The interplay of growth differentiation factor 15 (GDF15) expression and M2 macrophages during prostate carcinogenesis.

M2 (tumor-supportive) macrophages may upregulate growth differentiation factor 15 (GDF15), which is highly expressed in prostate tumors, but the combined utility of these markers as prognostic biomarkers are unclear. We retrospectively studied 90 prostate cancer cases that underwent radical prostatectomy as their primary treatment and were followed for biochemical recurrence (BCR). These cases also had a benign prostate biopsy at least 1 year or more before their prostate cancer surgery. Using computer algorithms to analyze digitalized immunohistochemically stained slides, GDF15 expression and the presence of M2 macrophages based on the relative density of CD204- and CD68-positive macrophages were measured in prostate: (i) benign biopsy, (ii) cancer and (iii) tumor-adjacent benign (TAB) tissue. Both M2 macrophages (P = 0.0004) and GDF15 (P < 0.0001) showed significant inter-region expression differences. Based on a Cox proportional hazards model, GDF15 expression was not associated with BCR but, in men where GDF15 expression differences between cancer and TAB were highest, the risk of BCR was significantly reduced (hazard ratio = 0.26; 95% confidence interval = 0.09-0.94). In addition, cases with high levels of M2 macrophages in prostate cancer had almost a 5-fold increased risk of BCR (P = 0.01). Expression of GDF15 in prostate TAB was associated with M2 macrophage levels in both prostate cancer and TAB and appeared to moderate M2-macrophage-associated BCR risk. In summary, the relationship of GDF15 expression and CD204-positive M2 macrophage levels is different in a prostate tumor environment compared with an earlier benign biopsy and, collectively, these markers may predict aggressive disease.

F-Actin is associated with a worsening qSOFA score and intensive care unit admission in emergency department patients at risk for sepsis.

Objective: We previously demonstrated that plasma levels of F-actin and Thymosin Beta 4 differs among patients with septic shock, non-infectious systemic inflammatory syndrome and healthy controls and may serve as biomarkers for the diagnosis of sepsis. The current study aims to determine if these proteins are associated with or predictive of illness severity in patients at risk for sepsis in the Emergency Department (ED).Methods: Prospective, biomarker study enrolling patients (>18 years) who met the Shock Precautions on Triage Sepsis rule placing them at-risk for sepsis.Results: In this study of 203 ED patients, F-actin plasma levels had a linear trend of increase when the quick Sequential Organ Failure Assessment (qSOFA) score increased. F-actin was also increased in patients who were admitted to the Intensive Care Unit (ICU) from the ED, and in those with positive urine cultures. Thymosin Beta 4 was not associated with or predictive of any significant outcome measures.Conclusion: Increased levels of plasma F-actin measured in the ED were associated with incremental illness severity as measured by the qSOFA score and need for ICU admission. F-actin may have utility in risk stratification of undifferentiated patients in the ED presenting with signs and symptoms of sepsis.

Potential effect of anti-inflammatory drug use on PSA kinetics and subsequent prostate cancer diagnosis: Risk stratification in black and white men with benign prostate biopsy.

BACKGROUND: Rising prostate-specific antigen (PSA) levels are associated with both increased risk of prostate cancer and prostatic inflammation. The confounding effects of inflammation on the utility of PSA kinetics to predict prostate cancer may be partially mitigated by anti-inflammatory drug use. We investigated the influence of anti-inflammatory drug use on the association of PSA kinetics with prostate cancer risk. METHODS: We studied 488 prostate cancer case-control pairs (290 white, 198 African American (AA)) nested in a retrospective cohort of men with a benign prostate biopsy. A series of multivariable models estimated prostate cancer risk associated with PSA velocity (PSAV) at different levels of anti-inflammatory drug use while adjusting for the presence of both clinical and histologic prostatitis. RESULTS: In men with one, two, or three or more courses of anti-inflammatory drug use, for each ng/mL/year increase in PSAV, prostate cancer risk increased 1.21-fold, 1.83-fold, and 1.97-fold, respectively ( P < 0.0001). In controls with histologic prostatitis, anti-inflammatory drug use was associated with a significantly lower PSAV ( P < 0.0001). This association was not observed in men with histologic prostatitis who were subsequently diagnosed with prostate cancer. A positive interaction between anti-inflammatory drug use and PSAV-associated prostate cancer risk was only observed in AA men, as well as a strong positive association between any anti-inflammatory drug use and clinical prostatitis ( P = 0.004). CONCLUSIONS: In men with benign prostate biopsy, accounting for the presence of histologic prostatitis and anti-inflammatory drug use, particularly in AA men, may help distinguish between men with rising PSA because of prostatitis vs undiagnosed cancer.

