Dysregulated Sulfide Metabolism in Multiple Sclerosis: Serum and Vascular Endothelial Inflammatory Responses.

Multiple sclerosis (MS) is a leading cause of neurodegenerative disability in younger individuals. When diagnosed early, MS can be managed more effectively, stabilizing clinical symptoms and delaying disease progression. The identification of specific serum biomarkers for early-stage MS could facilitate more successful treatment of this condition. Because MS is an inflammatory disease, we assessed changes in enzymes of the endothelial hydrogen sulfide (H2S) pathway in response to inflammatory cytokines. Blotting analysis was conducted to detect Cystathionine γ-lyase (CSE), Cystathionine beta synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MST) in human brain microvascular endothelial apical and basolateral microparticles (MPs) and cells following exposure to tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). CSE was increased in MPs and cells by exposure to TNF-α/IFN-γ; CBS was elevated in apical MPs but not in cells or basolateral MPs; MST was not significantly affected by cytokine exposure. To test how our findings relate to MS patients, we evaluated levels of CSE, CBS, and MST in serum samples from healthy control and MS patients. We found significantly decreased levels of CBS and MST (p = 0.0004, 0.009) in MS serum samples, whereas serum levels of CSE were marginally increased (p = 0.06). These observations support increased CSE and lower CBS and MST expression being associated with the vascular inflammation in MS. These changes in endothelial-derived sulfide enzymes at sites of inflammation in the brain may help to explain sulfide-dependent changes in vascular dysfunction/neuroinflammation underlying MS. These findings further support the use of serum samples to assess enzymatic biomarkers derived from circulating MPs. For example, "liquid biopsy" can be an important tool for allowing early diagnosis of MS, prior to the advanced progression of neurodegeneration associated with this disease.

Improved Healthcare Access Reduces Requirements for Surgery in Indigent IBD Patients Using Biologic Therapy: A 'Safety-Net' Hospital Experience.

Low socioeconomic status (SES) is associated with greater morbidity and increased healthcare resource utilization (HRU) in IBD. We examined whether a financial assistance program (FAP) to improve healthcare access affected outcomes and HRU in a cohort of indigent IBD patients requiring biologics. IBD patients (>18 years) receiving care at a 'safety-net' hospital who initiated biologics as outpatients between 1 January 2010 and 1 January 2019 were included. Patients were divided by FAP status. Patients without FAP had Medicare, Medicaid, or commercial insurance. Primary outcomes were steroid-free clinical remission at 6 and 12 months. Secondary outcomes were surgery, hospitalization, and ED utilization. Multivariate logistic regression was used to calculate odds ratio (OR) and 95% confidence interval (CI). Decision tree analysis (DTA) was also performed. We included 204 patients with 258 new biologic prescriptions. FAP patients had less complex Crohn's disease (50.7% vs. 70%, p = 0.033) than non-FAP patients. FAP records indicated fewer prior surgeries (19.6% vs. 38.4% p = 0.003). There were no statistically significant differences in remission rates, disease duration, or days between prescription and receipt of biologics. In multivariable logistic regression, adjusting for baseline demographics and disease severity variables, FAP patients were less likely to undergo surgery (OR: 0.28, 95% CI [0.08−0.91], p = 0.034). DTA suggests that imaging utilization may shed light on surgical differences. We found FAP enrollment was associated with fewer surgeries in a cohort of indigent IBD patients requiring biologics. Further studies are needed to identify interventions to address healthcare disparities in IBD.

Experimental Evaluation of the Elson Test Efficiency Following Central Slip Injury.

Purpose: The purpose of this article is to explore the amount of work, quantitated by flexion and extension cycles, that is needed to obtain a positive Elson test following a central slip injury. Methods: Thirteen frozen cadaveric fingers from individuals with an average age of 79.6 years were used. Testing was performed by imposing sinusoidal displacement of the 2 tendons, with loads ranging from 30 N to 2 N at 1 Hz. Following transection to the central slip, each finger was cycled 1,000 times using the same protocol adopted for the control. Following 100, 200, 300, and 1,000 cycles, we measured the extension angles of the metacarpophalangeal, proximal interphalangeal, and distal interphalangeal joints from the flexed position and the distance between landmarks of the extensor apparatus and simulated an Elson test. Results: In both the fingers, the range of motion of the metacarpophalangeal and distal interphalangeal joints measured in the controls remained unchanged, whereas the range of motion of the proximal interphalangeal joint was significantly reduced immediately after central slip transection. Combining both ring and middle fingers, for a displacement of 5 mm, the force measured in the control (1.05 ± 0.69 N) increased to the value of 2.36 ± 0.97 N at the 1,000th cycle. Although the middle finger has shown a significant difference in force at 100 cycles following central slip transection, 200 cycles were needed to observe a difference on the ring finger. Conclusions: In controlled conditions, there is a variation in resistance to flexion of the distal interphalangeal joint. However, the amplitude of the forces is so small that they are likely imperceptible clinically. Delayed testing should be considered to increase the sensitivity of the test or in patients experiencing pain. Type of study/level of evidence: Diagnostic V.

