Neuroanatomical and cognitive biomarkers of alpha-synuclein propagation in a mouse model of synucleinopathy prior to onset of motor symptoms.

Significant evidence suggests that misfolded alpha-synuclein (aSyn), a major component of Lewy bodies, propagates in a prion-like manner contributing to disease progression in Parkinson's disease (PD) and other synucleinopathies. In fact, timed inoculation of M83 hemizygous mice with recombinant human aSyn preformed fibrils (PFF) has shown symptomatic deficits after substantial spreading of pathogenic alpha-synuclein, as detected by markers for the phosphorylation of S129 of aSyn. However, whether accumulated toxicity impact human-relevant cognitive and structural neuroanatomical measures is not fully understood. Here we performed a single unilateral striatal PFF injection in M83 hemizygous mice, and using two assays with translational potential, ex vivo magnetic resonance imaging (MRI) and touchscreen testing, we examined the combined neuroanatomical and behavioral impact of aSyn propagation. In PFF-injected mice, we observed widespread atrophy in bilateral regions that project to or receive input from the injection site using MRI. We also identified early deficits in reversal learning prior to the emergence of motor symptoms. Our findings highlight a network of regions with related cellular correlates of pathology that follow the progression of aSyn spreading, and that affect brain areas relevant for reversal learning. Our experiments suggest that M83 hemizygous mice injected with human PFF provides a model to understand how misfolded aSyn affects human-relevant pre-clinical measures and suggest that these pre-clinical biomarkers could be used to detect early toxicity of aSyn and provide better translational measures between mice and human disease.

Pharmacists in zoos? A qualitative study investigating the potential for pharmacist involvement in wildlife health care in Aotearoa, New Zealand.

BACKGROUND: The importance of wildlife health has been critically emphasized by the current global pandemic. Pharmacists play a valuable role in the health care of companion animals and livestock; however, their involvement in exotic animal health is largely unexplored. OBJECTIVES: This project consulted with zoo vets in New Zealand and investigated their practices around prescribing and dispensing of medicines to explore the opportunities for the involvement of pharmacists. METHODS: A mixed methods approach was used where data were initially collected through an online survey distributed to 26 veterinarians and animal keepers working in zoos, wildlife parks, and sanctuaries. An optional semistructured interview followed the survey. RESULTS: The facilities surveyed housed New Zealand native animal species and 85% also housed exotic animals. Veterinarians dispensed 75% of medicines at their animal facility, whereas the remaining 25% were dispensed by veterinary nurses. On average, 5-10 medications were dispensed at each animal facility per day. Common medicines dispensed were antibiotics, pain relievers, and antifungals. Most respondents felt that they could benefit from working alongside pharmacists in veterinary care. Compounding, access to medicines and identification of tailored formulations were identified as areas where collaboration would be valued. Limitations in the knowledge of pharmacists in animal medicine were distinguished as an area enhancement to assist in collaborative relationships. CONCLUSIONS: There are opportunities for the skills of pharmacists to be incorporated into the care of animals in zoos and wildlife parks in New Zealand. Strengthening the pharmacist-veterinarian relationship can enhance the health outcomes of animals in animal facilities through this interprofessional interaction.

The oncogenic effect of sulfatase 2 in human hepatocellular carcinoma is mediated in part by glypican 3-dependent Wnt activation.

UNLABELLED: Heparan sulfate proteoglycans (HSPGs) act as coreceptors or storage sites for growth factors and cytokines such as fibroblast growth factor and Wnts. Glypican 3 (GPC3) is the most highly expressed HSPG in hepatocellular carcinoma (HCC). Sulfatase 2 (SULF2), an enzyme with 6-O-desulfatase activity on HSPGs, is up-regulated in 60% of primary HCCs and is associated with a worse prognosis. We have previously shown that the oncogenic effect of SULF2 in HCC may be mediated in part through up-regulation of GPC3. Here we demonstrate that GPC3 stimulates the Wnt/ß-catenin pathway and mediates the oncogenic function of SULF2 in HCC. Wnt signaling in vitro and in vivo was assessed in SULF2-negative Hep3B HCC cells transfected with SULF2 and in SULF2-expressing Huh7 cells transfected with short hairpin RNA targeting SULF2. The interaction between GPC3, SULF2, and Wnt3a was assessed by coimmunoprecipitation and flow cytometry. ß-catenin-dependent transcriptional activity was assessed with the TOPFLASH (T cell factor reporter plasmid) luciferase assay. In HCC cells, SULF2 increased cell surface GPC3 and Wnt3a expression, stabilized ß-catenin, and activated T cell factor transcription factor activity and expression of the Wnt/ß-catenin target gene cyclin D1. Opposite effects were observed in SULF2-knockdown models. In vivo, nude mouse xenografts established from SULF2-transfected Hep3B cells showed enhanced GPC3, Wnt3a, and ß-catenin levels. CONCLUSION: Together, these findings identify a novel mechanism mediating the oncogenic function of SULF2 in HCC that includes GPC3-mediated activation of Wnt signaling via the Wnt3a/glycogen synthase kinase 3 beta axis.

