Directed and acyclic synaptic connectivity in the human layer 2-3 cortical microcircuit.

The computational capabilities of neuronal networks are fundamentally constrained by their specific connectivity. Previous studies of cortical connectivity have mostly been carried out in rodents; whether the principles established therein also apply to the evolutionarily expanded human cortex is unclear. We studied network properties within the human temporal cortex using samples obtained from brain surgery. We analyzed multineuron patch-clamp recordings in layer 2-3 pyramidal neurons and identified substantial differences compared with rodents. Reciprocity showed random distribution, synaptic strength was independent from connection probability, and connectivity of the supragranular temporal cortex followed a directed and mostly acyclic graph topology. Application of these principles in neuronal models increased dimensionality of network dynamics, suggesting a critical role for cortical computation.

Mutual regulation of CD4+ T cells and intravascular fibrin in infections.

Innate myeloid cells especially neutrophils and their extracellular traps are known to promote intravascular coagulation and thrombosis formation in infections and various other conditions. Innate myeloid cell dependent fibrin formation can support systemic immunity while its dysregulation enhances the severity of infectious diseases. Less is known about the immune mechanisms preventing dysregulation of fibrin homeostasis in infection. During experimental systemic infections local fibrin deposits in the liver microcirculation cause rapid arrest of CD4+ T cells. Arrested T helper cells mostly represent Th17 cells that partially originate from the small intestine. Intravascular fibrin deposits activate mouse and human CD4+ T cells which can be mediated by direct fibrin - CD4+ T cell interactions. Activated CD4+ T cells suppress fibrin deposition and microvascular thrombosis by directly counteracting coagulation activation by neutrophils and classical monocytes. T cell activation, which is initially triggered by IL- 12p40- and MHC-II dependent mechanisms, enhances intravascular fibrinolysis via LFA-1. Moreover, CD4+ T cells disfavor the association of the fibrinolysis inhibitor TAFI with fibrin whereby fibrin deposition is increased by TAFI in the absence but not presence of T cells. In human infections thrombosis development is inversely related to microvascular levels of CD4+ T cells. Thus, fibrin promotes LFA-1 dependent T helper cell activation in infections which drives a negative feedback cycle that rapidly restricts intravascular fibrin and thrombosis development.

Predictive role of intracranial PD-L1 expression in a real-world cohort of NSCLC patients treated with immune checkpoint inhibition following brain metastasis resection.

BACKGROUND: Emerging evidence suggests that treatment of NSCLC brain metastases with immune checkpoint inhibitors (ICIs) is associated with response rates similar to those of extracranial disease. Programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) serves as a predictive biomarker for ICI response. However, the predictive value of brain metastasis-specific (intracranial) PD-L1 TPS is not established. We investigated the role of intra- and extracranial PD-L1 TPS in NSCLC patients treated with ICI following brain metastasis resection. METHODS: Clinical data from NSCLC patients treated with ICI following brain metastasis resection (n = 64) were analyzed. PD-L1 TPS of brain metastases (n = 64) and available matched extracranial tumor tissue (n = 44) were assessed via immunohistochemistry. Statistical analyses included cut point estimation via maximally selected rank statistics, Kaplan-Meier estimates, and multivariable Cox regression analysis for intracranial progression-free survival (icPFS), extracranial progression-free survival (ecPFS), and overall survival (OS). RESULTS: PD-L1 expression was found in 54.7% of brain metastases and 68.2% of extracranial tumor tissues, with a median intra- and extracranial PD-L1 TPS of 7.5% (0 - 50%, IQR) and 15.0% (0 - 80%, IQR), respectively. In matched tissue samples, extracranial PD-L1 TPS was significantly higher than intracranial PD-L1 TPS (p = 0.013). Optimal cut points for intracranial and extracranial PD-L1 TPS varied according to outcome parameter assessed. Notably, patients with a high intracranial PD-L1 TPS (> 40%) exhibited significantly longer icPFS as compared to patients with a low intracranial PD-L1 TPS (≤ 40%). The cut point of 40% for intracranial PD-L1 TPS was independently associated with OS, icPFS and ecPFS in multivariable analyses. CONCLUSION: Our study highlights the potential role of intracranial PD-L1 TPS in NSCLC, which could be used to predict ICI response in cases where extracranial tissue is not available for PD-L1 assessment as well as to specifically predict intracranial response.

