Growth Factor Binding Protein Research Articles

Hippocampal gene expression, serum cortisol level, and spatial memory in rats exposed to hypergravity.

Due to spatial disorientation reported in space, spatial memory and navigation performances could be more largely impaired by gravity changes. Hippocampus, a key structure for spatial memory, receives inputs from gravity-sensing otolith organs. To determine the key molecules in the rat hippocampus that contribute to an adaptation to altered gravity in terms of spatial memory performance. Gene expression of hippocampus and spatial memory after continuous two-weeks exposure to 2 G hypergravity (HG) were examined using a microarray analysis followed by real-time PCR methods and radial arm maze testing, respectively. Serum cortisol levels during HG load were measured as a stress marker. Accuracy to enter the correct arms in HG rats was significantly lower than that of controls, indicating an impaired spatial memory due to gravity changes. Microarray analysis followed by real-time PCR confirmed an upregulation of insulin like growth factor binding protein 2 (IGFBP2) gene. Serum cortisol level was the same level as controls at the last day of hypergravity, suggesting the adaptation to HG-induced stress. Given that the IGF systems are involved in neurotrophic and synaptic plasticity mechanisms, IGF system might contribute to the adaptation to altered gravity in terms of spatial memory.

Insulin-like growth factor binding protein-6 released from human mesenchymal stem cells confers neuronal protection through IGF-1R-mediated signaling

Human bone marrow-derived mesenchymal stem cells (hMSCs) are a desirable cell source for cell-based therapy to treat nervous system injuries due to their ability to differentiate into specific cell types. In addition to their multi-potency, hMSCs render the tissue microenvironment more favorable for tissue repair by secreting various growth factors. Our previous study demonstrated that hMSCs secrete several growth factors, including several insulin-like growth factor binding proteins (IGFBPs). Among these, IGFBP-6 binds with high affinity and inhibits insulin growth factor-2 (IGF-2) to inhibit the growth of IGF-2-dependent tumors. However, the function of IGFBP-6 in the nervous system remains to be fully elucidated. The present study investigated the protective effects of IGFBP-6 secreted by hMSCs on H2O2-injured primary cortical neuron cultures and lysolecithin-injured organotypic spinal cord slice cultures. Treatment of the H2O2-injured cortical neurons with conditioned media from hMSCs (hMSC-CM) increased the phosphorylation of Akt, reduced cell death and mitochondrial translocation of Bax, and regulated extracellular levels of IGF-1 and IGF-2. MTT assay, western blot analysis and ELISA were used to detect the cell viability and protein expression levels, respectively. An inhibitory antibody against IGFBP-6 eliminated this hMSC-CM-mediated neuroprotective effect in the injured cortical neuron cultures and spinal cord slice cultures. In addition, treatment with cyclolignan picropodophyllin, an inhibitor of IGF-1 receptor (IGF-1R), significantly inhibited neuronal protection by hMSC-CM. These findings demonstrated that hMSC-CM-mediated neuroprotection was attributed to IGF-1R-mediated signaling, potentiated via the inhibition of IGF-2 by IGFBP-6. The results of the present study provide insight into the mechanism by which hMSC administration may promote recovery from nerve injury.

Induction of Regulatory T Cells and Its Regulation with Insulin-like Growth Factor/Insulin-like Growth Factor Binding Protein-4 by Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (MSCs) are multipotent and exert anti-inflammatory effects, but the underlying mechanism remains to be elucidated. In the current study, we investigated the regulatory mechanism of regulatory T cell (Treg) induction through the growth factors released by human MSCs. Human naive CD4+ T cells were stimulated with anti-CD3/28 Abs and cocultured with human MSC culture supernatant for 48 h. The proliferation and cytokine production of CD4+ T cells and surface molecule expression on CD4+ T cells were evaluated. The proliferation of anti-CD3/28 Abs-stimulated CD4+ T cells was suppressed by the addition of human MSC culture supernatant; in addition, the production of IL-10 and IL-4 increased. The human MSC culture supernatant induced CD4+FOXP3+ Tregs that expressed CD25, CTLA-4, glucocorticoid-induced TNFR-related protein, insulin-like growth factor (IGF)-1R, and IGF-2R, showing antiproliferative activity against CD4+ T cells. In addition, the induction of Tregs by human MSC culture supernatant was enhanced by the addition of IGF and suppressed by the inhibition of IGF-1R. In contrast, a significant amount of IGF binding protein (IGFBP)-4, an inhibitor of IGF action, was detected in the human MSC culture supernatant. After neutralization of IGFBP-4 in the human MSC culture supernatant by anti-IGFBP-4 Ab, Treg numbers increased significantly. Thus, our results raise the possibility that human MSC actions also involve a negative-regulatory mechanism that suppresses Treg proliferation by releasing IGFBP-4. The results of this study suggest that regulation of IGF may be important for treatments using human MSCs.

