Genome-Wide Association Transethnic Meta-Analyses Identifies Novel Associations Regulating Coagulation Factor VIII and von Willebrand Factor Plasma Levels.

BACKGROUND: Factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are associated with risk of arterial and venous thrombosis and with hemorrhagic disorders. We aimed to identify and functionally test novel genetic associations regulating plasma FVIII and VWF. METHODS: We meta-analyzed genome-wide association results from 46 354 individuals of European, African, East Asian, and Hispanic ancestry. All studies performed linear regression analysis using an additive genetic model and associated ≈35 million imputed variants with natural log-transformed phenotype levels. In vitro gene silencing in cultured endothelial cells was performed for candidate genes to provide additional evidence on association and function. Two-sample Mendelian randomization analyses were applied to test the causal role of FVIII and VWF plasma levels on the risk of arterial and venous thrombotic events. RESULTS: We identified 13 novel genome-wide significant ( P≤2.5×10-8) associations, 7 with FVIII levels ( FCHO2/TMEM171/TNPO1, HLA, SOX17/RP1, LINC00583/NFIB, RAB5C-KAT2A, RPL3/TAB1/SYNGR1, and ARSA) and 11 with VWF levels ( PDHB/PXK/KCTD6, SLC39A8, FCHO2/TMEM171/TNPO1, HLA, GIMAP7/GIMAP4, OR13C5/NIPSNAP, DAB2IP, C2CD4B, RAB5C-KAT2A, TAB1/SYNGR1, and ARSA), beyond 10 previously reported associations with these phenotypes. Functional validation provided further evidence of association for all loci on VWF except ARSA and DAB2IP. Mendelian randomization suggested causal effects of plasma FVIII activity levels on venous thrombosis and coronary artery disease risk and plasma VWF levels on ischemic stroke risk. CONCLUSIONS: The meta-analysis identified 13 novel genetic loci regulating FVIII and VWF plasma levels, 10 of which we validated functionally. We provide some evidence for a causal role of these proteins in thrombotic events.

Acetylcholine Inhibits Platelet Activation.

Platelets are key mediators of thrombosis. Many agonists of platelet activation are known, but fewer endogenous inhibitors of platelets, such as prostacyclin and nitric oxide (NO), have been identified. Acetylcholinesterase inhibitors, such as donepezil, can cause bleeding in patients, but the underlying mechanisms are not well understood. We hypothesized that acetylcholine is an endogenous inhibitor of platelets. We measured the effect of acetylcholine or analogs of acetylcholine on human platelet activation ex vivo. Acetylcholine and analogs of acetylcholine inhibited platelet activation, as measured by P-selectin translocation and glycoprotein IIb IIIa conformational changes. Conversely, we found that antagonists of the acetylcholine receptor, such as pancuronium, enhance platelet activation. Furthermore, drugs inhibiting acetylcholinesterase, such as donepezil, also inhibit platelet activation, suggesting that platelets release acetylcholine. We found that NO mediates acetylcholine inhibition of platelets. Our data suggest that acetylcholine is an endogenous inhibitor of platelet activation. The cholinergic system may be a novel target for antithrombotic therapies.

Adjuvant Sunitinib in High-Risk Patients with Uveal Melanoma: Comparison with Institutional Controls.

