A multi-site feasibility study for personalized medicine in canines with osteosarcoma.

BACKGROUND: A successful therapeutic strategy, specifically tailored to the molecular constitution of an individual and their disease, is an ambitious objective of modern medicine. In this report, we highlight a feasibility study in canine osteosarcoma focused on refining the infrastructure and processes required for prospective clinical trials using a series of gene expression-based Personalized Medicine (PMed) algorithms to predict suitable therapies within 5 days of sample receipt. METHODS: Tumor tissue samples were collected immediately following limb amputation and shipped overnight from veterinary practices. Upon receipt (day 1), RNA was extracted from snap-frozen tissue, with an adjacent H&E section for pathological diagnosis. Samples passing RNA and pathology QC were shipped to a CLIA-certified laboratory for genomic profiling. After mapping of canine probe sets to human genes and normalization against a (normal) reference set, gene level Z-scores were submitted to the PMed algorithms. The resulting PMed report was immediately forwarded to the veterinarians. Upon receipt and review of the PMed report, feedback from the practicing veterinarians was captured. RESULTS: 20 subjects were enrolled over a 5 month period. Tissue from 13 subjects passed both histological and RNA QC and were submitted for genomic analysis and subsequent PMed analysis and report generation. 11 of the 13 samples for which PMed reports were produced were communicated to the veterinarian within the target 5 business days. Of the 7 samples that failed QC, 4 were due to poor RNA quality, whereas 2 were failed following pathological review. Comments from the practicing veterinarians were generally positive and constructive, highlighting a number of areas for improvement, including enhanced education regarding PMed report interpretation, drug availability, affordable pricing and suitable canine dosing. CONCLUSIONS: This feasibility trial demonstrated that with the appropriate infrastructure and processes it is possible to perform an in-depth molecular analysis of a patient's tumor in support of real time therapeutic decision making within 5 days of sample receipt. A number of areas for improvement have been identified that should reduce the level of sample attrition and support clinical decision making.

Genomic characterization of explant tumorgraft models derived from fresh patient tumor tissue.

BACKGROUND: There is resurgence within drug and biomarker development communities for the use of primary tumorgraft models as improved predictors of patient tumor response to novel therapeutic strategies. Despite perceived advantages over cell line derived xenograft models, there is limited data comparing the genotype and phenotype of tumorgrafts to the donor patient tumor, limiting the determination of molecular relevance of the tumorgraft model. This report directly compares the genomic characteristics of patient tumors and the derived tumorgraft models, including gene expression, and oncogenic mutation status. METHODS: Fresh tumor tissues from 182 cancer patients were implanted subcutaneously into immune-compromised mice for the development of primary patient tumorgraft models. Histological assessment was performed on both patient tumors and the resulting tumorgraft models. Somatic mutations in key oncogenes and gene expression levels of resulting tumorgrafts were compared to the matched patient tumors using the OncoCarta (Sequenom, San Diego, CA) and human gene microarray (Affymetrix, Santa Clara, CA) platforms respectively. The genomic stability of the established tumorgrafts was assessed across serial in vivo generations in a representative subset of models. The genomes of patient tumors that formed tumorgrafts were compared to those that did not to identify the possible molecular basis to successful engraftment or rejection. RESULTS: Fresh tumor tissues from 182 cancer patients were implanted into immune-compromised mice with forty-nine tumorgraft models that have been successfully established, exhibiting strong histological and genomic fidelity to the originating patient tumors. Comparison of the transcriptomes and oncogenic mutations between the tumorgrafts and the matched patient tumors were found to be stable across four tumorgraft generations. Not only did the various tumors retain the differentiation pattern, but supporting stromal elements were preserved. Those genes down-regulated specifically in tumorgrafts were enriched in biological pathways involved in host immune response, consistent with the immune deficiency status of the host. Patient tumors that successfully formed tumorgrafts were enriched for cell signaling, cell cycle, and cytoskeleton pathways and exhibited evidence of reduced immunogenicity. CONCLUSIONS: The preservation of the patient's tumor genomic profile and tumor microenvironment supports the view that primary patient tumorgrafts provide a relevant model to support the translation of new therapeutic strategies and personalized medicine approaches in oncology.