Obese women initiate autophagy in the endometrium but cannot sustain it: a mechanism for poor endometrial decidualization

To investigate the role of autophagy in obese women’s impaired ability to undergo in vitro endometrial decidualization. Basic Science Project Women with regular menses, proven fertility and exclusion criteria of endometriosis were enrolled at the time of elective tubal ligation. Patients underwent an endometrial biopsy and each sample was cultured for nine days to synchronize the samples and plated into a control and decidualzed group. The decidualized group were exposed to 1 mM medroxyprogesterone acetate and 0.5 mM cAMP for nine days. The mRNA expression levels of prolactin (PRL) and Insulin Like Growth Factor Binding Protein 1 (IGFBP1) were measured by quantitative RT-PCR. The fold change in gene expression between control and decidualized samples was calculated. ATP assays were performed to assess the ATP activity in control and decidualized cells, transmission electron microscopy was performed for quantitation of autophagosomes in each group and Western blot analysis was performed measuring accumulation of microtubule-associated protein light chain 3 (LC3)-II and ubiquitin-binding protein p62 which together determines autophagic flux. Thirty-seven patients were included, twenty three obese patients and fourteen non-obese patients. Obese patients exhibited a statistically significant (p<0.05) decrease in gene expression fold change of both markers of endometrial decidualization. Similarly, the normally sustained decrease in ATP that occurs in control cell decidualization to trigger autophagy, occurred but was not sustained in obese patients (p<0.05). On TEM there were more autophagosomes in obese patients compared to non-obese suggesting that autophagy was initiated. Western immunoblotting revealed an increase in LC3-II in obese patients contributing to the fact that autophagy was initiated however there was a statistically significant increase in p62 in Obese patients (p<0.05) compared to non-obese suggesting that although autophagosomes are formed, there is an impairment in their function. Autophagy is a contributing mechanism in obese women’s impaired ability to undergo in vitro decidualization and this impairment is at the level of cargo recycling as evidenced by Western immunoblotting of p62 and not in initiation of autophagy as evidenced by TEM of autophagosomes and Western immunoblotting of LC3-II.

WNK lysine deficient protein kinase 1 regulates human endometrial stromal cell decidualization, proliferation, and migration in part through mitogen-activated protein kinase 7.

The differentiation of endometrial stromal cells into decidual cells, termed decidualization, is an integral step in the establishment of pregnancy. The mitogen-activated protein kinase homolog, WNK lysine deficient protein kinase 1 (WNK1), is activated downstream of epidermal growth factor receptor during decidualization. Primary human endometrial stromal cells (HESCs) were subjected to small interfering RNA knockdown of WNK1 followed by in vitro decidualization. This abrogated expression of the decidual marker genes, insulin like growth factor binding protein 1 (IGFBP1) and prolactin (PRL), and prevented adoption of decidual cell morphology. Analysis of the WNK1-dependent transcriptome by RNA-Seq demonstrated that WNK1 regulates the expression of 1858 genes during decidualization. Gene ontology and upstream regulator pathway analysis showed that WNK1 regulates cell migration, differentiation, and proliferation. WNK1 was required for many of the gene expression changes that drive decidualization, including the induction of the inflammatory cytokines, C-C motif chemokine ligand 8 (CCL8), interleukin 1 beta (IL1B), and interleukin 15 (IL15), and the repression of transforming growth factor-beta (TGF-beta) pathway genes, including early growth response 2 (EGR2), SMAD family member 3 (SMAD3), integrin subunit alpha 2 (ITGA2), integrin subunit alpha 4 (ITGA4), and integrin subunit beta 3 (ITGB3). In addition to abrogating decidualization, WNK1 knockdown decreased the migration and proliferation of HESCs. Furthermore, mitogen-activated protein kinase 7 (MAPK7), a known downstream target of WNK1, was activated during decidualization in a WNK1-dependent manner. Small interfering RNA knockdown of MAPK7 demonstrated that MAPK7 regulates a subset of WNK1-regulated genes and controls the migration and proliferation of HESCs. These results indicate that WNK1 and MAPK7 promote migration and proliferation during decidualization and regulate the expression of inflammatory cytokines and TGF-beta pathway genes in HESCs.