PURPOSE: To compare overall survival in high-risk patients with primary uveal melanoma who received adjuvant sunitinib with institutional controls. DESIGN: Retrospective cohort. PARTICIPANTS: Selection criteria were (1) monosomy 3 and 8q amplification by cytogenetic or DecisionDx-UM Class 2 and (2) monosomy 3 and large tumor size (T3-4 by American Joint Committee on Cancer classification). Exclusion criteria were date of diagnosis before 2007 or after 2013 and age <18 years. METHODS: A cohort of patients who intended to receive adjuvant sunitinib for 6 months was compared with institutional historical controls with the same risk factors. Kaplan-Meier and Cox proportional hazards models were used to analyze the outcome. Propensity score was used to adjust for nonrandom assignment to sunitinib. MAIN OUTCOME MEASURES: Overall survival. RESULTS: From the Wills Eye Hospital Oncology Service Uveal Melanoma Cytogenetic Database (N = 1172), 128 patients fulfilled the selection and exclusion criteria. Median follow-up was 52.7 months (range, 0.26-108 months). A total of 54 patients received sunitinib. Their median age was 56 years (range, 29-81 years), and 48% were men. A total of 74 historical controls in the same risk category were identified. Their median age was 62 years (21-80 years), and 48% were men. Patients in the sunitinib group had worse cytogenetic or molecular features (monosomy 3 and 8q amplification or class 2 87% vs. 57%; P < 0.001), had smaller tumor sizes (T3-4 56% vs. 83%; P = 0.001), and were younger. There were 51 deaths, 14 (26%) in the sunitinib group and 37 (50%) in the control group. In the univariate analysis, the sunitinib group had longer overall survival (hazard ratio, 0.53; 95% confidence interval, 0.29-0.99; P = 0.041). In multivariate Cox regression analysis, interaction between use of sunitinib and age as a dichotomous variable was highly significant (P = 0.003). The following variables were statistically associated with prediction of overall survival: cytogenetic/molecular status (P = 0.015), T-size category (P = 0.022), gender (P = 0.040), and adjuvant sunitinib in patients aged <60 years (P = 0.004). Results were confirmed by propensity score analysis. CONCLUSIONS: In this retrospective study, the use of sunitinib in the adjuvant setting was associated with better overall survival.

Novel Thrombotic Function of a Human SNP in STXBP5 Revealed by CRISPR/Cas9 Gene Editing in Mice.

OBJECTIVE: To identify and characterize the effect of a SNP (single-nucleotide polymorphism) in the STXBP5 locus that is associated with altered thrombosis in humans. GWAS (genome-wide association studies) have identified numerous SNPs associated with human thrombotic phenotypes, but determining the functional significance of an individual candidate SNP can be challenging, particularly when in vivo modeling is required. Recent GWAS led to the discovery of STXBP5 as a regulator of platelet secretion in humans. Further clinical studies have identified genetic variants of STXBP5 that are linked to altered plasma von Willebrand factor levels and thrombosis in humans, but the functional significance of these variants in STXBP5 is not understood. APPROACH AND RESULTS: We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) techniques to produce a precise mouse model carrying a human coding SNP rs1039084 (encoding human p. N436S) in the STXBP5 locus associated with decreased thrombosis. Mice carrying the orthologous human mutation (encoding p. N437S in mouse STXBP5) have lower plasma von Willebrand factor levels, decreased thrombosis, and decreased platelet secretion compared with wild-type mice. This thrombosis phenotype recapitulates the phenotype of humans carrying the minor allele of rs1039084. Decreased plasma von Willebrand factor and platelet activation may partially explain the decreased thrombotic phenotype in mutant mice. CONCLUSIONS: Using precise mammalian genome editing, we have identified a human nonsynonymous SNP rs1039084 in the STXBP5 locus as a causal variant for a decreased thrombotic phenotype. CRISPR/Cas9 genetic editing facilitates the rapid and efficient generation of animals to study the function of human genetic variation in vascular diseases.

Establishment of an orthotopic patient-derived xenograft mouse model using uveal melanoma hepatic metastasis.

BACKGROUND: Metastatic uveal melanoma is a highly fatal disease; most patients die from their hepatic metastasis within 1 year. A major drawback in the development of new treatments for metastatic uveal melanoma is the difficulty in obtaining appropriate cell lines and the lack of appropriate animal models. Patient-derived xenograft (PDX) tumor models, bearing ectopically implanted tumors at a subcutaneous site, have been developed. However, these ectopically implanted PDX models have obstacles to translational research, including a low engraftment rate, slow tumor growth, and biological changes after multiple passages due to the different microenvironment. To overcome these limitations, we developed a new method to directly transplant biopsy specimens to the liver of immunocompromised mice. RESULTS: By using two metastatic uveal melanoma cell lines, we demonstrated that the liver provides a more suitable microenvironment for tumor growth compared to subcutaneous sites and that surgical orthotopic implantation (SOI) of tumor pieces allows the creation of a liver tumor in immunocompromised mice. Subsequently, 10 of 12 hepatic metastasis specimens from patients were successfully xenografted into the immunocompromised mice (83.3% success rate) using SOI, including 8 of 10 needle biopsy specimens (80%). Additionally, four cryopreserved PDX tumors were re-implanted to new mice and re-establishment of PDX tumors was confirmed in all four mice. The serially passaged xenograft tumors as well as the re-implanted tumors after cryopreservation were similar to the original patient tumors in histologic, genomic, and proteomic expression profiles. CT imaging was effective for detecting and monitoring PDX tumors in the liver of living mice. The expression of Ki67 in original patient tumors was a predictive factor for implanted tumor growth and the success of serial passages in PDX mice. CONCLUSIONS: Surgical orthotopic implantation of hepatic metastasis from uveal melanoma is highly successful in the establishment of orthotopic PDX models, enhancing their practical utility for research applications. By using CT scan, tumor growth can be monitored, which is beneficial to evaluate treatment effects in interventional studies.