Increased fall risk is associated with elevated co-contraction about the ankle during static balance challenges in older adults.

Falls are a leading contributor to disability in older adults. Increased muscle co-contraction in the lower extremities during static and dynamic balance challenges has been associated with aging, and also with a history of falling. Co-contraction during static balance challenges has not been previously linked with performance on clinical tests designed to ascertain fall risk. The purpose of this study was to investigate the relationship between co-contraction about the ankle during static balance challenges with fall risk on a commonly used dynamic balance assessment, the Four Square Step Test (FSST). Twenty-three volunteers (mean age 73 years) performed a series of five static balance challenges (Romberg eyes open/closed, Sharpened Romberg eyes open/closed, and Single Leg Standing) with continuous electromyography (EMG) of bilateral tibialis anterior and gastrocnemius muscles. Participants then completed the FSST and were categorized as 'at-risk' or 'not-at-risk' to fall based on a cutoff time of 12 s. Co-contraction was quantified with co-contraction index (CCI). CCI during narrow base conditions was positively correlated with time to complete FSST. High CCIs during all static balance challenges with the exception of Romberg stance with eyes closed were predictive of being at-risk to fall based on FSST time, odds ratio 19.3. The authors conclude that co-contraction about the ankle during static balance challenges can be predictive of performance on a dynamic balance test.

Met induces diverse mammary carcinomas in mice and is associated with human basal breast cancer.

Understanding the signaling pathways that drive aggressive breast cancers is critical to the development of effective therapeutics. The oncogene MET is associated with decreased survival in breast cancer, yet the role that MET plays in the various breast cancer subtypes is unclear. We describe a knockin mouse with mutationally activated Met (Met(mut)) that develops a high incidence of diverse mammary tumors with basal characteristics, including metaplasia, absence of progesterone receptor and ERBB2 expression, and expression of cytokeratin 5. With gene expression and tissue microarray analysis, we show that high MET expression in human breast cancers significantly correlated with estrogen receptor negative/ERBB2 negative tumors and with basal breast cancers. Few treatment options exist for breast cancers of the basal or trastuzumab-resistant ERBB2 subtypes. We conclude from these studies that MET may play a critical role in the development of the most aggressive breast cancers and may be a rational therapeutic target.

E-selectin inhibitor is superior to low-molecular-weight heparin for the treatment of experimental venous thrombosis.

BACKGROUND: This study evaluated E-selectin inhibition with GMI-1271 (Uproleselan [GMI]) alone and in combination with the standard of care low-molecular-weight heparin (LMWH) to improve vein recanalization, decrease vein wall inflammation and protect against adverse bleeding in a primate model. We sought to examine this novel treatment of venous thrombosis. METHODS: Using a well-documented primate animal model, iliac vein thrombosis was induced by balloon occlusion of the iliac vein for 6 hours. Starting on day 2 after thrombosis, animals began treatment in two phases. In phase one, nontreated controls received no treatment (n = 5) vs animals treated with the E-selectin inhibitor GMI, 25 mg/kg, subcutaneous (SC), once daily (n = 4) for 21 days (previously published data). In phase two, animals were treated with GMI plus a combination of LMWH 1.5 mg/kg or 40 mg (GMI + LMWHc) SC once daily (n = 8) for 19 days; and animals treated with LMWH 1.5 mg/kg or 40 mg (LMWHc) SC once daily (n = 6) for 19 days. Animals were evaluated by magnetic resonance venography for vein recanalization and inflammation by gadolinium extravasation, duplex ultrasound, coagulation tests (thromboelastography, bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen) and complete blood count at baseline, days 2, 7, 14, and 21 at euthanasia. Statistical analysis included using unpaired t test with Welch's correction for direct comparisons and one-way analysis of variance for comparison between the groups. RESULTS: Percent vein recanalization by magnetic resonance venography was highest in the GMI alone group followed by GMI + LMWHc, both significantly different from control. On ultrasound examination, animals treated with GMI alone had no decrease in open vein lumen by day 21, whereas decreases were observed in groups GMI + LMWHc (-26%), LMWHc (-27%), and controls (-80%). Vein wall inflammation decreased significantly in all treated groups. Intimal fibrosis and intimal thickness was best preserved in the GMI alone group. An analysis of total vein wall collagen revealed a trend in all treatment groups of decreasing vein wall collagen. No clinically significant bleeding events were noted in any group. The LMWH groups trended to have prolonged coagulation test values, whereas E-selectin inhibition with GMI did not cause clinically significant changes in coagulation measures. CONCLUSIONS: Treatment with E-selectin inhibition results in improved vein recanalization, a decrease in vein wall inflammation and vein wall intimal thickness and fibrosis, with no changes in markers of coagulation. E-selectin inhibition with GMI alone is superior to E-selectin inhibition combined with LMWH, LMWH alone, and no treatment in this deep vein thrombosis model of iliac vein thrombosis.

