Severe mpox in an immunocompromised patient complicated by deep tissue infection: A case report.

Objectives: We report prolonged mpox (>14 weeks) in a patient with HIV complicated by deep tissue MPXV infection despite two courses of tecovirimat treatment. Methods: MPXV-DNA levels in lesional swabs, blood and tissue were quantified by qPCR. Anti-MPXV antibodies were analyzed by IF and VNT. Infectivity was assessed by virus isolation. Sequencing was performed to assess for tecovirimat resistance mutations and quantitative results were obtained by digital SNP PCR (A288P). Results: The patient's clinical condition improved significantly during both tecovirimat treatment courses (each 14 days), yet we observed persistent MPXV-DNA in lesions accompanied by viremia (mean 1.4 × 104 copies/ml) for >14 weeks. A deep tissue infection driven by MPXV complicated the clinical course (week 9). Presence of infectious virus within the tissue and high infectious titers (>106 PFU/ml) were observed. The VP37 protein sequence revealed A288P substitutions. Digital PCR showed 1 % and less abundance (A288P) during first treatment course (blood and swabs), with increasing proportion during second course (week 8-9; 28 % in blood and swabs), however the mutation was absent in samples from deep tissue infection and MPXV isolates (week 9) indicating compartimentalization. Morphological fully enveloped MPXV partices visualized by TEM in necrotic areas suggesting tecovirimat treatment failure in the deep tissue compartment. Conclusion: Our data provide evidence that Tecovirimat treatment selects for compartimentalized viral mutations (A288P). While the patient clinically benefited from repeated tecovirimat course, emergence of viral muations and deep tissue infection emphasizes the challenge and importance of infectious disease monitoring in mpox patient management.

Lassa virus persistence with high viral titers following experimental infection in its natural reservoir host, Mastomys natalensis.

Lassa virus (LASV) outbreaks in West Africa pose a significant public health threat. We investigated the infection phenotype and transmission (horizontal and vertical) of LASV strain Ba366 in its natural host, Mastomys natalensis. Here we analyze viral RNA levels in body fluids, virus titers in organs and antibody presence in blood. In adults and 2-week-old animals, LASV causes transient infections with subsequent seroconversion. However, mice younger than two weeks exhibit persistent infections lasting up to 16 months despite antibody presence. LASV can be detected in various body fluids, organs, and cell types, primarily in lung, kidney, and gonadal epithelial cells. Despite the systemic virus presence, no pathological alterations in organs are observed. Infected animals efficiently transmit the virus throughout their lives. Our findings underscore the crucial role of persistently infected individuals, particularly infected females and their progeny, in LASV dissemination within the host population.

Cleavage site-directed antibodies reveal the prion protein in humans is shed by ADAM10 at Y226 and associates with misfolded protein deposits in neurodegenerative diseases.

Proteolytic cell surface release ('shedding') of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

Inefficient tissue immune response against MPXV in an immunocompromised mpox patient.

The recent outbreak of monkeypox virus (MPXV) was unprecedented in its size and distribution. Those living with uncontrolled HIV and low CD4 T cell counts might develop a fulminant clinical mpox course with increased mortality, secondary infections, and necrotizing lesions. Fatal cases display a high and widespread MPXV tissue burden. The underlying pathomechanisms are not fully understood. We report here the pathological findings of an MPXV-driven abscess in gastrocnemius muscle requiring surgery in an immunocompromised patient with severe mpox. Presence of virus particles and infectivity were confirmed by electron microscopy, expansion microscopy, and virus culture, respectively. MPXV tissue distribution by immunohistochemistry (IHC) showed a necrotic core with infection of different cell types. In contrast, at the lesion rim fibroblasts were mainly infected. Immune cells were almost absent in the necrotic core, but were abundant at the infection rim and predominantly macrophages. Further, we detected high amounts of alternatively activated GPNMB+-macrophages at the lesion border. Of note, macrophages only rarely colocalized with virus-infected cells. Insufficient clearance of infected cells and infection of lesion-associated fibroblasts sustained by the abundance of profibrotic macrophages might lead to the coalescing of lesions and the severe and persistent clinical mpox course observed in immunocompromised patients.

APOE3 Christchurch Heterozygosity and Autosomal Dominant Alzheimer's Disease.