Establishment and Characterization of Orthotopic Mouse Models for Human Uveal Melanoma Hepatic Colonization.

Uveal melanoma (UM) is a rare type of melanoma, although it is the most common primary ocular malignant tumor in adults. Nearly one-half the patients with primary UM subsequently develop systemic metastasis, preferentially to the liver. Currently, no treatment is effective for UM hepatic metastasis, and the prognosis is universally poor. The main challenge in designing a treatment strategy for UM hepatic metastasis is the lack of suitable animal models. We developed two orthotopic mouse models for human UM hepatic metastases: direct hepatic implantation model (intrahepatic dissemination model) and splenic-implantation model (hematogenous dissemination model) and investigated the tumorgenesis in the liver. A human UM cell line, established from a hepatic metastasis and nonobese diabetic severe combined immunodeficient γ mice, were used for development of in vivo tumor models. In the direct hepatic implantation model, a localized tumor developed in the liver in all cases and intrahepatic dissemination was subsequently seen in about one-half of cases. However, in the splenic implantation model, multiple hepatic metastases were observed after splenic implantation. Hepatic tumors subsequently seeded intra-abdominal metastasis; however, lung metastases were not seen. These findings are consistent with those observed in human UM hepatic metastases. These orthotopic mouse models offer useful tools to investigate the biological behavior of human UM cells in the liver.

Double-blinded, randomized phase II study using embolization with or without granulocyte-macrophage colony-stimulating factor in uveal melanoma with hepatic metastases.

PURPOSE: To investigate the effects of immunoembolization with granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with uveal melanoma (UM) with liver-only metastasis. MATERIALS AND METHODS: In this double-blind phase II clinical trial, patients were randomized to undergo immunoembolization or bland embolization (BE). Lobar treatment was performed with GM-CSF or normal saline solution mixed with ethiodized oil followed by embolization with gelatin sponge emulsified with iodinated contrast medium. Fifty-two patients (immunoembolization, n = 25; BE, n = 27) were enrolled. Response was assessed after every two treatments. The primary endpoint was overall response rate (ORR) of liver metastases. Progression-free survival (PFS), overall survival (OS), and immunologic responses were secondary endpoints. RESULTS: There were five partial responses in the immunoembolization group (ORR, 21.2%; 90% confidence interval [CI], 10.3%-30.5%) and three in the BE group (ORR, 16.7%; 90% CI, 6.3%-26.9%). Stable disease was seen in 12 patients in the immunoembolization group and 19 in the BE group. OS times were 21.5 months (95% CI, 18.5-24.8 mo) with immunoembolization and 17.2 months (95% CI, 11.9-22.4 mo) with BE. The degree of proinflammatory cytokine production was more robust after immunoembolization and correlated with time to "systemic" extrahepatic progression. In the immunoembolization group, interleukin (IL)-6 levels at 1 hour (P = .001) and IL-8 levels at 18 hours after the procedure (P < .001) were significant predictors of longer systemic PFS. Moreover, a dose-response pattern was evident between posttreatment serum cytokine concentrations and systemic PFS. CONCLUSIONS: Immunoembolization induced more robust inflammatory responses, which correlated with the delayed progression of extrahepatic systemic metastases.

