Characteristics of Consecutive Esophageal Motility Diagnoses After a Decade of Change

BACKGROUND AND AIMS Combined multichannel intraluminal impedance and esophageal manometry (MII-EM) measures concomitantly bolus transit and pressure changes allowing determination of the functional impact of esophageal motility abnormalities. Ten years ago our laboratory reported MII-EM results in 350 consecutive patients. Since then high-resolution impedance manometry (HRIM) became available and the definitions of ineffective esophageal motility (IEM) and nutcracker esophagus were revised. The aim of this study was to assess the impact of these developments on esophageal function testing. METHODS From August 2012 through May 2013, HRIM was performed in 350 patients referred for esophageal function testing. Each patient received 10 liquid and 10 viscous swallows. While taking advantage of the new technology and revised criteria, HRIM findings were classified according to the conventional criteria to allow more appropriate comparison with our earlier analysis. RESULTS Compared with the study performed 10 years ago, the prevalence of normal manometry (36% vs. 35%), achalasia (7% vs. 8%), scleroderma (1% vs. 1%), hypertensive lower esophageal sphincter (LES) (7% vs. 7%), and hypotensive LES (1% vs. 2%) remained the same, whereas the prevalence of distal esophageal spasm (9% vs. 3%), nutcracker esophagus (9% vs. 3%), and poorly relaxing LES (10% vs. 3%) decreased and the prevalence of IEM increased (20% vs. 31%) significantly. Compared with the early study, normal liquid bolus transit was significantly different in patients with hypertensive LES (96% vs. 57%) and poorly relaxing LES (55% vs. 100%). CONCLUSIONS This study brings to light the increase in prevalence of IEM. In addition, it suggests that the hypertensive LES and poorly relaxing LES may each affect bolus transit in about half of these patients.

The role of nitric oxide in small intestinal motility in a model of acute inflammation

Motility responses of the small intestine of iNOS deficient mice (iNOS −/−) and their wildtype littermates (iNOS+/+) to the inflammatory challenge of lipopolysaccharide (LPS) were investigated. LPS administration failed to attenuate intestinal transit in iNOS−/− mice but depressed transit in their iNOS+/+ littermates. Supporting an inhibitory role for sustained nitric oxide (NO) synthesis in the regulation of intestinal motility during inflammation, iNOS immunoreactivity was upregulated in all regions of the small intestine of iNOS+/+ mice. In contrast, neuronal NOS was barely affected. Cyclooxygenase activation was determined by prostaglandin E2 (PGE2) concentration. Following LPS challenge, PGE2 levels were elevated in all intestinal segments in both animal groups. Moreover, COX-1 and COX-2 protein levels were elevated in iNOS+/+ mice in response to LPS, while COX-2 levels were similarly increased in iNOS −/− intestine. However, no apparent relationship was observed between increased prostaglandin concentrations and attenuated intestinal transit. The presence of heme oxygenase 1 (HO-1) in the murine small intestine was also investigated. In both animal groups HO-1 immunoreactivity in the proximal intestine increased in response to treatment, while the constitutive protein levels detected in the middle and distal intestine were unresponsive to LPS administration. No apparent correlation of HO-1 to the suppression of small intestinal motility induced by LPS administration was detected. The presence of S-nitrosylated contractile proteins in the small intestine was determined. γ-smooth muscle actin was basally nitrosylated as well as in response to LPS, but myosin light chain kinase and myosin regulatory chain (MLC20) were not. In conclusion, in a model of acute intestinal inflammation, iNOS-produced NO plays a significant role in suppressing small intestinal motility while nNOS, COX-1, COX-2 and HO-1 do not participate in this event. S-nitrosylation of γ-smooth muscle actin is associated with elevated levels of nitric oxide in the smooth muscle of murine small intestine. ^

