Chromosome 3 cluster rs11385942 variant links complement activation with severe COVID-19.

BACKGROUND: Genetic variation at a multigene cluster at chromosome 3p21.31 and the ABO blood group have been associated with the risk of developing severe COVID-19, but the mechanism remains unclear. Complement activation has been associated with COVID-19 severity. OBJECTIVE: The aim of this study was to examine whether chromosome 3p21.31 and the ABO variants are linked to the activation of the complement cascade in COVID-19 patients. METHODS: We considered 72 unrelated European hospitalized patients with genetic data and evaluation of circulating C5a and soluble terminal complement complex C5b-9 (SC5b-9). Twenty-six (36.1%) patients carried the rs11385942 G>GA variant and 44 (66.1%) non-O blood group associated with increased risk of severe COVID-19. RESULTS: C5a and SC5-b9 plasma levels were higher in rs11385949 GA carriers than in non-carriers (P = 0.041 and P = 0.012, respectively), while C5a levels were higher in non-O group than in O group patients (P = 0.019). The association between rs11385949 and SC5b-9 remained significant after adjustment for ABO and disease severity (P = 0.004) and further correction for C5a (P = 0.018). There was a direct relationship between upper airways viral load and SC5b-9 in carriers of the rs11385949 risk allele (P = 0.032), which was not observed in non-carriers. CONCLUSIONS: The rs11385949 G>GA variant, tagging the chromosome 3 gene cluster variation and predisposing to severe COVID-19, is associated with enhanced complement activation, both with C5a and terminal complement complex, while non-O blood group with C5a levels. These findings provide a link between genetic susceptibility to more severe COVID-19 and complement activation.

Complement activation and endothelial perturbation parallel COVID-19 severity and activity.

BACKGROUND: Animal models and few clinical reports suggest the involvement of the complement system in the onset of severe manifestations of coronavirus disease-2019 (COVID-19). However, complement contribution to endotheliopathy and hypercoagulability has not been elucidated yet. OBJECTIVE: To evaluate the association among complement activation, endothelial damage and disease severity or activity in COVID-19 patients. METHODS: In this single-centre cohort study, 148 patients with COVID-19 of different severity were evaluated upon hospital admission and 30 days later. Markers of complement activation (SC5b-9 and C5a) and endothelial perturbation (von Willebrand factor [vWF], tissue-type plasminogen activator [t-PA], plasminogen activator inhibitor-1 [PAI-1], soluble thrombomodulin [sTM], and soluble endothelial selectin [sE-selectin]) were measured in plasma. RESULTS: The patients had high plasma levels of SC5b-9 and C5a (p = 0.0001 for both) and vWF, t-PA and PAI-1 (p = 0.0001 for all). Their SC5b-9 levels correlated with those of vWF (r = 0.517, p = 0.0001) and paralleled disease severity (severe vs mild p = 0.0001, severe vs moderate p = 0.026 and moderate vs mild p = 0.001). The levels of sE-selectin were significantly increased only in the patients with severe disease. After 30 days, plasma SC5b-9, C5a and vWF levels had significantly decreased (p = 0.0001 for all), and 43% of the evaluated patients had normal levels. CONCLUSIONS: Complement activation is boosted during the progression of COVID-19 and dampened during remission, thus indicating its role in the pathophysiology of the disease. The association between complement activation and the biomarkers of endothelial damage suggests that complement may contribute to tissue injury and could be the target of specific therapy.

Trends and risk factors of SARS-CoV-2 infection in asymptomatic blood donors.

BACKGROUND: A large proportion of SARS-CoV-2-infected individuals does not develop severe symptoms. Serological tests help in evaluating the spread of infection and disease immunization. The aim of this study was to prospectively examine the trends and risk factors of SARS-CoV-2 infection in blood donors. STUDY DESIGN AND METHODS: We screened 8798 asymptomatic donors presenting in Milan from July 2020 to February 2021 (10,680 presentations) before the vaccination campaign for anti-nucleoprotein (NP) antibodies, and for anti-spike receptor-binding domain (RBD) antibodies and nasopharyngeal swab PCR in those who tested positive. RESULTS: The prevalence of anti-NP+/RBD+ tests increased progressively with time up to ~15% (p < .0001), preceded by a peak of PCR+ tests. Anti-RBD titers were higher in anti-NP IgG+/IgM+ than in IgG+/IgM- individuals and in those with a history of infection (p < .0001); of these 197/630 (31.2%) displayed high titers (>80 AU/ml). Anti-RBD titers declined during follow-up, depending on baseline titers (p < .0001) and time (p = .025). Risk factors for seroconversion were a later presentation date and non-O ABO blood group (p < .001). A positive PCR was detected in 0.7% of participants in the absence of SARS-CoV-2 viremia. CONCLUSIONS: During the second wave of SARS-CoV-2 infection in Northern Italy, we detected an increase in seroprevalence in healthy blood donors from ~4% to ~15%, with a trend paralleling that observed in the general population. Seroconversion was more frequent in carriers of non-O blood groups. The persistence of anti-RBD antibodies was short-lived.