Carbon Nanodots Inhibit Oxidized Low Density Lipoprotein-Induced Injury and Monocyte Adhesion to Endothelial Cells Through Scavenging Reactive Oxygen Species.

Oxidized low density lipoprotein (Ox-LDL) is a known biomarker of inflammation and atherosclerosis, a leading cause of death worldwide. As a new class of nanomaterials, carbon nanodots (CNDs) are widely used in bioimaging, diagnostics, and drug delivery. However, there is no current report on how these CNDs affect the cardiovascular system, particularly their potential in mediating endothelial inflammatory dysfunction. This study examined effects of CNDs on Ox-LDL-mediated endothelial dysfunction. CNDs significantly inhibited Ox-LDL-mediated adhesion of monocytes to human microvascular endothelial cells (HMEC-1), in human microvascular endothelial cells (HMEC-1). CNDs significantly inhibited Ox-LDL-mediated adhesion of monocytes to endothelial cells, which is an essential step in the development of atherosclerosis. Further, CNDs significantly inhibited OxLDL-induced expression of interleukin-8 (IL-8), a vital cytokine on monocyte adhesion to the endothelial cells. These results demonstrate CNDs possess anti-inflammatory properties. CNDs also protect cells against Ox-LDL-induced cytotoxicity. Electron paramagnetic resonance (EPR) spectroscopy studies demonstrated direct reactive oxygen species-scavenging by CNDs. This result indicates that the anti-inflammatory properties of CNDs are most likely due to their direct scavenging of reactive oxygen species. Animal studies involving mice did not show any morphological or physical changes between the CNDs and control groups. Our study provides evidence of potential of CNDs in reducing Ox-LDL-mediated inflammation and cytotoxicity in HMEC-1.

A multilevel analysis of individual, household, and neighborhood correlates of intimate partner violence among low-income pregnant women in Jefferson county, Alabama.

OBJECTIVES: We examined individual, household, and neighborhood correlates of intimate partner violence (IPV) before and during pregnancy. METHODS: We used multilevel modeling to investigate IPV among 2887 pregnant women in 112 census tracts who sought prenatal care in 8 public clinics in Jefferson County, Alabama, from 1997 through 2001. Data were collected from the Perinatal Emphasis Research Center project, the 2000 Census, and the local Sheriff and Police Departments Uniform Crime Reports for 1997 through 2001. RESULTS: Participants were predominantly young, African American, on Medicaid, and residents of low-income neighborhoods. The prevalence of past-year male partner-perpetrated physical or sexual violence was 7.4%. Neighborhood residential stability, women performing most of the housework (lack of involvement among partners), being unmarried (being in an uncommitted relationship), and alcohol use were positively associated with elevated IPV risk. Significant protective factors for IPV included older age at first vaginal intercourse and a greater sense of mastery (e.g., the perception of oneself as an effective person). CONCLUSIONS: Both neighborhood contextual and individual and household compositional effects are associated with IPV among low-income pregnant women. The results imply that combined interventions to improve neighborhood conditions and strengthen families may effectively reduce IPV.

A new way to treat proximal deep venous thrombosis using E-selectin inhibition.