The Role of Raf Kinase Inhibitor Protein in Cell Motility

Raf Kinase Inhibitor Protein (RKIP) has been identified as a phosphatidylethanolamine-binding protein capable of inhibiting Raf-1 kinase, an enzyme significant in cell proliferation and cancer development. When properly functioning, RKIP can mediate the expression of Raf-1 kinase and help prevent uncontrolled cell division. RKIP also has suggested, but unclear, roles in spindle fiber formation during mitosis, regulation of apoptosis, and cell motility. The Fenteany laboratory in the Chemistry Department identified a new small molecule, named Locostatin, as a cell migration inhibitor in mammalian cells, with RKIP as its primary molecular target. Dictyostelium discoideum possess two RKIP proteins, RKIP-A and RKIP-B. In order to begin to study the function of RKIP in D. discoideum and its role in cell motility, I created a mutant cell line which lacks a functional RKIP-A gene. In this paper, we show that removal of RKIP-A does not affect vegetative motility, but impairs chemotaxis and development in the presence of drug. Interestingly, RKIP-A knockout mutants appear more resistant to drug effects on vegetative motility than wild-type cells. More research is needed to reconcile these seemingly contrasting results, and to better develop a model for RKIP-A’s role in cell motility.

Investigating the Affects of Cucurbitacin-I on Cellular Motility

Cellular migration is an integral component of many biological processes including immune function, wound healing and cancer cell metastasis. A complete model illustrating the mechanism by which cells accomplish movement is still lacking. Exploring the affects of various drugs on cell motility may be instrumental in discovering new proteins which mediate cell movement. This project aims ultimately to characterize the molecular target of the drug Cucurbitacin-I, a natural plant product. This drug has been shown to inhibit migration of epithelial sheets and may have anti-tumor activity. In this paper, we show that Cucurbitacin-I inhibits the migration of MDCK and B16F1 cells. The drug also affects the integrity of the actin cytoskeleton of these cells by indirectly stabilizing filamentous actin. Cucurbitacin-I does not, however, have an effect on the motility or cytoskeletal morphology of the soil amoeba, Dictyostelium discoidium.

Molecular mechanisms of response in gastrointestinal stromal tumors treated with imatinib mesylate

Gastrointestinal stromal tumors (GIST) represent 80% of sarcoma arising from the GI tract. The inciting event in tumor progression is mutation of the kit or, rarely, platelet derived growth factor receptor-α (PDGFR) gene. These mutations encode ligand independent, constitutively active proteins: Kit or PDGFR. ^ These tumors are notoriously chemo and radio resistant. Historically, patients with advanced disease realized a median overall survival of 9 months. However, with modern management of GIST with imatinib mesylate (Novartis), a small molecule inhibitor of the Kit, PDGFR, and Abl tyrosine kinases, patients now realize a median overall survival greater than 30 months. However, almost half of patients present with surgically resectable GIST and the utility of imatinib in this context has not been prospectively studied. Also, therapeutic benefit of imatinib is variable from patient to patient and alternative targeted therapy is emerging as potential alternatives to imatinib. Thus, elucidating prognostic factors for patients with GIST in the imatinib-era is crucial to providing optimal care to each particular patient. Moreover, the exact mechanism of action of imatinib in GIST is not fully understood. Therefore, physicians find difficulty in accurately predicting which patient will benefit from imatinib, how to assess response to therapy, and the time at which to assess response. ^ I have hypothesized that imatinib is tolerable and clinically beneficial in the context of surgery, VEGF expression and kit non-exon 11 genotypes portend poor survival on imatinib therapy, and imatinib's mechanism of action is in part due to anti-vascular effects and inhibition of the Kit/SCF signaling axis of tumor-associated endothelial cells. ^ Results herein demonstrate that imatinib is safe and increases the duration of disease-free survival when combined with surgery. Radiographic and molecular (namely, apoptosis) changes occur within 3 days of imatinib initiation. I illustrate that non-exon 11 mutant genotypes and VEGF are poor prognostic factors for patients treated with imatinib. These findings may allow for patient stratification to emerging therapies rather than imatinib. I show that imatinib has anti-vascular effects via inducing tumor endothelial cell apoptosis perhaps by abrogation of the Kit/SCF signaling axis. ^

Long-term gastrointestinal complications of Clostridium difficile associated diarrhea (CDAD)