(-)-Oleocanthal as a Dual c-MET-COX2 Inhibitor for the Control of Lung Cancer.

Lung cancer (LC) represents the topmost mortality-causing cancer in the U.S. LC patients have overall poor survival rate with limited available treatment options. Dysregulation of the mesenchymal epithelial transition factor (c-MET) and cyclooxygenase 2 (COX2) initiates aggressive LC profile in a subset of patients. The Mediterranean extra-virgin olive oil (EVOO)-rich diet already documented to reduce multiple malignancies incidence. (-)-Oleocanthal (OC) is a naturally occurring phenolic secoiridoid exclusively occurring in EVOO and showed documented anti-breast and other cancer activities via targeting c-MET. This study shows the novel ability of OC to suppress LC progression and metastasis through dual targeting of c-MET and COX-2. Western blot analysis and COX enzymatic assay showed significant reduction in the total and activated c-MET levels and inhibition of COX1/2 activity in the lung adenocarcinoma cells A549 and NCI-H322M, in vitro. In addition, OC treatment caused a dose-dependent inhibition of the HGF-induced LC cells migration. Daily oral treatment with 10 mg/kg OC for 8 weeks significantly suppressed the LC A549-Luc progression and prevented metastasis to brain and other organs in a nude mouse tail vein injection model. Further, microarray data of OC-treated lung tumors showed a distinct gene signature that confirmed the dual targeting of c-MET and COX2. Thus, the EVOO-based OC is an effective lead with translational potential for use as a prospective nutraceutical to control LC progression and metastasis.

Neurogranin Regulates Alcohol Sensitivity through AKT Pathway in the Nucleus Accumbens.

Dysfunction of glutamate neurotransmission in the nucleus accumbens (NAc) has been implicated in the pathophysiology of alcohol use disorders (AUD). Neurogranin (Ng) is exclusively expressed in the brain and mediates N-methyl-d-aspartate receptor (NMDAR) hypo-function by regulating the intracellular calcium-calmodulin (Ca2+ -CaM) pathway. Ng null mice (Ng-/- mice) demonstrate increased alcohol drinking compared to wild-type mice, while also showing less tolerance to the effect of alcohol. To identify the molecular mechanism related to alcohol seeking, both in vivo microdialysis and label-free quantification proteomics comparing Ng genotype and effects of alcohol treatment on the NAc are utilized. There is significant difference in glutamate and gamma-aminobutyric acid (GABA) neurotransmission between genotypes; however, alcohol administration normalizes both glutamate and GABA levels in the NAc. Using label-free proteomics, 427 protein expression changes are identified against alcohol treatment in the NAc among 4347 total proteins detected. Bioinformatics analyses reveal significant molecular differences in Ng null mice in response to acute alcohol treatment. Ingenuity pathway analysis found that the AKT network is altered significantly between genotypes, which may increase the sensitivity of alcohol in Ng null mice. The pharmacoproteomics results presented here illustrate a possible molecular basis of the alcohol sensitivity through Ng signaling in the NAc.

Two year follow-up of Crohn's patients substituted to certolizumab anti-TNFa therapy: SAVANT 2.

