COVID-19 Pathology in the Lung, Kidney, Heart and Brain: The Different Roles of T-Cells, Macrophages, and Microthrombosis.

Here, we aim to describe COVID-19 pathology across different tissues to clarify the disease's pathophysiology. Lungs, kidneys, hearts, and brains from nine COVID-19 autopsies were compared by using antibodies against SARS-CoV-2, macrophages-microglia, T-lymphocytes, B-lymphocytes, and activated platelets. Alzheimer's Disease pathology was also assessed. PCR techniques were used to verify the presence of viral RNA. COVID-19 cases had a short clinical course (0-32 days) and their mean age was 77.4 y/o. Hypoxic changes and inflammatory infiltrates were present across all tissues. The lymphocytic component in the lungs and kidneys was predominant over that of other tissues (p < 0.001), with a significantly greater presence of T-lymphocytes in the lungs (p = 0.020), which showed the greatest presence of viral antigens. The heart showed scant SARS-CoV-2 traces in the endothelium-endocardium, foci of activated macrophages, and rare lymphocytes. The brain showed scarce SARS-CoV-2 traces, prominent microglial activation, and rare lymphocytes. The pons exhibited the highest microglial activation (p = 0.017). Microthrombosis was significantly higher in COVID-19 lungs (p = 0.023) compared with controls. The most characteristic pathological features of COVID-19 were an abundance of T-lymphocytes and microthrombosis in the lung and relevant microglial hyperactivation in the brainstem. This study suggests that the long-term sequelae of COVID-19 derive from persistent inflammation, rather than persistent viral replication.

RNA-Seq profiling in peripheral blood mononuclear cells of amyotrophic lateral sclerosis patients and controls.

Coding and long non-coding RNA (lncRNA) metabolism is now revealing its crucial role in Amyotrophic Lateral Sclerosis (ALS) pathogenesis. In this work, we present a dataset obtained via Illumina RNA-seq analysis on Peripheral Blood Mononuclear Cells (PBMCs) from sporadic and mutated ALS patients (mutations in FUS, TARDBP, SOD1 and VCP genes) and healthy controls. This dataset allows the whole-transcriptome characterization of PBMCs content, both in terms of coding and non-coding RNAs, in order to compare the disease state to the healthy controls, both for sporadic patients and for mutated patients. Our dataset is a starting point for the omni-comprehensive analysis of coding and lncRNAs, from an easy to withdraw, manage and store tissue that shows to be a suitable model for RNA profiling in ALS.

A motor neuron strategy to save time and energy in neurodegeneration: adaptive protein stoichiometry.

Neurofilament proteins (Nf) are a biomarker of disease progression in amyotrophic lateral sclerosis (ALS). This study investigated whether there are major differences in expression from in vivo measurements of neurofilament isoforms, from the light chain, NfL (68 kDa), compared with larger proteins, the medium chain (NfM, 150 kDa) and the heavy (NfH, 200-210 kDa) chains in ALS patients and healthy controls. New immunological methods were combined with Nf subunit stoichiometry calculations and Monte Carlo simulations of a coarse-grained Nf brush model. Based on a physiological Nf subunit stoichiometry of 7 : 3 : 2 (NfL:NfM:NfH), we found an 'adaptive' Nf subunit stoichiometry of 24 : 2.4 : 1.6 in ALS. Adaptive Nf stoichiometry preserved NfL gyration radius in the Nf brush model. The energy and time requirements for Nf translation were 56 ± 27k ATP (5.6 h) in control subjects compared to 123 ± 102k (12.3 h) in ALS with 'adaptive' (24:2.4:1.6) Nf stoichiometry (not significant) and increased significantly to 355 ± 330k (35.5 h) with 'luxury' (7:3:2) Nf subunit stoichiometry (p < 0.0001 for each comparison). Longitudinal disease progression-related energy consumption was highest with a 'luxury' (7:3:2) Nf stoichiometry. Therefore, an energy and time-saving option for motor neurons is to shift protein expression from larger to smaller (cheaper) subunits, at little or no costs on a protein structural level, to compensate for increased energy demands.