OBJECTIVE: There is an inter-relationship between thrombosis and inflammation. Previously, we have shown the importance of P-selectin in thrombogenesis and thrombus resolution in many preclinical animal models. The role of E-selectin has been explored in rodent models and in a small pilot study of clinical calf vein deep venous thrombosis. The purpose of this study was to determine the role of E-selectin in thrombosis in a primate model of proximal iliac vein thrombosis, a model close to the human condition. METHODS: Iliac vein thrombosis was induced with a well-characterized primate model. Through a transplant incision, the hypogastric vein and iliac vein branches were ligated. Thrombus was induced by balloon occlusion of the proximal and distal iliac vein for 6 hours. The balloons were then deflated, and the primates recovered. Starting on postocclusion day 2, animals were treated with the E-selectin inhibitor GMI-1271, 25 mg/kg subcutaneously, once daily until day 21 (n = 4). Nontreated control animals received no treatment (n = 5). All animals were evaluated by magnetic resonance venography (MRV); evaluation of vessel area by ultrasound, protein analysis, hematology (complete blood count), and coagulation tests (bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen, and thromboelastography) were performed at baseline, day 2, day 7, day 14, and day 21 with euthanasia. In addition, platelet function and CD44 expression on leukocytes were determined. RESULTS: E-selectin inhibition by GMI-1271 significantly increased vein recanalization by MRV vs control animals on day 14 (P < .05) and day 21 (P < .0001). GMI-1271 significantly decreased vein wall inflammation by MRV with gadolinium vein wall enhancement vs control also on day 14 (P < .0001) and day 21 (P < .0001). The thromboelastographic measure of clot strength (maximum amplitude) showed significant decreases in animals treated with GMI-1271 vs controls at day 2 (P < .05) and day 7 (P < .05). Animals treated with GMI-1271 had significant vessel area increase by day 21 vs controls (P < .05) by ultrasound. Vein wall intimal thickening (P < .001) and intimal fibrosis (P < .05) scores were significantly decreased in GMI-1271-treated animals vs controls. Importantly, no significant differences in hematology or coagulation test results were noted between all groups, suggesting that E-selectin inhibition carries no bleeding potential. GMI-1271 did not affect platelet function or aggregation or CD44 expression on leukocytes. In addition, no episodes of bleeding were noted in either group. CONCLUSIONS: This study suggests that E-selectin modulates venous thrombus progression and that its inhibition will increase thrombus recanalization and decrease vein wall inflammation, without affecting coagulation. The use of an E-selectin inhibitor such as GMI-1271 could potentially change how we treat deep venous thrombosis.

Tissue inhibitor of metalloproteinase-1 is an early marker of acute endothelial dysfunction in a rodent model of venous oxidative injury.

Although there are extensive research data regarding arterial endothelial dysfunction, the effects of venous endothelial dysfunction are not well characterized. Matrix metalloproteinases (MMPs) have a defined role in vascular remodeling. MMPs are endopeptidases that are capable of degrading extracellular matrix proteins. We hypothesize that tissue inhibitor of metalloproteinase-1 (TIMP-1) can serve as an indicator of acute venous endothelial dysfunction in a rat model of oxidative injury. The experimental groups evaluated were as follows: rats not undergoing oxidative injury (controls), rats that received rose bengal but no laser (shams), and rats that received both rose bengal and laser illumination, resulting in an oxidative injury. Animals were evaluated at baseline (control, shams) and at 1 hr and 1 day post-oxidative injury. mRNA expression was determined by gene array technology and real-time polymerase chain reaction, plasma and vein wall TIMP-1 protein concentrations were determined by enzyme-linked immunosorbent assay, and vein wall morphometrics (cells/five high-power fields) were performed. B-cell lymphoma 2-like gene expression was upregulated at both 1 hr and 1 day post-injury. TIMP-1 protein and mRNA expression were significantly increased post-oxidative injury. One hour postinjury, vein wall polymorphonuclear leukocytes were present in significant numbers. Our results support the hypothesis that increased expression of TIMP-1 in venous endothelium and plasma may serve as an early indicator of endothelial dysfunction.

Myeloproliferative defects following targeting of the Drf1 gene encoding the mammalian diaphanous related formin mDia1.

