Host obesity alters the ovarian tumor immune microenvironment and impacts response to standard of care chemotherapy.

BACKGROUND: The majority of women with epithelial ovarian cancer (OvCa) are diagnosed with metastatic disease, resulting in a poor 5-year survival of 31%. Obesity is a recognized non-infectious pandemic that increases OvCa incidence, enhances metastatic success and reduces survival. We have previously demonstrated a link between obesity and OvCa metastatic success in a diet-induced obesity mouse model wherein a significantly enhanced tumor burden was associated with a decreased M1/M2 tumor-associated macrophage ratio (Liu Y et al. Can, Res. 2015; 75:5046-57). METHODS: The objective of this study was to use pre-clinical murine models of diet-induced obesity to evaluate the effect of a high fat diet (HFD) on response to standard of care chemotherapy and to assess obesity-associated changes in the tumor microenvironment. Archived tumor tissues from ovarian cancer patients of defined body mass index (BMI) were also evaluated using multiplexed immunofluorescence analysis of immune markers. RESULTS: We observed a significantly diminished response to standard of care paclitaxel/carboplatin chemotherapy in HFD mice relative to low fat diet (LFD) controls. A corresponding decrease in the M1/M2 macrophage ratio and enhanced tumor fibrosis were observed both in murine DIO studies and in human tumors from women with BMI > 30. CONCLUSIONS: Our data suggest that the reported negative impact of obesity on OvCa patient survival may be due in part to the effect of the altered M1/M2 tumor-associated macrophage ratio and enhanced fibrosis on chemosensitivity. These data demonstrate a contribution of host obesity to ovarian tumor progression and therapeutic response and support future combination strategies targeting macrophage polarization and/or fibrosis in the obese host.

Enhanced Lyn Activity Causes Severe, Progressive Emphysema and Lung Cancer.

The epidemiological patterns of incident chronic obstructive pulmonary disease (COPD) and lung adenocarcinoma are changing, with an increasing fraction of disease occurring in patients who are never-smokers or were not exposed to traditional risk factors. However, causative mechanism(s) are obscure. Overactivity of Src family kinases (SFKs) and myeloid cell-dependent inflammatory lung epithelial and endothelial damage are independent candidate mechanisms, but their pathogenic convergence has not been demonstrated. Here we present a novel preclinical model in which an activating mutation in Lyn, a nonreceptor SFK that is expressed in immune cells, epithelium, and endothelium-all strongly implicated in the pathogenesis of COPD-causes spontaneous inflammation, early-onset progressive emphysema, and lung adenocarcinoma. Surprisingly, even though activated macrophages, elastolytic enzymes, and proinflammatory cytokines were prominent, bone marrow chimeras formally demonstrated that myeloid cells were not disease initiators. Rather, lung disease arose from aberrant epithelial cell proliferation and differentiation, microvascular lesions within an activated endothelial microcirculation, and amplified EGFR (epidermal growth factor receptor) expression. In human bioinformatics analyses, LYN expression was increased in patients with COPD and was correlated with increased EGFR expression, a known lung oncogenic pathway, and LYN was linked to COPD. Our study shows that a singular molecular defect causes a spontaneous COPD-like immunopathology and lung adenocarcinoma. Furthermore, we identify Lyn and, by implication, its associated signaling pathways as new therapeutic targets for COPD and cancer. Moreover, our work may inform the development of molecular risk screening and intervention methods for disease susceptibility, progression, and prevention of these increasingly prevalent conditions.

Outcomes of the "BRCA Quality Improvement Dissemination Program": An initiative to improve patient receipt of cancer genetics services at five health systems.

OBJECTIVE: A quality improvement initiative (QII) was conducted with five community-based health systems' oncology care centers (sites A-E). The QII aimed to increase referrals, genetic counseling (GC), and germline genetic testing (GT) for patients with ovarian cancer (OC) and triple-negative breast cancer (TNBC). METHODS: QII activities occurred at sites over several years, all concluding by December 2020. Medical records of patients with OC and TNBC were reviewed, and rates of referral, GC, and GT of patients diagnosed during the 2 years before the QII were compared to those diagnosed during the QII. Outcomes were analyzed using descriptive statistics, two-sample t-test, chi-squared/Fisher's exact test, and logistic regression. RESULTS: For patients with OC, improvement was observed in the rate of referral (from 70% to 79%), GC (from 44% to 61%), GT (from 54% to 62%) and decreased time from diagnosis to GC and GT. For patients with TNBC, increased rates of referral (from 90% to 92%), GC (from 68% to 72%) and GT (81% to 86%) were observed. Effective interventions streamlined GC scheduling and standardized referral processes. CONCLUSION: A multi-year QII increased patient referral and uptake of recommended genetics services across five unique community-based oncology care settings.

Oleuropein Activates Neonatal Neocortical Proteasomes, but Proteasome Gene Targeting by AAV9 Is Variable in a Clinically Relevant Piglet Model of Brain Hypoxia-Ischemia and Hypothermia.