MET receptor serves as a promising target in melanoma brain metastases.

The development of brain metastases hallmarks disease progression in 20-40% of melanoma patients and is a serious obstacle to therapy. Understanding the processes involved in the development and maintenance of melanoma brain metastases (MBM) is critical for the discovery of novel therapeutic strategies. Here, we generated transcriptome and methylome profiles of MBM showing high or low abundance of infiltrated Iba1high tumor-associated microglia and macrophages (TAMs). Our survey identified potential prognostic markers of favorable disease course and response to immune checkpoint inhibitor (ICi) therapy, among them APBB1IP and the interferon-responsive gene ITGB7. In MBM with high ITGB7/APBB1IP levels, the accumulation of TAMs correlated significantly with the immune score. Signature-based deconvolution of MBM via single sample GSEA revealed enrichment of interferon-response and immune signatures and revealed inflammation, stress and MET receptor signaling. MET receptor phosphorylation/activation maybe elicited by inflammatory processes in brain metastatic melanoma cells via stroma cell-released HGF. We found phospho-METY1234/1235 in a subset of MBM and observed a marked response of brain metastasis-derived cell lines (BMCs) that lacked druggable BRAF mutations or developed resistance to BRAF inhibitors (BRAFi) in vivo to MET inhibitors PHA-665752 and ARQ197 (tivantinib). In summary, the activation of MET receptor in brain colonizing melanoma cells by stromal cell-released HGF may promote tumor self-maintenance and expansion and might counteract ICi therapy. Therefore, therapeutic targeting of MET possibly serves as a promising strategy to control intracranial progressive disease and improve patient survival.

Proteomic and transcriptomic profiling of brainstem, cerebellum and olfactory tissues in early- and late-phase COVID-19.

Neurological symptoms, including cognitive impairment and fatigue, can occur in both the acute infection phase of coronavirus disease 2019 (COVID-19) and at later stages, yet the mechanisms that contribute to this remain unclear. Here we profiled single-nucleus transcriptomes and proteomes of brainstem tissue from deceased individuals at various stages of COVID-19. We detected an inflammatory type I interferon response in acute COVID-19 cases, which resolves in the late disease phase. Integrating single-nucleus RNA sequencing and spatial transcriptomics, we could localize two patterns of reaction to severe systemic inflammation, one neuronal with a direct focus on cranial nerve nuclei and a separate diffuse pattern affecting the whole brainstem. The latter reflects a bystander effect of the respiratory infection that spreads throughout the vascular unit and alters the transcriptional state of mainly oligodendrocytes, microglia and astrocytes, while alterations of the brainstem nuclei could reflect the connection of the immune system and the central nervous system via, for example, the vagus nerve. Our results indicate that even without persistence of severe acute respiratory syndrome coronavirus 2 in the central nervous system, local immune reactions are prevailing, potentially causing functional disturbances that contribute to neurological complications of COVID-19.

KCNA2 IgG autoimmunity in neuropsychiatric diseases.

BACKGROUND: Autoantibodies against the potassium voltage-gated channel subfamily A member 2 (KCNA2) have been described in a few cases of neuropsychiatric disorders, but their diagnostic and pathophysiological role is currently unknown, imposing challenges to medical practice. DESIGN / METHODS: We retrospectively collected comprehensive clinical and paraclinical data of 35 patients with KCNA2 IgG autoantibodies detected in cell-based and tissue-based assays. Patients' sera and cerebrospinal fluid (CSF) were used for characterization of the antigen, clinical-serological correlations, and determination of IgG subclasses. RESULTS: KCNA2 autoantibody-positive patients (n = 35, median age at disease onset of 65 years, range of 16-83 years, 74 % male) mostly presented with cognitive impairment and/or epileptic seizures but also ataxia, gait disorder and personality changes. Serum autoantibodies belonged to IgG3 and IgG1 subclasses and titers ranged from 1:32 to 1:10,000. KCNA2 IgG was found in the CSF of 8/21 (38 %) patients and in the serum of 4/96 (4.2 %) healthy blood donors. KCNA2 autoantibodies bound to characteristic anatomical areas in the cerebellum and hippocampus of mammalian brain and juxtaparanodal regions of peripheral nerves but reacted exclusively with intracellular epitopes. A subset of four KCNA2 autoantibody-positive patients responded markedly to immunotherapy alongside with conversion to seronegativity, in particular those presenting an autoimmune encephalitis phenotype and receiving early immunotherapy. An available brain biopsy showed strong immune cell invasion. KCNA2 autoantibodies occurred in less than 10 % in association with an underlying tumor. CONCLUSION: Our data suggest that KCNA2 autoimmunity is clinically heterogeneous. Future studies should determine whether KCNA2 autoantibodies are directly pathogenic or develop secondarily. Early immunotherapy should be considered, in particular if autoantibodies occur in CSF or if clinical or diagnostic findings suggest ongoing inflammation. Suspicious clinical phenotypes include autoimmune encephalitis, atypical dementia, new-onset epilepsy and unexplained epileptic seizures.