Drug purchase price volatility in an academic medical center.

PURPOSE: Inpatient drug purchase price trends at an 811-bed academic medical center are described. SUMMARY: Recent highly publicized drug price increases by pharmaceutical manufacturers have generated public interest in regulatory solutions to reduce drug costs. Monitoring drug price changes through internal dashboards has been demonstrated to aid in purchasing decisions to reduce the impact of drug price changes on inpatient pharmacy drug budgets. In this research, University of Chicago Medicine created an internal dashboard to detail specific inpatient drug purchase price trends. Dashboard data input included all medications purchased through the organization's group purchasing organization over a 25-month time frame. A total of 69,245 drug purchases of 2,432 unique medications and/or dosage strengths were analyzed in the study. Within the 25-month time period, 706 medications (29%) had a net drug purchase price increase, while 898 (37%) had a net drug purchase price decrease. The range of net price percentage changes for medications with price increases was 0.01% to 733.6%; the range for medications with price decreases was 0.01% to 97.5%. CONCLUSION: Relative to previous purchase prices, drug purchase prices decreased or remained the same more often than they increased over a 25-month time frame. However, drug purchase price percentage changes were far greater for medications whose prices increased rather than decreased.

Methylation status of genes in non-neoplastic mucosa from patients with ulcerative colitis-associated colorectal cancer.

OBJECTIVES: Patients with ulcerative colitis (UC) are at increased risk for developing colorectal cancer (CRC). Surveillance in this at-risk population remains challenging. We assessed the methylation status of genes in the non-neoplastic mucosa of UC-CRC patients and controls to identify potential biomarkers of CRC. METHODS: We evaluated the methylation status of 10 genes (p16, p14, runt-related transcript factor-3 (RUNX3), cyclooxygenase-2 (COX-2), E-cadherin, methylated-in-tumor-1 (MINT1), MINT31, HPP1, estrogen receptor, SLC5A8) in UC-CRC tumors and non-neoplastic sections from both UC-CRC cases and UC controls (n=114 for each) using methylation-specific PCR. RESULTS: Amplification was successful for 96 UC controls, 83 tumors, and 66 non-adjacent, non-neoplastic samples. The prevalence of methylation was significantly greater in UC-CRC tumors for p16, RUNX3, MINT1, MINT31, and HPP1. Methylation of COX-2 and E-cadherin was greater in UC controls than in tumors. Univariate testing of these genes using non-adjacent, non-neoplastic sections from UC-CRC cases indicated that associations between p16, RUNX3, MINT1, MINT31, E-cadherin, and COX-2 and UC-CRC remained significant. In multivariable analysis of the six genes, only RUNX3, MINT1, and COX-2 remained significantly associated with the UC-CRC cases (odds ratio=12.6, 9.0, and 0.2, respectively). The results remained unaffected by the presence of PSC or severity of inflammation. Logistic regression modeling with the three genes showed interactions that increased the odds ratio for each gene. CONCLUSIONS: RUNX3, MINT1, and COX-2 are potential biomarkers for detecting the presence of CRC in patients with UC. These genes should be evaluated as biomarkers for colorectal dysplasia.

Sulfatase 2 up-regulates glypican 3, promotes fibroblast growth factor signaling, and decreases survival in hepatocellular carcinoma.