Lower motor neuron syndrome in a patient with HER2-positive metastatic breast cancer: A case report and review of the literature.

Paraneoplastic neurological syndromes are very rare and often associated to breast, ovarian and small cells lung cancers. Paraneoplastic motor neuron diseases (MNDs) are even rarer, and frequently described in patients with breast cancer. We presented the first case of patient affected by HER2-positive breast tumor and possible paraneoplastic lower motor neuron disease. In literature, few cases are reported but no one highlights the tumor receptors' profile. Instead, HER2-positive breast cancers are prone to be related to anti-Yo-associated paraneoplastic cerebellar disorders. Anti-onconeural antibodies positivity can be rarely found, confirming that paraneoplastic MND have no specific biomarkers. The presence of CSF oligoclonal bands (OBs) suggests the presence of immune-mediated mechanism, in absence of other possible OBs causes.

Lymphoblastoid cell lines as a model to understand amyotrophic lateral sclerosis disease mechanisms.

In the past, amyotrophic lateral sclerosis (ALS) has been considered a 'neurocentric' disease; however, new evidence suggests that it should instead be looked at from a 'multisystemic' or 'non-neurocentric' point of view. From 2006, we focused on the study of non-neural cells: ALS patients' peripheral blood mononuclear cells (PMBCs) and lymphoblastoid cell lines (LCLs). Here, we characterize LCLs of sporadic ALS (sALS) and patients carrying SOD1, TARDBP and FUS mutations to identify an ALS biologically relevant molecular signature, and determine whether and how mutations differentially affect ALS-linked pathways. Although LCLs are different from motor neurons (MNs), in LCLs we found some features typical of degenerating MNs in ALS, i.e. protein aggregation and mitochondrial dysfunction. Moreover, different gene mutations have different effects on ALS cellular mechanisms. TARDBP and FUS mutations imbalance mitochondrial dynamism toward increased fusion, whereas sALS and SOD1 mutations mainly affect fission. With regards to protein aggregation and/or mislocalization, TARDBP and SOD1 mutations show the presence of aggregates, whereas FUS mutation does not induce protein aggregation and/or mislocalization. Finally, all LCLs, independently from mutation, are not able to work in a condition of excessive energy request, suggesting that mitochondria from ALS patients are characterized by a significant metabolic defect. Taken together, these data indicate that LCLs could be a valid cellular model in ALS research in the identification and study of specific pathological pathways.

Predictors of outcome in a large retrospective cohort of patients with transverse myelitis.

BACKGROUND: Transverse myelitis (TM) is an inflammatory disorder that can be idiopathic or associated with central nervous system autoimmune/dysimmune inflammatory diseases, connective tissue autoimmune diseases, or post-infectious neurological syndromes. Prognosis of initial TM presentations is uncertain. OBJECTIVE: To identify outcome predictors in TM. METHODS: Retrospective study on isolated TM at onset. Scores ⩾3 on the modified Rankin scale (mRS) marked high disability. RESULTS: A total of 159 patients were identified. TM was classified as follows: idiopathic (I-TM, n = 53), post-infectious (PI-TM, n = 48), associated with multiple sclerosis (MS-TM, n = 51), or neuromyelitis optica spectrum disorders/connective tissue autoimmune diseases/neurosarcoidosis ( n = 7). At follow-up (median, 55 months; interquartile range, 32-80), 42 patients were severely disabled, and patients with I-TM or PI-TM showed the worst outcomes. Predictors of disability were infectious antecedents, sphincter and pyramidal symptoms, high mRS scores, blood-cerebrospinal fluid barrier damage, lumbar magnetic resonance imaging (MRI) lesions on univariate analysis, and older age (odds ratio (OR), 1.1; 95% confidence interval (CI), 1.0-1.1), overt/subclinical involvement of the peripheral nervous system (PNS) (OR, 9.4; 95% CI, 2.2-41.0), complete TM (OR, 10.8; 95% CI, 3.4-34.5) on multivariate analysis. CONCLUSION: Our findings help define prognosis and therapies in TM at onset. Infectious antecedents and PNS involvement associate with severe prognosis. Nerve conduction studies and lumbar MRI could improve the prognostic assessment of this condition.