P5865The new biomarkers in coronary artery disease - the diagnostic and prognostic value of insulin like growth factor binding protein 7 (IGFBP 7) and galectin 3 (Gal 3)

P5865The new biomarkers in coronary artery disease - the diagnostic and prognostic value of insulin like growth factor binding protein 7 (IGFBP 7) and galectin 3 (Gal 3)

Abstract NTOC-097: VACCINATION TARGETING INSULIN–LIKE GROWTH FACTOR BINDING PROTEIN–2 (IGFBP–2) IN ADVANCED OVARIAN CANCER: SAFETY, IMMUNOGENICITY, AND SURVEILLANCE, EPIDEMIOLOGY, AND END RESULTS (SEER) COMPARISON

Abstract BACKGROUND: Immunization against self-antigens can induce regulatory responses that inhibit desirable Type 1 antitumor immune responses. Deletion of epitopes that favor a regulatory phenotype may improve the efficacy of therapeutic vaccines. We have developed a novel IGFBP-2 targeting DNA plasmid vaccine that selectively induces Type 1 immunity. IGFBP-2 regulates invasiveness and metastases in ovarian cancer. Eradication of ovarian cancer cells expressing IGFBP-2 through effective immunization could prevent disease relapse or metastasis. METHODS: Twenty-five patients with advanced stage or recurrent ovarian cancer treated to complete remission after primary or salvage therapy received 3 monthly doses of an IGFBP-2 DNA vaccine in a single-arm, non-randomized study. ELISPOT and flow cytometry were used to characterize antigen specific T-cell responses. Serum antibodies were measured using ELISA and Western blot. The SEER database was reviewed to identify women diagnosed between 2006 and 2012 matched for age, year of diagnosis and stage of diagnosis. The difference between dates of diagnosis and enrollment (lead time) was calculated for each patient receiving vaccine. Only SEER patients who survived at least as long as the lead time of their matches plus an additional 6 months were kept for analysis. In cases where this resulted in no SEER matched patients, unmatched vaccine patients were excluded. Overall survival (OS) was analyzed using Cox models and the Kaplan-Meier method. RESULTS: 206 adverse events (AE) were recorded. Fatigue (12%) and injection site reactions (12%) were the most common. 97% of AE were grades 1-2, 3% grade 3, and no grades 4 or 5. In preliminary immune analysis (16 patients), IGFBP-2 specific T-cell precursor frequencies are significantly elevated over baseline levels at 4 (p&amp;lt;0.01) and 6 (p&amp;lt;0.001) months. T-regulatory cells were not increased over the levels measured in a control reference population. No patients developed new IGFBP-2 specific antibody responses after immunization suggesting a lack of Th2 augmentation. Median OS for the matched SEER group (n=754) was 11 months. Matched IGFBP-2 vaccinated patients (n=20) have yet to reach median OS, but the lower 95% confidence limit is 27.3 months (p&amp;lt;0.0001). CONCLUSIONS: IGFBP-2 Th1 selective immunization is well tolerated, generates significant Type I immunity, and may demonstrate clinical efficacy. Citation Format: John B. Liao, Denise L. Cecil, Yushe Dang, Kelsey K. Baker, Kelsie J. Ovenell, Jessica Reichow, Stephanie Parker, Doreen M. Higgins, Jennifer S. Childs, Elizabeth K. Broussard, Andrew L. Coveler, Lupe G. Salazar, Barbara A. Goff, Mary W. Redman, Mary L. Disis. VACCINATION TARGETING INSULIN–LIKE GROWTH FACTOR BINDING PROTEIN–2 (IGFBP–2) IN ADVANCED OVARIAN CANCER: SAFETY, IMMUNOGENICITY, AND SURVEILLANCE, EPIDEMIOLOGY, AND END RESULTS (SEER) COMPARISON [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-097.

