3 is a transcription factor involved in cell metabolism, survival, and inflammatory disease. However, mechanistic insight in Foxo3 effects is still limited. Here, we investigated the role of Foxo3 on natural killer (NK) cell responses and its effects in viral myocarditis. Methods and results: Effects of Foxo3 on viral load and immune responses were investigated in a model of coxsackie virus B3 myocarditis in wild-type (WT) and Foxo3 deficient mice. Reduced immune cell infiltration, viral titres, and pro-inflammatory cytokines in cardiac tissue were observed in Foxo3-/- mice 7 days post-infection (p.i.). Viral titres were also attenuated in hearts of Foxo3-/- mice at Day 3 while interferon-γ (IFNγ) and NKp46 expression were up-regulated suggesting early viral control by enhanced NK cell activity. CD69 expression of NK cells, frequencies of CD11b+CD27+ effector NK cells and cytotoxicity of Foxo3-/- mice was enhanced compared to WT littermates. Moreover, microRNA-155 expression, essential in NK cell activation, was elevated in Foxo3-/- NK cells while its inhibition led to diminished IFNγ production. Healthy humans carrying the longevity-associated FOXO3 single nucleotide polymorphism (SNP) rs12212067 exhibited reduced IFNγ and cytotoxic degranulation of NK cells. Viral inflammatory cardiomyopathy (viral CMI) patients with this SNP showed a poorer outcome due to less efficient virus control. Conclusion: Our results implicate Foxo3 in regulating NK cell function and suggest Foxo3 playing an important role in the antiviral innate immunity. Thus, enhanced FOXO3 activity such as in the polymorphism rs12212067 may be protective in chronic inflammation such as cancer and cardiovascular disease but disadvantageous to control acute viral infection.

Serological profiling of the EBV immune response in Chronic Fatigue Syndrome using a peptide microarray.

BACKGROUND: Epstein-Barr-Virus (EBV) plays an important role as trigger or cofactor for various autoimmune diseases. In a subset of patients with Chronic Fatigue Syndrome (CFS) disease starts with infectious mononucleosis as late primary EBV-infection, whereby altered levels of EBV-specific antibodies can be observed in another subset of patients. METHODS: We performed a comprehensive mapping of the IgG response against EBV comparing 50 healthy controls with 92 CFS patients using a microarray platform. Patients with multiple sclerosis (MS), systemic lupus erythematosus (SLE) and cancer-related fatigue served as controls. 3054 overlapping peptides were synthesised as 15-mers from 14 different EBV proteins. Array data was validated by ELISA for selected peptides. Prevalence of EBV serotypes was determined by qPCR from throat washing samples. RESULTS: EBV type 1 infections were found in patients and controls. EBV seroarray profiles between healthy controls and CFS were less divergent than that observed for MS or SLE. We found significantly enhanced IgG responses to several EBNA-6 peptides containing a repeat sequence in CFS patients compared to controls. EBNA-6 peptide IgG responses correlated well with EBNA-6 protein responses. The EBNA-6 repeat region showed sequence homologies to various human proteins. CONCLUSION: Patients with CFS had a quite similar EBV IgG antibody response pattern as healthy controls. Enhanced IgG reactivity against an EBNA-6 repeat sequence and against EBNA-6 protein is found in CFS patients. Homologous sequences of various human proteins with this EBNA-6 repeat sequence might be potential targets for antigenic mimicry.

Aluminum toxicity risk reduction as a result of reduced acid deposition in Adirondack lakes and ponds.

In 1990, the US Congress amended the Clean Air Act (CAA) to reduce regional-scale ecosystem degradation from SO x and NO x emissions which have been responsible for acid deposition in regions such as the Adirondack Mountains of New York State. An ecosystem assessment project was conducted from 1994 to 2012 by the Darrin Fresh Water Institute to determine the effect of these emission reduction policies on aquatic systems. The project investigated water chemistry and biota in 30 Adirondack lakes and ponded waters. Although regulatory changes made in response to the 1990 CAA amendments resulted in a reduction of acid deposition within the Adirondacks, the ecosystem response to these reductions is complicated. A statistical analysis of SO4, pH, Al, and DOC data collected during this project demonstrates positive change in response to decreased deposition. The changes in water chemistry also have lowered the risk of Al toxicity to brook trout (Salvelinus fontinalis [Mitchill]), which allowed the re-introduction of this species to Brooktrout Lake from which it had been extirpated. However, pH and labile aluminum (Alim) fluctuate and are not strongly correlated to changes in acid deposition. As such, toxicity to S. fontinalis also is cyclic and provides rationale for the difficulties inherent in re-establishing resident populations in impacted aquatic environments. Overall, aquatic ecosystems of the Adirondacks show a positive response to reduced deposition driven by changes in environmental policy, but the response is more complex and indicates an ecosystem-wide interaction between aquatic and watershed components of the ecosystem.

