Andrea Duchini

Etiopathogenesis of nonalcoholic steatohepatitis: role of obesity, insulin resistance and mechanisms of hepatotoxicity.

Incidence of nonalcoholic fatty liver disease is increasing with an estimated prevalence of 20–30% in developed nations. This is leading to increased incidence of chronic liver disease, cirrhosis, and hepatocellular cancer. It is critical to understand the etiology and pathogenesis of any disease to create therapeutic targets and develop new treatments. In this paper we discuss the etiology and pathogenesis of nonalcoholic steatohepatitis with special focus on obesity, role of insulin resistance, and molecular mechanisms of hepatotoxicity.

Increase in Mortality Rate of Liver Transplant Candidates Residing in Specific Geographic Areas: Analysis of UNOS Data

We sought to evaluate survival of liver transplant candidates living in geographic areas with limited access to specialized transplant centers (TxC). We analyzed survival outcome among candidates listed for liver transplant in United Network of Organ Sharing (UNOS) Region 4 from 2004 to 2010. Candidates were stratified into three groups according to the distance from the patients residence to the closest hospital with a liver transplant program: Group 1 (Gr 1) <30 miles (m), Group 2 (Gr 2) 30–60 m and Group 3 (Gr 3) >60 m. Of the 5673 patients included in the study, 49% resided >30 m from a TxC. Eight percent of the cohort experienced death or dropped out of the list due to medical condition deterioration, with worse outcomes for Gr 2 and Gr 3 (8.5% and 9.9%, respectively, vs. 6.5% for Gr 1 [p < 0.001]). Among patients with a MELD score <20, mortality was higher in Gr 2 and Gr 3 compared to Gr 1 (p < 0.001). We conclude that for Region 4, the mortality risk in patients living >30 m from a TxC is higher. We suggest that the variable “distance from a TxC” should be used to improve the estimate of the mortality risk for patients on the waiting list.

Evaluation of INK4A promoter methylation using pyrosequencing and circulating cell-free DNA from patients with hepatocellular carcinoma

Abstract Background: Hyper-methylation of CpG dinucleotides in the promoter region of inhibitor of cyclin-dependent kinase 4A (INK4A) has been reported in 60%–80% of hepatocellular carcinoma (HCC). As INK4A promoter hypermethylation event occurs early in HCC progression, the quantification of INK4A promoter methylation in blood sample may represent a useful biomarker for non-invasive diagnosis and prediction of response to therapy. Methods: We examined INK4A promoter methylation using circulating cell-free DNA (ccfDNA) in a total of 109 serum specimens, including 66 HCC and 43 benign chronic liver diseases. Methylation of the individual seven CpG sites was examined using pyrosequencing. Results: Our results showed that there were significantly higher levels of methylated INK4A in HCC specimens than controls and that the seven CpG sites had different levels of methylation and might exist in different PCR amplicons. The area under receiver operating characteristic (ROC) curve was 0.82, with 65.3% sensitivity and 87.2% specificity at 5% (LOD), 39.0% sensitivity and 96.5% specificity at 7% LOD, and 20.3% sensitivity and 98.8% specificity at 10% LOD, respectively. Conclusions: Our results support additional studies incorporating INK4A methylation testing of ccfDNA to further validate the diagnostic, predictive, and prognostic characteristics of this biomarker in HCC patients. The knowledge of the existence of epi-alleles should help improve assay design to maximize detection.

Non-Alcoholic Steatohepatitis in Myotonic Dystrophy: DMPK Gene Mutation, Insulin Resistance and Development of Steatohepatitis

Myotonic dystrophy is a multisystemic disorder characterized by repeat expansion mutations of the dystrophia myotonica protein kinase (DMPK) gene resulting in a defective muscular insulin receptor and insulin resistance. We describe a patient with myotonic dystrophy who developed biopsy-proven non-alcoholic steatohepatitis. We suggest that patients with myotonic dystrophy are at risk of developing steatohepatitis. The relationship between defective insulin receptor and development of steatohepatitis should be further investigated.

Pancreatic pleomorphic rhabdomyosarcoma.

Highlights • Pancreatic rhabdomyosarcoma is an extremely rare disease.• Pre-operative diagnosis is challenging due to unspecific presentation.• Surgical resection followed by chemotherapy and radiotherapy are the only possible treatments.• Long term prognosis is very poor.

