Mitotic Dysregulation at Tumor Initiation Creates a Therapeutic Vulnerability to Combination Anti-Mitotic and Pro-Apoptotic Agents for MYCN-Driven Neuroblastoma.

MYCN amplification occurs in approximately 20-30% of neuroblastoma patients and correlates with poor prognosis. The TH-MYCN transgenic mouse model mimics the development of human high-risk neuroblastoma and provides strong evidence for the oncogenic function of MYCN. In this study, we identified mitotic dysregulation as a hallmark of tumor initiation in the pre-cancerous ganglia from TH-MYCN mice that persists through tumor progression. Single-cell quantitative-PCR of coeliac ganglia from 10-day-old TH-MYCN mice revealed overexpression of mitotic genes in a subpopulation of premalignant neuroblasts at a level similar to single cells derived from established tumors. Prophylactic treatment using antimitotic agents barasertib and vincristine significantly delayed the onset of tumor formation, reduced pre-malignant neuroblast hyperplasia, and prolonged survival in TH-MYCN mice. Analysis of human neuroblastoma tumor cohorts showed a strong correlation between dysregulated mitosis and features of MYCN amplification, such as MYC(N) transcriptional activity, poor overall survival, and other clinical predictors of aggressive disease. To explore the therapeutic potential of targeting mitotic dysregulation, we showed that genetic and chemical inhibition of mitosis led to selective cell death in neuroblastoma cell lines with MYCN over-expression. Moreover, combination therapy with antimitotic compounds and BCL2 inhibitors exploited mitotic stress induced by antimitotics and was synergistically toxic to neuroblastoma cell lines. These results collectively suggest that mitotic dysregulation is a key component of tumorigenesis in early neuroblasts, which can be inhibited by the combination of antimitotic compounds and pro-apoptotic compounds in MYCN-driven neuroblastoma.

High incidence of common femoral Deep Vein Thrombosis in critically ill Covid19 patients.

Objective: To estimate the incidence and prevalence of deep venous thrombosis, and to evaluate the discriminative capacity of D-dimer in its diagnosis. METHODS: The prospective, observational study was conducted at the critical care unit of a tertiary care hospital in Pakistan from February to September 2021 and comprised consecutively admitted adult critically ill patients who were receiving therapeutic-dose anticoagulation therapy. All patients were screened on day one for deep venous thrombosis by colour doppler and compression ultrasonography. Patients who did not have deep venous thrombosis on the first scan were followed every 72 hours. Data was analysed using SPSS 26. RESULTS: Of the 142 patients, 99(69.7%) were male and 43(30.3%) were female. The overall mean age was 53.20+/-13.3 years. On the first scan, 25(17.6%) patients had deep venous thrombosis. Of the remaining 117 patients, 78(68.4%) were followed every 72 hours, and 23(29.48%) of them developed deep venous thrombosis. The most common site for DVT was the common femoral vein 46(95.8%) and most deep venous thrombosis cases were unilateral 28(58.33%). D-dimer levels showed no discriminative capacity for diagnosis of deep venous thrombosis (p=0.79). There were no significant risk factors for the development of deep venous thrombosis. Conclusion: There was a high incidence and prevalence of deep venous thrombosis despite therapeutic-dose anticoagulation therapy. The most common affected site was the common femoral vein and most deep venous thrombosis were unilateral. D-dimer levels had no discriminative capacity for the diagnosis of deep venous thrombosis DVT.

Transplantation sites for human and murine islets.

AIMS/HYPOTHESIS: Beta cell replacement is a potential cure for type 1 diabetes. In humans, islet transplants are currently infused into the liver via the portal vein, although this site has disadvantages. Here, we investigated alternative transplantation sites for human and murine islets in recipient mice, comparing the portal vein with quadriceps muscle and kidney, liver and spleen capsules. METHODS: Murine islets were isolated from C57BL6/J mice and transplanted into syngeneic recipients. Human islets were isolated and transplanted into either severe combined immunodeficiency (SCID) or recombination-activating gene 1 (RAG-1) immunodeficient recipient mice. All recipient mice were 8-12 weeks of age and had been rendered diabetic (defined as blood glucose concentrations ≥20 mmol/l on two consecutive days before transplantation) by alloxan tetrahydrate treatment. Islets were transplanted into five different sites (portal vein, quadriceps muscle, kidney, liver and spleen capsules). Blood glucose concentrations were monitored twice weekly until mice were killed. Dose-response studies were also performed to determine the minimum number of islets required to cure diabetes ('cure' is defined for this study as random fed blood glucose of <15 mmol/l). RESULTS: For transplantation of murine islets into the different sites, the kidney yielded 100% success, followed by muscle (70%), portal vein (60%), spleen capsule (29%) and liver capsule (0%). For human islets, transplantation into the kidney cured diabetes in 75-80% of recipient mice. Transplantation into muscle and portal vein had intermediate success (both 29% at 2000 islet equivalents), while transplantation into liver and spleen capsule failed (0%). With increased islet mass, success rates for muscle grafts improved to 52-56%. CONCLUSIONS/INTERPRETATION: For both human and murine islets, equivalent or superior glucose lowering results were obtained for transplantation into skeletal muscle, compared with the portal vein. Unfortunately, kidney grafts are not feasible in human recipients. Skeletal muscle offers easier access and greater potential for protocol biopsies. This study suggests that human trials of muscle as a transplant site may be warranted.