Osteopontin in cerebrospinal fluid as diagnostic biomarker for central nervous system lymphoma.

Central nervous system lymphoma (CNSL) is diagnostically challenging. The identification of reliable and easy to measure biomarkers is desirable to facilitate diagnosis. Here, we evaluated the value of cerebrospinal fluid (CSF) osteopontin (OPN) as a diagnostic biomarker for CNSL. OPN concentrations in CSF from 37 patients with CNSL (29 with primary CNSL and 8 with secondary CNS involvement of systemic lymphoma) and 36 controls [6 patients with inflammatory CNS disease other than multiple sclerosis (MS), 8 with MS, 9 with glioblastoma (GBM) and 13 healthy controls] were determined using an enzyme-linked immunosorbent assay. Non-parametric tests and receiver operating characteristic (ROC) curves were performed for determination of diagnostic accuracy. Median CSF OPN level in all CNSL patients was 620 ng/mL and higher than in patients with inflammatory CNS disease (356 ng/mL); P < .05, MS (163 ng/mL); P < .01, GBM (41 ng/mL); P < .01, or healthy controls (319 ng/mL); P < .01. The area under the ROC curve was 0.865 [95 % confidence interval (CI) 0.745-0.985] for differentiating CNSL and patients with inflammatory CNS disease; 0.956 (95 % CI 0.898-1.000) for CNSL and MS patients; 0.988 (95 % CI 0.964-1.000) for CNSL and GBM patients, and 0.915 (95 % CI 0.834-0.996) for CNSL patients and healthy controls. In multivariate analysis, high CSF OPN level was associated with shorter progression-free (HR 1.61, 95 % CI 1.13-2.31; P = .009) and overall survival (HR 1.52, 95 % CI 1.04-2.21; P = .029). CSF OPN is a potential biomarker in CNSL.

Cellular and humoral influenza-specific immune response upon vaccination in patients with common variable immunodeficiency and unclassified antibody deficiency.

BACKGROUND: Immunization against seasonal influenza with inactivated vaccine is recommended for patients with common variable immunodeficiency (CVID). However, humoral vaccine response in CVID patients is frequently impaired and current knowledge on T cell vaccine response in CVID and other patients with antibody deficiency is poor. OBJECTIVE: In the present study, we comparatively analyzed the antibody and T cellular immune response of patients with CVID and unclassified antibody deficiency to influenza vaccination in the season 2013-2014. METHODS: Eight patients with CVID, 8 patients with unclassified antibody deficiency and 9 healthy controls were vaccinated with a single dose of non-adjuvanted seasonal influenza vaccine. Before and 3 weeks after the vaccination antibody titers against the strains A/California/7/2009, A/Texas/50/2012, and B/Massachusetts/02/2012 included in the vaccine were measured by hemagglutination inhibition testing. Additionally, vaccine-specific T cell cytokine response was determined by stimulation with the complete vaccine in vitro. RESULTS: Whereas all healthy controls responded to vaccination with serum antibody titers, only 1/8 CVID patients and 4/8 patients with unclassified antibody deficiency showed a response against at least 1 of the 3 vaccine strains. However, 7/8 of the CVID and 6/8 of the patients with unclassified antibody deficiency had similar frequencies of vaccine-induced IFN-γ, TNF-α and IL-2 producing CD40L(+) T cells as the control group. CONCLUSION: Our data suggest that most CVID and unclassified antibody deficiency patients benefit from seasonal influenza vaccination by mounting a cellular response.

Antibodies to ß adrenergic and muscarinic cholinergic receptors in patients with Chronic Fatigue Syndrome.

Infection-triggered disease onset, chronic immune activation and autonomic dysregulation in CFS point to an autoimmune disease directed against neurotransmitter receptors. Autoantibodies against G-protein coupled receptors were shown to play a pathogenic role in several autoimmune diseases. Here, serum samples from a patient cohort from Berlin (n=268) and from Bergen with pre- and post-treatment samples from 25 patients treated within the KTS-2 rituximab trial were analysed for IgG against human α and ß adrenergic, muscarinic (M) 1-5 acetylcholine, dopamine, serotonin, angiotensin, and endothelin receptors by ELISA and compared to a healthy control cohort (n=108). Antibodies against ß2, M3 and M4 receptors were significantly elevated in CFS patients compared to controls. In contrast, levels of antibodies against α adrenergic, dopamine, serotonin, angiotensin, and endothelin receptors were not different between patients and controls. A high correlation was found between levels of autoantibodies and elevated IgG1-3 subclasses, but not with IgG4. Further patients with high ß2 antibodies had significantly more frequently activated HLA-DR+ T cells and more frequently thyreoperoxidase and anti-nuclear antibodies. In patients receiving rituximab maintenance treatment achieving prolonged B-cell depletion, elevated ß2 and M4 receptor autoantibodies significantly declined in clinical responder, but not in non-responder. We provide evidence that 29.5% of patients with CFS had elevated antibodies against one or more M acetylcholine and ß adrenergic receptors which are potential biomarkers for response to B-cell depleting therapy. The association of autoantibodies with immune markers suggests that they activate B and T cells expressing ß adrenergic and M acetylcholine receptors. Dysregulation of acetylcholine and adrenergic signalling could also explain various clinical symptoms of CFS.