Breast Milk Transforming Growth Factor ß Is Associated With Neonatal Gut Microbial Composition.

BACKGROUND AND OBJECTIVES: Breast milk is a complex bioactive fluid that varies across numerous maternal and environmental conditions. Although breast-feeding is known to affect neonatal gut microbiome, the milk components responsible for this effect are not well-characterized. Given the wide range of immunological activity breast milk cytokines engage in, we investigated 3 essential breast milk cytokines and their association with early life gut microbiota. METHODS: A total of 52 maternal-child pairs were drawn from a racially diverse birth cohort based in Detroit, Michigan. Breast milk and neonatal stool specimens were collected at 1-month postpartum. Breast milk transforming growth factor (TGF)ß1, TGFß2, and IL-10 were assayed using enzyme-linked immunosorbent assays, whereas neonatal gut microbiome was profiled using 16S rRNA sequencing. RESULTS: Individually, immunomodulators TGFß1 and TGFß2 were significantly associated with neonatal gut microbial composition (R = 0.024, P = 0.041; R = 0.026, P = 0.012, respectively) and increased richness, evenness, and diversity, but IL-10 was not. The effects of TGFß1 and TGFß2, however, were not independent of one another, and the effect of TGFß2 was stronger than that of TGFß1. Higher levels of TGFß2 were associated with the increased relative abundance of several bacteria, including members of Streptococcaceae and Ruminococcaceae, and lower relative abundance of distinct Staphylococcaceae taxa. CONCLUSIONS: Breast milk TGFß concentration explains a portion of variability in gut bacterial microbiota composition among breast-fed neonates. Whether TGFß acts in isolation or jointly with other bioactive components to alter bacterial composition requires further investigation. These findings contribute to an increased understanding of how breast-feeding affects the gut microbiome-and potentially immune development-in early life.

Plasma levels of F-actin and F:G-actin ratio as potential new biomarkers in patients with septic shock.

OBJECTIVE: To compare plasma levels of F-actin, G-actin and thymosin beta 4 (TB4) in humans with septic shock, noninfectious systemic inflammatory response syndrome (SIRS) and healthy controls. RESULTS: F-actin was significantly elevated in septic shock as compared with noninfectious SIRS and healthy controls. G-actin levels were greatest in the noninfectious SIRS group but significantly elevated in septic shock as compared with healthy controls. TB4 was not detectable in the septic shock or noninfectious SIRS group above the assay's lowest detection range (78 ng/ml). CONCLUSIONS: F-actin is significantly elevated in patients with septic shock as compared with noninfectious SIRS. F-actin and the F:G-actin ratio are potential biomarkers for the diagnosis of septic shock.

Transforming growth factor beta (TGFß1) in breast milk and indicators of infant atopy in a birth cohort.

BACKGROUND: The infant gut's ability to suppress immunologic reactions to food proteins could be influenced by levels of TGFß in breast milk. We hypothesized that lower levels of TGFß(1) in the breast milk (BM) of mothers in the WHEALS birth cohort are associated with atopy at infant age 2-3 yrs. METHODS: We used data collected during infancy in addition to the results of skin prick tests (SPT+) and measures of specific IgE >0.35 IU/ml (spIgE) to milk, egg, and peanut at infant age 2-3 years. Infants were classified as food allergic (FA) based on parental report of infant symptoms/diagnoses and information from clinical assessments. RESULTS: Data for 304 cohort members were analyzed. Among non-black infants, BM-TGFß(1) was lower for those classified as FA (vs. no FA) and those SPT+ (vs., SPT-), geometric mean = 1100 pg/ml vs. 1417pg/ml, p = 0.081; and 1100 pg/ml vs. 1415pg/ml, p = 0.064, respectively. Among infants of non-atopic mothers, BM-TGFß(1) was lower for those with elevated (vs. not elevated) sIgE, geometric mean = 1347 pg/ml vs. 1651 pg/ml, p = 0.047. Using logistic regression, adjusted odds ratios describing the association of BM-TGFß1 to the presence of atopic indicators in the infant were in the hypothesized direction only for non-black infants of non-atopic mothers: aORs for FA, sIgE and SPT+ were 0.08, 0.34, and 0.26 respectively; p = 0.091, 0.13, and 0.23. CONCLUSION: Immune benefit of BM-TGFß(1) could inform prevention strategies. Evidence of an association appears greatly influenced by infant race and maternal atopy. More research can determine if these relationships represent a modifiable risk factor for the development of food allergy in certain subgroups.