Chronic neuron- and age-selective down-regulation of TNF receptor expression in triple-transgenic Alzheimer disease mice leads to significant modulation of amyloid- and Tau-related pathologies.

Neuroinflammation, through production of proinflammatory molecules and activated glial cells, is implicated in Alzheimer's disease (AD) pathogenesis. One such proinflammatory mediator is tumor necrosis factor α (TNF-α), a multifunctional cytokine produced in excess and associated with amyloid ß-driven inflammation and cognitive decline. Long-term global inhibition of TNF receptor type I (TNF-RI) and TNF-RII signaling without cell or stage specificity in triple-transgenic AD mice exacerbates hallmark amyloid and neurofibrillary tangle pathology. These observations revealed that long-term pan anti-TNF-α inhibition accelerates disease, cautions against long-term use of anti-TNF-α therapeutics for AD, and urges more selective regulation of TNF signaling. We used adeno-associated virus vector-delivered siRNAs to selectively knock down neuronal TNF-R signaling. We demonstrate divergent roles for neuronal TNF-RI and TNF-RII where loss of opposing TNF-RII leads to TNF-RI-mediated exacerbation of amyloid ß and Tau pathology in aged triple-transgenic AD mice. Dampening of TNF-RII or TNF-RI+RII leads to a stage-independent increase in Iba-1-positive microglial staining, implying that neuronal TNF-RII may act nonautonomously on the microglial cell population. These results reveal that TNF-R signaling is complex, and it is unlikely that all cells and both receptors will respond positively to broad anti-TNF-α treatments at various stages of disease. In aggregate, these data further support the development of cell-, stage-, and/or receptor-specific anti-TNF-α therapeutics for AD.

Interleukin 6 mediates production of interleukin 10 in metastatic melanoma.

We previously reported that substantial amounts of IL-10, an immunomodulatory cytokine, are produced by cell suspensions of fresh human metastatic melanoma tissues. Production diminished with continuous culturing of cells, which suggests a pivotal interactive role between melanoma cells and the tumor microenvironment. In this study, we found that the culture media obtained from LPS-stimulated peripheral blood mononuclear cells induced IL-10 production by metastatic melanoma cells. Of the multiple cytokines present in the conditioned culture media, IL-6 was identified as the inducer of IL-10 production. A neutralizing antibody against IL-6 completely blocked the conditioned medium-induced IL-10 production. Metastatic melanoma cells that constitutively produce low amount of IL-10 increased IL-10 production in response to recombinant human IL-6 in a dose-dependent fashion. The response to exogenously added IL-6 was less significant in melanoma cells that produced high amounts of IL-6, probably due to pre-existing autocrine stimulation of IL-10 by endogenous IL-6. On the other hand, metastatic melanoma cells that do not constitutively produce IL-10 protein did not respond to exogenous IL-6. In IL-6-responsive melanoma cells, IL-6 increased STAT3 phosphorylation and inhibition of STAT3 signaling using siRNA or inhibitors for JAKs diminished IL-6-induced IL-10 production. In addition, inhibition of MEK and PI3K, but not mTOR, interfered with IL-10 production. Taken together, the data suggest that blocking of these signals leading to IL-10 production is a potential strategy to enhance an anti-melanoma immune response in metastatic melanoma.

Ablation of TNF-RI/RII expression in Alzheimer's disease mice leads to an unexpected enhancement of pathology: implications for chronic pan-TNF-α suppressive therapeutic strategies in the brain.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by severe memory loss and cognitive impairment. Neuroinflammation, including the extensive production of pro-inflammatory molecules and the activation of microglia, has been implicated in the disease process. Tumor necrosis factor (TNF)-α, a prototypic pro-inflammatory cytokine, is elevated in AD, is neurotoxic, and colocalizes with amyloid plaques in AD animal models and human brains. We previously demonstrated that the expression of TNF-α is increased in AD mice at ages preceding the development of hallmark amyloid and tau pathological features and that long-term expression of this cytokine in these mice leads to marked neuronal death. Such observations suggest that TNF-α signaling promotes AD pathogenesis and that therapeutics suppressing this cytokine's activity may be beneficial. To dissect TNF-α receptor signaling requirements in AD, we generated triple-transgenic AD mice (3xTg-AD) lacking both TNF-α receptor 1 (TNF-RI) and 2 (TNF-RII), 3xTg-ADxTNF-RI/RII knock out, the cognate receptors of TNF-α. These mice exhibit enhanced amyloid and tau-related pathological features by the age of 15 months, in stark contrast to age-matched 3xTg-AD counterparts. Moreover, 3xTg-ADxTNF-RI/RII knock out-derived primary microglia reveal reduced amyloid-ß phagocytic marker expression and phagocytosis activity, indicating that intact TNF-α receptor signaling is critical for microglial-mediated uptake of extracellular amyloid-ß peptide pools. Overall, our results demonstrate that globally ablated TNF receptor signaling exacerbates pathogenesis and argues against long-term use of pan-anti-TNF-α inhibitors for the treatment of AD.

