Increased circulating fibronectin, depletion of natural IgM and heightened EBV, HSV-1 reactivation in ME/CFS and long COVID.

Myalgic Encephalomyelitis/ Chronic Fatigue syndrome (ME/CFS) is a complex, debilitating, long-term illness without a diagnostic biomarker. ME/CFS patients share overlapping symptoms with long COVID patients, an observation which has strengthened the infectious origin hypothesis of ME/CFS. However, the exact sequence of events leading to disease development is largely unknown for both clinical conditions. Here we show antibody response to herpesvirus dUTPases, particularly to that of Epstein-Barr virus (EBV) and HSV-1, increased circulating fibronectin (FN1) levels in serum and depletion of natural IgM against fibronectin ((n)IgM-FN1) are common factors for both severe ME/CFS and long COVID. We provide evidence for herpesvirus dUTPases-mediated alterations in host cell cytoskeleton, mitochondrial dysfunction and OXPHOS. Our data show altered active immune complexes, immunoglobulin-mediated mitochondrial fragmentation as well as adaptive IgM production in ME/CFS patients. Our findings provide mechanistic insight into both ME/CFS and long COVID development. Finding of increased circulating FN1 and depletion of (n)IgM-FN1 as a biomarker for the severity of both ME/CFS and long COVID has an immediate implication in diagnostics and development of treatment modalities.

Endogenous Retroviral Elements Generate Pathologic Neutrophils in Pulmonary Arterial Hypertension.

Rationale: The role of neutrophils and their extracellular vesicles (EVs) in the pathogenesis of pulmonary arterial hypertension is unclear. Objectives: To relate functional abnormalities in pulmonary arterial hypertension neutrophils and their EVs to mechanisms uncovered by proteomic and transcriptomic profiling. Methods: Production of elastase, release of extracellular traps, adhesion, and migration were assessed in neutrophils from patients with pulmonary arterial hypertension and control subjects. Proteomic analyses were applied to explain functional perturbations, and transcriptomic data were used to find underlying mechanisms. CD66b-specific neutrophil EVs were isolated from plasma of patients with pulmonary arterial hypertension, and we determined whether they produce pulmonary hypertension in mice. Measurements and Main Results: Neutrophils from patients with pulmonary arterial hypertension produce and release increased neutrophil elastase, associated with enhanced extracellular traps. They exhibit reduced migration and increased adhesion attributed to elevated ß1-integrin and vinculin identified by proteomic analysis and previously linked to an antiviral response. This was substantiated by a transcriptomic IFN signature that we related to an increase in human endogenous retrovirus K envelope protein. Transfection of human endogenous retrovirus K envelope in a neutrophil cell line (HL-60) increases neutrophil elastase and IFN genes, whereas vinculin is increased by human endogenous retrovirus K deoxyuridine triphosphate diphosphatase that is elevated in patient plasma. Neutrophil EVs from patient plasma contain increased neutrophil elastase and human endogenous retrovirus K envelope and induce pulmonary hypertension in mice, mitigated by elafin, an elastase inhibitor. Conclusions: Elevated human endogenous retroviral elements and elastase link a neutrophil innate immune response to pulmonary arterial hypertension.

Examination of control asymptomatic cohorts reveals heightened anti-EBV and HHV-6 A/B dUTPase antibodies in the aging populations.

Members of the human Herpesviridae are found in high prevalence in the human virome. While these viruses are known to cause numerous disease pathologies in symptomatic individuals little is known concerning the role that these viruses may have in modulating the host immune system in asymptomatic "healthy" individuals, especially during the aging process. Examination of three cohorts of "healthy asymptomatic" individuals (n = 255) for the presence of antibodies against the herpesviruses deoxyuridine triphosphate nucleotidohydrolase (dUTPase) as a marker for lytic/abortive-lytic replication demonstrated that all cohorts exhibited differential anti-herpesvirus dUTPase antibodies positivity frequencies ranging from 40.4% to 84% with some individuals in these cohorts expressing antibodies to the dUTPases of multiple herpesviruses (17.2%-56%). Furthermore, our results demonstrate that there was a statistically significant difference in anti-human herpesvirus 6 A and 6B (HHV-6 A/B) dUTPase antibodies in Cohort 3 (age = 66.2 ± 15.02 years) versus Cohort 1 (age 46.88 ± 8.61 years), suggesting that reactivation of HHV-6 A/B is not attenuated by aging. It is well established/documented that herpesvirus dUTPases induce immune dysfunction, as such it is of critical importance that additional studies be performed to determine how these viral proteins alter immune responses in asymptomatic individuals.

Monocyte-released HERV-K dUTPase engages TLR4 and MCAM causing endothelial mesenchymal transition.

