Liza Konnikova

Knockdown of STAT3 expression by RNAi induces apoptosis in astrocytoma cells

BackgroundAstrocytomas are the most common type of primary central nervous system tumors. They are frequently associated with genetic mutations that deregulate cell cycle and render these tumors resistant to apoptosis. STAT3, signal transducer and activator of transcription 3, participates in several human cancers by inducing cell proliferation and inhibiting apoptosis and is frequently activated in astrocytomas.MethodsRNA interference was used to knockdown STAT3 expression in human astrocytes and astrocytoma cell lines. The effect of STAT3 knockdown on apoptosis, cell proliferation, and gene expression was then assessed by standard methods.ResultsWe have found that STAT3 is constitutively activated in several human astrocytoma cell lines. Knockdown of STAT3 expression by siRNA induces morphologic and biochemical changes consistent with apoptosis in several astrocytoma cell lines, but not in primary human astrocytes. Moreover, STAT3 is required for the expression of the antiapoptotic genes survivin and Bcl-xL in the A172 glioblastoma cell line.ConclusionThese results show that STAT3 is required for the survival of some astrocytomas. These studies suggest STAT3 siRNA could be a useful therapeutic agent for the treatment of astrocytomas.

Signal Transducer and Activator of Transcription 3 (STAT3) Regulates Human Telomerase Reverse Transcriptase (hTERT) Expression in Human Cancer and Primary Cells

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that plays a critical role in cytokine and growth factor signaling and is frequently activated in human tumors. Human telomerase reverse transcriptase (hTERT) is also often overexpressed in tumor cells and mediates cellular immortalization. Here we report that STAT3 directly regulates the expression of hTERT in a variety of human cancer cells. Moreover, STAT3 activity is required for the survival of many human tumors, and hTERT expression contributes to the survival of STAT3-dependent tumor cells. In addition, we find that growth factors and cytokines stimulate hTERT expression in primary human cells in a STAT3-dependent manner. Thus, STAT3 is a key regulator of hTERT expression in both normal and tumor cells.

The Role of Pulmonary Follow-up in Reducing Health Care Utilization in Infants With Bronchopulmonary Dysplasia

Objective To determine whether pulmonary follow-up affects rates of rehospitalization and visitations to emergency departments (EDs) in preterm infants with bronchopulmonary dysplasia (BPD). Methods In this retrospective cohort study, the authors identified all preterm infants born at ≤32 weeks’ gestation with at least one outpatient visit to a pulmonary follow-up clinic at Children’s Hospital Boston or a high-risk primary neurodevelopmental follow-up clinic for preterm infants. ED visits and rehospitalizations were identified through electronic medical records. Results Infants with pulmonary follow-up compared with infants without pulmonary follow-up were, respectively, younger (mean gestational age 26.3 ± 2.3 vs 28.3 ± 2.3 weeks, P < .0001), smaller at birth (birth weight <1200 g, 87.6% vs 57.2%, P < .0001), and needed more supplemental oxygen (55.7% vs 2.6%, P < .0001) and diuretics (65.8% vs 4.7%, P < .001) at the time of discharge from the neonatal intensive care unit. Although rates of rehospitalization were higher in infants with pulmonary follow-up, rates of visits to an ED for respiratory causes were not statistically significant. After controlling for baseline differences in both groups, the rates of rehospitalization or ED visits were the same for both groups. Conclusions Despite differences in lung disease status in infants with and without pulmonary follow-up, the rates of health care utilization were the same in both groups. Pulmonary follow-up may decrease the expected higher rates of ED visits and hospitalizations in preterm infants with more severe lung disease.

Impact of Antibiotics on Necrotizing Enterocolitis and Antibiotic-Associated Diarrhea

Antibiotic treatment alters the composition and metabolic function of the intestinal microbiota. These alterations may contribute to the pathogenesis of necrotizing enterocolitis (NEC) and antibiotic-associated diarrhea (AAD). Recent studies are beginning to unravel the contribution of specific groups of microbes and their metabolic pathways to these diseases. Probiotics or other microbiota-targeted therapies may provide effect strategies to prevent and treat NEC and AAD.

Enhanced TH17 Responses in Patients with IL10 Receptor Deficiency and Infantile-onset IBD

Background: IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4+ T-cell function. Methods: Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4+ T-cell subsets, naive T-cell proliferation, regulatory T cell (Treg)-mediated suppression, and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time polymerase chain reaction. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. Results: Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared with control subjects. In addition, in vitro Treg suppression of CD4+ T-cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naive cells exhibited higher proliferative capacity, a strong TH17 signature, and an increase in polarization toward TH17 cells, compared with controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1&bgr; leads to enhanced production of IL17A. Conclusions: IL10R signaling regulates TH17 polarization and T-cell proliferation in humans but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.