Regulatory T cells prevent inducible BALT formation by dampening neutrophilic inflammation.

Inducible BALT (iBALT) can amplify pulmonary or systemic inflammatory responses to the benefit or detriment of the host. We took advantage of the age-dependent formation of iBALT to interrogate the underlying mechanisms that give rise to this ectopic, tertiary lymphoid organ. In this study, we show that the reduced propensity for weanling as compared with neonatal mice to form iBALT in response to acute LPS exposure is associated with greater regulatory T cell expansion in the mediastinal lymph nodes. Ab- or transgene-mediated depletion of regulatory T cells in weanling mice upregulated the expression of IL-17A and CXCL9 in the lungs, induced a tissue neutrophilia, and increased the frequency of iBALT to that observed in neonatal mice. Remarkably, neutrophil depletion in neonatal mice decreased the expression of the B cell active cytokines, a proliferation-inducing ligand and IL-21, and attenuated LPS-induced iBALT formation. Taken together, our data implicate a role for neutrophils in lymphoid neogenesis. Neutrophilic inflammation is a common feature of many autoimmune diseases in which iBALT are present and pathogenic, and hence the targeting of neutrophils or their byproducts may serve to ameliorate detrimental lymphoid neogenesis in a variety of disease contexts.

Toll-like receptor 7 gene deficiency and early-life Pneumovirus infection interact to predispose toward the development of asthma-like pathology in mice.

BACKGROUND: Respiratory tract viruses are a major environmental risk factor for both the inception and exacerbations of asthma. Genetic defects in Toll-like receptor (TLR) 7-mediated signaling, impaired type I interferon responses, or both have been reported in asthmatic patients, although their contribution to the onset and exacerbation of asthma remains poorly understood. OBJECTIVE: We sought to determine whether Pneumovirus infection in the absence of TLR7 predisposes to bronchiolitis and the inception of asthma. METHODS: Wild-type and TLR7-deficient (TLR7(-/-)) mice were inoculated with the rodent-specific pathogen pneumonia virus of mice at 1 (primary), 7 (secondary), and 13 (tertiary) weeks of age, and pathologic features of bronchiolitis or asthma were assessed. In some experiments infected mice were exposed to low-dose cockroach antigen. RESULTS: TLR7 deficiency increased viral load in the airway epithelium, which became sloughed and necrotic, and promoted an IFN-α/ß(low), IL-12p70(low), IL-1ß(high), IL-25(high), and IL-33(high) cytokine microenvironment that was associated with the recruitment of type 2 innate lymphoid cells/nuocytes and increased TH2-type cytokine production. Viral challenge of TLR7(-/-) mice induced all of the cardinal pathophysiologic features of asthma, including tissue eosinophilia, mast cell hyperplasia, IgE production, airway smooth muscle alterations, and airways hyperreactivity in a memory CD4(+) T cell-dependent manner. Importantly, infections with pneumonia virus of mice promoted allergic sensitization to inhaled cockroach antigen in the absence but not the presence of TLR7. CONCLUSION: TLR7 gene defects and Pneumovirus infection interact to establish an aberrant adaptive response that might underlie virus-induced asthma exacerbations in later life.

Absence of Toll-IL-1 receptor 8/single immunoglobulin IL-1 receptor-related molecule reduces house dust mite-induced allergic airway inflammation in mice.