Polymorphism in COMT is associated with IgG3 subclass level and susceptibility to infection in patients with chronic fatigue syndrome.

BACKGROUND: Chronic fatigue syndrome (CFS) is considered as a neuroimmunological disease but the etiology and pathophysiology is poorly understood. Patients suffer from sustained exhaustion, cognitive impairment and an increased sensitivity to pain and sensory stimuli. A subset of patients has frequent respiratory tract infections (RRTI). Dysregulation of the sympathetic nervous system and an association with genetic variations in the catechol-O-methyltransferase (COMT) and glucocorticoid receptor genes influencing sympathetic and glucocorticoid metabolism were reported in CFS. Here, we analyzed the prevalence of SNPs of COMT and glucocorticoid receptor-associated genes in CFS patients and correlated them to immunoglobulin levels and susceptibility to RRTI. METHODS: We analyzed blood cells of 74 CFS patients and 76 healthy controls for polymorphisms in COMT, FKBP5 and CRHR1 by allelic discrimination PCR. Serum immunoglobulins were determined by immunoturbidimetric technique, cortisol levels by ECLIA. RESULTS: Contrary to previous reports, we found no difference between CFS patients and healthy controls in the prevalence of SNPs for COMT, FKBP5 and CRHR1. In patients with the Met/Met variant of COMT rs4680 we observed enhanced cortisol levels providing evidence for its functional relevance. Both enhanced IgE and diminished IgG3 levels and an increased susceptibility to RRTI were observed in CFS patients with the Met/Met variant. Such an association was not observed in 68 non-CFS patients with RRTI. CONCLUSION: Our results indicate a relationship of COMT polymorphism rs4680 with immune dysregulation in CFS providing a potential link for the association between stress and infection susceptibility in CFS.

Human bone marrow contains a subset of quiescent early memory CD8(+) T cells characterized by high CD127 expression and efflux capacity.

Even today it is still not completely understood how CD8(+) T-cell memory is maintained long term. Since bone marrow (BM) is a niche for immunological memory, we sought to identify long-lasting early memory CD8(+) T cells in this compartment. To achieve this, we looked for CD8(+) T cells that are able to efflux Rhodamine 123, a typical property of stem cells. Indeed, we identified a distinct subset of CD8(+) T cells in BM, with the capacity to efflux and high CD127 expression. These CD127(hi) effluxers are conventional CD8(+) T cells exhibiting a broad TCR-Vß repertoire and are generated in response to viral peptides in vitro. CD127(hi) effluxer CD8(+) T cells have an early memory phenotype defined by preferential TNF-α production and a Bcl-2(hi) , KLRG-1(low) profile. This population has long telomeres and shows constitutively low frequencies of Ki-67 expression ex vivo, but has a high proliferative and differentiation capacity in vitro. However, IL-15 downmodulates CD127 in CD127(hi) effluxer CD8(+) T cells in vitro. Consequently, the CD127(low) effluxer subset may comprise cells recently exposed to IL-15. Taken together, CD127(hi) effluxer CD8(+) T cells represent a novel population of early memory T cells resident in BM with properties required for long-lived memory.

Deficient EBV-specific B- and T-cell response in patients with chronic fatigue syndrome.

Epstein-Barr virus (EBV) has long been discussed as a possible cause or trigger of Chronic Fatigue Syndrome (CFS). In a subset of patients the disease starts with infectious mononucleosis and both enhanced and diminished EBV-specific antibody titers have been reported. In this study, we comprehensively analyzed the EBV-specific memory B- and T-cell response in patients with CFS. While we observed no difference in viral capsid antigen (VCA)-IgG antibodies, EBV nuclear antigen (EBNA)-IgG titers were low or absent in 10% of CFS patients. Remarkably, when analyzing the EBV-specific memory B-cell reservoir in vitro a diminished or absent number of EBNA-1- and VCA-antibody secreting cells was found in up to 76% of patients. Moreover, the ex vivo EBV-induced secretion of TNF-α and IFN-γ was significantly lower in patients. Multicolor flow cytometry revealed that the frequencies of EBNA-1-specific triple TNF-α/IFN-γ/IL-2 producing CD4(+) and CD8(+) T-cell subsets were significantly diminished whereas no difference could be detected for HCMV-specific T-cell responses. When comparing EBV load in blood immune cells, we found more frequently EBER-DNA but not BZLF-1 RNA in CFS patients compared to healthy controls suggesting more frequent latent replication. Taken together, our findings give evidence for a deficient EBV-specific B- and T-cell memory response in CFS patients and suggest an impaired ability to control early steps of EBV reactivation. In addition the diminished EBV response might be suitable to develop diagnostic marker in CFS.