Lymph node ratio is an important and independent prognostic factor for patients with stage III melanoma.

INTRODUCTION: The incidence of melanoma is dramatically increasing worldwide. We hypothesized that the ratio of metastatic to examined lymph node ratio (LNR) would be the most important prognostic factor for stage III patients. METHODS: We retrospectively reviewed our institutional database of melanoma patients and identified 168 patients who underwent lymph node dissection (LND) for stage III disease between 1993 and 2007. Patients were divided into three groups based on LNR (≤10%, n = 93; 10-≤25%, n = 45; and >25%, n = 30). Univariate and multivariate analysis was performed using Cox proportional hazards model. RESULTS: The median survival time of the entire group of patients was 34 months. The median number of positive nodes was 2 (range = 1, 55), and the median number of examined nodes was 22 (range = 5-123). Tumor characteristics of the primary melanoma (such as thickness, ulceration, and primary site) were not significant predictors of survival in this analysis. By univariate analysis, LNR was an important prognostic factor. Patients with LNR 10-25% and >25% had decreased survival compared to those patients with LNR ≤10% (HR = hazard ratio = 2.0 and 3.1, respectively; P ≤ 0.005). The number of positive lymph nodes also impacted on survival (P = 0.001). In multivariate analysis, LNR of 10-25% and >25% predicted survival (HR = 2.5 and 4.0, respectively). CONCLUSION: LNR is an important prognostic factor in patients undergoing LND for stage III melanoma. It can be used to stratify patients being considered for adjuvant therapy trials and should be evaluated using a larger prospective database.

Tumor-derived interleukin-10 as a prognostic factor in stage III patients undergoing adjuvant treatment with an autologous melanoma cell vaccine.

OBJECTIVES: Interleukin-10 (IL-10) downregulates T-cell-mediated immune responses. We studied the association between IL-10 production by freshly isolated melanoma cell suspensions in vitro and overall survival in patients undergoing adjuvant treatment with a vaccine prepared from the same autologous melanoma cells modified with a hapten, dinitrophenyl (DNP). METHODS: Forty-four patients with cutaneous melanoma (29 stage III and 15 stage IV) were prospectively evaluated. Tumor cells were extracted from metastatic deposits for production of DNP-modified autologous melanoma cell vaccine. Small aliquots of the melanoma cell suspensions were separated prior to vaccine processing and cultured overnight for IL-10 production. Based on a blind assessment of the distribution of IL-10 levels in the culture supernatants, a cutoff of 200 pg/ml was used to define high versus low IL-10 producers. Cox regression model was used for multivariate analysis. Overall survival was calculated using the Kaplan-Meier method, and survival curves were compared with the log-rank test. RESULTS: Out of 44 patients, 29 were low and 15 were high IL-10 producers. The median OS was significantly worse for high compared with low IL-10 producers (10.5 months vs. 42 months; P = 0.022). In stage III patients, the multivariate hazard ratio for high versus low IL-10 producers was 2.92 (95% CI, 1.04-8.20; P = 0.041). The corresponding hazard ratio in stage IV patients was 0.92 (95% CI, 1.04-8.20; P = 0.888). CONCLUSIONS: High IL-10 production in the tumor microenvironment could be a determinant of clinical outcomes in stage III melanoma patients receiving autologous melanoma cell vaccine.

In vitro migration of cytotoxic T lymphocyte derived from a colon carcinoma patient is dependent on CCL2 and CCR2.