The effectiveness of ant-TNF 'biologic' therapy in is well supported in the management of moderate to severe Crohn's Disease (CD). Our first 'SAVANT' study was to our knowledge the first study report one- year outcomes in patients (n=60) who switched from previous anti-TNFa treatment to Cimzia/Certolizumab. This current study (SAVANT 2) follows up on longer term outcomes and provides additional clinical and biochemical data that may contribute to therapeutic responses. This IRB approved study was a retrospective analysis of the initial patients included in SAVANT 1. Patients who were switched to TNF antagonist Certolizumab as an alternative biologic were followed an additional year. Retrospective consideration of immunomodulator use, smoking status and clinical data were also evaluated. Of 60 patients with moderate-severe CD who participated in the SAVANT 1 study, 15 patients were excluded due to inadequate follow up. 45 patients were studied for a total of two years following substitution with Certolizumab from prior anti TNF agent therapy. Clinical remission at 1 year was 75% (45/60) and 55% (25/45) at the second year. At the second year, 5 more patients had discontinued Certolizumab due to worse disease or adverse events, indicating a cumulative two-year failure rate of 33% (20/60). Smoking and concomitant use of immunomodulators were similar between 'success' and 'failure' groups. SAVANT 2, the first study to report long term outcomes of switching from Infliximab or Adalimumab to Certolizumab showed that at 2 years, 25 patient's maintained clinical remission. The discontinuation rates were 25 and 11% at years 1 & 2 respectively. The 5 patients who lost responsiveness after the first year were women, the majority of smoked. Additional prospective studies to assess the appropriateness and feasibility of biologic substitution are still needed.

Substitution with Alternative Anti-TNFα Therapy (SAVANT)-Outcomes of a Crohn's Disease Cohort Undergoing Substitution Therapy with Certolizumab.

BACKGROUND: Biological therapy targeting tumor necrosis factor-alfa has revolutionized the treatment of Crohn's disease (CD). Our study retrospectively reviewed clinical outcomes of 60 patients administratively substituted from Infliximab or Adalimumab to Certolizumab. Maintenance of disease and failure rates after substitution of anti-tumor necrosis factor-alfa agents in CD patients were monitored over 1 year, and this is the first outcomes study of patients maintained on Infliximab or Adalimumab substituted to Certolizumab. METHODS: A hospital pharmacy directive required all patients on biological therapy to be administratively substituted to Certolizumab therapy. This single-center retrospective analysis initially included 68 CD patients presenting at Louisiana State University Health Sciences Center-Shreveport. Clinical, endoscopic, and serologic data were compared at baseline and at 4 intervals over 1 year. RESULTS: Of 60 enrolled CD patients, 45 (75%) successfully transitioned to Certolizumab and had stable disease at 1 year. Of the 15 (25%) patients who "failed" substitution at 1 year, 5 were returned to Adalimumab and 7 to Infliximab; 3 were maintained on steroids awaiting subsequent therapy. Importantly, when patients were segregated on the basis of initial disease control, it was found that 3 (12.5%) previously well-controlled patients failed therapy, whereas 12 (33.3%) who initially had active disease failed Certolizumab substitution. CONCLUSIONS: Our study found that 25% of CD patients substituted to Cimzia failed substitution, whereas 75% still exhibited a good clinical response with stable disease at 1 year. Our findings indicate that disease status and behavior at the time of biological substitution may predict therapeutic responsiveness.

Gastric cancer in women: A regional health-center seven year retrospective study.

AIM: To investigate whether regional geography influences ethnic and gender trends for the development of gastric cancer (GC). METHODS: This retrospective analysis of the INVISION patient database at Louisiana State University Health Sciences Center-Shreveport (LSUHSC-S), a southern United States regional hospital, was performed from 2005-2011. Using the international statistical classification of diseases 9 (ICD-9), inpatient, day surgery outpatient, and emergency outpatient diagnosis codes entered into medical records were used to identify GC patients. For each study year, the patients were evaluated for age, ethnicity, and gender, and each patient was counted only once throughout the study. Subsequent patient encounters were counted as visits and separated by inpatient and clinic visits. Complex or severe disease may require more frequent and intensive clinical management; therefore, we evaluated annual clinic visits as "surrogate markers" of disease severity. Finally, we studied the primary diagnosis for Helicobacter pylori (H. pylori) infection (ICD-9 code 41.86) as an additional factor that might increase the risk of GC. RESULTS: A total of 285 patients were diagnosed with GC at LSUHSC-S between 2005 and 2011. African Americans (181 patients, 89 males and 92 females, 63.5% of total patients) had significantly higher frequencies of GC diagnosis compared with non-Hispanic whites (104 patients, 54 males and 50 females, 36.5% of total patients), at a ratio of 1.74 (P = 0.002). Within each ethnic group, men and women were diagnosed at approximately equal annual rates. Our findings differed significantly from United States national trends, which found that African American females and white females had lower risks for GC than their corresponding male counterparts. The United States national trend between 2005 and 2011 showed that African Americans males had a higher incidence of GC, with an annual mean (per 100000) of 16.31 ± 0.76 compared with white males (9 ± 0.1, P < 0.001), African American females (8.7 ± 0.34, P < 0.001) and white females (4.05 ± 0.07, P < 0.001). Among the GC patients, the number of clinic visits was highest among African American males (195.1 ± 28.1), who had significantly more clinic visits than African Americans females (123 ± 13.02, P < 0.05), white males (41.57 ± 4.74, P < 0.001) and white females (35 ± 8.9, P < 0.001). Similar trends were found for inpatient visits, with an annual mean of 11.43 ± 1.5 for African American males, followed by African American females (7.29 ± 1.36), white males (2.57 ± 0.69) and white females (1.57 ± 0.612). African American males had significantly more inpatient visits than white males (P < 0.001), and African American females had more inpatient visits than white females (P < 0.01). African American patients showed the highest frequency of H. pylori positive status, with approximately 72% vs 28% for the white patients. CONCLUSION: Increase in GC diagnoses among women at LSUHSC-S is significantly higher than United States national averages, suggesting local geographic and socioeconomic influences may alter GC disease course.