Mo1002 MELD Score Is a Risk for First Episode of Spontaneous Bacterial Peritonitis in Patients With Cirrhosis

Introduction: Nodular Regenerative Hyperplasia (NRH) is a rare noncirrhotic portal hypertensive liver disease characterized by the diffuse transformation of hepatic parenchyma into regenerative nodules without fibrosis. Although the specific etiology has yet to be elucidated, NRH has been associated with various diseases, conditions and medications. Thus, whether NRH occurs from a single unifying pathophysiologic process or multiple different processes is still uncertain. Aims: To characterize biochemical, radiological, and histologic markers of liver disease in patients (pts) with and without NRH in multiple disease cohorts. Methods: Pts with chronic granulomatous disease (CGD), sickle cell disease (SCD), systemic mast cell disease (SMCD), common variable immunodeficiency disease (CVID) and sporadic cases of NRH underwent hepatologic evaluation and liver biopsy for clinical care purposes were evaluated with biomarkers of liver disease, radiologic imaging and histopathologic analysis. Results: 137 pts were evaluated and 56 (41%) were diagnosed with NRH histologically. Of those with NRH, 11 (20%) had CGD, 22 (39%) SCD, 7 (13%) SMCD, 12 (21%) CVID and 4 (7%) sporadic disease. Age at biopsy across all NRH cohorts was significantly older compared to the non-NRH population (mean 40 vs. 33 years, p=0.0128). Patients with NRH had significantly higher alkaline phosphatase (197 vs. 190 U/L, p=0.01), increased liver volumes (2152 vs. 1166 cm3, p=0.01) and increased spleen volumes (after excluding SCD) (1071 vs. 410 cm3, p=0.01), which negatively correlated with platelets ( ρ -0.58, p= 0.002). In the SCD cohort, pts with NRH had increased gamma-glutamyltransferase (128 vs. 67 U/L, p=0.03) and increased lactate dehydrogenase (432 vs. 322 U/L, p=0.01). On histologic evaluation, SCD-NRH pts had increased lobular inflammation (p=0.04). SMCDNRH pts had increased portal and peri-portal inflammation (p=0.005 and p=0.01, respectively), veno-occlusive changes (p=0.04), portal venopathy (p=0.001) and sinusoidal dilation (p=0.03). Conclusions: Despite parallels in clinical disease presentation, histopathologic examination of NRH in separate disease cohorts suggests different mechanistic pathways. Given the potential for multiple etiologies, NRH may be more common than previously thought. Further investigation should focus on the underlying disease process as it may provide a window into biology.