BACKGROUND: Infiltration of colorectal carcinomas (CRC) with T-cells has been associated with good prognosis. There are some indications that chemokines could be involved in T-cell infiltration of tumors. Selective modulation of chemokine activity at the tumor site could attract immune cells resulting in tumor growth inhibition. In mouse tumor model systems, gene therapy with chemokines or administration of antibody (Ab)-chemokine fusion proteins have provided potent immune mediated tumor rejection which was mediated by infiltrating T cells at the tumor site. To develop such immunotherapeutic strategies for cancer patients, one must identify chemokines and their receptors involved in T-cell migration toward tumor cells. METHODS: To identify chemokine and chemokine receptors involved in T-cell migration toward CRC cells, we have used our previously published three-dimensional organotypic CRC culture system. Organotypic culture was initiated with a layer of fetal fibroblast cells mixed with collagen matrix in a 24 well tissue culture plate. A layer of CRC cells was placed on top of the fibroblast-collagen layer which was followed by a separating layer of fibroblasts in collagen matrix. Anti-CRC specific cytotoxic T lymphocytes (CTLs) mixed with fibroblasts in collagen matrix were placed on top of the separating layer. Excess chemokine ligand (CCL) or Abs to chemokine or chemokine receptor (CCR) were used in migration inhibition assays to identify the chemokine and the receptor involved in CTL migration. RESULTS: Inclusion of excess CCL2 in T-cell layer or Ab to CCL2 in separating layer of collagen fibroblasts blocked the migration of CTLs toward tumor cells and in turn significantly inhibited tumor cell apoptosis. Also, Ab to CCR2 in the separating layer of collagen and fibroblasts blocked the migration of CTLs toward tumor cells and subsequently inhibited tumor cell apoptosis. Expression of CCR2 in four additional CRC patients' lymphocytes isolated from infiltrating tumor tissues suggests their role in migration in other CRC patients. CONCLUSIONS: Our data suggest that CCL2 secreted by tumor cells and CCR2 receptors on CTLs are involved in migration of CTLs towards tumor. Gene therapy of tumor cells with CCL2 or CCL2/anti-tumor Ab fusion proteins may attract CTLs that potentially could inhibit tumor growth.

Early oligodendrocyte/myelin pathology in Alzheimer's disease mice constitutes a novel therapeutic target.

The detection of myelin disruptions in Alzheimer's disease (AD)-affected brain raises the possibility that oligodendrocytes undergo pathophysiological assault over the protracted course of this neurodegenerative disease. Oligodendrocyte compromise arising from direct toxic effects imparted by pathological amyloid-beta peptides and/or through signals derived from degenerating neurons could play an important role in the disease process. We previously demonstrated that 3xTg-AD mice, which harbor the human amyloid precursor protein Swedish mutant transgene, presenilin knock-in mutation, and tau P301L mutant transgene, exhibit significant alterations in overall myelination patterns and oligodendrocyte status at time points preceding the appearance of amyloid and tau pathology. Herein, we demonstrate that Abeta(1-42) leads to increased caspase-3 expression and apoptotic cell death of both nondifferentiated and differentiated mouse oligodendrocyte precursor (mOP) cells in vitro. Through use of a recombinant adeno-associated virus serotype-2 (rAAV2) vector expressing an Abeta(1-42)-specific intracellular antibody (intrabody), oligodendrocyte and myelin marker expression, as well as myelin integrity, were restored in the vector-infused brain regions of 3xTg-AD mice. Overall, this work provides further insights into the impact of Abeta(1-42)-mediated toxicity on the temporal and spatial progression of subtle myelin disruption during the early presymptomatic stages of AD and may help to validate new therapeutic options designed to avert these early impairments.

Abeta-directed single-chain antibody delivery via a serotype-1 AAV vector improves learning behavior and pathology in Alzheimer's disease mice.