EphB2 stem-related and EphA2 progression-related miRNA-based networks in progressive stages of CRC evolution: clinical significance and potential miRNA drivers.

EphB2 and EphA2 control stemness and differentiation in the intestinal mucosa, but the way they cooperate with the complex mechanisms underlying tumor heterogeneity and how they affect the therapeutic outcome in colorectal cancer (CRC) patients, remain unclear. MicroRNA (miRNA) expression profiling along with pathway analysis provide comprehensive information on the dysregulation of multiple crucial pathways in CRC.Through a network-based approach founded on the characterization of progressive miRNAomes centered on EphA2/EphB2 signaling during tumor development in the AOM/DSS murine model, we found a miRNA-dependent orchestration of EphB2-specific stem-like properties in earlier phases of colorectal tumorigenesis and the EphA2-specific control of tumor progression in the latest CRC phases. Furthermore, two transcriptional signatures that are specifically dependent on the EphA2/EphB2 signaling pathways were identified, namely EphA2, miR-423-5p, CREB1, ADAMTS14, and EphB2, miR-31-5p, mir-31-3p, CRK, CXCL12, ARPC5, SRC.EphA2- and EphB2-related signatures were validated for their expression and clinical value in 1663 CRC patients. In multivariate analysis, both signatures were predictive of survival and tumor progression.The early dysregulation of miRs-31, as observed in the murine samples, was also confirmed on 49 human tissue samples including preneoplastic lesions and tumors. In light of these findings, miRs-31 emerged as novel potential drivers of CRC initiation.Our study evidenced a miRNA-dependent orchestration of EphB2 stem-related networks at the onset and EphA2-related cancer-progression networks in advanced stages of CRC evolution, suggesting new predictive biomarkers and potential therapeutic targets.

The synthetic purine reversine selectively induces cell death of cancer cells.

The synthetic purine reversine has been shown to possess a dual activity as it promotes the de-differentiation of adult cells, including fibroblasts, into stem-cell-like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti-cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced.

NEU4L sialidase overexpression promotes ß-catenin signaling in neuroblastoma cells, enhancing stem-like malignant cell growth.

Neuroblastoma (NB) is a frequently lethal tumor that occurs in childhood and originates from embryonic neural crest cells. The malignant and aggressive phenotype of NB is strictly related to the deregulation of pivotal pathways governing the proliferation/differentiation status of neural crest precursor cells, such as MYCN, Delta/Notch and Wnt/ß-catenin (CTNNB1) signaling. In this article, we demonstrate that sialidase NEU4 long (NEU4L) influences the differentiation/proliferation behavior of NB SK-N-BE cells by determining hyperactivation of the Wnt/ß-catenin signaling pathway. NEU4L overexpression in SK-N-BE cells induced significant increases in active, nonphosphorylated ß-catenin content, ß-catenin/TCF transcriptional activity and ß-catenin gene target expression including MYCN, MYC, CCND2 (cyclin D2) and CDC25A. In turn, these molecular features strongly modified the behavior of NEU4L SK-N-BE overexpressing cells, promoting the following: (1) an enhanced proliferation rate, mainly due to a faster transition from G1 to S phase in the cell cycle; (2) a more undifferentiated cell phenotype, which was similar to stem-like NB cells and possibly mediated by an increase of the expression of the pluripotency genes, MYC, NANOG, OCT-4, CD133 and NES (nestin); (3) the failure of NB cell differentiation after serum withdrawal. The molecular link between NEU4L and Wnt/ß-catenin signaling appeared to rely most likely on the capability of the enzyme to modify the sialylation level of cell glycoproteins. These findings could provide a new candidate for therapeutic treatment.