Cerebral hypoxia-ischemia (HI) compromises the proteasome in a clinically relevant neonatal piglet model. Protecting and activating proteasomes could be an adjunct therapy to hypothermia. We investigated whether chymotrypsin-like proteasome activity differs regionally and developmentally in the neonatal brain. We also tested whether neonatal brain proteasomes can be modulated by oleuropein, an experimental pleiotropic neuroprotective drug, or by targeting a proteasome subunit gene using recombinant adeno-associated virus-9 (AAV). During post-HI hypothermia, we treated piglets with oleuropein, used AAV-short hairpin RNA (shRNA) to knock down proteasome activator 28γ (PA28γ), or enforced PA28γ using AAV-PA28γ with green fluorescent protein (GFP). Neonatal neocortex and subcortical white matter had greater proteasome activity than did liver and kidney. Neonatal white matter had higher proteasome activity than did juvenile white matter. Lower arterial pH 1 h after HI correlated with greater subsequent cortical proteasome activity. With increasing brain homogenate protein input into the assay, the initial proteasome activity increased only among shams, whereas HI increased total kinetic proteasome activity. OLE increased the initial neocortical proteasome activity after hypothermia. AAV drove GFP expression, and white matter PA28γ levels correlated with proteasome activity and subunit levels. However, AAV proteasome modulation varied. Thus, neonatal neocortical proteasomes can be pharmacologically activated. HI slows the initial proteasome performance, but then augments ongoing catalytic activity. AAV-mediated genetic manipulation in the piglet brain holds promise, though proteasome gene targeting requires further development.

Improved detection of flaviviruses in Australian mosquito populations via replicative intermediates.

Mosquito-borne flaviviruses are significant contributors to the arboviral disease burdens both in Australia and globally. While routine arbovirus surveillance remains a vital exercise to identify known flaviviruses in mosquito populations, novel or divergent and emerging species can be missed by these traditional methods. The MAVRIC (monoclonal antibodies to viral RNA intermediates in cells) system is an ELISA-based method for broad-spectrum isolation of positive-sense and double-stranded RNA (dsRNA) viruses based on detection of dsRNA in infected cells. While the MAVRIC ELISA has successfully been used to detect known and novel flaviviruses in Australian mosquitoes, we previously reported that dsRNA could not be detected in dengue virus-infected cells using this method. In this study we identified additional flaviviruses which evade detection of dsRNA by the MAVRIC ELISA. Utilising chimeric flaviviruses we demonstrated that this outcome may be dictated by the non-structural proteins and/or untranslated regions of the flaviviral genome. In addition, we report a modified fixation method that enables improved detection of flavivirus dsRNA and inactivation of non-enveloped viruses from mosquito populations using the MAVRIC system. This study demonstrates the utility of anti-dsRNA monoclonal antibodies for identifying viral replication in insect and vertebrate cell systems and highlights a unique characteristic of flavivirus replication.

Novel monoclonal antibodies against Australian strains of negeviruses and insights into virus structure, replication and host -restriction.

We report the isolation of Australian strains of Bustos virus and Ngewotan virus, two insect-specific viruses in the newly identified taxon Negevirus, originally isolated from Southeast Asian mosquitoes. Consistent with the expected insect-specific tropism of negeviruses, these isolates of Ngewotan and Bustos viruses, alongside the Australian negevirus Castlerea virus, replicated exclusively in mosquito cells but not in vertebrate cells, even when their temperature was reduced to 34 °C. Our data confirmed the existence of two structural proteins, putatively one membrane protein forming the majority of the virus particle, and one glycoprotein forming a projection on the apex of the virions. We generated and characterized 71 monoclonal antibodies to both structural proteins of the two viruses, most of which were neutralizing. Overall, these data increase our knowledge of negevirus mechanisms of infection and replication in vitro.

Mechanical Characteristics of Hybrid Composites with ±45° Glass and 0°/90° Stainless Steel Fibers.

Lack of energy dissipation is one of the shortcomings of conventional glass fiber reinforced composites. The addition of steel fibers to the conventional FRP composite to create a hybrid composite has been recently investigated as an option to address this limitation. The current literature is limited to composites reinforced with metal and non-metal fibers of the same alignment. In this study, hybrid and nonhybrid FRP composites of different layups, fiber content, and weave type were manufactured and subjected to hysteretic tensile loads. Woven glass fabrics in ±45° orientation were hybridized with unidirectional stainless steel fabrics in 0° and 90° orientations. This put the glass and steel layers in in-plane shear and normal stresses, respectively. The nonlinear stress⁻strain relationship, residual plastic strains, energy dissipation capability, and failure mechanisms of hybrid and nonhybrid composite type were compared. The hybrid composites presented improved energy dissipation, tensile strength, and stiffness when compared to nonhybrid ones. The applicability of an existing constitutive model that was originally developed for in-plane shear of conventional composites was investigated and refinements were proposed to present the hysteretic stress⁻strain relationship after addition of steel fibers. The refined model captured the increased plastic strain values and energy dissipation because of stainless steel fibers in the hybrid composite samples. An Armstrong⁻Frederick plasticity model was implemented to model the stress⁻strain relationship of the stainless steel composite samples.

Mechanical Behavior of Stainless Steel Fiber-Reinforced Composites Exposed to Accelerated Corrosion.