Adiponectin modulates NK-cell function.

Adiponectin (APN) has been shown to exert antiinflammatory effects in various disease models but little is known concerning its regulation of NK-cell function. Here, we show that the majority of human CD56(dim) NK cells express surface Adiponectin receptor (AdipoR) 1 and 2 while most CD56(high) NK cells are AdipoR-negative. Toll-like receptor (TLR) ligand-induced IFN-γ production was diminished by APN while it had no influence on NK-cell cytotoxicity. In contrast only a small subpopulation of murine NK cells expresses surface AdipoRs, but about 90% store them intracellularly. APN-deficient knockout (KO) mice had elevated frequencies of NK cells. However, cytotoxic degranulation of NK cells was decreased in APN knockout (APN-KO) animals. Accordingly, frequencies of CD11b(high) CD27(high) and CD94(high) effector NK cells and expression of NKG2D were lower in APN-KO mice. Upon CVB3 infection NK-cell function was restored in APN-KO mice. Our data suggest that in addition to its antiinflammatory effects APN also influences the numerical and differentiation status of NK cells, which may further impact the outcome of immune-mediated diseases in APN-KO mice.

A frequent Toll-like receptor 1 gene polymorphism affects NK- and T-cell IFN-γ production and is associated with Helicobacter pylori-induced gastric disease.

BACKGROUND: Helicobacter pylori infects approximately 50% of the world population. Among the infected individuals, only 10-20% develop peptic ulcers and <3% progress to gastric cancer (GC). Th1-predominant immune responses have been suggested to underlie H. pylori-induced gastric diseases. However, the reason for a strong inter-individual variation of susceptibility and course of the disease is currently far from being understood. It has been shown that H. pylori stimulates the host's Toll-like receptor (TLR) 2/1 complex. Furthermore, the single nucleotide polymorphism (SNP) I602S of TLR1 alters the inflammatory cytokine response of monocytes. Therefore, we hypothesized an association of this TLR1 SNP with H. pylori-mediated gastric pathologies. MATERIALS AND METHODS: Subjects with different TLR1 genotypes were analyzed for their IFN-γ response of NK- and T-cells. We further genotyped 548 patients with gastric diseases for this SNP and compared patients with gastritis with those having ulcer, and patients with high-risk gastritis versus patients with GC. RESULTS: Homozygous 602S allele carriers exhibited impaired in vitro IFN-γ responses to the TLR2/1 agonist Pam(3) CSK(4). The TLR1 I602S SNP is significantly associated with GC (p = .002) and gastric ulcer (p = .051). Odds ratios showed significantly reduced risk regarding GC and peptic ulcer for the homozygous mutated genotype. The odds ratios were 0.4 (95% CI, 0.22-0.72) and 0.588 (95% CI, 0.35-1.00), respectively. CONCLUSION: In conclusion, our results suggest that the nonfunctional TLR1 602S/S genotype is associated with a reduced risk of H. pylori-induced gastric diseases, probably via diminished Th1 responses.

CCN1: a novel inflammation-regulated biphasic immune cell migration modulator.

In this study, we performed a comprehensive analysis of the effect of CCN1 on the migration of human immune cells. The molecule CCN1, produced by fibroblasts and endothelial cells, is considered as an important matrix protein promoting tissue repair and immune cell adhesion by binding various integrins. We recently reported that CCN1 therapy is able to suppress acute inflammation in vivo. Here, we show that CCN1 binds to various immune cells including T cells, B cells, NK cells, and monocytes. The addition of CCN1 in vitro enhances both actin polymerization and transwell migration. Prolonged incubation with CCN1, however, results in the inhibition of migration of immune cells by a mechanism that involves downregulation of PI3Kγ, p38, and Akt activation. Furthermore, we observed that immune cells themselves produce constitutively CCN1 and secretion is induced by pro-inflammatory stimuli. In line with this finding, patients suffering from acute inflammation had enhanced serum levels of CCN1. These findings extend the classical concept of CCN1 as a locally produced cell matrix adhesion molecule and suggest that CCN1 plays an important role in regulating immune cell trafficking by attracting and locally immobilizing immune cells.