UNLABELLED: It has been shown that the heparin-degrading endosulfatase, sulfatase 1 (SULF1), functions as a liver tumor suppressor, but the role of the related sulfatase, sulfatase 2 (SULF2), in liver carcinogenesis remains to be elucidated. We investigated the effect of SULF2 on liver tumorigenesis. Expression of SULF2 was increased in 79 (57%) of 139 hepatocellular carcinomas (HCCs) and 8 (73%) of 11 HCC cell lines. Forced expression of SULF2 increased HCC cell growth and migration, whereas knockdown of SULF2 using short hairpin RNA targeting SULF2 abrogated HCC cell proliferation and migration in vitro. Because SULF1 and SULF2 desulfate heparan sulfate proteoglycans (HSPGs) and the HSPG glypican 3 (GPC3) is up-regulated in HCC, we investigated the effects of SULF2 on GPC3 expression and the association of SULF2 with GPC3. SULF2-mediated cell growth was associated with increased binding of fibroblast growth factor 2 (FGF2), phosphorylation of extracellular signal-regulated kinase and AKT, and expression of GPC3. Knockdown of GPC3 attenuated FGF2 binding in SULF2-expressing HCC cells. The effects of SULF2 on up-regulation of GPC3 and tumor growth were confirmed in nude mouse xenografts. Moreover, HCC patients with increased SULF2 expression in resected HCC tissues had a worse prognosis and a higher rate of recurrence after surgery. CONCLUSION: In contrast to the tumor suppressor effect of SULF1, SULF2 has an oncogenic effect in HCC mediated in part through up-regulation of FGF signaling and GPC3 expression.

A Biomarker Panel to Detect Synchronous Neoplasm in Non-neoplastic Surveillance Biopsies from Patients with Ulcerative Colitis.

BACKGROUND: Patients with ulcerative colitis (UC) are at risk for colorectal neoplasia. Challenges associated with surveillance colonoscopy with random biopsies for detection of dysplasia/cancer are well-documented. This study extended our findings in UC-associated colorectal cancer to include low-grade dysplasia (LGD) patients, testing whether our biomarker panel detects any UC-associated neoplasm. METHODS: DNA from the LGD area and the corresponding nonadjacent, non-dysplastic section from 171 UC-LGD patients was extracted. TaqMan SNP Genotyping Assays for TNF-α, IL-1ß, and IL23R were used to evaluate polymorphisms for each gene. Bisulfite-treated DNA was used for methylation testing of RUNX3, COX2, and MINT1. LGD data were combined with UC-cancer patient data for statistical testing. Logistic regression analyses determined associations between genetic/epigenetic/clinical variables and UC-associated neoplasia. Receiver operating characteristic analyses were performed to determine the final synchronous neoplasm detection panel. RESULTS: Comparison of nonadjacent, non-dysplastic DNA from UC-neoplasm patients versus UC-controls indicated that TNF-α, IL-1ß, and methylation of RUNX3, MINT1, and COX2 were significantly different (P < 0.0001). In multivariable analysis, all remained significant with an area under the curve of 0.85, exceeding the clinical variable panel area under the curve. Combining clinical and experimental variables yielded a neoplasm biomarker panel with an area under the curve of 0.95 (sensitivity and specificity of 82% and 91%, respectively). Analysis of DNA from LGD with known progression compared with LGD without progression indicated a significant difference in RUNX3 methylation. CONCLUSIONS: A combined clinical, genetic, and epigenetic model for detecting synchronous neoplasm by testing of non-neoplastic colonic tissue had favorable operating characteristics and could complement current patient care.

Methylated eyes absent 4 (EYA4) gene promotor in non-neoplastic mucosa of ulcerative colitis patients with colorectal cancer: evidence for a field effect.

BACKGROUND: Aberrant methylation of the EYA4 gene (mEYA4) highly discriminates ulcerative colitis (UC) cases with colorectal neoplasia from UC controls in both tissue and stool. It is not known if mEYA4 is also present in nonadjacent non-neoplastic mucosa (NNM) of UC patients with colorectal neoplasia. METHODS: Formalin-fixed tissues from 25 UC cases with colorectal cancer (CRC) and 25 UC controls with neither CRC nor dysplasia were matched on gender, age, disease duration, disease extent, and coexistence of primary sclerosing cholangitis. DNA was extracted from sections of CRC and NNM from cases and UC control mucosae. Bisulfite-treated DNA was amplified using real-time methylation-specific PCR. The Wilcoxon rank-sum test assessed differences in mEYA4 levels from CRC, NNM, and control samples. Logistic regression was used to estimate sensitivity and specificity. RESULTS: Sufficient DNA was available for 20 cases and 20 controls. The median mEYA4 level (with interquartile range) was 2 (0-5.7) in control mucosae but 93 (38.5-306) in CRC (P < 0.0001) and 27.4 (3-140) in NNM (P = 0.0009). At 95% specificity, mEYA4 was present in 80% of CRC and 50% of NNM tissue samples. The odds ratio of mEYA4 in NNM as an indicator of synchronous CRC was 19 (95% confidence interval, 2-170). CONCLUSIONS: The authors demonstrate significantly higher mEYA4 levels in NNM and synchronous CRC from UC cases than in colorectal mucosae of UC controls without neoplasia. The finding of this CRC-associated field change has important implications to the use of mEYA4 as a potential UC surveillance marker in tissue or stool.