Meningeal melanomatosis: a challenge for timely diagnosis.

Neoplastic meningitis is a central nervous system complication that occurs in 3-5% of patients with cancer. Although most commonly seen in patients with disseminated disease, in a small percentage of patients, it may be the initial manifestation of cancer or even primitive in origin. In the absence of cancer history, the diagnosis of neoplastic meningitis may be challenging even for expert neurologists. Prognosis is poor, with a median overall survival of weeks from diagnosis. In the retrospective study herein, we described three cases of meningeal melanomatosis in patients without previous cancer history. The patients were diagnosed with significant delay (17 to 47 weeks from symptom onset) mainly due to the deferral in performing the appropriate testing. Even when the diagnosis was suspected, investigations by MRI, cerebrospinal fluid, or both proved at times unhelpful for confirmation. Prognosis was dismal, with a median survival of 4 weeks after diagnosis. Our observations are a cue to analyze the main pitfalls in the diagnosis of neoplastic meningitis in patients without cancer history and emphasize key elements that may facilitate early diagnosis.

Herpes simplex encephalitis in glioma patients: a challenging diagnosis.

OBJECTIVES: In recent years, herpes simplex encephalitis (HSE) has been reported with increasing frequency in settings of immunosuppression, such as acquired immunodeficiency, transplantation and cancer. As observed, in immunocompromised individuals HSE presents peculiar clinical and paraclinical features, and poorer prognosis. METHODS: Here we describe a retrospective series of seven cases of HSE in patients with high-grade glioma (HGG), collected among three institutions in a 5-year period (during this time, a total of 1750 patients with HGG were treated). RESULTS: Diagnosis of the condition was particularly challenging due to the confounding clinical presentation and the atypical biological findings. As a result, antiviral treatment was started with a sharp delay compared with immunocompetent hosts. Prognosis was poor, with high short-term mortality and severe residual disability in survivors. CONCLUSIONS: The substantial incidence of HSE observed in our centres together with the difficulty in diagnosing the condition suggest that the incidence of this complication may be highly underestimated. The aim of our report is to strengthen the observation of HSE in patients with HGG and outline the key elements that may allow its diagnosis.

Facing contrast-enhancing gliomas: perfusion MRI in grade III and grade IV gliomas according to tumor area.

Tumoral neoangiogenesis characterizes high grade gliomas. Relative Cerebral Blood Volume (rCBV), calculated with Dynamic Susceptibility Contrast (DSC) Perfusion-Weighted Imaging (PWI), allows for the estimation of vascular density over the tumor bed. The aim of the study was to characterize putative tumoral neoangiogenesis via the study of maximal rCBV with a Region of Interest (ROI) approach in three tumor areas-the contrast-enhancing area, the nonenhancing tumor, and the high perfusion area on CBV map-in patients affected by contrast-enhancing glioma (grades III and IV). Twenty-one patients were included: 15 were affected by grade IV and 6 by grade III glioma. Maximal rCBV values for each patient were averaged according to glioma grade. Although rCBV from contrast-enhancement and from nonenhancing tumor areas was higher in grade IV glioma than in grade III (5.58 and 2.68; 3.01 and 2.2, resp.), the differences were not significant. Instead, rCBV recorded in the high perfusion area on CBV map, independently of tumor compartment, was significantly higher in grade IV glioma than in grade III (7.51 versus 3.78, P = 0.036). In conclusion, neoangiogenesis encompasses different tumor compartments and CBV maps appear capable of best characterizing the degree of neovascularization. Facing contrast-enhancing brain tumors, areas of high perfusion on CBV maps should be considered as the reference areas to be targeted for glioma grading.

Prognostic value of CD109+ circulating endothelial cells in recurrent glioblastomas treated with bevacizumab and irinotecan.