Rho GTPase-effector mammalian diaphanous (mDia)-related formins assemble nonbranched actin filaments as part of cellular processes, including cell division, filopodia assembly, and intracellular trafficking. Whereas recent efforts have led to thorough characterization of formins in cytoskeletal remodeling and actin assembly in vitro, little is known about the role of mDia proteins in vivo. To fill this knowledge gap, the Drf1 gene, which encodes the canonical formin mDia1, was targeted by homologous recombination. Upon birth, Drf1+/- and Drf1-/- mice were developmentally and morphologically indistinguishable from their wild-type littermates. However, both Drf1+/- and Drf1-/- developed age-dependent myeloproliferative defects. The phenotype included splenomegaly, fibrotic and hypercellular bone marrow, extramedullary hematopoiesis in both spleen and liver, and the presence of immature myeloid progenitor cells with high nucleus-to-cytoplasm ratios. Analysis of cell surface markers showed an age-dependent increase in the percentage of CD11b+-activated and CD14+-activated monocytes/macrophages in both spleen and bone marrow in Drf1+/- and Drf1-/- animals. Analysis of the erythroid compartment showed a significant increase in the proportion of splenic cells in S phase and an expansion of erythroid precursors (TER-119+ and CD71+) in Drf1-targeted mice. Overall, knocking out mDia1 expression in mice leads to a phenotype similar to human myeloproliferative syndrome (MPS) and myelodysplastic syndromes (MDS). These observations suggest that defective DRF1 expression or mDia1 function may contribute to myeloid malignancies and point to mDia1 as an attractive therapeutic target in MDS and MPS.

Inactivation of Apc in the mouse prostate causes prostate carcinoma.

Alterations of the Wnt/beta-catenin signaling pathway are positively associated with the development and progression of human cancer, including carcinoma of the prostate. To determine the role of activated Wnt/beta-catenin signaling in mouse prostate carcinogenesis, we created a mouse prostate tumor model using probasin-Cre-mediated deletion of Apc. Prostate tumors induced by the deletion of Apc have elevated levels of beta-catenin protein and are highly proliferative. Tumor formation is fully penetrant and follows a consistent pattern of progression. Hyperplasia is observed as early as 4.5 weeks of age, and adenocarcinoma is observed by 7 months. Continued tumor growth usually necessitated sacrifice between 12 and 15 months of age. Despite the high proliferation rate, we have not observed metastasis of these tumors to the lymph nodes or other organs. Surgical castration of 6-week-old mice inhibited tumor formation, and castration of mice with more advanced tumors resulted in the partial regression of specific prostate glands. However, significant areas of carcinoma remained 2 months postcastration, suggesting that tumors induced by Apc loss of function are capable of growth under conditions of androgen depletion. We conclude that the prostate-specific deletion of Apc and the increased expression of beta-catenin associated with prostate carcinoma suggests a role for beta-catenin in prostate cancer and offers an appropriate animal model to investigate the interaction of Wnt signaling with other genetic and epigenetic signals in prostate carcinogenesis.

A highly invasive human glioblastoma pre-clinical model for testing therapeutics.

Animal models greatly facilitate understanding of cancer and importantly, serve pre-clinically for evaluating potential anti-cancer therapies. We developed an invasive orthotopic human glioblastoma multiforme (GBM) mouse model that enables real-time tumor ultrasound imaging and pre-clinical evaluation of anti-neoplastic drugs such as 17-(allylamino)-17-demethoxy geldanamycin (17AAG). Clinically, GBM metastasis rarely happen, but unexpectedly most human GBM tumor cell lines intrinsically possess metastatic potential. We used an experimental lung metastasis assay (ELM) to enrich for metastatic cells and three of four commonly used GBM lines were highly metastatic after repeated ELM selection (M2). These GBM-M2 lines grew more aggressively orthotopically and all showed dramatic multifold increases in IL6, IL8, MCP-1 and GM-CSF expression, cytokines and factors that are associated with GBM and poor prognosis. DBM2 cells, which were derived from the DBTRG-05MG cell line were used to test the efficacy of 17AAG for treatment of intracranial tumors. The DMB2 orthotopic xenografts form highly invasive tumors with areas of central necrosis, vascular hyperplasia and intracranial dissemination. In addition, the orthotopic tumors caused osteolysis and the skull opening correlated to the tumor size, permitting the use of real-time ultrasound imaging to evaluate antitumor drug activity. We show that 17AAG significantly inhibits DBM2 tumor growth with significant drug responses in subcutaneous, lung and orthotopic tumor locations. This model has multiple unique features for investigating the pathobiology of intracranial tumor growth and for monitoring systemic and intracranial responses to antitumor agents.