BACKGROUND: Variants in APOE and PSEN1 (encoding apolipoprotein E and presenilin 1, respectively) alter the risk of Alzheimer's disease. We previously reported a delay of cognitive impairment in a person with autosomal dominant Alzheimer's disease caused by the PSEN1 E280A variant who also had two copies of the apolipoprotein E3 Christchurch variant (APOE3 Ch). Heterozygosity for the APOE3 Ch variant may influence the age at which the onset of cognitive impairment occurs. We assessed this hypothesis in a population in which the PSEN1 E280A variant is prevalent. METHODS: We analyzed data from 27 participants with one copy of the APOE3 Ch variant among 1077 carriers of the PSEN1 E280A variant in a kindred from Antioquia, Colombia, to estimate the age at the onset of cognitive impairment and dementia in this group as compared with persons without the APOE3 Ch variant. Two participants underwent brain imaging, and autopsy was performed in four participants. RESULTS: Among carriers of PSEN1 E280A who were heterozygous for the APOE3 Ch variant, the median age at the onset of cognitive impairment was 52 years (95% confidence interval [CI], 51 to 58), in contrast to a matched group of PSEN1 E280A carriers without the APOE3 Ch variant, among whom the median age at the onset was 47 years (95% CI, 47 to 49). In two participants with the APOE3 Ch and PSEN1 E280A variants who underwent brain imaging, 18F-fluorodeoxyglucose positron-emission tomographic (PET) imaging showed relatively preserved metabolic activity in areas typically involved in Alzheimer's disease. In one of these participants, who underwent 18F-flortaucipir PET imaging, tau findings were limited as compared with persons with PSEN1 E280A in whom cognitive impairment occurred at the typical age in this kindred. Four studies of autopsy material obtained from persons with the APOE3 Ch and PSEN1 E280A variants showed fewer vascular amyloid pathologic features than were seen in material obtained from persons who had the PSEN1 E280A variant but not the APOE3 Ch variant. CONCLUSIONS: Clinical data supported a delayed onset of cognitive impairment in persons who were heterozygous for the APOE3 Ch variant in a kindred with a high prevalence of autosomal dominant Alzheimer's disease. (Funded by Good Ventures and others.).

APOE3 Christchurch modulates ß-catenin/Wnt signaling in iPS cell-derived cerebral organoids from Alzheimer's cases.

A patient with the PSEN1 E280A mutation and homozygous for APOE3 Christchurch (APOE3Ch) displayed extreme resistance to Alzheimer's disease (AD) cognitive decline and tauopathy, despite having a high amyloid burden. To further investigate the differences in biological processes attributed to APOE3Ch, we generated induced pluripotent stem (iPS) cell-derived cerebral organoids from this resistant case and a non-protected control, using CRISPR/Cas9 gene editing to modulate APOE3Ch expression. In the APOE3Ch cerebral organoids, we observed a protective pattern from early tau phosphorylation. ScRNA sequencing revealed regulation of Cadherin and Wnt signaling pathways by APOE3Ch, with immunostaining indicating elevated ß-catenin protein levels. Further in vitro reporter assays unexpectedly demonstrated that ApoE3Ch functions as a Wnt3a signaling enhancer. This work uncovered a neomorphic molecular mechanism of protection of ApoE3 Christchurch, which may serve as the foundation for the future development of protected case-inspired therapeutics targeting AD and tauopathies.

Genetic modifiers of cognitive decline in PSEN1 E280A Alzheimer's disease.

INTRODUCTION: Rate of cognitive decline (RCD) in Alzheimer's disease (AD) determines the degree of impairment for patients and of burden for caretakers. We studied the association of RCD with genetic variants in AD. METHODS: RCD was evaluated in 62 familial AD (FAD) and 53 sporadic AD (SAD) cases, and analyzed by whole-exome sequencing for association with common exonic functional variants. Findings were validated in post mortem brain tissue. RESULTS: One hundred seventy-two gene variants in FAD, and 227 gene variants in SAD associated with RCD. In FAD, performance decline of the immediate recall of the Rey-Osterrieth figure test associated with 122 genetic variants. Olfactory receptor OR51B6 showed the highest number of associated variants. Its expression was detected in temporal cortex neurons. DISCUSSION: Impaired olfactory function has been associated with cognitive impairment in AD. Genetic variants in these or other genes could help to identify risk of faster memory decline in FAD and SAD patients.

Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation.