BACKGROUND: Recent data suggest that circulating endothelial and progenitor cells (CECs and CEPs, respectively) may have predictive potential in cancer patients treated with bevacizumab, the antibody recognizing vascular endothelial growth factor (VEGF). Here we report on CECs and CEPs investigated in 68 patients affected by recurrent glioblastoma (rGBM) treated with bevacizumab and irinotecan and two Independent Datasets of rGBM patients respectively treated with bevacizumab alone (n=32, independent dataset A: IDA) and classical antiblastic chemotherapy (n=14, independent dataset B: IDB). METHODS: rGBM patients with KPS ≥50 were treated until progression, as defined by MRI with RANO criteria. CECs expressing CD109, a marker of tumor endothelial cells, as well as other CEC and CEP subtypes, were investigated by six-color flow cytometry. RESULTS: A baseline count of CD109+ CEC higher than 41.1/ml (1(st) quartile) was associated with increased progression free survival (PFS; 20 versus 9 weeks, P=0.008) and overall survival (OS; 32 versus 23 weeks, P=0.03). Longer PFS (25 versus 8 weeks, P=0.02) and OS (27 versus 17 weeks, P=0.03) were also confirmed in IDA with CD109+ CECs higher than 41.1/ml but not in IDB. Patients treated with bevacizumab with or without irinotecan that were free from MRI progression after two months of treatment had significant decrease of CD109+ CECs: median PFS was 19 weeks; median OS 29 weeks. The presence of two non-contiguous lesions (distant disease) at baseline was an independent predictor of shorter PFS and OS (P<0.001). CONCLUSIONS: Data encourage further studies on the predictive potential of CD109+ CECs in GBM patients treated with bevacizumab.

Acute late-onset encephalopathy after radiotherapy: an unusual life-threatening complication.

Unusual late-onset complications of brain irradiation, characterized by reversible neurologic focal signs, seizures, and MRI alterations, have recently been reported and classified as stroke-like migraine attacks after radiation therapy (SMART)(1) and peri-ictal pseudoprogression (PIPG).(2.)

Posttranscriptional regulation of SOD1 gene expression under oxidative stress: Potential role of ELAV proteins in sporadic ALS.

Increased levels of SOD1 mRNA have been observed in sporadic ALS patients (SALS) compared to controls. Hence, the understanding of the mechanisms by which SOD1 gene expression is modulated may shed new light on SOD1 involvement in ALS. Of interest, some adenine/uracil-rich elements (AREs) in SOD1 3'-untranslated region have been identified. These sequences represent the docking sites for several RNA-binding proteins such as ELAV proteins (ELAVs), positive regulators of gene expression. We first investigated in SH-SY5Y cells whether SOD1 mRNA represents a target of ELAVs. Results from RNA Electrophoretic Mobility Shift and RNA-immunoprecipitation assays showed a molecular interaction between ELAVs and SOD1 mRNA. We also observed that the treatment with H2O2 induced a significant increase of the amount of SOD1 mRNA bound by ELAVs and an up-regulation of SOD1 protein levels. We found a specific increase in ELAV/HuR phosphorylation, suggesting activation of this protein, in peripheral blood mononuclear cells from SALS patients compared to controls. Finally, we found increased levels of ELAV proteins in the motor cortex and spinal cord from SALS patients compared to controls, in parallel with SOD1 up-regulation in the same areas. This study suggests, for the first time, that ELAVs are involved in the regulation of SOD1 gene expression at post-transcriptional level and that these proteins are more activated in ALS pathology. The link between ELAVs and SOD1 may open novel perspectives for ALS research, paving the way for new therapeutic options.

Postinfectious neurologic syndromes: a prospective cohort study.