The neurobiology of SARS-CoV-2 infection.

Worldwide, over 694 million people have been infected with SARS-CoV-2, with an estimated 55-60% of those infected developing COVID-19. Since the beginning of the pandemic in December 2019, different variants of concern have appeared and continue to occur. With the emergence of different variants, an increasing rate of vaccination and previous infections, the acute neurological symptomatology of COVID-19 changed. Moreover, 10-45% of individuals with a history of SARS-CoV-2 infection experience symptoms even 3 months after disease onset, a condition that has been defined as 'post-COVID-19' by the World Health Organization and that occurs independently of the virus variant. The pathomechanisms of COVID-19-related neurological complaints have become clearer during the past 3 years. To date, there is no overt - that is, truly convincing - evidence for SARS-CoV-2 particles in the brain. In this Review, we put special emphasis on discussing the  methodological difficulties of viral detection in CNS tissue and discuss immune-based (systemic and central) effects contributing to COVID-19-related CNS affection. We sequentially review the reported changes to CNS cells in COVID-19, starting with the blood-brain barrier and blood-cerebrospinal fluid barrier - as systemic factors from the periphery appear to primarily influence barriers and conduits - before we describe changes in brain parenchymal cells, including microglia, astrocytes, neurons and oligodendrocytes as well as cerebral lymphocytes. These findings are critical to understanding CNS affection in acute COVID-19 and post-COVID-19 in order to translate these findings into treatment options, which are still very limited.

Post-COVID exercise intolerance is associated with capillary alterations and immune dysregulations in skeletal muscles.

The SARS-CoV-2 pandemic not only resulted in millions of acute infections worldwide, but also in many cases of post-infectious syndromes, colloquially referred to as "long COVID". Due to the heterogeneous nature of symptoms and scarcity of available tissue samples, little is known about the underlying mechanisms. We present an in-depth analysis of skeletal muscle biopsies obtained from eleven patients suffering from enduring fatigue and post-exertional malaise after an infection with SARS-CoV-2. Compared to two independent historical control cohorts, patients with post-COVID exertion intolerance had fewer capillaries, thicker capillary basement membranes and increased numbers of CD169+ macrophages. SARS-CoV-2 RNA could not be detected in the muscle tissues. In addition, complement system related proteins were more abundant in the serum of patients with PCS, matching observations on the transcriptomic level in the muscle tissue. We hypothesize that the initial viral infection may have caused immune-mediated structural changes of the microvasculature, potentially explaining the exercise-dependent fatigue and muscle pain.

Chimeric autoantibody receptor T cells deplete NMDA receptor-specific B cells.