WASP-mediated regulation of anti-inflammatory macrophages is IL-10 dependent and is critical for intestinal homeostasis

Mutations in Wiskott–Aldrich syndrome protein (WASP) cause autoimmune sequelae including colitis. Yet, how WASP mediates mucosal homeostasis is not fully understood. Here we show that WASP-mediated regulation of anti-inflammatory macrophages is critical for mucosal homeostasis and immune tolerance. The generation and function of anti-inflammatory macrophages are defective in both human and mice in the absence of WASP. Expression of WASP specifically in macrophages, but not in dendritic cells, is critical for regulation of colitis development. Importantly, transfer of WT anti-inflammatory macrophages prevents the development of colitis. DOCK8-deficient macrophages phenocopy the altered macrophage properties associated with WASP deficiency. Mechanistically, we show that both WASP and DOCK8 regulates macrophage function by modulating IL-10-dependent STAT3 phosphorylation. Overall, our study indicates that anti-inflammatory macrophage function and mucosal immune tolerance require both WASP and DOCK8, and that IL-10 signalling modulates a WASP-DOCK8 complex.Deficiency in Wiskott-Aldrich syndrome protein (WASP) has been associated with autoimmune colitis, but the underlying mechanism is still unclear. Here the authors show that WASP deficiency is associated with defective WASP/DOCK8 complex formation, altered IL-10 signalling, and impaired anti-inflammatory macrophage functions.

An algorithm for the classification of mRNA patterns in eosinophilic esophagitis: Integration of machine learning

Background: Diagnostic evaluation of eosinophilic esophagitis (EoE) remains difficult, particularly the assessment of the patients allergic status. Objective: This study sought to establish an automated medical algorithm to assist in the evaluation of EoE. Methods: Machine learning techniques were used to establish a diagnostic probability score for EoE, p(EoE), based on esophageal mRNA transcript patterns from biopsies of patients with EoE, gastroesophageal reflux disease and controls. Dimensionality reduction in the training set established weighted factors, which were confirmed by immunohistochemistry. Following weighted factor analysis, p(EoE) was determined by random forest classification. Accuracy was tested in an external test set, and predictive power was assessed with equivocal patients. Esophageal IgE production was quantified with epsilon germ line (IGHE) transcripts and correlated with serum IgE and the Th2‐type mRNA profile to establish an IGHE score for tissue allergy. Results: In the primary analysis, a 3‐class statistical model generated a p(EoE) score based on common characteristics of the inflammatory EoE profile. A p(EoE) ≥ 25 successfully identified EoE with high accuracy (sensitivity: 90.9%, specificity: 93.2%, area under the curve: 0.985) and improved diagnosis of equivocal cases by 84.6%. The p(EoE) changed in response to therapy. A secondary analysis loop in EoE patients defined an IGHE score of ≥37.5 for a patient subpopulation with increased esophageal allergic inflammation. Conclusions: The development of intelligent data analysis from a machine learning perspective provides exciting opportunities to improve diagnostic precision and improve patient care in EoE. The p(EoE) and the IGHE score are steps toward the development of decision trees to define EoE subpopulations and, consequently, will facilitate individualized therapy. GRAPHICAL ABSTRACT Figure. No caption available.

High-dimensional immune phenotyping and transcriptional analyses reveal robust recovery of viable human immune and epithelial cells from frozen gastrointestinal tissue

Simultaneous analyses of peripheral and mucosal immune compartments can yield insight into the pathogenesis of mucosal-associated diseases. Although methods to preserve peripheral immune cells are well established, studies involving mucosal immune cells have been hampered by lack of simple storage techniques. We provide a cryopreservation protocol allowing for storage of gastrointestinal (GI) tissue with preservation of viability and functionality of both immune and epithelial cells. These methods will facilitate translational studies allowing for batch analysis of mucosal tissue to investigate disease pathogenesis, biomarker discovery and treatment responsiveness.

The Role of Nutrition in Health and Disease in Premature Infants: Current Knowledge Gaps and Defining the Research Agenda

Mounting evidence demonstrates that nutrition and growth in early life has long-lasting effects, particularly for premature infants. The life course approach to neonatal nutrition and growth incorporates several key concepts that shape our current understanding of the role of nutrition and growth in the neonatal period and identify gaps in knowledge (Fig. 8.1).