Myeloperoxidase immunohistochemistry as a measure of disease activity in ulcerative colitis: association with ulcerative colitis-colorectal cancer, tumor necrosis factor polymorphism and RUNX3 methylation.

BACKGROUND: Patients with extensive, longstanding ulcerative colitis (UC), a disease of chronic colonic inflammation, are at risk for colorectal cancer (CRC). Elucidating the mechanism and fully characterizing the nature of this chronic inflammation offers the potential to identify those at greatest risk. We performed a case-control study comparing histologic disease activity (HDA; neutrophils on hematoxylin and eosin [H&E]-stained slides) with immunohistochemistry (IHC) directed against specific cell types. We correlated IHC results with data previously generated on methylation status of RUNX3 and single nucleotide polymorphisms (SNPs) in tumor necrosis factor alpha (TNF-α). METHODS: A nonadjacent, nonneoplastic section of bowel wall was identified for each UC-CRC case. HDA was assessed for UC-CRC cases (n = 50) and UC-controls (n = 50). Sections were immunostained using antibodies against macrophages (CD68), neutrophils/monocytes (myeloperoxidase, MPO), and T cells (CD3). Slides were scored using ImageJ and results reported as the percent area positive for each marker. RESULTS: HDA did not correlate with infiltrate levels as measured by IHC and increasing HDA was inversely related to UC-CRC risk. Conversely, the percent area positive for CD68 and MPO was significantly elevated in UC-CRC cases versus controls (P = 0.04 and < 0.0001, respectively). In areas designated inactive, MPO staining remained significantly higher in UC-CRC cases versus controls (P = 0.002). Increased MPO staining was associated with methylation of RUNX3 and the TNF-α -308G>A SNP. CONCLUSIONS: HDA is less sensitive than IHC and may underestimate inflammatory cell populations associated with UC-CRC. The epigenetic/genetic associations related to elevated MPO staining in UC-CRC may offer new methods for risk stratification and adjunctive screening tools.

Upregulation of DNA methyltransferase-mediated gene silencing, anchorage-independent growth, and migration of colon cancer cells by interleukin-6.

Inflammatory bowel disease is characterized by chronic inflammation which predisposes to colorectal cancer. The mechanisms by which inflammation promotes tumorigenesis are not fully known. We aimed to investigate the links between colonic inflammation and tumorigenesis via epigenetic gene silencing. Colon cancer specimens were assessed for the expression of DNA methyltransferase-1 (DNMT-1) using immunohistochemistry. Colorectal carcinoma cell lines were assessed for DNMT1 expression, methylcytosine content, promoter methylation, gene expression, and tumorigenesis in response to interleukin (IL)-6. DNMT1 was expressed at higher levels in both the peritumoral stroma and tumor in inflammatory bowel disease-associated cancers compared with sporadic colon cancers. IL-6 treatment of colon cancer cells resulted in an increase in DNMT1 expression, independent of de novo gene expression. IL-6 increased the methylation of promoter regions of genes associated with tumor suppression, adhesion, and apoptosis resistance. Expression of a subset of these genes was downregulated by IL-6, an effect that was prevented by preincubation with 5-azadeoxycytidine, a DNMT1 inhibitor. Anchorage-independent growth and migration of colon cancer cells was also increased by IL-6 in a 5-azadeoxycytidine-sensitive manner. Our results indicate that DNMT-mediated gene silencing may play a role in inflammation-associated colon tumorigenesis.

Tumor necrosis factor-alpha polymorphisms in ulcerative colitis-associated colorectal cancer.