Differentiating asthma phenotypes in young adults through polyclonal cytokine profiles.

BACKGROUND: Recent research has emphasized the need to better discriminate asthma phenotypes and consider underlying mechanistic endotypes in epidemiologic and clinical studies. Although allergic asthma and nonallergic asthma are frequently combined into 1 disease category in observational research and clinical trials, few studies have investigated the extent to which these 2 separate phenotypes are associated with distinct cytokine immunologic profiles in a representative young adult population. OBJECTIVE: To investigate the cytokine production-based endotypes underlying the clinical phenotypes of allergic and nonallergic asthma in a population-based birth cohort evaluated as young adults. METHODS: Participants included 18- to 21-year-old members (n = 540) of a suburban Detroit birth cohort study, the Childhood Allergy Study. Phorbol myristate acetate-stimulated whole blood interleukin (IL)-4, IL-5, IL-10, IL-12, IL-13, IL-17A, IL-17F, IL-22, and interferon-γ secretory responses were analyzed for associations comparing participants with allergic vs nonallergic asthma phenotypes with those without asthma. RESULTS: T-helper cell type (TH) 2-polarized responses, measured as higher mean IL-5 and IL-13 secretions and lower ratios of interferon-γ and IL-12 to 3 TH2 cytokines (IL-4, IL-5, or IL-13), were observed only in participants with allergic asthma. Nonallergic asthma was associated with TH1-polarized responses, including higher adjusted interferon-γ secretion compared with participants with allergic asthma and, surprisingly, those without asthma (odds ratio 2.5, confidence interval 1.2-5.1, P < .01). CONCLUSION: As expected, young adults with a history of an allergic asthma phenotype exhibited a TH2-polarized cytokine response after polyclonal stimulation. However, TH1 polarization was observed in patients with a history of nonallergic asthma. Allergic and nonallergic asthma are associated with etiologically distinct immune endotypes, underscoring the importance of discriminating these endotypes in research analyses and clinical management.

Dissociation of Hypertension and Renal Damage After Cessation of High-Salt Diet in Dahl Rats.

BACKGROUND: Every year, thousands of patients with hypertension reduce salt consumption in an effort to control their blood pressure. However, hypertension has a self-sustaining character in a significant part of the population. We hypothesized that chronic hypertension leads to irreversible renal damage that remains after removing the trigger, causing an elevation of the initial blood pressure. METHODS: Dahl salt-sensitive rat model was used for chronic, continuous observation of blood pressure. Rats were fed a high salt diet to induce hypertension, and then the diet was switched back to normal sodium content. RESULTS: We found that developed hypertension was irreversible by salt cessation: after a short period of reduction, blood pressure grew even higher than in the high-salt phase. Notably, the self-sustaining phase of hypertension was sensitive to benzamil treatment due to sustaining epithelial sodium channel hyperactivity, as shown with patch-clamp analysis. Glomerular damage and proteinuria were also irreversible. In contrast, some mechanisms, contributing to the development of salt-sensitive hypertension, normalized after salt restriction. Thus, flow cytometry demonstrated that dietary salt reduction in hypertensive animals decreased the number of total CD45+, CD3+CD4+, and CD3+CD8+ cells in renal tissues. Also, we found tubular recovery and improvement of glomerular filtration rate in the postsalt period versus a high-salt diet. CONCLUSIONS: Based on earlier publications and current data, poor response to salt restriction is due to the differential contribution of the factors recognized in the developmental phase of hypertension. We suggest that proteinuria or electrolyte transport can be prioritized over therapeutic targets of inflammatory response.

Patterns of B-cell lymphocyte expression changes in pre- and post-malignant prostate tissue are associated with prostate cancer progression.