OBJECTIVES: Postinfectious neurologic syndromes (PINSs) of the CNS include heterogeneous disorders, sometimes relapsing. In this study, we aimed to a) describe the spectrum of PINSs; b) define predictors of outcome in PINSs; and c) assess the clinical/paraclinical features that help differentiate PINSs from multiple sclerosis (MS). METHODS: In this prospective cohort study, adult inpatients with PINSs underwent extensive diagnostic assessment and therapeutic protocols at inclusion and during a minimum 2-year follow-up. We compared them with newly diagnosed, treatment-naive patients with MS, also prospectively recruited. RESULTS: The study sample comprised 176 patients with PINSs aged 59.9 ± 17.25 years (range: 18-80 years) divided into 2 groups: group 1 (CNS syndromes, 64%)-encephalitis, encephalomyelitis, or myelitis; and group 2 (CNS + peripheral nervous system [PNS] syndromes, 36%)-encephalomyeloradiculoneuritis or myeloradiculoneuritis. We observed the patients for 24 to 170 months (median 69 months). Relapses, almost invariably involving the spinal cord, occurred in 30.5%. PNS involvement was an independent risk factor for relapses (hazard ratio 2.8). The outcome was poor in 43% of patients; risk factors included older age, greater neurologic disability at onset, higher serum-CSF albumin percentage transfer, myelitis, and PNS involvement. Steroid resistance occurred in 30% of the patients, half of whom responded favorably to IV immunoglobulins. Compared with MS, PINSs were characterized by older age, lower tendency to relapse, and distinct CSF findings. CONCLUSIONS: The category of PINSs should be revised: most of the clinical variants have a poor prognosis and are not readily classifiable on the basis of current knowledge. PNS involvement has a critical role in relapses, which seem to affect the spine only.

COL4A1-related disease: raised creatine kinase and cerebral calcification as useful pointers.

BACKGROUND: Mutations in COL4A1 are responsible for a spectrum of clinical phenotypes characterized by neurological, ocular, and renal involvement. Neurological features are the most prominent but as such are rather nonspecific. CASE PRESENTATION: Here, we report three new cases that, like five patients we previously described, show the novel common finding of raised creatine kinase (CK) concentration. CONCLUSION: Raised CK concentration, in addition to intracranial calcification, is to be considered another useful pointer to a final diagnosis of COL4A1-related disease.

An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1.

Recent studies suggest that Cu/Zn superoxide dismutase (SOD1) could be pathogenic in both familial and sporadic amyotrophic lateral sclerosis (ALS) through either inheritable or nonheritable modifications. The presence of a misfolded WT SOD1 in patients with sporadic ALS, along with the recently reported evidence that reducing SOD1 levels in astrocytes derived from sporadic patients inhibits astrocyte-mediated toxicity on motor neurons, suggest that WT SOD1 may acquire toxic properties similar to familial ALS-linked mutant SOD1, perhaps through posttranslational modifications. Using patients' lymphoblasts, we show here that indeed WT SOD1 is modified posttranslationally in sporadic ALS and is iper-oxidized (i.e., above baseline oxidation levels) in a subset of patients with bulbar onset. Derivatization analysis of oxidized carbonyl compounds performed on immunoprecipitated SOD1 identified an iper-oxidized SOD1 that recapitulates mutant SOD1-like properties and damages mitochondria by forming a toxic complex with mitochondrial Bcl-2. This study conclusively demonstrates the existence of an iper-oxidized SOD1 with toxic properties in patient-derived cells and identifies a common SOD1-dependent toxicity between mutant SOD1-linked familial ALS and a subset of sporadic ALS, providing an opportunity to develop biomarkers to subclassify ALS and devise SOD1-based therapies that go beyond the small group of patients with mutant SOD1.

Systemic sclerosis in aquaporin-4 antibody-positive longitudinally extensive transverse myelitis.

We report on the first patient with a relapsing, anti-aquaporin-4 (AQP-4) antibody-positive, longitudinally extensive transverse myelitis (LETM) who developed systemic sclerosis (SSc). A 62-year-old woman, who presented with bilateral, distal lower limb and perineal numbness, developed clinical manifestations and paraclinical features of SSc. Spinal cord imaging revealed lesions that were consistent with LETM. Patient's serum was positive for neuromyelitis optica (NMO)-IgG/AQP-4 antibodies. High-dose intravenous corticosteroids improved the neurological symptoms. The present case expands the list of autoimmune systemic diseases that occur in neuromyelitis optica spectrum disorders associated with NMO-IgG/AQP-4 antibodies.