Dose-dependent thrombus resolution due to oral plaminogen activator inhibitor (PAI)-1 inhibition with tiplaxtinin in a rat stenosis model of venous thrombosis.

This study aimed to evaluate a small-molecule PAI-1 inhibitor (PAI-039; tiplaxtinin) in a rodent stenosis model of venous thrombosis in a two-phase experiment. Phase 1 determined the efficacy of tiplaxtinin against Lovenox (LOV), while phase 2 determined the dose-dependent efficacy. For both phases, drug treatment began 24 hours after surgically induced venous thrombosis and continued for four days. Phase 1 animals (n = 24) receiving low-dose (LD; 1 mg/kg oral gavage) PAI-1 inhibitor demonstrated a 52% decrease in thrombus weight (TW) versus controls (p < 0.05) with significant reductions in active plasma PAI-1, while the high-dose (HD; 10 mg/kg oral gavage) group demonstrated a 23% reduction in TW versus controls. Animals treated subcutaneously with LOV (3 mg/kg) showed a 39% decrease in TW versus controls (p < 0.05). Coagulation tests (aPTT and TCT) were significantly different in LOV compared to PAI-1 inhibitor groups. PAI-039 treatment was also associated with significantly increased return of inferior vena cava blood flow four days post-thrombosis versus controls (p < 0.05). In phase 2 (n = 30), TW was reduced from the 0.5 mg/kg to 5 mg/kg experimental groups, with the 10 mg/kg group demonstrating a paradoxical increase. The 5 mg/kg group showed statistically significant decreases in TW versus controls after four treatment days (p < 0.05). This is the first study to demonstrate dose related effects of PAI-039 on increasing thrombus resolution and inferior vena cava blood flow without adverse effects on anti-coagulation in a rat stenosis model of venous thrombosis.

Vitreous Levels of Luteinizing Hormone and VEGF are Strongly Correlated in Healthy Mammalian Eyes.

Purpose/Aim: Luteinizing hormone (LH) is known to function as a key regulator of vascular endothelial growth factor (VEGF) expression in reproductive organs. In recent years, LH has also been detected in human vitreous and LH receptors have been identified in human retina. This study was aimed to investigate a potential correlation between LH and VEGF levels in healthy mammalian eyes to provide supporting evidence of LH's potential involvement in intraocular VEGF regulation. METHODS: 18 bovine and 30 porcine eyes were procured from an abattoir and VEGF and LH levels were measured in the vitreous extracted from these eyes by commercially available bovine & porcine ELISA assay kits. Total protein of the vitreous was measured by using Micro BSA protein assay kit. RESULTS: After total protein normalization, the Pearson Correlation Coefficients (PCC) showed a strong and significant correlation between LH and VEGF levels. (Bovine LH/VEGF PCC: 0.89, p < 0.001; Porcine LH/VEGF PCC: 0.80, p < 0.001). Linear regression analyses, adjusted for gender, showed significant linear relationships between LH and VEGF levels in both bovine and porcine vitreous. (Bovine: t-value = 7.69, p < 0.0001, adjusted r2 = .79; Porcine: t-value = 6.71, p < 0.001, adjusted r2 = .62) Conclusions: We show that VEGF and LH are strongly correlated in healthy, adult mammalian eyes. The robustness of the correlation is shown both by its strength of association and reproducibility in two species. Given that LH is well known to regulate VEGF levels in several tissue types, the LH/VEGF linear relationship in vitreous potentially implicates LH in homeostatic VEGF regulation of the eye. Because we also found that the correlation between LH and VEGF only became manifest when our targeted analytes were normalized by total amount of protein, preclinical and clinical investigators should consider normalizing analytes in vitreous by total protein when assessing potential correlations among them.