Anti-NMDA receptor (NMDAR) autoantibodies cause NMDAR encephalitis, the most common autoimmune encephalitis, leading to psychosis, seizures, and autonomic dysfunction. Current treatments comprise broad immunosuppression or non-selective antibody removal. We developed NMDAR-specific chimeric autoantibody receptor (NMDAR-CAAR) T cells to selectively eliminate anti-NMDAR B cells and disease-causing autoantibodies. NMDAR-CAARs consist of an extracellular multi-subunit NMDAR autoantigen fused to intracellular 4-1BB/CD3ζ domains. NMDAR-CAAR T cells recognize a large panel of human patient-derived autoantibodies, release effector molecules, proliferate, and selectively kill antigen-specific target cell lines even in the presence of high autoantibody concentrations. In a passive transfer mouse model, NMDAR-CAAR T cells led to depletion of an anti-NMDAR B cell line and sustained reduction of autoantibody levels without notable off-target toxicity. Treatment of patients may reduce side effects, prevent relapses, and improve long-term prognosis. Our preclinical work paves the way for CAAR T cell phase I/II trials in NMDAR encephalitis and further autoantibody-mediated diseases.

Detection of recurrent high-grade glioma using microstructure characteristics of distinct metabolic compartments in a multimodal and integrative 18F-FET PET/fast-DKI approach.

OBJECTIVES: Differentiation between high-grade glioma (HGG) and post-treatment-related effects (PTRE) is challenging, but advanced imaging techniques were shown to provide benefit. We aim to investigate microstructure characteristics of metabolic compartments identified from amino acid PET and to evaluate the diagnostic potential of this multimodal and integrative O-(2-18F-fluoroethyl)-L-tyrosine-(FET)-PET and fast diffusion kurtosis imaging (DKI) approach for the detection of recurrence and IDH genotyping. METHODS: Fifty-nine participants with neuropathologically confirmed recurrent HGG (n = 39) or PTRE (n = 20) were investigated using static 18F-FET PET and a fast-DKI variant. PET and advanced diffusion metrics of metabolically defined (80-100% and 60-75% areas of 18F-FET uptake) compartments were assessed. Comparative analysis was performed using Mann-Whitney U tests with Holm-Sídák multiple-comparison test and Wilcoxon signed-rank test. Receiver operating characteristic (ROC) curves, regression, and Spearman's correlation analysis were used for statistical evaluations. RESULTS: Compared to PTRE, recurrent HGG presented increased 18F-FET uptake and diffusivity (MD60), but lower (relative) mean kurtosis tensor (rMKT60) and fractional anisotropy (FA60) (respectively p < .05). Diffusion metrics determined from the metabolic periphery showed improved diagnostic performance - most pronounced for FA60 (AUC = 0.86, p < .001), which presented similar benefit to 18F-FET PET (AUC = 0.86, p < .001) and was negatively correlated with amino acid uptake (rs = - 0.46, p < .001). When PET and DKI metrics were evaluated in a multimodal biparametric approach, TBRmax + FA60 showed highest diagnostic accuracy (AUC = 0.93, p < .001), which improved the detection of relapse compared to PET alone (difference in AUC = 0.069, p = .04). FA60 and MD60 distinguished the IDH genotype in the post-treatment setting. CONCLUSION: Detection of glioma recurrence benefits from a multimodal and integrative PET/DKI approach, which presented significant diagnostic advantage to the assessment based on PET alone. CLINICAL RELEVANCE STATEMENT: A multimodal and integrative 18F-FET PET/fast-DKI approach for the non-invasive microstructural characterization of metabolic compartments provided improved diagnostic capability for differentiation between recurrent glioma and post-treatment-related changes, suggesting a role for the diagnostic workup of patients in post-treatment settings. KEY POINTS: • Multimodal PET/MRI with integrative analysis of 18F-FET PET and fast-DKI presents clinical benefit for the assessment of CNS cancer, particularly for the detection of recurrent high-grade glioma. • Microstructure markers of the metabolic periphery yielded biologically pertinent estimates characterising the tumour microenvironment, and, thereby, presented improved diagnostic accuracy with similar accuracy to amino acid PET. • Combined 18F-FET PET/fast-DKI achieved the best diagnostic performance for detection of high-grade glioma relapse with significant benefit to the assessment based on PET alone.

Whole genome sequencing reveals insights into hepatitis E virus genome diversity, and virus compartmentalization in chronic hepatitis E.