Alzheimer's disease (AD) is a progressive dementing disorder characterized by age-related amyloid-beta (Abeta) deposition, neurofibrillary tangles, and synapse and neuronal loss. It is widely recognized that Abeta is a principal pathogenic mediator of AD. Our goal was to develop an immunotherapeutic approach, which would specifically lead to the clearance and/or neutralization of Abeta in the triple transgenic mouse model (3xTg-AD). These mice develop the amyloid and tangle pathologies and synaptic dysfunction reminiscent of human AD. Using a human single-chain variable fragment (scFv) antibody phage display library, a novel scFv antibody specific to Abeta was isolated, its activity characterized in vitro, and its open reading frame subsequently cloned into a recombinant adeno-associated virus (rAAV) vector. Three-month-old 3xTg-AD mice were intrahippocampally infused with serotype-1 rAAV vectors encoding Abeta-scFv or a control vector using convection-enhanced delivery (CED). Mice receiving rAAV1-Abeta-scFv harbored lower levels of insoluble Abeta and hyperphosphorylated tau, and exhibited improved cognitive function as measured by the Morris Water Maze (MWM) spatial memory task. These results underscore the potential of gene-based passive vaccination for AD, and provide further rationale for the development of Abeta-targeting strategies for this debilitating disease.

Interferon-{gamma} differentially affects Alzheimer's disease pathologies and induces neurogenesis in triple transgenic-AD mice.

Inflammatory processes, including the episodic and/ or chronic elaboration of cytokines, have been identified as playing key roles in a number of neurological disorders. Whether these activities impart a disease-resolving and/or contributory outcome depends at least in part on the disease context, stage of pathogenesis, and cellular milieu in which these factors are released. Interferon-gamma (IFNgamma) is one such cytokine that produces pleiotropic effects in the brain. It is protective by ensuring maintenance of virus latency after infection, yet deleterious by recruiting and activating microglia that secrete potentially damaging factors at sites of brain injury. Using the triple-transgenic mouse model of Alzheimer's disease (3xTg-AD), which develops amyloid and tau pathologies in a pattern reminiscent of human Alzheimer's disease, we initiated chronic intrahippocampal expression of IFNgamma through delivery of a serotype-1 recombinant adeno-associated virus vector (rAAV1-IFNgamma). Ten months of IFNgamma expression led to an increase in microglial activation, steady-state levels of proinflammatory cytokine and chemokine transcripts, and severity of amyloid-related pathology. In contrast, these rAAV1-IFNgamma-treated 3xTg-AD mice also exhibited diminished phospho-tau pathology and evidence of increased neurogenesis. Overall, IFNgamma mediates what seem to be diametrically opposed functions in the setting of AD-related neurodegeneration. Gaining an understanding as to how these apparently divergent functions are interrelated and controlled could elucidate new therapeutic strategies designed to harness the neuroprotective activity of IFNgamma.

Generating differentially targeted amyloid-beta specific intrabodies as a passive vaccination strategy for Alzheimer's disease.

Amyloid-beta (A beta) has been identified as a key component in Alzheimer's disease (AD). Significant in vitro and human pathological data suggest that intraneuronal accumulation of A beta peptides plays an early role in the neurodegenerative cascade. We hypothesized that targeting an antibody-based therapeutic to specifically abrogate intracellular A beta accumulation could prevent or slow disease onset. A beta 42-specific intracellular antibodies (intrabodies) with and without an intracellular trafficking signal were engineered from a previously characterized single-chain variable fragment (scFv) antibody. The intrabodies, one with an endoplasmic reticulum (ER) targeting signal and one devoid of a targeting sequence, were assessed in cells harboring a doxycycline (Dox)-regulated mutant human amyloid precursor protein Swedish mutant (hAPP(swe)) transcription unit for their abilities to prevent A beta peptide egress. Adeno-associated virus (AAV) vectors expressing the engineered intrabodies were administered to young adult 3xTg-AD mice, a model that develops amyloid and Tau pathologies, prior to the initial appearance of intraneuronal A beta. Chronic expression of the ER-targeted intrabody (IB) led to partial clearance of A beta 42 deposits and interestingly, in reduced staining for a pathologic phospho-Tau epitope (Thr231). This approach may provide insights into the functional relevance of intraneuronal A beta accumulation in early AD and potentially lead to the development of new therapeutics.

Malignant melanoma presenting as obstructive jaundice secondary to metastasis to the Ampulla of Vater.