BACKROUND: Inflammation characterized by the presence of T and B cells is often observed in prostate cancer, but it is unclear how T- and B-cell levels change during carcinogenesis and whether such changes influence disease progression. METHODS: The study used a retrospective sample of 73 prostate cancer cases (45 whites and 28 African Americans) that underwent surgery as their primary treatment and had a benign prostate biopsy at least 1 year before diagnosis. CD3+, CD4+, and CD20+ lymphocytes were quantified by immunohistochemistry in paired pre- and post-diagnostic benign prostate biopsy and tumor surgical specimens, respectively. Clusters of similar trends of expression across two different timepoints and three distinct prostate regions-benign biopsy glands (BBG), tumor-adjacent benign glands (TAG), and malignant tumor glandular (MTG) regions-were identified using Time-series Anytime Density Peaks Clustering (TADPole). A Cox proportional hazards model was used to estimate the hazard ratio (HR) of time to biochemical recurrence associated with region-specific lymphocyte counts and regional trends. RESULTS: The risk of biochemical recurrence was significantly reduced in men with an elevated CD20+ count in TAG (HR = 0.81, p = 0.01) after adjusting for covariates. Four distinct patterns of expression change across the BBG-TAG-MTG regions were identified for each marker. For CD20+, men with low expression in BBG and higher expression in TAG compared to MTG had an adjusted HR of 3.06 (p = 0.03) compared to the reference group that had nominal differences in CD20+ expression across all three regions. The two CD3+ expression patterns that featured lower CD3+ expression in the BBG compared to the TAG and MTG regions had elevated HRs ranging from 3.03 to 4.82 but did not reach statistical significance. CONCLUSIONS: Longitudinal and spatial expression patterns of both CD3+ and CD20+ suggest that increased expression in benign glands during prostate carcinogenesis is associated with an aggressive disease course.

Inflammatory endotype of odontogenic sinusitis.

BACKGROUND: Odontogenic sinusitis (ODS) is distinct from non-odontogenic rhinosinusitis with regard to clinical features as well as diagnostic and therapeutic approaches. While numerous studies have explored immune profiles of chronic rhinosinusitis, very few studies have explored the inflammatory endotype of ODS. METHODS: Odontogenic sinusitis was diagnosed by confirming infectious sinusitis adjacent to infectious maxillary odontogenic pathology. Maxillary sinus cultures and mucosal biopsies were obtained during endoscopic endonasal surgery in ODS and control patients. Controls were patients undergoing endoscopic skull base surgery with no sinus disease. Specimens were snap frozen in liquid nitrogen and stored at -80°C. Analysis was performed using a multiplex assay to measure Th-1 (TNFα, IFNγ, IL-2,12,18), Th-2 (IL-4,5,9,13), Th-17 (IL-17A,17F,22), and innate (CCL5,CXCL9,CXCL10, IL-6,8,10,12,23,27) immune pathways. Groups were compared via independent sample t-tests; if assumptions were violated, nonparametric Wilcoxon ranked sum tests were performed. RESULTS: Specimens from 22 ODS patients were compared to nine controls. ODS mucosal tissue was sampled in the setting of the following dental pathologies: post-dental extraction (n = 15), untreated apical periodontitis (n = 2), apical periodontitis after root canal therapy (n = 2), and maxillary sinus bone grafting with or without dental implantation (n = 3). The following cytokines were significantly elevated in ODS compared to controls: IFNγ, TNFα, IL-6, 8, 10, 27, and CXCL9. IL-17 levels were similar in both ODS and controls. Therefore, ODS demonstrated heightened innate and Th1 immune activity. CONCLUSION: ODS demonstrated both innate immune and Th1 inflammatory endotypes. Further studies are needed to explore ODS immunopathobiology and its potential impact on ODS management.

Treatment with N-acetyl-seryl-aspartyl-lysyl-proline prevents experimental autoimmune myocarditis in rats.

Myocarditis is commonly associated with cardiotropic infections and has been linked to development of autoimmunity. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring tetrapeptide that prevents inflammation and fibrosis in hypertension and other cardiovascular diseases; however, its effect on autoimmune-mediated cardiac diseases remains unknown. We studied the effects of Ac-SDKP in experimental autoimmune myocarditis (EAM), a model of T cell-mediated autoimmune disease. This study was conducted to test the hypothesis that Ac-SDKP prevents autoimmune myocardial injury by modulating the immune responses. Lewis rats were immunized with porcine cardiac myosin and treated with Ac-SDKP or vehicle. In EAM, Ac-SDKP prevented both systolic and diastolic cardiac dysfunction, remodeling as shown by hypertrophy and fibrosis, and cell-mediated immune responses without affecting myosin-specific autoantibodies or antigen-specific T cell responses. In addition, Ac-SDKP reduced cardiac infiltration by macrophages, dendritic cells, and T cells, pro-inflammatory cytokines [interleukin (IL)-1α, tumor necrosis factor-α, IL-2, IL-17] and chemokines (cytokine-induced neutrophil chemoattractant-1, interferon-γ-induced protein 10), cell adhesion molecules (intercellular adhesion molecule-1, L-selectin), and matrix metalloproteinases (MMP). Ac-SDKP prevents autoimmune cardiac dysfunction and remodeling without reducing the production of autoantibodies or T cell responses to cardiac myosin. The protective effects of Ac-SDKP in autoimmune myocardial injury are most likely mediated by inhibition of 1) innate and adaptive immune cell infiltration and 2) expression of proinflammatory mediators such as cytokines, chemokines, adhesion molecules, and MMPs.