Genome-wide DNA methylation as an epigenetic consequence of Epstein-Barr virus infection of immortalized keratinocytes.

UNLABELLED: The oral cavity is a persistent reservoir for Epstein-Barr virus (EBV) with lifelong infection of resident epithelial and B cells. Infection of these cell types results in distinct EBV gene expression patterns regulated by epigenetic modifications involving DNA methylation and chromatin structure. Regulation of EBV gene expression relies on viral manipulation of the host epigenetic machinery that may result in long-lasting host epigenetic reprogramming. To identify epigenetic events following EBV infection, a transient infection model was established to map epigenetic changes in telomerase-immortalized oral keratinocytes. EBV-infected oral keratinocytes exhibited a predominantly latent viral gene expression program with some lytic or abortive replication. Calcium and methylcellulose-induced differentiation was delayed in EBV-positive clones and in clones that lost EBV compared to uninfected controls, indicating a functional consequence of EBV epigenetic modifications. Analysis of global cellular DNA methylation identified over 13,000 differentially methylated CpG residues in cells exposed to EBV compared to uninfected controls, with CpG island hypermethylation observed at several cellular genes. Although the vast majority of the DNA methylation changes were silent, 65 cellular genes that acquired CpG methylation showed altered transcript levels. Genes with increased transcript levels frequently acquired DNA methylation within the gene body while those with decreased transcript levels acquired DNA methylation near the transcription start site. Treatment with the DNA methyltransferase inhibitor, decitabine, restored expression of some hypermethylated genes in EBV-infected and EBV-negative transiently infected clones. Overall, these observations suggested that EBV infection of keratinocytes leaves a lasting epigenetic imprint that can enhance the tumorigenic phenotype of infected cells. IMPORTANCE: Here, we show that EBV infection of oral keratinocytes led to CpG island hypermethylation as an epigenetic scar of prior EBV infection that was retained after loss of the virus. Such EBV-induced epigenetic modification recapitulated the hypermethylated CpG island methylator phenotype (CIMP) observed in EBV-associated carcinomas. These epigenetic alterations not only impacted gene expression but also resulted in delayed calcium and methylcellulose-induced keratinocyte differentiation. Importantly, these epigenetic changes occurred in cells that were not as genetically unstable as carcinoma cells, indicating that EBV infection induced an epigenetic mutator phenotype. The impact of this work is that we have provided a mechanistic framework for how a tumor virus using the epigenetic machinery can act in a "hit-and-run" fashion, with retention of epigenetic alterations after loss of the virus. Unlike genetic alterations, these virally induced epigenetic changes can be reversed pharmacologically, providing therapeutic interventions to EBV-associated malignancies.

Identification of genes correlated with early-stage bladder cancer progression.