Evaluation of Phosphorylated Insulin like Growth Factor Binding Protein-1 and Sonographically Measured Cervical Length for Prediction of Preterm Birth in Threatened Preterm Labor: A Clinical Approach

Evaluation of Phosphorylated Insulin like Growth Factor Binding Protein-1 and Sonographically Measured Cervical Length for Prediction of Preterm Birth in Threatened Preterm Labor: A Clinical Approach

Su1262 - Serum Levels of Insulin-Like Growth Factor 1 (IGF-1) and Insulin Like Growth Factor Binding Protein 2 (IGFBP-2) in Patients with Pancreatic Adenocarcinoma (PDAC)

Su1262 - Serum Levels of Insulin-Like Growth Factor 1 (IGF-1) and Insulin Like Growth Factor Binding Protein 2 (IGFBP-2) in Patients with Pancreatic Adenocarcinoma (PDAC)

Elevated insulin and reduced insulin like growth factor binding protein-3/prostate specific antigen ratio with increase in prostate size in Benign Prostatic Hyperplasia

BackgroundInsulin and insulin like growth factor-1 (IGF-1) have growth promoting effects, while insulin like growth factor binding protein-3 (IGFBP-3) has growth inhibitory effects. The present study was designed to assess the concentrations of insulin, IGF-1, IGFBP-3 and their association with prostate size in patients with BPH. MethodsNinety 90 BPH cases and 90 controls were enrolled in the study. Insulin, IGF-1, IGFBP-3, PSA, testosterone and estradiol were estimated in both the groups. ResultsInsulin, IGF-1 and estradiol were increased and IGFBP-3/PSA was decreased in BPH cases when compared with controls. Insulin (r=0.64, p=0.001) and IGF-1 (r=0.22, p=0.03) were positively correlated and IGFBP-3/PSA (r=−0.316, p=0.002) were negatively correlated with prostate size in BPH. Multivariate analysis showed that insulin (p=0.001) and IGFBP-3/PSA (p=0.004) predicts the prostate size in patients with BPH. Insulin was increased and IGFBP-3/PSA was reduced in BPH patients with increased prostate size. At a cutoff concentration of 527.52, IGFBP-3/PSA ratio was found to differentiate benign growth of prostate from normal prostate with 96% sensitivity and 96% specificity. ConclusionInsulin is elevated and IGFBP-3/PSA is reduced with increase prostate size in BPH cases.

Insulin-Like Growth Factor Binding Protein 4 Fragments Provide Incremental Prognostic Information on Cardiovascular Events in Patients With ST-Segment Elevation Myocardial Infarction.

BackgroundFragments of insulin‐like growth factor binding protein 4 (IGFBP‐4) are potential new biomarkers for cardiac risk assessment. The fragments are generated on specific cleavage by pregnancy‐associated plasma protein‐A, which exerts proatherogenic activity. This study investigated the prognostic value of IGFBP‐4 fragments in patients with ST‐segment elevation myocardial infarction.Methods and ResultsWe prospectively included 656 patients with ST‐segment elevation myocardial infarction treated with percutaneous coronary intervention from September 2006 to December 2008. Blood samples were drawn before percutaneous coronary intervention, and levels of intact IGFBP‐4 and N‐terminal and C‐terminal IGFBP‐4 fragments were measured by specific assays. End points were 5‐year all‐cause and cardiovascular mortality and the combined end point of major adverse cardiac events. Prognostic potential was evaluated on top of a clinical model in terms of discrimination, calibration, and reclassification analysis. During follow‐up, 166 patients experienced a major adverse cardiac event and 136 patients died, of whom 69 died from cardiovascular causes. Both IGFBP‐4 fragments were associated with all end points (P<0.001). After multivariable adjustments, both N‐terminal and C‐terminal IGFBP‐4 fragment levels remained associated with all end points, including cardiovascular mortality with hazard ratios per doubling in protein concentration of 2.54 (95% CI 1.59–4.07; P<0.001) and 2.07 (95% CI 1.41–3.04; P<0.001), respectively. Incorporation of IGFBP‐4 fragments into a clinical model with 15 risk factors improved C‐statistics and model calibration and provided incremental prognostic contribution, as assessed by net reclassification improvement and integrated discrimination improvement.Conclusions IGFBP‐4 fragments are associated with increased risk of all‐cause mortality, cardiovascular mortality, and major adverse cardiac events in patients with ST‐segment elevation myocardial infarction.

Abstract OT3-01-03: A phase I trial of the safety and immunogenicity of a DNA plasmid based vaccine (WOKVAC) encoding epitopes derived from three breast cancer antigens (IGFBP2, HER2, and IGF1R) in patients with breast cancer