We previously reported heightened expression of the human endogenous retroviral protein HERV-K deoxyuridine triphosphate nucleotidohydrolase (dUTPase) in circulating monocytes and pulmonary arterial (PA) adventitial macrophages of patients with PA hypertension (PAH). Furthermore, recombinant HERV-K dUTPase increased IL-6 in PA endothelial cells (PAECs) and caused pulmonary hypertension in rats. Here we show that monocytes overexpressing HERV-K dUTPase, as opposed to GFP, can release HERV-K dUTPase in extracellular vesicles (EVs) that cause pulmonary hypertension in mice in association with endothelial mesenchymal transition (EndMT) related to induction of SNAIL/SLUG and proinflammatory molecules IL-6 as well as VCAM1. In PAECs, HERV-K dUTPase requires TLR4-myeloid differentiation primary response-88 to increase IL-6 and SNAIL/SLUG, and HERV-K dUTPase interaction with melanoma cell adhesion molecule (MCAM) is necessary to upregulate VCAM1. TLR4 engagement induces p-p38 activation of NF-κB in addition to p-pSMAD3 required for SNAIL and pSTAT1 for IL-6. HERV-K dUTPase interaction with MCAM also induces p-p38 activation of NF-κB in addition to pERK1/2-activating transcription factor-2 (ATF2) to increase VCAM1. Thus in PAH, monocytes or macrophages can release HERV-K dUTPase in EVs, and HERV-K dUTPase can engage dual receptors and signaling pathways to subvert PAEC transcriptional machinery to induce EndMT and associated proinflammatory molecules.

Upregulation of Human Endogenous Retrovirus-K Is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension.

BACKGROUND: Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. METHODS: Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat. RESULTS: SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1ß, and tumor necrosis factor α) in circulating monocytes, pulmonary arterial endothelial cells, and also activated B cells. Vulnerability of pulmonary arterial endothelial cells (PAEC) to apoptosis was increased by HERV-K deoxyuridine triphosphate nucleotidohydrolase in an interleukin 6-independent manner. Furthermore, 3 weekly injections of HERV-K deoxyuridine triphosphate nucleotidohydrolase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8) and elevated interleukin 6. CONCLUSIONS: Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.

Current and potential immune therapies and vaccines in the management of psoriasis.

Psoriasis is a chronic, immune skin disease associated with significant morbidity. Development of psoriasis is influenced by numerous genes, one allele is HLA-CW*0602. Other genes and single nucleotide polymorphisms affect immunologic pathways and antimicrobial peptide synthesis. Dendritic cells initiate psoriasis by activating T-cells toward a Th1 and Th17 response, with increased cytokines including TNF-α, IL-6, -12, -17, -22, and -23. IL-22 appears to promote keratinocyte dedifferentiation and increased antimicrobial peptide synthesis while TNF-α and IL-17 induce leukocyte localization within the psoriatic plaque. These recent insights identifying key cytokine pathways have led to the development of inhibitors with significant efficacy in the treatment of psoriasis. While a strategy for vaccine modulation of the immune response in psoriasis is in progress, with new technology they may provide a cost-effective long-term treatment that may induce tolerance or targeted self-inhibition for patients with autoimmune disorders, such as psoriasis.

Evidence for the role of Epstein Barr Virus infections in the pathogenesis of acute coronary events.

BACKGROUND: The role of viral infections in the pathogenesis of atherosclerosis remains controversial largely due to inconsistent detection of the virus in atherosclerotic lesions. However, viral infections elicit a pro-inflammatory cascade known to be atherogenic and to precipitate acute ischemic events. We have published in vitro data that provide the foundation for a mechanism that reconciles these conflicting observations. To determine the relation between an early viral protein, deoxyuridine triphosphate nucleotidohydrolase (dUTPase), produced following reactivation of Epstein Barr Virus (EBV) to circulating pro-inflammatory cytokines, intercellular adhesion molecule-1 (ICAM-1) and acute coronary events. METHODOLOGY/PRINCIPAL FINDINGS: Blood samples were obtained from 299 patients undergoing percutaneous coronary intervention for stable angina (SA), unstable angina (UA), or acute myocardial infarction (AMI). Plasma concentrations of pro-inflammatory cytokines and neutralizing antibody against EBV-encoded dUTPase were compared in the three patient groups. AMI was associated with the highest measures of interleukin-6 (ANOVA p<0.05; 4.6 ± 2.6 pg/mL in patients with AMI vs. 3.2 ± 2.3 pg/mL in SA). ICAM-1 was significantly higher in patients with AMI (ANOVA p<0.05; 304 ± 116 pg/mL in AMI vs. 265 ± 86 pg/mL SA). The highest values of ICAM-1 were found in patients having an AMI and who were antibody positive for dUTPase (ANOVA p=0.008; 369 ± 183 pg/mL in AMI and positive for dUTPase vs. 249 ± 70 pg/mL in SA negative for dUTPase antibody). CONCLUSIONS/SIGNIFICANCE: These clinical data support a model, based on in vitro studies, by which EBV may precipitate AMI even under conditions of low viral load through the pro-inflammatory action of the early protein dUTPase that is produced even during incomplete viral replication. They further support the putative role of viral infections in the pathogenesis of atherosclerosis and coronary artery events.