BACKGROUND: Pulmonary hypertension (PH) is a chronic vascular disease characterized, among other abnormalities, by hyperproliferative smooth muscle cells and a perturbed cellular redox and metabolic balance. Oxidants induce cell cycle arrest to halt proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 (cyclin-dependent kinase 4) and investigate its role in cell proliferation and PH. METHODS: Oxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells and human pulmonary arterial endothelial cells. Site-directed mutagenesis, tandem mass-spectrometry, cell-based experiments, in vitro kinase activity assays, in silico structural modeling, and a novel redox-dead constitutive knock-in mouse were utilized to investigate the nature and definitively establish the importance of CDK4 cysteine modification in pulmonary vascular cell proliferation. Furthermore, the cyclin D-CDK4 oxidation was assessed in vivo in the pulmonary arteries and isolated human pulmonary arterial smooth muscle cells of patients with pulmonary arterial hypertension and in 3 preclinical models of PH. RESULTS: Cyclin D-CDK4 forms a reversible oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively, in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity, decrease Rb (retinoblastoma) protein phosphorylation, and induce cell cycle arrest. Mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases cell proliferation rate and alleviates disease phenotype in an experimental mouse PH model, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and human pulmonary arterial smooth muscle cells from patients with pulmonary arterial hypertension display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in human pulmonary arterial hypertension. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental PH models by mitigating pulmonary vascular remodeling. CONCLUSIONS: A novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for the design of a selective covalent inhibitor predicted to be beneficial in PH.

Using autopsies to dissect COVID-19 pathogenesis.

The emergence of SARS-CoV-2 has resulted in millions of deaths as a result of COVID-19. Suitable models were missing at the beginning of the pandemic, and studies investigating disease pathogenesis relied on patients who had succumbed to COVID-19. Since then, autopsies of patients have substantially contributed to our understanding of the pathogenesis of COVID-19 and associated major organ complications. Here we summarize how autopsies have complemented experimental studies, mainly in animal models, and how they have facilitated critical knowledge of COVID-19 to improve daily clinical practice and develop therapeutic interventions. Employing advanced histopathologic and molecular genetic methods in post-mortem tissues, the COVID-19 pandemic has highlighted the importance of autopsies for virology research and clinical practice in current and emerging infectious diseases.

APOE4 impairs the microglial response in Alzheimer's disease by inducing TGFß-mediated checkpoints.

The APOE4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). The contribution of microglial APOE4 to AD pathogenesis is unknown, although APOE has the most enriched gene expression in neurodegenerative microglia (MGnD). Here, we show in mice and humans a negative role of microglial APOE4 in the induction of the MGnD response to neurodegeneration. Deletion of microglial APOE4 restores the MGnD phenotype associated with neuroprotection in P301S tau transgenic mice and decreases pathology in APP/PS1 mice. MGnD-astrocyte cross-talk associated with ß-amyloid (Aß) plaque encapsulation and clearance are mediated via LGALS3 signaling following microglial APOE4 deletion. In the brains of AD donors carrying the APOE4 allele, we found a sex-dependent reciprocal induction of AD risk factors associated with suppression of MGnD genes in females, including LGALS3, compared to individuals homozygous for the APOE3 allele. Mechanistically, APOE4-mediated induction of ITGB8-transforming growth factor-ß (TGFß) signaling impairs the MGnD response via upregulation of microglial homeostatic checkpoints, including Inpp5d, in mice. Deletion of Inpp5d in microglia restores MGnD-astrocyte cross-talk and facilitates plaque clearance in APP/PS1 mice. We identify the microglial APOE4-ITGB8-TGFß pathway as a negative regulator of microglial response to AD pathology, and restoring the MGnD phenotype via blocking ITGB8-TGFß signaling provides a promising therapeutic intervention for AD.

CYP19A1 mediates severe SARS-CoV-2 disease outcome in males.

Male sex represents one of the major risk factors for severe COVID-19 outcome. However, underlying mechanisms that mediate sex-dependent disease outcome are as yet unknown. Here, we identify the CYP19A1 gene encoding for the testosterone-to-estradiol metabolizing enzyme CYP19A1 (also known as aromatase) as a host factor that contributes to worsened disease outcome in SARS-CoV-2-infected males. We analyzed exome sequencing data obtained from a human COVID-19 cohort (n = 2,866) using a machine-learning approach and identify a CYP19A1-activity-increasing mutation to be associated with the development of severe disease in men but not women. We further analyzed human autopsy-derived lungs (n = 86) and detect increased pulmonary CYP19A1 expression at the time point of death in men compared with women. In the golden hamster model, we show that SARS-CoV-2 infection causes increased CYP19A1 expression in the lung that is associated with dysregulated plasma sex hormone levels and reduced long-term pulmonary function in males but not females. Treatment of SARS-CoV-2-infected hamsters with a clinically approved CYP19A1 inhibitor (letrozole) improves impaired lung function and supports recovery of imbalanced sex hormones specifically in males. Our study identifies CYP19A1 as a contributor to sex-specific SARS-CoV-2 disease outcome in males. Furthermore, inhibition of CYP19A1 by the clinically approved drug letrozole may furnish a new therapeutic strategy for individualized patient management and treatment.