Abstract Background: Three proteins insulin like growth factor binding protein 2 (IGFBP2), human epidermal growth factor receptor 2 (HER2), and insulin like growth factor receptor-1 (IGF1R) are overexpressed in in pre-invasive and high risk breast lesions and are associated with progression to invasive breast cancer. These proteins are immunogenic and elicit both humoral and cellular immunity in breast cancer patients. It is hypothesized that immunization with a plasmid vaccine (WOKVAC) targeting antigens from these proteins will be safe and immunogenic. WOKVAC has been designed to include extended sequences of the immunizing antigens that are predominantly associated with eliciting Type I immune responses. Type I immunity results in immune cells called T-cells secreting high levels of inflammatory cytokines (called Th1) that stimulate tumor destruction as well as the generation of cytotoxic T-cells that can directly kill the tumors. Trial design: Phase I dose escalation study of 3 doses of WOKVAC admixed with 100mcg of GM-CSF. Patients will be assigned sequentially to one of three arms (10 patients/arm): Arm 1=150mcg, Arm 2=300mcg, Arm 3=600mcg. Each dose arm will have a staggered enrollment to assess toxicity. If the Arm 1 dose is determined to be safe, Arm 2 patients can be enrolled. If the Arm 2 dose is safe and immunologically more efficacious than Arm 1 then Arm 3 patients can be enrolled. Study treatment includes 3 monthly vaccines, two evaluations at 1 and 6 months post vaccine and a 5 year follow-up to collect reports from the patient's primary oncologist. Toxicity is assessed at baseline through the end of the study. Serial blood draws for immunologic monitoring is done. Eligibility criteria: Patients with non-metastatic, node positive, HER2 negative breast cancer that is in remission and defined as no evidence of disease. Patients must have a good performance status, be at least 28 days from last cytotoxic chemotherapy and/or radiotherapy and 28 days from any use of systemic steroids. Specific aims: (1) Determine safety of 3 escalating doses of WOKVAC, (2) Determine the most immunogenic dose, (3) Determine whether a WOKVAC Th1 polyepitope plasmid based vaccine elicits a persistent memory T-cell and (4) Determine whether WOKVAC vaccination modulates T regulatory cells and myeloid derived suppressor cells. Statistical methods: (1) Safety will be assessed per NCI CTCAE v. 4.0, (2) Immunogenicity will be defined by the magnitude of the Th1 IFN-gamma antigen specific immune response. Successful immunization is a protein specific IFN-g precursor frequency greater than 1:20,000 PBMC for each antigen or 2 fold increase if baseline immune response (3) The IGFBP2, HER2, and IGF1R specific IFN-g/IL-10 ratios by ELISPOT will be evaluated to determine that a predominantly Th1 immune response is stimulated, and (4) Humoral immune response will be measured by ELISA and serum antibody avidity for IGFBP2, HER2, and IGF1R to determine an avidity index (AI) before and after vaccination. Targeted Accrual: 30 patients Contact information: University of Washington: 866-392-8588/TrialTVG@uw.edu University of Wisconsin: 608-265-2493/prevention@uwcarbone.wisc.edu. Citation Format: Childs JS, Higgins DM, DeShong K, Heckman-Stoddard BM, Wojtowicz ME, Stanton SE, Bailey HH, Wisinski KB, Disis ML. A phase I trial of the safety and immunogenicity of a DNA plasmid based vaccine (WOKVAC) encoding epitopes derived from three breast cancer antigens (IGFBP2, HER2, and IGF1R) in patients with breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT3-01-03.

子宮体癌細胞株(HHUA)におけるアポトーシスとEGFによる細胞増殖に対するInsulin-like Growth Factor Binding Protein-3 (IGFBP-3)の作用(杏林大学学位論文要旨および審査要旨)

子宮体癌細胞株(HHUA)におけるアポトーシスとEGFによる細胞増殖に対するInsulin-like Growth Factor Binding Protein-3 (IGFBP-3)の作用(杏林大学学位論文要旨および審査要旨)

Customized biomaterials to augment chondrocyte gene therapy.

The present manuscript focuses on the enhancement of chondrocyte gene therapy through the modification of scaffold materials to enhance the retention of targeted gene products. This study combined tissue engineering and gene therapy, where customized biomaterials augmented the action of IGF-I by enhancing the retention of protein produced by transfection of the IGF-I gene. This approach enabled tuning of binding of IGF-I to alginate, which increased GAG and HYPRO production by transfected chondrocytes. To our knowledge, peptide-based modification of materials to augment growth factor-targeted gene therapy has not been reported previously.