Effects of epidermal growth factor receptor blockade on ependymoma stem cells in vitro and in orthotopic mouse models.

Some lines of evidence suggest that tumors, including ependymoma, might arise from a subpopulation of cells, termed cancer stem cells (CSCs), with self-renewal and tumor-initiation properties. Given the strict dependence of CSCs on epidermal growth factor (EGF) through EGF receptor (EGFR), we investigated the effects of EGFR inhibitors in ependymoma-stem cells (SCs) in vitro and in orthotopic mouse models. We established two ependymoma-SC lines from two recurrent pediatric ependymoma. Both lines expressed markers of radial glia--the candidate SCs of ependymoma--and showed renewal ability, multipotency, and tumorigenicity after orthotopic implantation, despite markedly different expression of CD133 (94 vs. 6%). High phosphorylated-EGFR/EGFR ratio was detected, which decreased after differentiation. EGFR inhibitors (gefitinib and AEE788) reduced clonogenicity, proliferation and survival of ependymoma-SC lines dose-dependently, and blocked EGF-induced activation of EGFR, Akt and extracellular signal-regulated kinase 1/2. Overall, AEE788 was more effective than gefitinib. EGFR blockade as well as differentiation strongly reduced CD133 expression. However, ex vivo treatment with AEE788 did not impair orthotopic tumor engraftment, whereas ex vivo differentiation did, suggesting that CD133 does not absolutely segregate for tumorigenicity in ependymoma-SCs. Orally administered AEE788 prolonged survival of mice bearing ependymoma-SC-driven orthotopic xenografts from 56 to 63 days, close to statistical significance (log-rank p=0.06). Our study describes for the first time EGFR signaling in ependymoma-SCs and the effects of EGFR blockade in complementary in vitro and in vivo systems. The experimental models we developed can be used to further investigate the activity of EGFR inhibitors or other antineoplastic agents in this tumor.

Mutant SOD1 and mitochondrial damage alter expression and splicing of genes controlling neuritogenesis in models of neurodegeneration.

Mitochondrial dysfunction has been implicated in the pathogenesis of a number of neurodegenerative disorders including Parkinson, Alzheimer, and Amyotrophic Lateral Sclerosis (ALS). In addition, aberrant mRNA splicing has been documented in neurodegeneration. To characterize the cellular response to mitochondrial perturbations at the level of gene expression and alternative pre-mRNA splicing we used splicing-sensitive microarrays to profile human neuroblastoma SH-SY5Y cells treated with paraquat, a neurotoxic herbicide that induces the formation of reactive oxygen species and causes mitochondrial damage in animal models, and SH-SY5Y cells stably expressing the mutant G93A-SOD1 protein, one of the genetic causes of ALS. In both models we identified a common set of genes whose expression and alternative splicing are deregulated. Pathway analysis of the deregulated genes revealed enrichment in genes involved in neuritogenesis, axon growth and guidance, and synaptogenesis. Alterations in transcription and pre-mRNA splicing of candidate genes were confirmed experimentally in the cell line models as well as in brain and spinal cord of transgenic mice carrying the G93A-SOD1 mutation. Our findings expand the realm of the pathways implicated in neurodegeneration and suggest that alterations of axonal function may descend directly from mitochondrial damage.

Impact of SARS-CoV-2 infection on the recovery of peripheral blood mononuclear cells by density gradient.

SARS-CoV-2 virus infection is responsible for coronavirus disease (COVID-19), which is characterised by a hyperinflammatory response that plays a major role in determining the respiratory and immune-mediated complications of this condition. While isolating peripheral blood mononuclear cells (PBMCs) from whole blood of COVID-19 patients by density gradient centrifugation, we noticed some changes in the floating properties and in the sedimentation of the cells on density medium. Investigating this further, we found that in early phase COVID-19 patients, characterised by reduced circulating lymphocytes and monocytes, the PBMC fraction contained surprisingly high levels of neutrophils. Furthermore, the neutrophil population exhibited alterations in the cell size and in the internal complexity, consistent with the presence of low density neutrophils (LDNs) and immature forms, which may explain the shift seen in the floating abilities and that may be predictive of the severity of the disease. The percentage of this subset of neutrophils found in the PBMC band was rather spread (35.4 ± 27.2%, with a median 28.8% and IQR 11.6-56.1, Welch's t-test early phase COVID-19 versus blood donor healthy controls P < 0.0001). Results confirm the presence of an increased number of LDNs in patients with early stage COVID-19, which correlates with disease severity and may be recovered by centrifugation on a density gradient together with PBMCs.