Background. The incidence of Clostridium difficile -associated diarrhea (CDAD) is increasing worldwide likely because of increased use of broad spectrum antibiotics and the introduction of a clonal hyper-virulent strain called the BI strain. Short-term complications of CDAD include recurrent disease, requirement for colectomy, and persistent disease. However, data on the long-term consequences of CDAD are scarce. Among other infectious diseases (Shigella, Salmonella, and Campylobacter), long-term consequences such as irritable bowel syndrome (IBS), chronic dyspepsia/diarrhea, and other GI effects have been noted. Since the mechanism of action of these agents is similar to C.difficile, we hypothesized that patients with CDAD have greater likelihood of developing IBS and other functional gastrointestinal disorders (FGIDs) in the long-term as compared to a general sample of recently hospitalized patients. ^ Objective. To evaluate the long-term gastrointestinal complications of CDAD, (IBS, functional diarrhea, functional abdominal bloating, functional constipation and functional abdominal pain syndrome). ^ Methods. The current study was a secondary analysis of a previously completed observational case-control outcome study. Adult CDAD patients at St. Luke's Episcopal Hospital, Houston (SLEH) were followed up and interviewed by telephone six months after the initial diagnosis thereafter evaluated for the development of IBS and other FGIDs. A total of 46 patients with CDAD infection were recruited at SLEH between May-November 2007. The comparators were patients hospitalized in SLEH within one month before or after the admission of the reference case, hospital length of stay within one week longer or shorter than reference case, and age within 10 years more or less than the reference case. Cases and comparators were compared using Fisher's exact test. A p<0.05 was considered significant. ^ Results. Thirty CDAD patients responded to the questionnaires and were compared to 40 comparators. No comparator developed a FGID, while 3 (10%) CDAD patients developed new onset IBS (p=0.07), 4 (13.3%) developed new onset Functional Diarrhea (p=0.03), and 3 (10%) developed new onset Functional Constipation (p=0.07). No patient developed Functional Abdominal Bloating and Functional Abdominal Pain Syndrome. ^ Conclusion. In this study, new onset functional diarrhea was significantly more common in patients CDAD within six months after initial infection compared to matched controls.^

Survival of gastrointestinal and stromal tumor patients at UT M.D. Anderson Cancer Center

Objective. Gastrointestinal Stromal Tumors (GISTs) are rare mesenchymal tumors of the gastrointestinal (GI) tract with spindled cell, epithelioid, or occasionally pleomorphic morphology. The primary objective of this paper is to describe the demographic and clinical characteristics and survival among GIST patients registered at the University of Texas M.D. Anderson Cancer Center (MDACC). ^ Methods. This cohort study includes 783 consecutive patients diagnosed with GIST from 1995 to 2007. Demographic, clinical and survival information were obtained from the MDACC cancer registry. ^ Statistical Analysis. Kaplan-Meier survival curves, univariate and multivariate Cox proportional hazards analysis were conducted to estimate survival and identify prognostic clinical factors associated with survival. Results. The age at diagnosis of MDACC GIST cases ranged from 17 to 91 with a mean of 57 years and a male-to-female ratio of 1.3:1. The racial distribution was whites 77%, African-Americans 9.5%, Hispanics 9.3% and other races 4.2%. Fifty per cent of the GISTs arose from stomach, 35% small intestine, 7% retroperitoneal space, 6% colorectal and 2% were omentum and mesentery. About half of the tumors were less than 10 cm in size. Fifty eight per cent of the tumors were localized whereas 36% were metastatic. MDACC GIST patients were generally comparable to SEER patients, but, on the average, were 7 years younger than SEER patients and were predominantly whites. ^ Stratification of 783 GIST cases by year of diagnosis based on the introduction of imatinib treatment in 2000 revealed that 60% of the GIST cases were first diagnosed between 2000 and 2007 whereas, 40% were first diagnosed between 1995 and 1999. There was a significant difference between the two cohorts in the distribution of race, GIST symptom, tumor size, tumor site, and stage of the tumor at diagnosis. The 1- and 5-year survival was 93% and 59% in the 1995–2007 cohort. Multivariate Cox regression analysis identified age at diagnosis (p<0.001), female sex (p=0.047), tumor size (p=0.07), multiple cancers (p=0.002), and GIST diagnosed between 2000 and 2007 (p<0.001) were significantly associated with survival. Approximately, 58% of the cases were treated with imatinib whereas 42% did not receive imatinib in 2000–2005 cohort. There was a significant difference in survival between imatinib and non-imatinib groups and in the distribution of tumor size categories, stage of the tumor at diagnosis and cancers before the diagnosis of GIST. The 1- and 5-year survival for imatinib patients was 99% and 73% and was 91% and 63% for non-imatinib patients. Multivariate Cox regression analysis of the 2000–2007 cohort identified, age at diagnosis and tumor stage as possible prognostic factors associated with survival.^