Allergic asthma is a chronic inflammatory disease predominately associated with the activation of CD4(+) T helper Type 2 (Th2) cells. Innate pattern recognition receptors are widely acknowledged to shape the adaptive immune response. For example, the activation of airway epithelial Toll-like receptor-4 (TLR4) is necessary for the generation of house dust mite (HDM)-specific Th2 responses and the development of asthma in mice. Here we sought to determine whether the absence of Toll-interleukin-1 receptor (TIR)-8, a negative regulator of TLR4 signaling that is highly expressed in airway epithelial cells, would exacerbate HDM-induced asthma in a murine model. We found that Th2 but not Th1 or Th17 cytokine expression was significantly reduced in the lung and draining lymph nodes in HDM-sensitized/challenged TIR8 gene-deleted mice. Mucus-producing goblet cells, HDM-specific IgG1, and airway hyperreactivity were also significantly reduced in HDM-exposed, TIR8-deficient mice. Consistent with the attenuated Th2 response, eotaxin-2/CCL24 expression and airway and peribronchial eosinophils were significantly reduced in the absence of TIR8. In contrast, IL-17A-responsive chemokines and neutrophil numbers were unaffected. Similar findings were obtained for cockroach allergen. HDM sensitization alone up-regulated the expression of IL-1F5, a putative TIR8 ligand and inducer of IL-4. Of note, innate IL-4, IL-5, IL-13, and IL-33 cytokine expression was reduced during HDM sensitization in the absence of TIR8, as was the recruitment of conventional dendritic cells and basophils to the draining lymph nodes. Our findings suggest that TIR8 enhances the development of HDM-induced innate and adaptive Th2, but not Th1 or Th17 type immunity.

Plasmacytoid dendritic cells promote host defense against acute pneumovirus infection via the TLR7-MyD88-dependent signaling pathway.

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants. In human infants, plasmacytoid dendritic cells (pDC) are recruited to the nasal compartment during infection and initiate host defense through the secretion of type I IFN, IL-12, and IL-6. However, RSV-infected pDC are refractory to TLR7-mediated activation. In this study, we used the rodent-specific pathogen, pneumonia virus of mice (PVM), to determine the contribution of pDC and TLR7 signaling to the development of the innate inflammatory and early adaptive immune response. In wild-type, but not TLR7- or MyD88-deficient mice, PVM inoculation led to a marked infiltration of pDC and increased expression of type I, II, and III IFNs. The delayed induction of IFNs in the absence of TLR7 or MyD88 was associated with a diminished innate inflammatory response and augmented virus recovery from lung tissue. In the absence of TLR7, PVM-specific CD8(+) T cell cytokine production was abrogated. The adoptive transfer of TLR7-sufficient, but not TLR7-deficient pDC to TLR7 gene-deleted mice recapitulated the antiviral responses observed in wild-type mice and promoted virus clearance. In summary, TLR7-mediated signaling by pDC is required for appropriate innate responses to acute pneumovirus infection. It is conceivable that as-yet-unidentified defects in the TLR7 signaling pathway may be associated with elevated levels of RSV-associated morbidity and mortality among otherwise healthy human infants.

Network Theoretical Approach to Explore Factors Affecting Signal Propagation and Stability in Dementia's Protein-Protein Interaction Network.

Dementia-a syndrome affecting human cognition-is a major public health concern given to its rising prevalence worldwide. Though multiple research studies have analyzed disorders such as Alzheimer's disease and Frontotemporal dementia using a systems biology approach, a similar approach to dementia syndrome as a whole is required. In this study, we try to find the high-impact core regulating processes and factors involved in dementia's protein-protein interaction network. We also explore various aspects related to its stability and signal propagation. Using gene interaction databases such as STRING and GeneMANIA, a principal dementia network (PDN) consisting of 881 genes and 59,085 interactions was achieved. It was assortative in nature with hierarchical, scale-free topology enriched in various gene ontology (GO) categories and KEGG pathways, such as negative and positive regulation of apoptotic processes, macroautophagy, aging, response to drug, protein binding, etc. Using a clustering algorithm (Louvain method of modularity maximization) iteratively, we found a number of communities at different levels of hierarchy in PDN consisting of 95 "motif-localized hubs", out of which, 7 were present at deepest level and hence were key regulators (KRs) of PDN (HSP90AA1, HSP90AB1, EGFR, FYN, JUN, CELF2 and CTNNA3). In order to explore aspects of network's resilience, a knockout (of motif-localized hubs) experiment was carried out. It changed the network's topology from a hierarchal scale-free topology to scale-free, where independent clusters exhibited greater control. Additionally, network experiments on interaction of druggable genome and motif-localized hubs were carried out where UBC, EGFR, APP, CTNNB1, NTRK1, FN1, HSP90AA1, MDM2, VCP, CTNNA1 and GRB2 were identified as hubs in the resultant network (RN). We finally concluded that stability and resilience of PDN highly relies on motif-localized hubs (especially those present at deeper levels), making them important therapeutic intervention candidates. HSP90AA1, involved in heat shock response (and its master regulator, i.e., HSF1), and EGFR are most important genes in pathology of dementia apart from KRs, given their presence as KRs as well as hubs in RN.

ß Cell Hypoxia-Inducible Factor-1α Is Required for the Prevention of Type 1 Diabetes.