The entirely carbohydrate immunogen Tn-PS A1 induces a cancer cell selective immune response and cytokine IL-17.

The tumor-associated carbohydrate antigen/hapten Thomsen-nouveau (Tn; a-D-GalpNAc-ONH2) was conjugated to a zwitterionic capsular polysaccharide, PS A1, from commensal anaerobe Bacteroides fragilis ATCC 25285/NCTC 9343 for the development of an entirely carbohydrate cancer vaccine construct and probed for immunogenicity. This communication discloses that murine anti-Tn IgG3 antibodies both bind to and recognize human tumor cells that display the Tn hapten. Furthermore, the sera from immunization of mice with Tn-PS A1 contain cytokine interleukin 17 (IL-17A), which is known to possess anti-tumor function and represents a striking difference to an IL-2, and IL-6 profile obtained with anti-PS A1 sera.

Racial differences in the systemic inflammatory response to prostate cancer.

Systemic inflammation may increase risk for prostate cancer progression, but the role it plays in prostate cancer susceptibility is unknown. From a cohort of over 10,000 men who had either a prostate biopsy or transurethral resection that yielded a benign finding, we analyzed 517 incident prostate cancer cases identified during follow-up and 373 controls with one or more white blood cell tests during a follow-up period between one and 18 years. Multilevel, multivariable longitudinal models were fit to two measures of systemic inflammation, neutrophil-to-lymphocyte ratio (NLR) and monocyte-to-lymphocyte ratio (MLR), to determine NLR and MLR trajectories associated with increased risk for prostate cancer. For both measures, we found no significant differences in the trajectories by case/control status, however in modeling NLR trajectories there was a significant interaction between race (white or Black and case-control status. In race specific models, NLR and MLR values were consistently higher over time among white controls than white cases while case-control differences in NLR and MLR trajectories were not apparent among Black men. When cases were classified as aggressive as compared to non-aggressive, the case-control differences in NLR and MLR values over time among white men were most apparent for non-aggressive cases. For NLR among white men, significant case-control differences were observed for the entire duration of observation for men who had inflammation in their initial prostate specimen. It is possible that, among white men, monitoring of NLR and MLR trajectories after an initial negative biopsy may be useful in monitoring prostate cancer risk.

Growth and differentiation factor 15 and NF-κB expression in benign prostatic biopsies and risk of subsequent prostate cancer detection.

Growth and differentiation factor 15 (GDF-15), also known as macrophage inhibitory cytokine 1 (MIC-1), may act as both a tumor suppressor and promotor and, by regulating NF-κB and macrophage signaling, promote early prostate carcinogenesis. To determine whether expression of these two inflammation-related proteins affect prostate cancer susceptibility, dual immunostaining of benign prostate biopsies for GDF-15 and NF-κB was done in a study of 503 case-control pairs matched on date, age, and race, nested within a historical cohort of 10,478 men. GDF-15 and NF-κB expression levels were positively correlated (r = 0.39; p < 0.0001), and both were significantly lower in African American (AA) compared with White men. In adjusted models that included both markers, the odds ratio (OR) for NF-κB expression was statistically significant, OR =0.87; p = 0.03; 95% confidence interval (CI) =0.77-0.99, while GDF-15 expression was associated with a nominally increased risk, OR =1.06; p = 0.27; 95% CI =0.96-1.17. When modeling expression levels by quartiles, the highest quartile of NF-κB expression was associated with almost a fifty percent reduction in prostate cancer risk (OR =0.51; p = 0.03; 95% CI =0.29-0.92). In stratified models, NF-κB had the strongest negative association with prostate cancer in non-aggressive cases (p = 0.03), older men (p = 0.03), and in case-control pairs with longer follow-up (p = 0.02). Risk associated with GDF-15 expression was best fit using nonlinear regression modeling where both first (p = 0.02) and second (p = 0.03) order GDF-15 risk terms were associated with significantly increased risk. This modeling approach also revealed significantly increased risk associated with GDF-15 expression for subsamples defined by AA race, aggressive disease, younger age, and in case-control pairs with longer follow-up. Therefore, although positively correlated in benign prostatic biopsies, NF-κB and GDF-15 expression appear to exert opposite effects on risk of prostate tumor development.