Early Phases of COVID-19 Are Characterized by a Reduction in Lymphocyte Populations and the Presence of Atypical Monocytes.

Background: Severe acute respiratory syndrome coronavirus 2 is a recently discovered pathogen responsible of coronavirus disease 2019 (COVID-19). The immunological changes associated with this infection are largely unknown. Methods: We evaluated the peripheral blood mononuclear cells profile of 63 patients with COVID-19 at diagnosis. We also assessed the presence of association with inflammatory biomarkers and the 28-day mortality. Results: Lymphocytopenia was present in 51 of 63 (80.9%) patients, with a median value of 720 lymphocytes/µl (IQR 520-1,135). This reduction was mirrored also on CD8+ (128 cells/µl, IQR 55-215), natural killer (67 cells/µl, IQR 35-158) and natural killer T (31 cells/µl, IQR 11-78) cells. Monocytes were preserved in total number but displayed among them a subpopulation with a higher forward and side scatter properties, composed mainly of cells with a reduced expression of both CD14 and HLA-DR. Patients who died in the 28 days from admission (N=10, 15.9%), when compared to those who did not, displayed lower mean values of CD3+ (337.4 cells/µl vs 585.9 cells/µl; p=0.028) and CD4+ cells (232.2 cells/µl vs 381.1 cells/µl; p=0.042) and an higher percentage of CD8+/CD38+/HLA-DR+ lymphocytes (13.5% vs 7.6%; p=0.026). Discussion: The early phases of COVID-19 are characterized by lymphocytopenia, predominance of Th2-like lymphocytes and monocytes with altered immune profile, which include atypical mononuclear cells.

Green fluorescent protein transgene driven by Kit regulatory sequences is expressed in hematopoietic stem cells.

BACKGROUND: The transcriptional regulation of stem cell genes is still poorly understood. Kit, encoding the stem cell factor receptor, is a pivotal molecule for multiple types of stem/progenitor cells. We previously generated mouse lines expressing transgenic green fluorescent protein under the control of Kit promoter/first intron regulatory elements, and we demonstrated expression in hematopoietic progenitors. DESIGN AND METHODS: In the present work we investigated whether the transgene is also expressed in hematopoietic stem cells of adult bone marrow and fetal liver. To this purpose, we tested, in long-term repopulating assays, cell fractions expressing different levels of green fluorescent protein within Kit-positive or SLAM-selected populations. RESULTS: The experiments demonstrated transgene expression in both fetal and adult hematopoietic stem cells and indicated that the transgene is transcribed at distinctly lower levels in hematopoietic stem cells than in pluripotent and committed progenitors. CONCLUSIONS: These results, together with previous data, show that a limited subset of DNA sequences drives gene expression in number of stem cell types (hematopoietic stem cells, primordial germ cells, cardiac stem cells). Additionally, our results might help to further improve high level purification of hematopoietic stem cells for experimental purposes. Finally, as the Kit/green fluorescent protein transgene is expressed in multiple stem cell types, our transgenic model provides powerful in vivo system to track these cells during development and tissue regeneration.

Production and characterization of human induced pluripotent stem cells (iPSC) CSSi007-A (4383) from Joubert Syndrome.

Joubert syndrome (JS) is an autosomal recessive neurodevelopmental disorder, characterized by congenital cerebellar and brainstem defects, belonging to the group of disorders known as ciliopathies, which are caused by mutations in genes encoding proteins of the primary cilium and basal body. Human induced pluripotent stem cells (hiPSCs) from a patient carrying a homozygous missense mutation (c.2168G > A) in AHI1, the first gene to be associated with JS, were produced using a virus-free protocol.

Generation of induced pluripotent stem cell line CSSi008-A (4698) from a patient affected by advanced stage of Dentato-Rubral-Pallidoluysian atrophy (DRPLA).

Dentato-Rubral-pallidoluysian atrophy (DRPLA) is a rare autosomal, dominant, progressive neurodegenerative disease that causes involuntary movements, mental and emotional problems. DRPLA is caused by a mutation in the ATN1 gene that encodes for an abnormal polyglutamine stretch in the atrophin-1 protein. DRPLA is most common in the Japanese population, where it has an estimated incidence of 2 to 7 per million people. This condition has also been seen in families from North America and Europe. We obtained a reprogrammed iPSC line from a Caucasian patient with a juvenile onset of the disease, carrying 64 CAG repeat expansion in the ATN1 gene.