Diagnostic utility of leptin and insulin-like growth factor binding protein-2 in hepatocellular carcinoma of diabetic and non-diabetic Egyptian patients

Purpose: To elucidate the possible diagnostic utility of adipokines and insulin growth factor binding proteins in hepatocellular carcinoma (HCC) diabetic subjects. Methods: Seventy five patients were divided equally into 3 groups as follows: healthy normal control (NC), non-diabetic hepatocellular carcinoma (HCC) and diabetic hepatocellular carcinoma (HCC-DM). Serum levels of leptin, insulin growth factor binding protein-2 (IGFBP-2) and alpha fetoprotein (AFP) were measured. Correlation and receiver operating characteristics (ROC) analysis was carried out. Results: HCC and HCC-DM groups showed changes in body mass index (BMI, p > 0.05 and p < 0.001 respectively), glucose, insulin, homeostatic model assessment-insulin resistance (HOMA-IR), liver function tests and AFP (p < 0.001). Leptin levels increased significantly in both HCC and HCC-DM (p < 0.001). Furthermore, IGFBP-2 showed significant increase in both groups (p < 0.001). Both leptin and insulin-like growth factor binding protein-2 (IGFBP-2) displayed significant positive correlation with AFP (p < 0.001). ROC analysis indicate different diagnostic accuracies for the tested markers for the various groups. Conclusion: Leptin and IGFBP-2 demonstrate significant potentials as diagnostic tools for HCC patients, especially diabetic cases, with IGFBP-2 displaying the highest diagnostic accuracy for HCC and HCC-DM groups. Keywords: Hepatocellular carcinoma, Diabetes mellitus, Leptin, Insulin-like growth factor-binding proteins-2, Adipokines

SALIVARY INSULIN LIKE GROWTH FACTOR BINDING PROTEIN-3 AND TRANSFERRIN LEVELS IN PATIENTS WITH ORAL SQUAMOUS CELL CARCINOMA VERSUS ORAL LICHEN PLANUS

Objective: To gather preliminary data concerning local salivary levels of insulin like growth factor binding protein-3 (IGFBP-3) and its binding protein transferrin (Tf) in patients suffering from oral squamous cell carcinoma (OSCC) and oral lichen planus (OLP) compared to normal controls and to correlate it with clinical picture in OLp and histologic grading in OSCC. Materials and methods: Salivary samples from 40 patients suffering from OSCC (20 poorly and 20 moderately differentiated), 40 patients suffering from OLP (clinically evaluated using REU scoring system) and 40 controls were collected and TF and IGFbP-3 levels were estimated using enzyme linked immunosorbent assay (ELISA), for statistical analysis, ANOVA followed by Tukey’s post hoc and Pearson correlation tests were used. Results: Significantly higher salivary levels of IGFBP-3 and Tf were recorded in different histological grades of SCC in comparison to Olp. Control group recorded the lowest significant values compared to OLP and OSCC (p<0.0001). A week positive correlation was recorded between levels of Tf and IGFBP-3 in all groups. As well, a week positive correlation between IGFBP-3 and Tf levels with REU in OLP and histological grades in OSCC was shown, except for the correlation between TF and OSCC histologic grading that was strong positive. Conclusions: IGFBP-3 and Tf seems to play a role in pathogenesis of both OSCC and Olp, and could be considered as a reliable marker in diagnosis of OSCC and OLP, However, further studies are required .

Identification of Genomic Regions Related to pH in Nellore Beef Cattle

ObjectivesThe aim of this study was to identify genomic regions that potentially have association with pH in Nellore cattle.Materials and MethodsPost mortem muscle metabolism (pH) was measured on samples of Longissimus thoracis muscle collected 24 h post mortem using a portable pHmeter (Hanna HI9963) from 1208 feedlot Nellore steers slaughthered at 2 yr of age. Genotypic data of all animals was used for genome wide association studies through the ssGWAS method. Those animals were genotyped with the Illumina Bovine beadchip HD GGPi (74K). Based on another Nellore population genotyped for Illumina beadchip BovineHD (777K), genotypes were imputed using the FImpute software. Analyses were performed using a pedigree composed by 6276 animals. Single step analyses were performed using the Blupf90 family of programs considering windows of 10 markers to estimate the SNP effects. This procedure enables the identification of regions associated with pH along the chromosomes. After quality control (MAF < 0.05%, call rate < 90%), 463,995 SNPs in autosomal chromosomes were used in the association analyses.ResultsThe current investigation revelead 15 regions located in 10 different chromosomes (2, 3, 4, 8, 9, 11, 12, 16, 17, and 21), which explained more than 1% of the additive variance (Fig. 1). Gene identification was performed using the BioMart do Ensembl Genome Browser tool (www.ensembl.org). The following genes were identified in 6 distinct regions: WD repeat, sterile ɑ motif and U-box domain containing 1; palmdelphin; insulin like growth factor binding protein 1; Bos taurus charged multivesicular body protein 7; tumor necrosis factor receptor superfamily, member 10d, decoy with truncated death domain; rho-related BTB domain-containing protein 2; spectrin repeat containing nuclear envelope protein 1 e ubiquitin specific peptidase like 1.ConclusionThe ssGWAS method, using high density panel, allowed the identification of regions and genes related to pH in Nellore beef cattle. Further investigation combining information of the reported genes and its biological pathways is required to better evaluate their importance for meat quality traits.