Elimination of Signaling by the Luteinizing Hormone Receptor Reduces Ocular VEGF and Retinal Vascularization during Mouse Eye Development.

Purpose/Aim: Vascular endothelial growth factor (VEGF) dysregulation is implicated in the pathogenesis of retinopathy of prematurity (ROP). Identifying the factors that contribute to VEGF regulation during normal retinal vascularization is the key to ROP prevention. Currently, physiologic hypoxia is thought to be responsible for retinal VEGF regulation in utero. However, a potential hormonal contribution to VEGF regulation during eye development has not been fully investigated. The placental hormone, human chorionic gonadotropin and the pituitary hormone, and luteinizing hormone (LH) induce VEGF expression in several tissue types. Both of these gonadotropins activate the same LH receptor (LHR) in the human body; LHRs are expressed in the retina. In this study, we aimed to show that LHR signaling participates in VEGF regulation in the developing eye. METHODS: When offspring from breeding pairs of LHR knockout mice (lhrkos) reached 21 days old, eyes and serum were extracted from homozygote lhrkos and wildtype (WT) siblings. VEGF levels were measured using Mouse VEGF Quantikine immunoassay kit. Retinas were incubated with isolectin for endothelial cell staining, flat mounted and imaged by confocal microscopy. Retinal vascular density was quantified using Imaris software. Some eyes were sectioned and stained for histopathologic review. RESULTS: Ocular VEGF and retinal vascular volumes were significantly reduced by ~ 15% in lhrko eyes. Serum VEGF was not changed. The lhrko retinas did not display any anomalies. CONCLUSIONS: We provide evidence that LHR signaling plays a role in VEGF regulation and vascularization in the developing eye. Given that human preterm infants may have altered LHR-activity, the effect of gonadotropins on eye development should be further studied to identify novel strategies for ROP prevention.

Confirmation of Luteinizing Hormone (LH) in Living Human Vitreous and the Effect of LH Receptor Reduction on Murine Electroretinogram.

Luteinizing hormone (LH), produced in the anterior pituitary, has been detected in cadaver eyes and LH receptors (LHRs) have been identified in the retina, with the highest density in cone photoreceptors. Our aim was to confirm the presence of LH in the living, human eye as well as to examine the potential impact of a reduction in LHR signaling on visual processing. Vitreous samples were collected from 40 patients (23 diabetics, 17 non-diabetics) who were undergoing vitrectomies for various indications. LH concentration was quantified in each sample via an electro-chemiluminescence immunoassay and Meso Scale Discovery platform and normalized to total protein. In addition, full-field electroretinography (ERG) was performed on 11 adult LHR knockout heterozygous mice (B6;129X1-Lhcgrtm1Zmlei/J) and 11 wild types using the Celeris-Diagnosys system. The median LH values (pg/mg total protein) for non-diabetics, diabetics without proliferative diabetic retinopathy (PDR) and diabetics with PDR were 40.7, 41.9 and 167.8 respectively. LH levels were significantly higher in diabetics with PDR. In our ERG investigation, heterozygous LHRKOs were found to have significantly reduced amplitudes of a-wave and b-waves at high stimulus intensities with no significant change in a-wave or b-wave amplitudes at lower intensities; this is consistent with a selective impairment of cone-mediated responses. Our findings confirm LH is present in the adult human eye. Our findings also suggest that a reduction in LH receptor signaling negatively impacts visual processing of the cone photoreceptors. Overall, our study results support the theory that LH likely plays a physiologic role in the eye.

Bacterial and Pneumocystis Infections in the Lungs of Gene-Knockout Rabbits with Severe Combined Immunodeficiency.

Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (-/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.

E-selectin inhibition with GMI-1271 decreases venous thrombosis without profoundly affecting tail vein bleeding in a mouse model.