BACKGROUND: Hepatitis E virus (HEV) is a leading cause of acute hepatitis and can cause chronic infections in immunocompromised patients. Although HEV infections can be treated with ribavirin, antiviral efficacy is hampered by resistance mutations, normally detected by virus sequencing. OBJECTIVES: High-throughput sequencing (HTS) allows for cost-effective complete viral genome sequencing. This enables the discovery and delineation of new subtypes, and revised the recognition of quasispecies and putative resistance mutations. However, HTS is challenged by factors including low viral load, sample degradation, high host background, and high viral diversity. STUDY DESIGN: We apply complete genome sequencing strategies for HEV, including a targeted enrichment approach. These approaches were used to investigate sequence diversity in HEV RNA-positive animal and human samples and intra-host diversity in a chronically infected patient. RESULTS: Here, we describe the identification of potential novel subtypes in a blood donation (genotype 3) and in an ancient livestock sample (genotype 7). In a chronically infected patient, we successfully investigated intra-host virus diversity, including the presence of ribavirin resistance mutations. Furthermore, we found convincing evidence for HEV compartmentalization, including the central nervous system, in this patient. CONCLUSIONS: Targeted enrichment of viral sequences enables the generation of complete genome sequences from a variety of difficult sample materials. Moreover, it enables the generation of greater sequence coverage allowing more advanced analyses. This is key for a better understanding of virus diversity. Investigation of existing ribavirin resistance, in the context of minorities or compartmentalization, may be critical in treatment strategies of HEV patients.

Predictive IDH Genotyping Based on the Evaluation of Spatial Metabolic Heterogeneity by Compartmental Uptake Characteristics in Preoperative Glioma Using 18F-FET PET.

Molecular markers are of increasing importance for classifying, treating, and determining the prognosis for central nervous system tumors. Isocitrate dehydrogenase (IDH) is a critical regulator of glucose and amino acid metabolism. Our objective was to investigate metabolic reprogramming of glioma using compartmental uptake (CU) characteristics in O-(2-18F-fluoroethyl)-l-tyrosine (FET) PET and to evaluate its diagnostic potential for IDH genotyping. Methods: Between 2017 and 2022, patients with confirmed glioma were preoperatively investigated using static 18F-FET PET. Metabolic tumor volume (MTV), MTV for 60%-100% uptake (MTV60), and T2-weighted and contrast-enhancing lesion volumes were automatically segmented using U-Net neural architecture and isocontouring. Volume intersections were determined using the Dice coefficient. Uptake characteristics were determined for metabolically defined compartments (central [80%-100%] and peripheral [60%-75%] areas of 18F-FET uptake). CU ratio was defined as the fraction between the peripheral and central compartments. Mean target-to-background ratio was calculated. Comparisons were performed using parametric and nonparametric tests. Receiver-operating-characteristic curves, regression, and correlation were used for statistical analysis. Results: In total, 52 participants (male, 27, female, 25; mean age ± SD, 51 ± 16 y) were evaluated. MTV60 was greater and distinct from contrast-enhancing lesion volume (P = 0.046). IDH-mutated tumors presented a greater volumetric CU ratio and SUV CU ratio than IDH wild-type tumors (P < 0.05). Volumetric CU ratio determined IDH genotype with excellent diagnostic performance (area under the curve [AUC], 0.88; P < 0.001) at more than 5.49 (sensitivity, 86%, specificity, 90%), because IDH-mutated tumors presented a greater peripheral metabolic compartment than IDH wild-type tumors (P = 0.045). MTV60 and MTV were not suitable for IDH classification (P > 0.05). SUV CU ratio (AUC, 0.72; P = 0.005) and target-to-background ratio (AUC, 0.68; P = 0.016) achieved modest diagnostic performance-inferior to the volumetric CU ratio. Furthermore, the classification of loss of heterozygosity of chromosomes 1p and 19q (AUC, 0.75; P = 0.019), MGMT promoter methylation (AUC, 0.70; P = 0.011), and ATRX loss (AUC, 0.73; P = 0.004) by amino acid PET was evaluated. Conclusion: We proposed parametric 18F-FET PET as a noninvasive metabolic biomarker for the evaluation of CU characteristics, which differentiated IDH genotype with excellent diagnostic performance, establishing a critical association between spatial metabolic heterogeneity, mitochondrial tricarboxylic acid cycle, and genomic features with critical implications for clinical management and the diagnostic workup of patients with central nervous system cancer.