Roles of insulin-like growth factor binding protein-3 in gastrointestinal stromal tumors

Imatinib mesylate, a selective inhibitor of KIT, PDGFR, and Abl kinases, has shown significant success as a therapy for patients with advanced gastrointestinal stromal tumors (GISTs). However, the underlying mechanisms of imatinib-induced cytotoxicity are not well understood. Using gene expression profiling and real-time PCR for target validation, we identified insulin-like growth factor binding protein-3 (IGFBP3) to be to be up-regulated after imatinib treatment in imatinib-sensitive GISTs. IGFBP3 is a multifunctional protein that regulates cell proliferation and survival and mediates the effects of a variety of anti-cancer agents through IGF-dependent and IGF-independent mechanisms. Therefore, we hypothesized that IGFBP3 mediates GIST cell response to imatinib. To test this hypothesis, we manipulated IGFBP3 protein levels in two KIT mutant, imatinib-sensitive GIST cell lines and assessed the resultant changes in cell viability, survival, and imatinib sensitivity. In GIST882 cells, endogenous IGFBP3 was required for cell viability. However, inhibiting imatinib-induced IGFBP3 up-regulation by RNA interference or neutralization resulted in reduced drug sensitivity, suggesting that IGFBP3 sensitizes GIST882 cells to imatinib. GIST-T1 cells, on the other hand, had no detectable levels of endogenous IGFBP3, nor did imatinib induce IGFBP3 up-regulation, in contrast to our previous findings. IGFBP3 overexpression in GIST-T1 cells reduced viability but did not induce cell death; rather, the cells became polyploid through a mechanism that may involve attenuated Cdc20 expression and securin degradation. Moreover, IGFBP3 overexpression resulted in a loss of KIT activation and decreased levels of mature KIT. Consistent with this, GIST-T1 cells overexpressing IGFBP3 were less sensitive to imatinib. Furthermore, as neither GIST882 cells nor GIST-T1 cells expressed detectable levels of IGF-1R, IGFBP3 is likely not exerting its effects by modulating IGF signaling through IGF-1R or IR/IGF-1R hybrid receptors in these cell lines. Collectively, these findings demonstrate that IGFBP3 has cell-dependent effects and would, therefore, not be an ideal marker for identifying imatinib response in GISTs. Nevertheless, our results provide preliminary evidence that IGFBP3 may have some therapeutic benefits in GISTs. ^

MODULATION OF HOST INTESTINAL MYOELECTRIC ACTIVITY BY LOCAL ANAPHYLAXIS: A COMPONENT OF PROTECTIVE IMMUNITY TO AN ENTERIC PARASITE (TRICHINELLA SPIRALIS, EIMERIA NIESCHULZI, BOWEL MOTILITY)

The hypothesis tested was that rapid rejection of Trichinella spiralis infective larvae from immunized rats following a challenge infection is associated with a local anaphylactic reaction, and this response should be reflected in altered small intestinal motility. The objective was to determine if altered gut smooth muscle function accompanies worm rejection based on the assumption that anaphylaxis in vivo could be detected by changes in intestinal smooth muscle contractile activity (ie. an equivalent of the Schultz-Dale reaction or in vitro anaphylaxis). The aims were to (1) characterize motility changes by monitoring intestinal myoelectric activity in conscious rats during the enteric phase of T. spiralis infection in immunized hosts, (2) detect the onset and magnitude of myoelectric changes caused by challenge infection in immunized rats, (3) determine the parasite stimulus causing changes, and (4) determine the specificity of host response to stimulation. Electrical slow wave frequency, spiking activity, normal interdigestive migrating myoelectric complexes and abnormal migrating action potential complexes were measured. Changes in myoelectric parameters induced by larvae inoculated into the duodenum of immune hosts differed from those associated with primary infection with respect to time of onset, magnitude and duration. Myoelectric changes elicited by live larvae could not be reproduced by inoculation of hosts with dead larvae, larval excretory-secretory products, or by challenge with a heterologous parasite, Eimeria nieschulzi. These results indicate that (1) local anaphylaxis is a component of the initial response to T. spiralis in immune hosts, since the rapid onset of altered smooth muscle function parallels in time the expression of rapid rejection of infective larvae, and (2) an active mucosal penetration attempt by the worm is necessary to elicit this host response. These findings provide evidence that worm rejection is a consequence of, or sequel to, an immediate hypersensitivity reaction elicited when parasites attempt to invade the gut mucosa of immunized hosts. ^