Selectins, such as E-selectin (CD62E), function in venous thrombosis by binding and activating immune cells to initiate the coagulation cascade. GMI-1271 is a small molecule antagonist that inhibits E-selectin activity. Here we determine whether inhibition of E-selectin is sufficient to decrease acute venous thrombosis and associated inflammatory events in both prophylactic and treatment protocols without significantly affecting haemostasis. Male C57BL/6 mice underwent surgery for experimental thrombosis induction and were harvested at peak thrombus formation in our animal model, two days post induction. Groups included non-thrombosed true controls, shams, controls, and prophylactic or treatment groups of GMI-1271 (10 mg/kg intraperitoneal BID (twice a day) and low-molecular-weight heparin (LMWH, Lovenox 6 mg/kg subcutaneously (SC), once a day (SID). Compared with control animals, prophylaxis or treatment with LMWH and GMI-1271 in a dose-dependent manner significantly decreased thrombosis. GMI-1271 significantly lowered tail bleeding times when compared to LMWH. GMI-1271 and LMWH prophylactically administered significantly decreased vein wall neutrophil cell extravasation. However, all treatment and prophylactic therapies significantly decreased vein wall monocyte extravasation versus controls. GMI-1271 prophylactic therapy significantly decreased intra-thrombus cell counts versus control animals and other treatment groups. Immunohistochemistry confirmed that both treatments with GMI-1271 and LMWH significantly decreased activated leukocyte migration. GMI-1271 therapy significantly decreased thrombus weight and resulted in significantly lower bleeding times than LMWH. GMI-1271 treated mice showed decreased local and systemic inflammatory effects while modulating neutrophil activation, suggesting that GMI-1271 is a viable therapeutic candidate for venous thrombosis prophylaxis and treatment.

Methionine-deficient diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-I and insulin levels, and increases hepatocyte MIF levels and stress resistance.

A diet deficient in the amino acid methionine has previously been shown to extend lifespan in several stocks of inbred rats. We report here that a methionine-deficient (Meth-R) diet also increases maximal lifespan in (BALB/cJ x C57BL/6 J)F1 mice. Compared with controls, Meth-R mice have significantly lower levels of serum IGF-I, insulin, glucose and thyroid hormone. Meth-R mice also have higher levels of liver mRNA for MIF (macrophage migration inhibition factor), known to be higher in several other mouse models of extended longevity. Meth-R mice are significantly slower to develop lens turbidity and to show age-related changes in T-cell subsets. They are also dramatically more resistant to oxidative liver cell injury induced by injection of toxic doses of acetaminophen. The spectrum of terminal illnesses in the Meth-R group is similar to that seen in control mice. Studies of the cellular and molecular biology of methionine-deprived mice may, in parallel to studies of calorie-restricted mice, provide insights into the way in which nutritional factors modulate longevity and late-life illnesses.

Renal transitional cell carcinoma and choristoma in a degu (Octodon degus).

A 4.5-year-old female degu (Octodon degus) was minimally responsive with a poor body condition, a rough haircoat, and moderate dehydration. Blood was present around its urethral orifice and on the cage bedding. Laboratory analyses revealed leukocytosis with neutrophilia and anemia; hypoproteinemia and hypoalbuminemia; hyperglycemia, hyperphosphatemia, and elevated alanine aminotransferase, blood urea nitrogen, and creatinine; and hematuria and pyuria with occasional squamous and transitional epithelial cells. A urine culture was positive for coagulase-negative Staphylococcus sp. On gross necropsy, the right kidney was enlarged, cystic, and greenish-brown, with a 10-mm, hemorrhagic, granular mass extending from the renal pelvis into the cranial cortex. Only a small amount of renal cortex appeared normal. The urinary bladder had focal areas of hemorrhage and contained frank blood. Histologically, the papillary mass in the right renal pelvis comprised basophilic, moderately anaplastic, clustered epithelial transition cells consistent with a transitional cell carcinoma. Internally, the tumor showed squamous metaplasia and moderate multifocal interstitial fibrosis. The right kidney cortex contained a choristoma comprising trabecular bone, mature adipocytes, and cellular infiltrates suggestive of osteocytes, lymphocytes, and plasma cells. The urinary bladder had mild to moderate, focal, hemorrhage with neutrophilic inflammation and contained focal areas of mild transitional cell epithelial hyperplasia; these changes may have been secondary to irritation by hemorrhage in the renal pelvis. There was no evidence of metastasis. Renal transitional cell tumors are rare in rodents. This is the first report of both a renal transitional cell carcinoma and a renal choristoma in a degu.