The development of autoimmune disease type 1 diabetes (T1D) is determined by both genetic background and environmental factors. Environmental triggers include RNA viruses, particularly coxsackievirus (CV), but how they induce T1D is not understood. Here, we demonstrate that deletion of the transcription factor hypoxia-inducible factor-1α (HIF-1α) from ß cells increases the susceptibility of non-obese diabetic (NOD) mice to environmentally triggered T1D from coxsackieviruses and the ß cell toxin streptozotocin. Similarly, knockdown of HIF-1α in human islets leads to a poorer response to coxsackievirus infection. Studies in coxsackievirus-infected islets demonstrate that lack of HIF-1α leads to impaired viral clearance, increased viral load, inflammation, pancreatitis, and loss of ß cell mass. These findings show an important role for ß cells and, specifically, lack of ß cell HIF-1α in the development of T1D. These data suggest new strategies for the prevention of T1D.

Network Modeling of microRNA-mRNA Interactions in Neuroblastoma Tumorigenesis Identifies miR-204 as a Direct Inhibitor of MYCN.

Neuroblastoma is a pediatric cancer of the sympathetic nervous system where MYCN amplification is a key indicator of poor prognosis. However, mechanisms by which MYCN promotes neuroblastoma tumorigenesis are not fully understood. In this study, we analyzed global miRNA and mRNA expression profiles of tissues at different stages of tumorigenesis from TH-MYCN transgenic mice, a model of MYCN-driven neuroblastoma. On the basis of a Bayesian learning network model in which we compared pretumor ganglia from TH-MYCN+/+ mice to age-matched wild-type controls, we devised a predicted miRNA-mRNA interaction network. Among the miRNA-mRNA interactions operating during human neuroblastoma tumorigenesis, we identified miR-204 as a tumor suppressor miRNA that inhibited a subnetwork of oncogenes strongly associated with MYCN-amplified neuroblastoma and poor patient outcome. MYCN bound to the miR-204 promoter and repressed miR-204 transcription. Conversely, miR-204 directly bound MYCN mRNA and repressed MYCN expression. miR-204 overexpression significantly inhibited neuroblastoma cell proliferation in vitro and tumorigenesis in vivo Together, these findings identify novel tumorigenic miRNA gene networks and miR-204 as a tumor suppressor that regulates MYCN expression in neuroblastoma tumorigenesis.Significance: Network modeling of miRNA-mRNA regulatory interactions in a mouse model of neuroblastoma identifies miR-204 as a tumor suppressor and negative regulator of MYCN. Cancer Res; 78(12); 3122-34. ©2018 AACR.

Deletion of ARNT (Aryl hydrocarbon receptor nuclear translocator) in ß-cells causes islet transplant failure with impaired ß-cell function.

BACKGROUND: Replacing ß-cells by islet-transplantation can cure type 1 diabetes, but up to 70% of ß-cells die within 10 days of transplantation. ARNT (Aryl hydrocarbon Receptor Nuclear Translocator) regulates ß-cell function, and potentially survival. Lack of ARNT impairs the ability of ß-cells to respond to physiological stress and potentiates the onset of diabetes, but the exact role of ARNT in graft outcome is unknown. AIM: To investigate the effect of ß-cell deletion of ARNT on graft outcomes. METHODS: Islets were isolated from donor mice which had ß-cell specific ARNT-deletion (ß-ARNT) or littermate floxed controls. The islets were transplanted into diabetic SCID recipients in ratios of (a) 3 donors: 1 recipient, (b) 1 donor: 1 recipient or (c) ½ of the islets from 1 donor: 1 recipient. After 28 days, the kidney containing the graft was removed (nephrectomy) to exclude regeneration of the endogenous pancreas. RESULTS: In the supra-physiological-mass model (3:1), both groups achieved reasonable glycaemia, with slightly higher levels in ß-ARNT-recipients. In adequate-mass model (1:1), ß-ARNT recipients had poor glucose control versus floxed-control recipients and versus the ß-ARNT donors. In the low-ß-cell-mass model (½:1) ß-ARNT transplants completely failed, whereas controls had good outcomes. Unexpectedly, there was no difference in the graft insulin content or ß-cell mass between groups indicating that the defect was not due to early altered ß-cell survival. CONCLUSION: Outcomes for islet transplants lacking ß-cell ARNT were poor, unless markedly supra-physiological masses of islets were transplanted. In the 1:1 transplant model, there was no difference in ß-cell volume. This is surprising because transplants of islets lacking one of the ARNT-partners HIF-1α have increased apoptosis and decreased islet volume. ARNT also partners HIF-2α and AhR (aryl hydrocarbon receptor) to form active transcriptional complexes, and further work to understand the roles of HIF-2α and AhR in transplant outcomes is needed.