CONTEXT: Malignant melanoma commonly metastasizes to the small intestine where it can cause pain, bleeding, and obstruction. However, jaundice from metastatic melanoma is relatively uncommon. CASE REPORT: A case of known malignant melanoma presenting as new onset obstructive jaundice as a result of a rarely reported metastasis to the ampulla of Vater. CONCLUSION: Multidisciplinary management of patients with metastatic melanoma is essential.

Unique Geospatial Accumulations of Uveal Melanoma.

PURPOSE: The main purpose of this paper was to describe the unique accumulation of cases of uveal melanoma (UM). All patients were white and did not have known occupational risk factors. From the authors' standpoint, there were no lifestyle factors in common in the reported cases. Results of more extensive analyses, including geospatial analysis, are currently being conducted and will be presented in a separate paper. DESIGN: Observational case series. METHODS: Descriptive data from medical records, patient interviews, and questionnaires were obtained from 5 patients from North Carolina, 6 patients from Alabama, and 14 patients from New York. Standard incidence ratio (SIR) calculations were provided by the respective states' cancer registries. UM is the most common primary malignant eye tumor in adults, although it is rare, with 2,500 cases diagnosed annually in the United States. Despite a growing understanding of the molecular characteristics, there remains uncertainty regarding epidemiologic trends and environmental risk factors. This study identified 3 geographic accumulations of UM: 1) Huntersville, NC; 2) Auburn, AL; and 3) Broome and Tioga Counties, New York. Investigation of these groups will guide ongoing efforts to discover potential risk factor and assist with future treatment and prevention. RESULTS: In North Carolina, 5 females who were identified as living in Huntersville, NC, were diagnosed with UM at ages 20, 22, 24, 30, and 31. The SIR calculations considering the observed and expected incidence ratios was 0.7 (95% confidence interval [CI], 0.5-0.9) in Mecklenburg County. In Alabama, 6 individuals who were identified as either attending Auburn University or employed there from 1989 to 1993 had diagnoses of UM. Initial SIR calculations for white females of all ages was 1.15 (95% CI, 0.989-1.328). In New York, SIR for Broome and Tioga counties were 0.93 and not significant. However, in Tioga county, for males and females and females alone, SIRs were 2.00 (P = .04) and 3.33 (P = .006). CONCLUSIONS: Although most of the conclusions that the SIR does not meet statistical criteria that defines these accumulations as true "cancer clusters," considering the incidence and demographics of UM, these accumulations of cases is unexpected and worth additional exploration. Further investigation into these cases with additional geospatial analyses and blood and tumor testing is ongoing. Information learned from the study of these unique populations may inform a better understanding of the pathogenesis of UM.

An Outcome Assessment of a Single Institution's Longitudinal Experience with Uveal Melanoma Patients with Liver Metastasis.

There is no FDA-approved treatment for metastatic uveal melanoma (UM) and overall outcomes are generally poor for those who develop liver metastasis. We performed a retrospective single-institution chart review on consecutive series of UM patients with liver metastasis who were treated at Thomas Jefferson University Hospital between 1971-1993 (Cohort 1, n = 80), 1998-2007 (Cohort 2, n = 198), and 2008-2017 (Cohort 3, n = 452). In total, 70% of patients in Cohort 1 received only systemic therapies as their treatment modality for liver metastasis, while 98% of patients in Cohort 2 and Cohort 3 received liver-directed treatment either alone or with systemic therapy. Median Mets-to-Death OS was shortest in Cohort 1 (5.3 months, 95% CI: 4.2-7.0), longer in Cohort 2 (13.6 months, 95% CI: 12.2-16.6) and longest in Cohort 3 (17.8 months, 95% CI: 16.6-19.4). Median Eye Tx-to-Death OS was shortest in Cohort 1 (40.8 months, 95% CI: 37.1-56.9), and similar in Cohort 2 (62.6 months, 95% CI: 54.6-71.5) and Cohort 3 (59.4 months, 95% CI: 56.2-64.7). It is speculated that this could be due to the shift of treatment modalities from DTIC-based chemotherapy to liver-directed therapies. Combination of liver-directed and newly developed systemic treatments may further improve the survival of these patients.