Intestinal IL-1ß Plays a Role in Protecting against SARS-CoV-2 Infection.

The intestine is constantly balancing the maintenance of a homeostatic microbiome and the protection of the host against pathogens such as viruses. Many cytokines mediate protective inflammatory responses in the intestine, among them IL-1ß. IL-1ß is a proinflammatory cytokine typically activated upon specific danger signals sensed by the inflammasome. SARS-CoV-2 is capable of infecting multiple organs, including the intestinal tract. Severe cases of COVID-19 were shown to be associated with a dysregulated immune response, and blocking of proinflammatory pathways was demonstrated to improve patient survival. Indeed, anakinra, an Ab against the receptor of IL-1ß, has recently been approved to treat patients with severe COVID-19. However, the role of IL-1ß during intestinal SARS-CoV-2 infection has not yet been investigated. Here, we analyzed postmortem intestinal and blood samples from patients who died of COVID-19. We demonstrated that high levels of intestinal IL-1ß were associated with longer survival time and lower intestinal SARS-CoV-2 RNA loads. Concurrently, type I IFN expression positively correlated with IL-1ß levels in the intestine. Using human intestinal organoids, we showed that autocrine IL-1ß sustains RNA expression of IFN type I by the intestinal epithelial layer. These results outline a previously unrecognized key role of intestinal IL-1ß during SARS-CoV-2 infection.

Vagus nerve inflammation contributes to dysautonomia in COVID-19.

Dysautonomia has substantially impacted acute COVID-19 severity as well as symptom burden after recovery from COVID-19 (long COVID), yet the underlying causes remain unknown. Here, we hypothesized that vagus nerves are affected in COVID-19 which might contribute to autonomic dysfunction. We performed a histopathological characterization of postmortem vagus nerves from COVID-19 patients and controls, and detected SARS-CoV-2 RNA together with inflammatory cell infiltration composed primarily of monocytes. Furthermore, we performed RNA sequencing which revealed a strong inflammatory response of neurons, endothelial cells, and Schwann cells which correlated with SARS-CoV-2 RNA load. Lastly, we screened a clinical cohort of 323 patients to detect a clinical phenotype of vagus nerve affection and found a decreased respiratory rate in non-survivors of critical COVID-19. Our data suggest that SARS-CoV-2 induces vagus nerve inflammation followed by autonomic dysfunction which contributes to critical disease courses and might contribute to dysautonomia observed in long COVID.

Resilience to autosomal dominant Alzheimer's disease in a Reelin-COLBOS heterozygous man.

We characterized the world's second case with ascertained extreme resilience to autosomal dominant Alzheimer's disease (ADAD). Side-by-side comparisons of this male case and the previously reported female case with ADAD homozygote for the APOE3 Christchurch (APOECh) variant allowed us to discern common features. The male remained cognitively intact until 67 years of age despite carrying a PSEN1-E280A mutation. Like the APOECh carrier, he had extremely elevated amyloid plaque burden and limited entorhinal Tau tangle burden. He did not carry the APOECh variant but was heterozygous for a rare variant in RELN (H3447R, termed COLBOS after the Colombia-Boston biomarker research study), a ligand that like apolipoprotein E binds to the VLDLr and APOEr2 receptors. RELN-COLBOS is a gain-of-function variant showing stronger ability to activate its canonical protein target Dab1 and reduce human Tau phosphorylation in a knockin mouse. A genetic variant in a case protected from ADAD suggests a role for RELN signaling in resilience to dementia.

Expansion-enhanced super-resolution radial fluctuations enable nanoscale molecular profiling of pathology specimens.

Expansion microscopy physically enlarges biological specimens to achieve nanoscale resolution using diffraction-limited microscopy systems1. However, optimal performance is usually reached using laser-based systems (for example, confocal microscopy), restricting its broad applicability in clinical pathology, as most centres have access only to light-emitting diode (LED)-based widefield systems. As a possible alternative, a computational method for image resolution enhancement, namely, super-resolution radial fluctuations (SRRF)2,3, has recently been developed. However, this method has not been explored in pathology specimens to date, because on its own, it does not achieve sufficient resolution for routine clinical use. Here, we report expansion-enhanced super-resolution radial fluctuations (ExSRRF), a simple, robust, scalable and accessible workflow that provides a resolution of up to 25 nm using LED-based widefield microscopy. ExSRRF enables molecular profiling of subcellular structures from archival formalin-fixed paraffin-embedded tissues in complex clinical and experimental specimens, including ischaemic, degenerative, neoplastic, genetic and immune-mediated disorders. Furthermore, as examples of its potential application to experimental and clinical pathology, we show that ExSRRF can be used to identify and quantify classical features of endoplasmic reticulum stress in the murine ischaemic kidney and diagnostic ultrastructural features in human kidney biopsies.