Asthma and its relationship to mitochondrial copy number: Results from the Asthma Translational Genomics Collaborative (ATGC) of the Trans-Omics for Precision Medicine (TOPMed) program.

BACKGROUND: Mitochondria support critical cellular functions, such as energy production through oxidative phosphorylation, regulation of reactive oxygen species, apoptosis, and calcium homeostasis. OBJECTIVE: Given the heightened level of cellular activity in patients with asthma, we sought to determine whether mitochondrial DNA (mtDNA) copy number measured in peripheral blood differed between individuals with and without asthma. METHODS: Whole genome sequence data was generated as part of the Trans-Omics for Precision Medicine (TOPMed) Program on participants from the Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-ethnicity (SAPPHIRE) and the Study of African Americans, Asthma, Genes, & Environment II (SAGE II). We restricted our analysis to individuals who self-identified as African American (3,651 asthma cases and 1,344 controls). Mitochondrial copy number was estimated using the sequencing read depth ratio for the mitochondrial and nuclear genomes. Respiratory complex expression was assessed using RNA-sequencing. RESULTS: Average mitochondrial copy number was significantly higher among individuals with asthma when compared with controls (SAPPHIRE: 218.60 vs. 200.47, P<0.001; SAGE II: 235.99 vs. 223.07, P<0.001). Asthma status was significantly associated with mitochondrial copy number after accounting for potential explanatory variables, such as participant age, sex, leukocyte counts, and mitochondrial haplogroup. Despite the consistent relationship between asthma status and mitochondrial copy number, the latter was not associated with time-to-exacerbation or patient-reported asthma control. Mitochondrial respiratory complex gene expression was disproportionately lower in individuals with asthma when compared with individuals without asthma and other protein-encoding genes. CONCLUSIONS: We observed a robust association between asthma and higher mitochondrial copy number. Asthma having an effect on mitochondria function was also supported by lower respiratory complex gene expression in this group.

SDF-1 and CXCR4 are up-regulated by VEGF and contribute to glioma cell invasion.

Glioma cells produce vascular endothelial growth factor (VEGF) to induce vascularization and thereby supply the malignant tissue with oxygen and nutrients. However, little is known about the direct effects of VEGF on tumor cells. In this study, we investigate the ability of VEGF to promote proliferation and invasion of human glioma cells (U251n). Since the chemokine and its receptor, SDF-1/CXCR4, promote glioma cell proliferation and are up-regulated in human glioblastomas, we also tested the effects of VEGF on SDF-1 and CXCR4 mRNA expression. Using cell culture, the effect of VEGF on proliferation of U251n cells was measured using ELISA to detect incorporated BrdU as a marker of DNA syntheses. The effects of VEGF and SDF-1 on U251n cell invasion and proliferation were measured using inhibitors to VEGF receptor1 and receptor2, DC101 and MF1, respectively, and a CXCR4 antagonist (AMD3100). SDF-1 and CXCR4 mRNA expression in U251n and U87MG cells were measured using quantitative PCR. VEGF antisense phosphorothioate oligodeoxynucleotide (AS-VEGF) was also used to down-regulate VEGF expression in U251n cells. VEGF significantly increased U251n cell proliferation and invasion in a dose-dependent manner. These effects were blocked by the VEGF receptor inhibitors, DC101/MF1. The CXCR4 antagonist AMD3100 blocked U251n increased invasion, but not proliferation. CXCR4 and SDF-1 mRNA were up-regulated when U251n and U87MG cells were treated with VEGF. Eight micrometer VEGF antisense phosphorothioate oligodeoxynucleotide (AS-VEGF) down-regulated CXCR4 and SDF-1 mRNA levels in U251n cells. VEGF has a direct effect on U251n glioma cell proliferation and invasion. VEGF up-regulates SDF-1 and CXCR4 mRNA expression, and contributes to U251n cell invasion.