Clinical and economic outcomes of serious postoperative infections following resection of common respiratory and gastrointestinal solid tumors

Unlike infections occurring during periods of chemotherapy-induced neutropenia, postoperative infections in patients with solid malignancy remain largely understudied. The purpose of this population-based study was to evaluate the clinical and economic burden, as well as the relationship of hospital surgical volume and outcomes associated with serious postoperative infection (SPI) – i.e., bacteremia/sepsis, pneumonia, and wound infection – following resection of common solid tumors.^ From the Texas Discharge Data Research File, we identified all Texas residents who underwent resection of cancer of the lung, esophagus, stomach, pancreas, colon, or rectum between 2002 and 2006. From their billing records, we identified ICD-9 codes indicating SPI and also subsequent SPI-related readmissions occurring within 30 days of surgery. Random-effects logistic regression was used to calculate the impact of SPI on mortality, as well as the association between surgical volume and SPI, adjusting for case-mix, hospital characteristics, and clustering of multiple surgical admissions within the same patient and patients within the same hospital. Excess bed days and costs were calculated by subtracting values for patients without infections from those with infections computed using multilevel mixed-effects generalized linear model by fitting a gamma distribution to the data using log link.^ Serious postoperative infection occurred following 9.4% of the 37,582 eligible tumor resections and was independently associated with an 11-fold increase in the odds of in-hospital mortality (95% Confidence Interval [95% CI], 6.7-18.5, P < 0.001). Patients with SPI required 6.3 additional hospital days (95% CI, 6.1 - 6.5) at an incremental cost of $16,396 (95% CI, $15,927–$16,875). There was a significant trend toward lower overall rates of SPI with higher surgical volume (P=0.037). ^ Due to the substantial morbidity, mortality, and excess costs associated with SPI following solid tumor resections and given that, under current reimbursement practices, most of this heavy burden is borne by acute care providers, it is imperative for hospitals to identify more effective prophylactic measures, so that these potentially preventable infections and their associated expenditures can be averted. Additional volume-outcomes research is also needed to identify infection prevention processes that can be transferred from higher- to lower-volume providers.^

SOCIAL AND BIOLOGICAL DETERMINANTS OF SHORT-TERM GROWTH VELOCITY AMONG PRESCHOOLERS OF RURAL GUATEMALA: A PATH ANALYSIS APPROACH (WEIGHT-GAIN, IRON DEFICIENCY, GASTROINTESTINAL SYMPTOMS)

This dissertation develops and tests through path analysis a theoretical model to explain how socioeconomic, socioenvironmental, and biologic risk factors simultaneously influence each other to further produce short-term, depressed growth in preschoolers. Three areas of risk factors were identified: child's proximal environment, maturational stage, and biological vulnerability. The theoretical model represented both the conceptual framework and the nature and direction of the hypotheses. Original research completed in 1978-80 and in 1982 provided the background data. It was analyzed first by nested-analysis of variance, followed by path analysis. The study provided evidence of mild iron deficiency and gastrointestinal symptomatology in the etiology of depressed, short-term weight gain. Also, there was evidence suggesting that family resources for material and social survival significantly contribute to the variability of short-term, age-adjusted growth velocity. These results challenge current views of unifocal intervention, whether for prevention or control. For policy formulations, though, the mechanisms underlying any set of interlaced relationships must be decoded. Theoretical formulations here proposed should be reassessed under a more extensive research design. It is suggested that studies should be undertaken where social changes are actually in progress; otherwise, nutritional epidemiology in developing countries operates somewhere between social reality and research concepts, with little grasp of its real potential. The study stresses that there is a connection between substantive theory, empirical observation, and policy issues. ^