New Postmortem Perspective on Emerging SARS-CoV-2 Variants of Concern, Germany.

We performed autopsies on persons in Germany who died from COVID-19 and observed higher nasopharyngeal SARS-CoV-2 viral loads for variants of concern (VOC) compared with non-VOC lineages. Pulmonary inflammation and damage appeared higher in non-VOC than VOC lineages until adjusted for vaccination status, suggesting COVID-19 vaccination may mitigate pulmonary damage.

Extracellular Matrix Profiling and Disease Modelling in Engineered Vascular Smooth Muscle Cell Tissues.

Aortic smooth muscle cells (SMCs) have an intrinsic role in regulating vessel homeostasis and pathological remodelling. In two-dimensional (2D) cell culture formats, however, SMCs are not embedded in their physiological extracellular matrix (ECM) environment. To overcome the limitations of conventional 2D SMC cultures, we established a 3D in vitro model of engineered vascular smooth muscle cell tissues (EVTs). EVTs were casted from primary murine aortic SMCs by suspending a SMC-fibrin master mix between two flexible silicon-posts at day 0 before prolonged culture up to 14 days. Immunohistochemical analysis of EVT longitudinal sections demonstrated that SMCs were aligned, viable and secretory. Mass spectrometry-based proteomics analysis of murine EVT lysates was performed and identified 135 matrisome proteins. Proteoglycans, including the large aggregating proteoglycan versican, accumulated within EVTs by day 7 of culture. This was followed by the deposition of collagens, elastin-binding proteins and matrix regulators up to day 14 of culture. In contrast to 2D SMC controls, accumulation of versican occurred in parallel to an increase in versikine, a cleavage product mediated by proteases of the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family. Next, we tested the response of EVTs to stimulation with transforming growth factor beta-1 (TGFß-1). EVTs contracted in response to TGFß-1 stimulation with altered ECM composition. In contrast, treatment with the pharmacological activin-like kinase inhibitor (ALKi) SB 431542 suppressed ECM secretion. As a disease stimulus, we performed calcification assays. The ECM acts as a nidus for calcium phosphate deposition in the arterial wall. We compared the onset and extent of calcification in EVTs and 2D SMCs cultured under high calcium and phosphate conditions for 7 days. Calcified EVTs displayed increased tissue stiffness by up to 30 % compared to non-calcified controls. Unlike the rapid calcification of SMCs in 2D cultures, EVTs sustained expression of the calcification inhibitor matrix Gla protein and allowed for better discrimination of the calcification propensity between independent biological replicates. In summary, EVTs are an intuitive and versatile model to investigate ECM synthesis and turnover by SMCs in a 3D environment. Unlike conventional 2D cultures, EVTs provide a more relevant pathophysiological model for retention of the nascent ECM produced by SMCs.

Loss of Homeostatic Microglia Signature in Prion Diseases.

Prion diseases are neurodegenerative diseases that affect humans and animals. They are always fatal and, to date, no treatment exists. The hallmark of prion disease pathophysiology is the misfolding of an endogenous protein, the cellular prion protein (PrPC), into its disease-associated isoform PrPSc. Besides the aggregation and deposition of misfolded PrPSc, prion diseases are characterized by spongiform lesions and the activation of astrocytes and microglia. Microglia are the innate immune cells of the brain. Activated microglia and astrocytes represent a common pathological feature in neurodegenerative disorders. The role of activated microglia has already been studied in prion disease mouse models; however, it is still not fully clear how they contribute to disease progression. Moreover, the role of microglia in human prion diseases has not been thoroughly investigated thus far, and specific molecular pathways are still undetermined. Here, we review the current knowledge on the different roles of microglia in prion pathophysiology. We discuss microglia markers that are also dysregulated in other neurodegenerative diseases including microglia homeostasis markers. Data on murine and human brain tissues show that microglia are highly dysregulated in prion diseases. We highlight here that the loss of homeostatic markers may especially stand out.