Small Molecule Based Ex Vivo Expansion of CD34+CD90+CD49f+ Hematopoietic Stem & Progenitor Cells from Non-Enriched Umbilical Cord Blood Mononucleated Cells

Umbilical cord blood (UCB) transplantation in adults have slow hematopoietic recovery compared to bone marrow (BM) or peripheral blood grafts mainly due to low number of total nucleated cells (TNC) and hematopoietic stem & progenitor cells (HSPC). Current investigational clinical strategies focus on increasing HSPC dosage by expanding CD34 enriched grafts that have resulted in early neutrophil recovery followed by long term hematopoietic reconstitution.In an effort to expand HSPC, specifically those expressing primitive phenotype (CD34+CD90+CD49f+) from non-enriched UCB, we developed a proprietary library of 50 small molecules using structure-activity-relationship studies. Freshly-thawed UCB-mononucleated cells (MNC), were cultured in serum or animal component free expansion medium supplemented with optimal concentration of respective compound. The effects of the expansion protocol were measured based on phenotypic and functional assays. Cell cultures with basal cytokines served as control.Screening of the small molecule library showed negligible acute adverse effects on CD45+ leukocyte population and its viability within 72 hours compared to cytokine control. In long term expansion cultures lasting up to 11 days, one specific structural analog, C7, resulted in 1195.8±71.7-folds increase of absolute CD45+CD34+CD38-CD45RA- progenitors which was at least 9.2-folds higher than control cultures (P<0.01; n=4). Colony forming unit assay showed significant increase of granulocyte-macrophage colonies from C7 treated cells compared to cytokine control (P<0.01; n=6) although TNC expansion was comparable between the culture conditions. It was necessary for the cytokine cocktail to comprise of at least stem cell factor, thrombopoietin and Fms-related tyrosine kinase 3 ligand for mediating HSPC expansion in presence of C7, although further addition of insulin like growth factor binding protein 2 marginally boosted expansion (P<0.001; n=3). Majority of HSPC expansion occurred between the seventh and tenth day of the culture period. In MNC initiated cultures, addition of C7 boosted primitive HSPC (CD45+CD34+CD38-CD45RA-CD90+CD49f+) by 633.3±8.5-folds over 10 days which was at least 7.4-folds higher than control cultures (P<0.001; n=3). In cultures initiated with purified CD34+CD38- cells, there was at least 15.9-folds higher expansion of HSPC defined by CD45+CD34+CD38-CD45RA-CD90+ in presence of C7 compared to cytokine cultures (P<0.05; n=3). Expansion of HSPC by C7 was at least 2-folds higher in comparison to mesenchymal stromal co-culture system which is the only known clinical protocol that allows for UCB expansion without prior CD34/CD133 selection (P<0.001; n=6). Transplantation of C7 expanded UCB grafts (n=11) at equivalent dosage of 2.5x107 cells/kg to sub-lethally irradiated NOD SCID Gamma (NSG) mice resulted in 3.21- and 2.09-folds higher engraftment of human CD45+ cells in the peripheral blood by day 21 compared to non-expanded (P=0.0030; n=6) and cytokine expanded grafts (P=0.0005; n=12) respectively. The frequency of SCID repopulating cells contributing to early peripheral blood engraftment was 2.48-folds higher in C7 expanded graft compared to unmanipulated graft. C7 expanded graft sustained human cell engraftment over 19 weeks which were primarily myeloid cells (CD33+/CD15+) as opposed to non-expanded graft which consisted of CD3+ T cells. Analysis of NSG BM at week 19 post-transplantation, showed significantly better (P<0.0001) chimerism of human CD45 cells in female (n=15) recipient compared to male (n=14) irrespective of graft type (transplantation dosage: 2.5x107 cells/kg). C7 expanded graft gave comparable level of human CD45 and CD34 progenitor cell engraftment as that of the non-expanded grafts. Multi-lineage reconstitution of NSG BM comprising of both myeloid and lymphoid human cells could be achieved with the C7 expanded graft. At higher transplantation dosage of 5.0x107 cells/kg, the expanded grafts had a higher survival rate of 75% compared to 50% for non-expanded graft mainly due to lower incidence of graft-versus-host-disease.In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment or stromal cell co-culture. The expanded UCB consists of phenotypically defined primitive HSPC that maintains in vitro and in vivo functionality. DisclosuresHwang:Celgene Corporation: Honoraria, Other: Travel Support; Roche Singapore: Honoraria, Other: Travel Support; Pfizer Singapore: Honoraria, Other: Travel Support; Novartis International AG: Honoraria, Other: Travel Support; Bristol-Myers Squibb Pte Ltd: Honoraria, Other: Travel Support; MSD Pharma (Singapore): Honoraria, Other: Travel Support; Sanofi Aventis Singapore: Honoraria, Other: Travel Support; Janssen-Cilag Singapore: Honoraria, Other: Travel Support.