BIM MEDIATES IMATINIB-INDUCED APOPTOSIS OF GASTROINTESTINAL STROMAL TUMORS: TRANSLATIONAL IMPLICATIONS

Gastrointestinal stromal tumors (GISTs) are oncogene-addicted cancers driven by activating mutations in the genes encoding receptor tyrosine kinases KIT and PDGFR-α. Imatinib mesylate, a specific inhibitor of KIT and PDGFR-α signaling, delays progression of GIST, but is incapable of achieving cure. Thus, most patients who initially respond to imatinib therapy eventually experience tumor progression, and have limited therapeutic options thereafter. To address imatinib-resistance and tumor progression, these studies sought to understand the molecular mechanisms that regulate apoptosis in GIST, and evaluate combination therapies that kill GISTs cells via complementary, but independent, mechanisms. BIM (Bcl-2 interacting mediator of apoptosis), a pro-apoptotic member of the Bcl-2 family, effects apoptosis in oncogene-addicted malignancies treated with targeted therapies, and was recently shown to mediate imatinib-induced apoptosis in GIST. This dissertation examined the molecular mechanism of BIM upregulation and its cytotoxic effect in GIST cells harboring clinically-representative KIT mutations. Additionally, imatinib-induced alterations in BIM and pro-survival Bcl-2 proteins were studied in specimens from patients with GIST, and correlated to apoptosis, FDG-PET response, and survival. Further, the intrinsic pathway of apoptosis was targeted therapeutically in GIST cells with the Bcl-2 inhibitor ABT-737. These studies show that BIM is upregulated in GIST cells and patient tumors after imatinib exposure, and correlates with induction of apoptosis, response by FDG-PET, and disease-free survival. These studies contribute to the mechanistic understanding of imatinib-induced apoptosis in clinically-relevant models of GIST, and may facilitate prediction of resistance and disease progression in patients. Further, combining inhibition of KIT and Bcl-2 induces apoptosis synergistically and overcomes imatinib-resistance in GIST cells. Given that imatinib-resistance and GIST progression may reflect inadequate BIM-mediated inhibition of pro-survival Bcl-2 proteins, the preclinical evidence presented here suggests that direct engagement of apoptosis may be an effective approach to enhance the cytotoxicity of imatinib and overcome resistance.

ANTI-TUMOR EFFECTS OF THE NOTCH PATHWAY IN GASTROINTESTINAL STROMAL TUMORS

Gastrointestinal Stromal Tumors (GIST) are sarcomas driven by gain-of-function mutations of KIT or PDGFRA. Although, the introduction of tyrosine kinase inhibitors has dramatically changed the history of this disease, evidences emerge that inhibition of KIT or PDGFRA are not sufficient to cure patients. The developmental pathway Notch has a critical role in the cell fate, regulating cell proliferation and differentiation. Dysregulation of Notch pathway has been implicated in a wide variety of cancers functioning as a tumor promoter or a tumor suppressor in a cell context dependent manner. Given that Notch activation deregulates the morphogenesis of mesenchymal cells in the GI track, that Notch acts as a tumor suppressor in neuroendocrine tumors, and finally that the cell of origin of GIST are the Interstitial Cell of Cajal that arise from a mesenchymal origin with some neuroendocrine features, we hypothesized that Notch pathway signaling may play a role in growth, survival and differentiation of GIST cells. To test this hypothesis, we genetically and pharmacologically manipulated the Notch pathway in human GIST cells. In this study, we demonstrated that constitutively active intracellular domain of Notch1 (ICN-1) expression potently induced growth arrest and downregulated KIT expression. We have performed a retrospective analysis of 15 primary GIST patients and found that high mRNA level of Hes1, a major target gene of Notch pathway, correlated with a significantly longer relapse-free survival. Therefore, we have established that treatment with the FDA approved histone deacetylase inhibitor SAHA (Vorinostat) caused dose-dependent upregulation of Notch1 expression and a parallel decrease in viability in these cells. Retroviral silencing of downstream targets of Notch with dominant negative Hes-1 as well as pharmacological inhibition of Notch pathway with a γ-secretase inhibitor partially rescued GIST cells from SAHA treatment. Taken together these results identify anti-tumor effect of Notch1 and a negative cross-talk between Notch1 and KIT pathways in GIST. Consequently, we propose that activation of this pathway with HDAC inhibitors may be a potential therapeutic strategy for GIST patients.