Background: Efficacy of treatment for small intestinal bacterial overgrowth (SIBO) is typically measured by subjective response to antibiotics.However, validated patient-reported outcome measures have not yet been adapted for the management of SIBO.Aim: To prospectively investigate clinical response to treatment for patients with SIBO using validated patientreported outcomes measures instruments.Methods: This was a longitudinal study of adults undergoing lactulose breath test (LBT) for SIBO at a tertiary center in 9/2018-11/2019.Validated symptom questionnaires were prospectively collected at the time of LBT, including the GI Patient Reported Outcomes Measurement Information System (GI-PROMIS) scales for belly pain, gas/bloating, nausea, constipation, and diarrhea; a visual analog scale (VAS) for GI symptom severity, and the 12-item short form health survey (SF-12v2) measuring health-related quality of life (HR-QOL).Patients who tested positive on LBT, defined as ‡20 ppm rise in hydrogen within 90 minutes and/or methane level ‡10 ppm, were included for follow-up.All patients underwent antibiotic therapy in response to positive LBT per their clinicians' recommendations.At 1-year post-LBT, all patients completed GI-PROMIS questions in their predominant symptom domain, VAS, and SF-12v2.Raw GI-PROMIS scores were scaled to standardized T-scores.Statistical analyses were performed using Pearson's correlation.Results: 69 patients (81% female, mean age 49.7 y) tested positive on LBT and completed symptom inventories pre-LBT and at 1 year post-LBT.Post-treatment physical component scale (PCS) on SF-12v2 was inversely associated with post-treatment GI-PROMIS score, suggesting that improvement in physical HRQOL is associated decreased symptoms in the predominant symptom domain (R=-0.41,p=0.002).Post-treatment mental component scale (MCS) on SF-12v2 had similar negative association with post-treatment GI-PROMIS score (R=-0.31,p=0.02), suggesting improved mental HRQOL with decreased symptoms.Similarly, pre/post treatment change in MCS inversely correlated to pre/post treatment change in GI-PROMIS score (R=-0.36,p=0.01).There was a strong positive linear correlation between post-treatment VAS and GI-PROMIS score (R=0.70,p<0.0001).Likewise, there was high degree of correlation between pre/post treatment change in VAS and pre/post treatment change in GI-PROMIS score (R=0.58,p<0.0001).Conclusion: There were high degrees of agreement among multiple patient reported outcome measures in assessing response to treatment for SIBO patients at 1 year follow up.These validated symptom instruments (GI-PROMIS, SF-12v2, and VAS) reflect both overall and GI specific symptom severity and HR-QOL, and may be used in the clinical setting to aide in management of conditions with wide array of symptoms and presentations such as SIBO.

Breast cancer cell-response to inflammatory cytokines such as interleukin-6 (IL-6) and oncostatin M (OSM) may affect the course of clinical disease in a cancer subtype-dependent manner. Furthermore, vascular endothelial growth factor A (VEGF) secretion induced by IL-6 and OSM may also be subtype-dependent. Utilizing datasets from Oncomine, we show that poor survival of invasive ductal carcinoma (IDC) breast cancer patients is correlated with both high VEGF expression and high cytokine or cytokine receptor expression in tumors. Importantly, epidermal growth factor receptor-negative (HER2-), but not HER2-positive (HER2+), patient survival is significantly lower with high tumor co-expression of VEGF and OSM, OSMRβ, IL-6, or IL-6Rα compared to low co-expression. Furthermore, assessment of HER2- breast cancer cells in vitro identified unique signaling differences regulating cytokine-induced VEGF secretion. The levels of VEGF secretion were analyzed by ELISA with siRNAs for hypoxia inducible factor 1 α (HIF1α) and signal transducer and activator of transcription 3 (STAT3). Specifically, we found that estrogen receptor-negative (ER-) MDA-MB-231 cells respond only to OSM through STAT3 signaling, while ER+ T47D cells respond to both OSM and IL-6, though to IL-6 to a lesser extent. Additionally, in the ER+ T47D cells, OSM signals through both STAT3 and HIF1α. These results highlight that the survival of breast cancer patients with high co-expression of VEGF and IL-6 family cytokines is dependent on breast cancer subtype. Thus, the heterogeneity of human breast cancer in relation to IL-6 family cytokines and VEGF may have important implications in clinical treatment options, disease progression, and ultimately patient prognosis.

Bone Morphogenetic Protein 1 (BMP1) inhibition is a potential method for treating fibrosis because BMP1, a member of the zinc metalloprotease family, is required to convert pro-collagen to collagen. A novel class of reverse hydroxamate BMP1 inhibitors was discovered, and cocrystal structures with BMP1 were obtained. The observed binding mode is unique in that the small molecule occupies the nonprime side of the metalloprotease pocket providing an opportunity to build in metalloprotease selectivity. Structure-guided modification of the initial hit led to the identification of an oral in vivo tool compound with selectivity over other metalloproteases. Due to irreversible inhibition of cytochrome P450 3A4 for this chemical class, the risk of potential drug-drug interactions was managed by optimizing the series for subcutaneous injection.