Recent advancements in metal fibers have introduced a promising new type of stainless steel fiber with high stiffness, high failure strain, and a thickness < 100 µm (<0.00394 in.) that can be utilized in a steel fiber-reinforced polymer. However, stainless steel is known to be susceptible to pitting corrosion. The main goal of this study is to compare the impact of corrosion on the mechanical properties of steel fiber-reinforced composites with those of conventional types of stainless steel. By providing experimental evidences, this study may promote the application of steel fiber-reinforced composite as a viable alternative to conventional metals. Samples of steel fiber-reinforced polymer and four different types of stainless steel were subjected to 144 and 288 h of corrosion in ferric chloride solution to simulate accelerated corrosion conditions. The weight losses due to corrosion were recorded. The corroded and control samples were tested under monotonic tensile loading to measure the ultimate stresses and strains. The effect of corrosion on the mechanical properties of the different materials was evaluated. The digital image correlation (DIC) technique was used to investigate the failure mechanism of the corrosion-damaged specimens. Overall, steel fiber-reinforced composites had the greatest corrosion resistance.

Heterogeneity of lower airway inflammation in children with severe-persistent asthma.

RATIONALE: The treatment of children with severe-persistent asthma remains problematic. Recent studies suggest that stratification of this cohort by inflammatory type may be useful in designing effective treatment strategies. In this study, we examined the inflammatory profile in bronchoalveolar lavage fluid from children with severe-persistent asthma and compared this profile with serum IgE levels. METHODS: The inflammatory profile in the bronchoalveolar fluid from 32 children who met criteria for severe-persistent asthma as defined by the Severe Asthma Research Program (SARP) were analyzed retrospectively. Inflammatory patterns were classified as neutrophilic, eosinophilic, mixed, or pauci-granulocytic. Serum total IgE was measured prior to bronchoscopy and determined by ELISA at each hospital's lab by standard procedures. RESULTS: The most common pattern of inflammation in this cohort was neutrophilic (37.5%) followed by eosinophilic (28.1%), mixed (21.9%), and pauci-granulocytic (11.1%). The odds ratio of an eosinophilic BAL pattern for patients with an elevated serum IgE was 4.67 (CI 0.78-28, P = 0.12). A correlation between serum IgE levels and BAL eosinophil percentages was present (P = 0.04). CONCLUSIONS: To our knowledge, ours is one of few studies to systematically investigate the pattern of lower airway inflammation in children with severe-persistent asthma. Our results differ from a recent investigation in children, showing more heterogeneity and a greater proportion of neutrophilic inflammation. Further investigation is required to determine whether specific inflammatory patterns are associated with specific etiologies, and whether individualized therapy is warranted.

West nile virus-induced activation of mammalian target of rapamycin complex 1 supports viral growth and viral protein expression.

UNLABELLED: Since its introduction in New York City, NY, in 1999, West Nile virus (WNV) has spread to all 48 contiguous states of the United States and is now the leading cause of epidemic encephalitis in North America. As a member of the family Flaviviridae, WNV is part of a group of clinically important human pathogens, including dengue virus and Japanese encephalitis virus. The members of this family of positive-sense, single-stranded RNA viruses have limited coding capacity and are therefore obligated to co-opt a significant amount of cellular factors to translate their genomes effectively. Our previous work has shown that WNV growth was independent of macroautophagy activation, but the role of the evolutionarily conserved mammalian target of rapamycin (mTOR) pathway during WNV infection was not well understood. mTOR is a serine/threonine kinase that acts as a central cellular censor of nutrient status and exercises control of vital anabolic and catabolic cellular responses such as protein synthesis and autophagy, respectively. We now show that WNV activates mTOR and cognate downstream activators of cap-dependent protein synthesis at early time points postinfection and that pharmacologic inhibition of mTOR (KU0063794) significantly reduced WNV growth. We used an inducible Raptor and Rictor knockout mouse embryonic fibroblast (MEF) system to further define the role of mTOR complexes 1 and 2 in WNV growth and viral protein synthesis. Following inducible genetic knockout of the major mTOR cofactors raptor (TOR complex 1 [TORC1]) and rictor (TORC2), we now show that TORC1 supports flavivirus protein synthesis via cap-dependent protein synthesis pathways and supports subsequent WNV growth. IMPORTANCE: Since its introduction in New York City, NY, in 1999, West Nile virus (WNV) has spread to all 48 contiguous states in the United States and is now the leading cause of epidemic encephalitis in North America. Currently, the mechanism by which flaviviruses such as WNV translate their genomes in host cells is incompletely understood. Elucidation of the host mechanisms required to support WNV genome translation will provide broad understanding for the basic mechanisms required to translate capped viral RNAs. We now show that WNV activates mTOR and cognate downstream activators of cap-dependent protein synthesis at early time points postinfection. Following inducible genetic knockout of the major mTOR complex cofactors raptor (TORC1) and rictor (TORC2), we now show that TORC1 supports WNV growth and protein synthesis. This study demonstrates the requirement for TORC1 function in support of WNV RNA translation and provides insight into the mechanisms underlying flaviviral RNA translation in mammalian cells.