Hypertrophic Pachymeningitis with Persistent Intrathecal Inflammation Secondary to Neurosarcoidosis Treated with Intraventricular Chemotherapy: A Case Report.

Hypertrophic pachymeningitis (HP) is a rare immune-mediated disease characterized by thickening of the dura mater with consecutive cranial neuropathy. While HP is usually treated with systemic immunotherapies, response to therapy is variable and may be limited by insufficient drug concentrations in the brain. We report on a 57-year-old patient with HP manifesting with vision and hearing loss who had sustained clinical progression despite various systemic immunotherapies. Intraventricular chemotherapy with methotrexate, cytarabine, and dexamethasone was initiated. We present clinical, imaging and cerebrospinal fluid (CSF) findings, including cytokine levels before and after intraventricular treatment: rapid decrease of cell count, lactate and profibrotic cytokine levels in the CSF following intraventricular chemotherapy was paralleled by a mild reduction of dura thickness in MRI. The already severely impaired visual acuity and hearing loss did not progress further. Treatment was complicated by exacerbation of previously subtle psychiatric symptoms. Follow-up was terminated after 6 months as the patient suffered from a fatal ischemic stroke. Autopsy revealed neurosarcoidosis as the underlying cause of HP. This case report suggests that intrathecal chemotherapy can reduce the inflammatory milieu in the CNS and should be considered for treatment-refractory HP before irreversible damage of cranial nerves has occurred.

HIF prolyl hydroxylase 2/3 deletion disrupts astrocytic integrity and exacerbates neuroinflammation.

Astrocytes constitute the parenchymal border of the blood-brain barrier (BBB), modulate the exchange of soluble and cellular elements, and are essential for neuronal metabolic support. Thus, astrocytes critically influence neuronal network integrity. In hypoxia, astrocytes upregulate a transcriptional program that has been shown to boost neuroprotection in several models of neurological diseases. We investigated transgenic mice with astrocyte-specific activation of the hypoxia-response program by deleting the oxygen sensors, HIF prolyl-hydroxylase domains 2 and 3 (Phd2/3). We induced astrocytic Phd2/3 deletion after onset of clinical signs in experimental autoimmune encephalomyelitis (EAE) that led to an exacerbation of the disease mediated by massive immune cell infiltration. We found that Phd2/3-ko astrocytes, though expressing a neuroprotective signature, exhibited a gradual loss of gap-junctional Connexin-43 (Cx43), which was induced by vascular endothelial growth factor-alpha (Vegf-a) expression. These results provide mechanistic insights into astrocyte biology, their critical role in hypoxic states, and in chronic inflammatory CNS diseases.

Distinct tissue niches direct lung immunopathology via CCL18 and CCL21 in severe COVID-19.

Prolonged lung pathology has been associated with COVID-19, yet the cellular and molecular mechanisms behind this chronic inflammatory disease are poorly understood. In this study, we combine advanced imaging and spatial transcriptomics to shed light on the local immune response in severe COVID-19. We show that activated adventitial niches are crucial microenvironments contributing to the orchestration of prolonged lung immunopathology. Up-regulation of the chemokines CCL21 and CCL18 associates to endothelial-to-mesenchymal transition and tissue fibrosis within these niches. CCL21 over-expression additionally links to the local accumulation of T cells expressing the cognate receptor CCR7. These T cells are imprinted with an exhausted phenotype and form lymphoid aggregates that can organize in ectopic lymphoid structures. Our work proposes immune-stromal interaction mechanisms promoting a self-sustained and non-resolving local immune response that extends beyond active viral infection and perpetuates tissue remodeling.

The brain reacting to COVID-19: analysis of the cerebrospinal fluid proteome, RNA and inflammation.