Early pregnancy vitamin D and patterns of antenatal inflammation in African-American women.

Vitamin D is essential for the health of both mother and fetus during pregnancy. In the nonpregnant state, vitamin D demonstrates anti-inflammatory effects, but little is known about this relationship during pregnancy. African-American women are at a higher risk of vitamin D deficiency and for altered inflammatory responses during pregnancy. Therefore, we investigated the association of early pregnancy vitamin D nutrition, as assessed by serum 25-hydroxyvitamin D (25-OHD), with second-trimester inflammatory biomarkers (high-sensitivity C-reactive protein, IL-6, IL-10, IL-1ß and TNF-α) in 178 pregnant African-American women. Mean serum 25-OHD was 13.4±8.4 ng/ml, and most women (n=147, 82.6%) had inadequate or deficient levels of 25-OHD (<20 ng/ml). Both serum 25-OHD and some inflammatory cytokines (IL-1ß and TNF-α) demonstrated significant seasonal variation. In univariate models, log transformed 25-OHD was significantly and inversely associated with log transformed IL-1ß (p=0.002) and log transformed IL-6 (p=0.032). After adjusting for covariates, including seasonality, only the inverse association with IL-1ß remained statistically significant (p=0.027). Early pregnancy vitamin D nutrition is associated with some inflammatory biomarkers in mid-pregnancy. Additional studies are needed to determine if low vitamin D nutrition is associated with birth outcomes via an inflammatory-mediated pathway.

Mesenchymal stem cells deliver synthetic microRNA mimics to glioma cells and glioma stem cells and inhibit their cell migration and self-renewal.

MicroRNAs (miRNAs) have emerged as potential cancer therapeutics; however, their clinical use is hindered by lack of effective delivery mechanisms to tumor sites. Mesenchymal stem cells (MSCs) have been shown to migrate to experimental glioma and to exert anti-tumor effects by delivering cytotoxic compounds. Here, we examined the ability of MSCs derived from bone marrow, adipose tissue, placenta and umbilical cord to deliver synthetic miRNA mimics to glioma cells and glioma stem cells (GSCs). We examined the delivery of miR-124 and miR-145 mimics as glioma cells and GSCs express very low levels of these miRNAs. Using fluorescently labeled miRNA mimics and in situ hybridization, we demonstrated that all the MSCs examined delivered miR-124 and miR-145 mimics to co-cultured glioma cells and GSCs via gap junction- dependent and independent processes. The delivered miR-124 and miR-145 mimics significantly decreased the luciferase activity of their respected reporter target genes, SCP-1 and Sox2, and decreased the migration of glioma cells and the self-renewal of GSCs. Moreover, MSCs delivered Cy3-miR-124 mimic to glioma xenografts when administered intracranially. These results suggest that MSCs can deliver synthetic exogenous miRNA mimics to glioma cells and GSCs and may provide an efficient route of therapeutic miRNA delivery in vivo.

Synthesis and Biological Evaluation of Novel N-phenyl-5-carboxamidyl Isoxazoles as Potential Chemotherapeutic Agents for Colon Cancer.

A new series of isoxazole derivatives, N-phenyl-5-carboxamidyl isoxazoles, was investigated for their anticancer activity with solid tumor selectivity. Six N-phenyl-5-carboxamidylisoxazoles were chemically synthesized and evaluated by the in vitro disk-diffusion assay and IC50 cytotoxicity determination. The results showed that one of the derivatives, compound 3,N-(4-chlorophenyl)-5-carboxamidyl isoxazole, was the most active against colon 38 and CT-26 mouse colon tumor cells with an IC50 of 2.5 µg/mL for both cell lines. Western blot analysis showed that compound 3 significantly down-regulated the expression of phosphorylated STAT3 in both human and mouse colon cancer cells indicating that the mechanism of action for compound 3 may involve the inhibition of JAK3/STAT3 signaling pathways. Flow cytometric analysis with Annexin V staining showed that the death induced by compound 3 is mediated through cell necrosis and not apoptotic pathway. In summary, our results show that compound 3 is a new N-phenyl-5-carboxamidyl isoxazole with potential anticancer activity. Compound 3 inhibits the phosphorylation of STAT3, a novel target for chemotherapeutic drugs, and is worthy of further investigation as a potential chemotherapeutic agent for treating colon cancer.