OBJECTIVES: Ulcerative colitis (UC) is characterized by chronic recurrent mucosal inflammation. Genetic studies in UC have indicated linkage to chromosome 6 in the region of the tumor necrosis factor-alpha (TNF-alpha) gene, a potent proinflammatory cytokine. TNF-alpha production is influenced by multiple factors including the type of immune cell and its level of activation. However, several single nucleotide polymorphisms (SNP) in the promoter region of TNF-alpha have been correlated with either increased or decreased production, indicating that regulation of TNF-alpha is in part genetic. Because UC patients are at increased risk for developing colorectal cancer (CRC), we investigated if there was an association between SNPs in the promoter of the TNF-alpha gene and UC-CRC. METHODS: DNA was extracted from formalin-fixed, paraffin-embedded tissue from 114 UC-CRC cases and 114 UC-no CRC controls. Controls were frequency matched on duration and extent of colitis, age, ethnicity, and gender. All 228 tissue samples were analyzed for five TNF-alpha promoter polymorphisms (-238[G-->A], -308[G-->A], -857[C-->T], -863[C-->A], and -1031[T-->C]) using PCR and sequencing. RESULTS: The -308(G-->A) SNP was significantly associated with UC-CRC cases at both the allele and genotype level (P < 0.0001). No other SNPs were significantly associated with UC-CRC. CONCLUSION: We report a novel finding of a strong association between the -308(G-->A) SNP and UC-CRC. Complete elucidation of the mechanism of UC-CRC carcinogenesis will require investigation of other genes involved in modulating inflammation, but our results suggest that some UC patients may have additional genetic predispositions toward developing UC-CRC.

Alterations in cell proliferation and apoptosis in colon cancers with microsatellite instability.

Colon cancers with microsatellite instability (MSI) demonstrate a host immune response characterized by tumor infiltrating lymphocytes (TILs) that may exert effects upon tumor cell apoptosis and cell proliferation. Accordingly, we compared rates of apoptosis and cell proliferation in colon cancers with defective DNA mismatch repair and their association with phenotypic features and clinical outcome. Primary Astler-Coller stage B2 and C colon carcinomas (n = 329) were analyzed for MSI and for hMLH1 and hMSH2 protein expression. Apoptosis (TUNEL assay) and p53 expression were also analyzed by immunohistochemistry, and TILs were quantified by morphology. DNA ploidy and proliferation (PI: S phase + G(2)M) were evaluated using flow cytometry. MSI-H (n = 58) colon cancers showed increased TILs that were significantly associated with increased apoptosis, higher apoptosis to proliferation (AI/PI) ratios, reduced proliferative indices (PI) and diploid DNA content. Increased TILs (p = 0.036) and reduced PI (p = 0.042), but not AI or AI/PI, were associated with improved disease-free survival. Tumors with MSI-H (p = 0.032) or loss of hMLH1 or hMSH2 proteins (p = 0.040), or diploidy (p = 0.0015), had better adjusted overall survival rates. Interestingly, similar rates of cell turnover and overlapping survival rates were found in diploid MSS/MSI-L tumors and in MSI-H cases. In conclusion, higher apoptosis/proliferation ratios and reduced cell proliferation are phenotypic features of MSI-H tumors that are associated with increased TILs, indicating an activated immune response that may contribute to their favorable survival rates.

SULF1 inhibits tumor growth and potentiates the effects of histone deacetylase inhibitors in hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the third most common cause of cancer death worldwide. Improved treatments for advanced HCC are urgently needed. The recently identified human sulfatase 1 enzyme (SULF1) desulfates cell surface heparan sulfate glycosaminoglycans and down-regulates cell growth signaling in HCC cells in vitro. While investigating the epigenetic regulation of SULF1, we discovered that histone H4 acetylation is up-regulated by SULF1 in HCC cells. Histone deacetylase (HDAC) inhibitors reprogram cellular gene expression through the acetylation of nucleosomal histones and promote cell growth arrest and apoptosis. Hence, they are a promising modality for cancer treatment. METHODS: To explore the interaction between SULF1 expression and HDAC inhibitor action, we examined the effects of SULF1 expression on HCC cells and xenografts treated with HDAC inhibitors. RESULTS: (1) Forced expression of SULF1 significantly delayed the growth of Huh7 and Hep3B xenografts in nude mice in vivo. (2) SULF1 increased histone H4 acetylation by modulation of cellular HDAC and histone acetyltransferase activities. (3) SULF1 enhanced the induction of apoptosis by the HDAC inhibitors apicidin and scriptaid. (4) SULF1 enhanced the inhibition of tumor growth, migration, and angiogenesis by HDAC inhibitors. We also demonstrate that knockdown of SULF1 with shRNA constructs up-regulates phosphorylation of AKT and Erk and attenuates apicidin-induced apoptosis. The interaction between SULF1 and apicidin was confirmed in vivo in Huh7 and Hep3B xenografts. CONCLUSIONS: These results show that SULF1 promotes histone H4 acetylation, potentiates the effects of HDAC inhibitors, and inhibits HCC tumorigenesis.