A novel, immortal, and multipotent human neural stem cell line generating functional neurons and oligodendrocytes.

The discovery and study of neural stem cells have revolutionized our understanding of the neurogenetic process, and their inherent ability to adopt expansive growth behavior in vitro is of paramount importance for the development of novel therapeutics based on neural cell replacement. Recent advances in high-throughput assays for drug development and gene discovery dictate the need for rapid, reproducible, long-term expansion of human neural stem cells (hNSCs). In this view, the complement of wild-type cell lines currently available is insufficient. Here we report the establishment of a stable human neural stem cell line (immortalized human NSCs [IhNSCs]) by v-myc-mediated immortalization of previously derived wild-type hNSCs. These cells demonstrate three- to fourfold faster proliferation than wild-type cells in response to growth factors but retain rather similar properties, including multipotentiality. By molecular biology, biochemistry, immunocytochemistry, fluorescence microscopy, and electrophysiology, we show that upon growth factor removal, IhNSCs completely downregulate v-myc expression, cease proliferation, and differentiate terminally into three major neural lineages: astrocytes, oligodendrocytes, and neurons. The latter are functional, mature cells displaying clear-cut morphological and physiological features of terminally differentiated neurons, encompassing mostly the GABAergic, glutamatergic, and cholinergic phenotypes. Finally, IhNSCs produce bona fide oligodendrocytes in fractions up to 20% of total cell number. This is in contrast to the negligible propensity of hNSCs to generate oligodendroglia reported so far. Thus, we describe an immortalized hNSC line endowed with the properties of normal hNSCs and suitable for developing the novel, reliable assays and reproducible high-throughput gene and drug screening that are essential in both diagnostics and cell therapy studies.

Production and characterization of CSSI003 (2961) human induced pluripotent stem cells (iPSCs) carrying a novel puntiform mutation in RAI1 gene, Causative of Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is a complex genetic disorder characterized by developmental delay, behavioural problems and circadian rhythm dysregulation. About 90% of SMS cases are due to a 17p11.2 deletion containing retinoic acid induced1 (RAI1) gene, 10% are due to heterozygous mutations affecting RAI1 coding region. Little is known about RAI1 role.

Generation of induced pluripotent stem cell line, CSSi004-A (2962), from a patient diagnosed with Huntington's disease at the presymptomatic stage.

Huntington's disease (HD) is an incurable, autosomal dominant, hereditary neurodegenerative disorder that typically manifests itself in midlife. This pathology is linked to the deregulation of multiple, as yet unknown, cellular processes starting before HD onset. A human iPS cell line was generated from skin fibroblasts of a subject at the presymptomatic life stage, carrying a polyglutamine expansion in HTT gene codifying Huntingtin protein. The iPSC line contained the expected CAG expansion, expressed the expected pluripotency markers, displayed in vivo differentiation potential to the three germ layers and had a normal karyotype.

Production and characterization of human induced pluripotent stem cells (iPSCs) from Joubert Syndrome: CSSi001-A (2850).

Joubert Syndrome (JS) is a rare autosomal recessive or X-linked condition characterized by a peculiar cerebellar malformation, known as the molar tooth sign (MTS), associated with other neurological phenotypes and multiorgan involvement. JS is a ciliopathy, a spectrum of disorders whose causative genes encode proteins involved in the primary cilium apparatus. In order to elucidate ciliopathy-associated molecular mechanisms, human induced pluripotent stem cells (hiPSCs) were derived from a patient affected by JS carrying a homozygous missense mutation in the AHI1 gene (p.H896R) that encodes a protein named Jouberin.

Generation of induced pluripotent stem cell line, CSSi002-A (2851), from a patient with juvenile Huntington Disease.

Huntington Disease (HD) is an autosomal dominant disorder characterized by motor, cognitive and behavioral features caused by a CAG expansion in the HTT gene beyond 35 repeats. The juvenile form (JHD) may begin before the age of 20years and is associated with expanded alleles as long as 60 or more CAG repeats. In this study, induced pluripotent stem cells were generated from skin fibroblasts of a 8-year-old child carrying a large size mutation of 84 CAG repeats in the HTT gene. HD appeared at age 3 with mixed psychiatric (i.e. autistic spectrum disorder) and motor (i.e. dystonia) manifestations.