Patients with COVID-19 can have a variety of neurological symptoms, but the active involvement of central nervous system (CNS) in COVID-19 remains unclear. While routine cerebrospinal fluid (CSF) analyses in patients with neurological manifestations of COVID-19 generally show no or only mild inflammation, more detailed data on inflammatory mediators in the CSF of patients with COVID-19 are scarce. We studied the inflammatory response in paired CSF and serum samples of patients with COVID-19 (n = 38). Patients with herpes simplex virus encephalitis (HSVE, n = 10) and patients with non-inflammatory, non-neurodegenerative neurological diseases (n = 28) served as controls. We used proteomics, enzyme-linked immunoassays, and semiquantitative cytokine arrays to characterize inflammatory proteins. Autoantibody screening was performed with cell-based assays and native tissue staining. RNA sequencing of long-non-coding RNA and circular RNA was done to study the transcriptome. Proteomics on single protein level and subsequent pathway analysis showed similar yet strongly attenuated inflammatory changes in the CSF of COVID-19 patients compared to HSVE patients with, e.g., downregulation of the apolipoproteins and extracellular matrix proteins. Protein upregulation of the complement system, the serpin proteins pathways, and other proteins including glycoproteins alpha-2 and alpha-1 acid. Importantly, calculation of interleukin-6, interleukin-16, and CXCL10 CSF/serum indices suggest that these inflammatory mediators reach the CSF from the systemic circulation, rather than being produced within the CNS. Antibody screening revealed no pathological levels of known neuronal autoantibodies. When stratifying COVID-19 patients into those with and without bacterial superinfection as indicated by elevated procalcitonin levels, inflammatory markers were significantly (p < 0.01) higher in those with bacterial superinfection. RNA sequencing in the CSF revealed 101 linear RNAs comprising messenger RNAs, and two circRNAs being significantly differentially expressed in COVID-19 than in non-neuroinflammatory controls and neurodegenerative patients. Our findings may explain the absence of signs of intrathecal inflammation upon routine CSF testing despite the presence of SARS-CoV2 infection-associated neurological symptoms. The relevance of blood-derived mediators of inflammation in the CSF for neurological COVID-19 and post-COVID-19 symptoms deserves further investigation.

Functional proteomic profiling links deficient DNA clearance with increased mortality in individuals with severe COVID-19 pneumonia.

The factors that influence survival during severe infection are unclear. Extracellular chromatin drives pathology, but the mechanisms enabling its accumulation remain elusive. Here, we show that in murine sepsis models, splenocyte death interferes with chromatin clearance through the release of the DNase I inhibitor actin. Actin-mediated inhibition was compensated by upregulation of DNase I or the actin scavenger gelsolin. Splenocyte death and neutrophil extracellular trap (NET) clearance deficiencies were prevalent in individuals with severe COVID-19 pneumonia or microbial sepsis. Activity tracing by plasma proteomic profiling uncovered an association between low NET clearance and increased COVID-19 pathology and mortality. Low NET clearance activity with comparable proteome associations was prevalent in healthy donors with low-grade inflammation, implicating defective chromatin clearance in the development of cardiovascular disease and linking COVID-19 susceptibility to pre-existing conditions. Hence, the combination of aberrant chromatin release with defects in protective clearance mechanisms lead to poor survival outcomes.

A ubiquitous bone marrow reservoir of preexisting SARS-CoV-2-reactive memory CD4+ T lymphocytes in unexposed individuals.

Circulating, blood-borne SARS-CoV-2-reactive memory T cells in persons so far unexposed to SARS-CoV-2 or the vaccines have been described in 20-100% of the adult population. They are credited with determining the efficacy of the immune response in COVID-19. Here, we demonstrate the presence of preexisting memory CD4+ T cells reacting to peptides of the spike, membrane, or nucleocapsid proteins of SARS-CoV-2 in the bone marrow of all 17 persons investigated that had previously not been exposed to SARS-CoV-2 or one of the vaccines targeting it, with only 15 of these persons also having such cells detectable circulating in the blood. The preexisting SARS-CoV-2-reactive memory CD4+ T cells of the bone marrow are abundant and polyfunctional, with the phenotype of central memory T cells. They are tissue-resident, at least in those persons who do not have such cells in the blood, and about 30% of them express CD69. Bone marrow resident SARS-CoV-2-reactive memory CD4+ memory T cells are also abundant in vaccinated persons analyzed 10-168 days after 1°-4° vaccination. Apart from securing the bone marrow, preexisting cross-reactive memory CD4+ T cells may play an important role in shaping the systemic immune response to SARS-CoV-2 and the vaccines, and contribute essentially to the rapid establishment of long-lasting immunity provided by memory plasma cells, already upon primary infection.