Antibody to Epstein-Barr virus deoxyuridine triphosphate nucleotidohydrolase and deoxyribonucleotide polymerase in a chronic fatigue syndrome subset.

BACKGROUND: A defined diagnostic panel differentiated patients who had been diagnosed with chronic fatigue syndrome (CFS), based upon Fukuda/Carruthers criteria. This diagnostic panel identified an Epstein-Barr virus (EBV) subset of patients (6), excluding for the first time other similar "clinical" conditions such as cytomegalovirus (CMV), human herpesvirus 6 (HHV6), babesiosis, ehrlichiosis, borreliosis, Mycoplasma pneumoniae, Chlamydia pneumoniae, and adult rheumatic fever, which may be mistakenly called CFS. CFS patients were treated with valacyclovir (14.3 mg/kg q6h) for ≥ 12 months. Each patient improved, based upon the Functional Activity Appraisal: Energy Index Score Healthcare Worker Assessment (EIPS), which is a validated (FSS-9), item scale with high degree of internal consistency measured by Cronbach's alpha. METHODS: Antibody to EBV viral capsid antigen (VCA) IgM, EBV Diffuse Early Antigen EA(D), and neutralizing antibodies against EBV-encoded DNA polymerase and EBV-encoded dUTPase were assayed serially approximately every three months for 13-16 months from sera obtained from patients with CFS (6) and from sera obtained from twenty patients who had no history of CFS. RESULTS: Antibodies to EBV EA(D) and neutralizing antibodies against the encoded-proteins EBV DNA polymerase and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) were present in the EBV subset CFS patients. Of the sera samples obtained from patients with CFS 93.9% were positive for EA(D), while 31.6% of the control patients were positive for EBV EA(D). Serum samples were positive for neutralizing antibodies against the EBV-encoded dUTPase (23/52; 44.2%) and DNA polymerase (41/52; 78.8%) in EBV subset CFS patients, but negative in sera of controls. CONCLUSIONS: There is prolonged elevated antibody level against the encoded proteins EBV dUTPase and EBV DNA polymerase in a subset of CFS patients, suggesting that this antibody panel could be used to identify these patients, if these preliminary findings are corroborated by studies with a larger number of EBV subset CFS patients.

Silver nanocolloids disrupt medaka embryogenesis through vital gene expressions.

Silver nanomaterials are the major components of healthcare products largely because of their antimicrobial effects. However, their unintended toxicity to biological organisms and its mechanism are not well understood. Using medaka fish embryo model, the toxic effects and corresponding mechanisms of silver nanocolloids (SNC, particle size 3.8 ± 1.0-diameter nm) were investigated. SNC caused morphological changes in embryos including cardiovascular malformations, ischemia, underdeveloped central nervous system and eyes, and kyphosis at exposures of 0.5 mg/L. Interestingly, SNC were observed inside the eggs at a level of 786.1 ± 32.5 pg/mg egg weight, and TEM analysis showed that SNC adhered to the surface and inside of the chorion. Meanwhile, medaka oligo DNA microarray and qRT-PCR were used for gene expression analysis in the embryos exposed to 0.05 mg/L SNC for 48 h. As a result, expressions of six of the oxidative stress-, embryogenesis- and morphogenesis-related genes, ctsL, tpm1, rbp, mt, atp2a1, and hox6b6, were affected by the SNC exposure, and these genes' involvement in those malformations was implied. Thus, SNC could potentially cause malformations in the cardiovascular and central nervous systems in developing medaka embryo through SNC-induced differential expression of the genes related to oxidative stress, embryonic cellular proliferation, and morphological development.

Death-signal-induced relocalization of cyclin-dependent kinase 11 to mitochondria.

Fas receptor-Fas ligand interaction appears to be important in carcinogenesis, tumour outgrowth and metastasis. Emerging evidence suggests that CDK11 (cyclin-dependent kinase 11) plays a role in apoptosis and melanoma development. Here, we show that CDK11p110 protein kinase was cleaved after induction of apoptosis by Fas. The N-terminal portion of CDK11p110, CDK11p60, was translocated from the nucleus to the mitochondria. The targeting of CDK11p60 to mitochondria occurred as early as 12 h after treatment. Overexpression of EGFP (enhanced green fluorescent protein)-tagged CDK11p60 could partially break down the mitochondrial membrane potential, induce cytochrome c release and promote apoptosis. Reduction of endogenous CDK11p110 protein levels with siRNA (small interfering RNA) resulted in the suppression of both cytochrome c release and apoptosis. In addition, subcellular fractionation studies of Fas-mediated apoptosis demonstrated that CDK11p60 was associated with the mitochondrial import motor, mitochondrial heat shock protein 70. Taken together, our data suggest that CDK11p60 can contribute to apoptosis by direct signalling at the mitochondria, thereby amplifying Fas-induced apoptosis in melanoma cells.