Pituitary growth hormone (GH) secretion is partially rescued in HIV-infected patients with GH deficiency (GHD) compared to hypopituitary patients.

Biochemical growth hormone deficiency is prevalent among human immunodeficiency virus-infected patients, but if this condition is clinically relevant remains challenging. The aim is to prospectively compare the growth hormone deficiency/insulin-like growth factor-1 status of 71 human immunodeficiency virus-infected patients with impaired growth hormone response to growth hormone releasing hormone + Arginine with that of 65 hypopituitary patients affected by a true growth hormone deficiency secondary to pituitary disease. The main outcomes were: basal serum growth hormone, insulin-like growth factor-1, insulin-like growth factor binding protein 3, growth hormone peak and area under the curve after growth hormone response to growth hormone releasing hormone + Arginine test, body mass index, waist and hip circumference, and body composition by dual energy X-ray absorptiometry. Insulin-like growth factor-1 binding protein 3, basal growth hormone (p < 0.005), growth hormone peak and area under the curve after growth hormone response to growth hormone releasing hormone + Arginine, waist to hip ratio, insulin-like growth factor-1, fasting glucose, insulin, and triglycerides (p < 0.0001) were lower in hypopituitary than human immunodeficiency virus-infected patients. Total and trunk fat mass by dual energy X-ray absorptiometry were higher in hypopituitary than in human immunodeficiency virus-infected patients (p < 0.0001). In all the patients total body fat was associated with both growth hormone peak and area under the curve at stepwise linear regression analysis. The degree of growth hormone deficiency is more severe in hypopituitary than in human immunodeficiency virus-infected patients, suggesting that the function of growth hormone/insulin-like growth factor-1 axis is partially rescued in the latter thanks to a preserved pituitary secretory reserve. Data from the current study suggest that human immunodeficiency virus-infected patients with peak growth hormone < 9 mg/L may have partial growth hormone deficiency and clinicians should be cautious before prescribing recombinant human growth hormone replacement treatment to patients living with human immunodeficiency virus.

Adiponectin Deficiency Leads to Female Subfertility and Ovarian Dysfunctions in Mice.

Adipose tissue plays an important role in regulating female fertility, owing to not only its energy stores but also the endocrine actions of secreted adipokines. As one of the adipokines, adiponectin is almost exclusively secreted from the fat, and its circulating concentration is paradoxically reduced in obesity. Although recent studies implied a purported positive role of adiponectin in ovarian functions, definitive in vivo evidence has been sorely lacking. We have consistently observed subfertility in female adiponectin null mice and therefore postulated a protective role of adiponectin in ovarian functions. Female adiponectin null mice displayed impaired fertility, reduced retrieval of oocytes, disrupted estrous cycle, elevated number of atretic follicles, and impaired late folliculogenesis. Analysis of their sera revealed a significant decrease in estradiol and FSH but an increase in LH and testosterone at proestrus. In addition, we found marked reduction of progesterone levels at diestrus, a significant decrease in LH receptor expression as well as in the number of GnRH immunoreactive neurons. Adiponectin deficiency also altered the peak concentrations of LH surge and led to lower expression of Cytochrome P450 family 11 subfamily A member 1 (P450scc), an enzyme critical for progesterone synthesis, as well as an increase in BCL2 associated X, apoptosis regulator and Insulin like growth factor binding protein 4 in atretic follicles. These physiological and molecular events were independent of insulin sensitivity. Thus, we have revealed a novel mechanism linking adiponectin and female fertility that entails regulation of reproductive hormone balance and ovarian follicle development.