Cerebrospinal BAFF and Epstein-Barr virus-specific oligoclonal bands in multiple sclerosis and other inflammatory demyelinating neurological diseases.

We measured circulating serum and cerebrospinal fluid (CSF) concentrations of B lymphocyte activating factor of the tumour necrosis factor superfamily (BAFF), and determined total and Epstein-Barr virus (EBV)-specific oligoclonal IgG bands (OCBs) in 43 patients with multiple sclerosis (MS), 23 patients with other inflammatory demyelinating neurological diseases, and 20 patients with non-inflammatory neurological diseases. Serum and CSF BAFF concentrations did not differ in the three studied groups. In MS, the highest BAFF concentrations were found in the CSF samples with more than 6 OCBs (233.1 ± 129.5 vs 79.2 ± 51.6 pg/mL in the samples with less than 7 OCBs, p<0.0001). Irrespectively from BAFF levels, EBV-specific OCBs were detected in MS and in the other non-inflammatory and inflammatory demyelinating neurological diseases, with a similar frequency, and as a 'mirror pattern' in 30 of 33 EBV-specific OCB-positive cases (p<0.0001). These results indicate that circulating CSF BAFF concentrations cannot help differentiate MS from other inflammatory demyelinating neurological diseases, but positively associates with the qualitative expression of elevated intrathecal IgG production in MS, and that the oligoclonal EBV-specific antibody response, when present, is mostly systemic in all the studied neurological patients, and not preferentially restricted to MS.

G93A SOD1 alters cell cycle in a cellular model of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative multifactorial disease characterized, like other diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) or frontotemporal dementia (FTD), by the degeneration of specific neuronal cell populations. Motor neuron loss is distinctive of ALS. However, the causes of onset and progression of motor neuron death are still largely unknown. In about 2% of all cases, mutations in the gene encoding for the Cu/Zn superoxide dismutase (SOD1) are implicated in the disease. Several alterations in the expression or activation of cell cycle proteins have been described in the neurodegenerative diseases and related to cell death. In this work we show that mutant SOD1 can alter cell cycle in a cellular model of ALS. Our findings suggest that modifications in the cell cycle progression could be due to an increased interaction between mutant G93A SOD1 and Bcl-2 through the cyclins regulator p27. As previously described in post mitotic neurons, cell cycle alterations could fatally lead to cell death.

Mutations of FUS gene in sporadic amyotrophic lateral sclerosis.

BACKGROUND: Mutations in the FUS gene have recently been discovered to be a major cause of familial amyotrophic lateral sclerosis (FALS). OBJECTIVE: To determine the identity and frequency of FUS gene mutations in a large cohort of Italian patients enriched in sporadic cases (SALS). METHODS: Exons 5, 6, 14 and 15 of the FUS gene were screened for mutations in 1009 patients (45 FALS and 964 SALS). The genetic analysis was extended to the entire coding sequence of FUS in all the FALS and 293 of the SALS patients. RESULTS: Seven missense mutations (p.G191S, p.R216C, p.G225V, p.G230C, p.R234C, p.G507D and p.R521C) were identified in nine patients (seven SALS and two FALS), and none in 500 healthy Italian controls. All mutations are novel except for the p.R521C mutation identified in one SALS and one FALS case. Both patients showed a similar unusual presentation, with proximal, mostly symmetrical, upper limb weakness, with neck and axial involvement. With the exception of p.G507D and p.R521C, the mutations identified in SALS patients are all localised in the glycine-rich region encoded by exon 6. In addition, eight different in-frame deletions in two polyglycine motifs were detected, the frequency of which was not significantly different in patients and controls. CONCLUSIONS: The results show that FUS missense mutations are present in 0.7% of Italian SALS cases, and confirm the previous mutational frequency reported in FALS (4.4%). An unusual proximal and axial clinical presentation seems to be associated with the presence of the p.R521C mutation.