The Poloxamer family of surfactants are commonly used in the biopharmaceutical industry as cell culture media additives to protect cells from the turbulent environment of sparged bioreactors. Despite the widespread use of poloxamers in cell culture, their performance as cell protectants varies depending on their physical structure, molecular weight, and batch-to-batch composition. In this study, the interfacial properties of Poloxamer 188 (P188), Poloxamer 407 (P407), and a mixture of P188 and P407 were characterized to investigate the mechanism of surfactant-mediated shear protection of mammalian cells. The foam stability and equilibrium surface tension of these surfactant systems correlated with their ability to mitigate physical damage to cells in a turbulent environment. We demonstrate that while P188 can function as highly effective shear protectant, the presence of a surface-active contaminant can greatly hinder its protective characteristics. P407 was found to function as such an interfacially active "impurity," disrupting shear protection when mixed with P188 by preferentially adsorbing to the gas-liquid and membrane-liquid interface. Addition of surface-active impurities altered the interfacial properties of the surfactant system and could be detected using an equilibrium surface tension assay. The mechanism of disruption by P407 was determined to be independent of cell-to-bubble attachment, suggesting that poloxamer adsorption to and subsequent reinforcement of the cell membrane may play a key role in protecting cells in high shear environments. This investigation contributes to our understanding of the mechanism of surfactant-mediated shear protection of cells and demonstrates that a surface tension assay can be utilized as a screening tool to ensure that poloxamer lots are free of surface active impurities.

Glycoprotein 130 (gp130) is a glycosylated type 1 membrane protein and functions as a cytokine receptor modulating activity of the interleukin-6 (IL-6) family cytokines, which include IL-6, IL-11, IL-27, oncostatin M (OSM), leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin 1 (CT-1), cardiotrophin-like cytokine (CLC), and neuropoietin (NP). This study was designed to determine differential expression of VEGF in breast cancer cells by IL-6 family cytokines, and to elucidate the molecular mechanisms and signaling pathways involved in cytokine-mediated VEGF induction. Triple negative breast cancer (TNBC) MDA-MB-231 and estrogen receptor positive/progesterone receptor positive/Her2 negative (ER+/PR+/Her2-) T47D cells were treated with three cytokines (25 ng/mL), OSM, IL-6, and LIF, and analyzed. Our results showed that all three cytokines increased levels of HIF1α by Western blot analysis. OSM lead to a greater production of VEGF compared to IL-6 and LIF, as measured by enzyme-linked immunosorbent assay (ELISA) and as seen by luciferase reporter assay. Of the three cytokines, OSM alone induced VEGF in TNBC MDA-MB-231 cells, and this effect was mediated by signal transducer activator of transcription 3 (STAT3) signaling. However, both OSM and IL-6 increased VEGF secretion levels in ER+/PR+/Her2- T47D breast cancer cells, and this signaling was mediated by both HIF1α and pSTAT3, utilizing ELISA and Western blot analysis. These results implicate OSM as the key gp130 cytokine inducing VEGF secretion, and also suggest that this induction is not always HIF1α dependent and is cell-line specific. Citation Format: Danielle S. Hedeen, Ken Tawara, Madhuri Nandakumar, Ryan Fox, David Chang, Alex Ide, Andrew Oler, Dollie LaJoie, Cheryl L. Jorcyk. Differential expression of VEGF in breast cancer cells induced by gp130 cytokines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4183. doi:10.1158/1538-7445.AM2015-4183

Abstract Oncostatin M (OSM) is an interleukin-6 (IL-6) family cytokine that has been shown to induce expression of vascular endothelial growth factor (VEGF) in astroglioma and in hepatocellular and breast carcinoma. It is also known to induce VEGF in non-cancerous cells such as cardiac myocytes, hepatocytes, and smooth muscle cells. Under hypoxic conditions, VEGF expression is known to be dependent on the transcription factor hypoxia-inducible factor 1 alpha (HIF1α). Our current studies were designed to determine whether VEGF expression induced by OSM and other IL-6 family members, including IL-6 and leukemia inhibitory factor (LIF), is likewise dependent on HIF1α in human breast cancer cells. To analyze VEGF expression levels, MDA-MB-231 and T47D human breast cancer cells were cultured in the presence or absence of OSM, IL-6, and LIF, with or without a HIF1α siRNA, for 12 to 72 hrs. All three cytokines significantly induced HIF1α expression by Western blot analysis at each time point in both MDA-MD-231 and T47D cells. Conditioned media from these experiments was also analyzed using ELISA to determine HIF1α-induced VEGF expression. Treatment with OSM resulted in a two- to three-fold induction of secreted VEGF, while treatment with IL-6 and LIF showed no significant induction. In addition, OSM-induced VEGF expression was independent of HIF1α in MDA-MB-231 cells. This was indicated by unchanged secreted VEGF levels despite the reduction of HIF1α expression in siRNA treated groups. In T47D cells however, HIF1α siRNA-treatment for 72 hrs reduced VEGF levels by 40% suggesting that OSM-induced HIF1α may be partially responsible for VEGF expression in this cell line. To summarize, it is important to note that IL-6 and LIF did not induce VEGF expression in two commonly used human breast cancer cell lines, MDA-MB-231 and T47D. Furthermore, OSM-induced VEGF was not dependent on HIF1α in MDA-MB-231 cells, suggesting regulation of VEGF by an alternative molecular mechanism. Investigations are in progress to better understand mechanisms utilized by breast cancer cells for OSM-induced VEGF expression. Future results could lead to the potential design of OSM-targeted therapeutics. Funded by NIH R15CA137510, ACS RSG-09-276-01-CSM, Susan G. Komen for the Cure KG100513, and NIH/NCRR P20RR016454. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1580. doi:10.1158/1538-7445.AM2011-1580