Transitional cell carcinoma (TCC) of the bladder ranks fourth in incidence of all cancers in the developed world, yet the mechanisms of its origin and progression remain poorly understood. There are also few useful diagnostic or prognostic biomarkers for this disease. We have combined a transgenic mouse model for invasive bladder cancer (UPII-SV40Tag mice) with DNA microarray technology to determine molecular mechanisms involved in early TCC development and to identify new biomarkers for detection, diagnosis, and prognosis of TCC. We have identified genes that are differentially expressed between the bladders of UPII-SV40Tag mice and their age-matched wild-type littermates at 3, 6, 20, and 30 weeks of age. These are ages that correspond to premalignant, carcinoma in situ, and early-stage and later stage invasive TCC, respectively. Our preliminary analysis of the microarray data sets has revealed approximately 1,900 unique genes differentially expressed (> or =3-fold difference at one or more time points) between wild-type and UPII-SV40Tag urothelium during the time course of tumor development. Among these, there were a high proportion of cell cycle regulatory genes and a proliferation signaling genes that are more strongly expressed in the UPII-SV40Tag bladder urothelium. We show that several of the genes upregulated in UPII-SV40Tag urothelium, including RacGAP1, PCNA, and Hmmr, are expressed at high levels in superficial bladder TCC patient samples. These findings provide insight into the earliest events in the development of bladder TCC as well as identify several promising early-stage biomarkers.

Identification of the B-Raf/Mek/Erk MAP kinase pathway as a target for all-trans retinoic acid during skin cancer promotion.

BACKGROUND: Retinoids have been studied extensively for their potential as therapeutic and chemopreventive agents for a variety of cancers, including nonmelanoma skin cancer (NMSC). Despite their use for many years, the mechanism of action of retinoids in the prevention of NMSC is still unclear. In this study we have attempted to understand the chemopreventive mechanism of all-trans retinoic acid (ATRA), a primary biologically active retinoid, in order to more efficiently utilize retinoids in the clinic. RESULTS: We have used the 2-stage dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) mouse skin carcinogenesis model to investigate the chemopreventive effects of ATRA. We have compared the gene expression profiles of control skin to skin subjected to the 2-stage protocol, with or without ATRA, using Affymetrix 430 2.0 DNA microarrays. Approximately 49% of the genes showing altered expression with TPA treatment are conversely affected when ATRA is co-administered. The activity of these genes, which we refer to as 'counter-regulated', may contribute to chemoprevention by ATRA. The counter-regulated genes have been clustered into functional categories and bioinformatic analysis has identified the B-Raf/Mek/Erk branch of the MAP kinase pathway as one containing several genes whose upregulation by TPA is blocked by ATRA. We also show that ATRA blocks signaling through this pathway, as revealed by immunohistochemistry and Western blotting. Finally, we found that blocking the B-Raf/Mek/Erk pathway with a pharmacological inhibitor, Sorafenib (BAY43-9006), induces squamous differentiation of existing skin SCCs formed in the 2-stage model. CONCLUSION: These results indicate that ATRA targets the B-Raf/Mek/Erk signaling pathway in the 2-stage mouse skin carcinogenesis model and this activity coincides with its chemopreventive action. This demonstrates the potential for targeting the B-Raf/Mek/Erk pathway for chemoprevention and therapy of skin SCC in humans. In addition our DNA microarray results provide the first expression signature for the chemopreventive effect of ATRA in a mouse skin cancer model. This is a potential source for novel targets for ATRA and other chemopreventive and therapeutic agents that can eventually be tested in the clinic.

Retinoids and skin: microarrays shed new light on chemopreventive action of all-trans retinoic acid.

Despite the use of retinoids in the clinic for many years, their mode of action in the prevention of skin cancer is still unclear. Recent microarray analyses of the chemopreventive effect of all-trans retinoic acid (ATRA), one of the primary naturally occurring biologically active retinoids, in the two-stage mouse skin chemical carcinogenesis model have provided novel insight into their action. Comparison of the gene expression profiles of control skin to skin subjected to the two-stage protocol for 3 wk, with or without ATRA, has shown that approximately half of the genes regulated by 12-o-tetradecanoylphorbol-13-acetate (TPA) are oppositely regulated when ATRA is coadministered with TPA. It was further shown the Raf/Mek/Erk branch of mitogen-activated protein (MAP) kinase pathway contains a disproportionate number of oppositely regulated genes, thereby implicating it as one of the key pathways involved in tumor promotion by TPA, that is blocked by ATRA. This result has pointed the way toward the detailed study of Raf/Mek/Erk pathway signaling in skin cancer development and its potential as a target pathway for chemoprevention by ATRA and other chemopreventive drugs.