Hepatitis A Vaccination in healthcare workers

Mark S. Kindy Department of Neurosciences, Medical University of South Carolina, and the Ralph H. Johnson VA Medical Center, USA Recently, interest in semiallogeneic vaccines has been increasing, as shown by the publication of successful preclinical and clinical studies by us and others that validate this immunotherapeutic approach to cancer, viral diseases such as the acquired immunodefi ciency syndrome and neurological diseases. Th ese reports indicate that treatment with semiallogeneic vaccines can induce a specifi c immune response against various tumors and against the human immunodefi ciency virus. Experimental studies using inbred mice and their syngeneic tumors initially established that 1) inoculation of semiallogeneic cell hybrids (derived from the fusion of syngeneic tumor cells with an allogeneic cell line) protects the animal host from subsequent lethal challenges with unmodifi ed syngeneic tumor cells; 2) adoptive transfer of immunity required T lymphocytes; and 3) the enhanced immunity was not the result of an allogeneic eff ect per se, because the tumor-associated antigens and alloantigens needed to be on the same cell (the hybrid). Human studies with semiallogeneic cell hybrids have focused on the use of therapeutic vaccines for cancer. Because of the many technical problems associated with obtaining suffi cient amounts of autologous tumor cells for the preparation of patient-specifi c cancer vaccines, and because of evidence that allogeneic eff ects may enhance the antitumor immune response, it seemed reasonable to combine the best of both approaches (autologous and allogeneic), because semiallogeneic hybrids focus allorecognition and major histocompatibility complex self-restricted recognition on the same cell and, therefore, in the same microenvironment. Our studies demonstrate the feasibility of using semiallogenic vaccines to treat a variety of tumors and neurological disease.Daming Zhu Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Disease, National Institutes of Health, USA Malaria is one of most deadly infectious diseases in the world, with an estimated 350-500 million people suff ering from malaria episodes and nearly 1 million deaths per year. Most of these episodes are caused by Plasmodium falciparum and P. vivax parasites. Th e world is in urgent need of a vaccine to combat against malaria. Th e quality control is one of the most important processes in vaccine research and development because it directly relates to the safety and effi cacy of the vaccines. Th is presentation will focus on the quality control evaluation of malaria sub-unit vaccines. Th e biophysical/biochemical/biological methods involved in evaluating vaccine purity, identity, integrity, stability and potency will be discussed. Th ese methods should have general applications for the quality control evaluation of subunit vaccines.Olga Borges University of Coimbra, Portugal Th e development of oral and nasal formulations for under-utilized vaccines in order to simplify their transport, storage and administration in poor countries is considered a challenge [1]. In fact, for eff ective oral immunization, antigens must be protected from the acidic and proteolytic environment of the gastrointestinal tract, effi ciently taken up by cells of the gut associated lymphoid tissue (GALT) and an appropriate immune response must be induced [2]. With this in mind we have been working on the design of chitosan-based particles as adjuvant for mucosal vaccination and recently a new delivery system was evaluated as adjuvant for oral administration of the recombinant hepatitis B antigen (HBsAg) [3]. In this previous work we obtained some promising results, like high titers of anti-HBsAg IgG in serum and antiHBsAg sIgA in mice intestinal washings. However, a high percentage of non-responder mice were observed. Th erefore, the main objective of the group is to develop more sophisticated chitosan-based delivery systems associating to the particles not only the antigens but also an immunopotentiator. Th ree diff erent chitosan-based formulations were developed associating to them aluminum salts, a mast cell activator (c48/80 compound) and CpGODN. An overview of the work done by group on the development of a mucosal hepatitis B vaccine, as well as in vitro results of these new adjuvants will be present during the congress.Yasuyuki Ishi RIKEN Research Center for Allergy and Immunology, Japan Invariant natural killer T (iNKT) cells having Th e invariant T cell antigen receptor (TCR)  chain are a unique population to regulate the immune response between the innate and acquired immunity. iNKT cells interact with glycolipids presented via CD1d molecule on antigen-presenting cells (APCs), resulting in the activation of killing activity and the production of a variety of cytokines. KRN7000, alpha-galacosyl ceramide (-GalCer), among glycolipids functions as the powerful ligand for not only rodent but also primate iNKT cells. As iNKT cells potentially have two bi-directional functions, one is immune activation and the other is immune suppression, it was expected that KRN7000 would be utilized as an adjuvant for various vaccines of cancer, infectious and immune diseases. However, the pharmaceutical development of KRN7000 is not successful because of the bi-directional functions of iNKT cells. In our recent studies, two functions of immune activation and suppression are dissected by the in vivo delivery system of KRN7000. Dendritic cells (DCs), the professional APCs, could be used as delivery cells of KRN7000 for immune activation. Systemic administration of KRN7000-pulsed DCs preferentially enhanced protective immunity against tumors and infection by microbes. In contrast, liposomal formulation of KRN7000 could be delivered to the marginal zone B220positive cells in a spleen, induce regulatory T cells (Treg) and diminish immune responses in model animals of autoimmunity, allergic diseases and graft versus host diseases (GvHD).Immunology and Biotechnology Unit, Zoology Department, Faculty of Science, Tanta University, Egypt Th e success of anti-tumor immunity depends on the generation of functionally eff ective T cells. Adoptive cell therapy (ACT) of autologous tumor-reactive T cells aft er chemotherapy a nd followed by vaccination is a promising approach for generation of functional T cells for cancer immunotherapy. Th is ACT modality consists of in vitro stimulation of T cells from a host own peripheral blood or tumor and then infusing them back to the same host blood followed by vaccination regimen such as peptide or peptide-pulsed dendritic cells (DCs). Th e host is irradiated or treated with chemotherapeutic drug such as cyclophosphamide (CTX) prior ACT to induce lymphopenia. Th e cellular and molecular mechanisms underlying the benefi cial eff ects of lymphodepletion in the context of adoptive T cell therapy and vaccination, however, are not well understood. Defi ning these mechanisms would signifi cantly improve the application of lymphodepletion to ACT. Our recent studies have identifi ed expansion of DCs, the central player of immune response, as a potential mechanism. We have utilized the toll-like receptor 3 (TLR3) agonist poly(I:C), a synthetic viral mimic double-stranded RNA, to induce the full activation of DCs expanded in vivo aft er chemotherapy and during vaccination with defi ned tumor antigens, resulting in effi cacious therapeutic anti-tumor responses. Our results reveal that the combination of T cell therapy and vaccination in the presence of a potent adjuvant such as a TLR agonist at precise timing post chemotherapy opens a new avenue for cancer immunotherapy that can be translated into the clinical setting to cure diff erent cancers.

An Unusual Case of Ulcerative Duodenitis

Question: A 51-year-old Caucasian woman with a past medical history of gastroesophageal reflux disease presented to the emergency department with a 3-day history of worsening generalized abdominal pain, associated with nausea, non-bloody vomiting, 2–3 episodes of small volume hematochezia, and joint pains in hands and feet. Patient denied nonsteroid antiinflammatory or aspirin use, and recent alcohol intake, before such episodes. She had a normal screening colonoscopy 1 month ago. Two weeks before presentation, she was treated with a 1-week course of cephalexin for a urinary tract infection. Physical examination was significant for diffuse abdominal tenderness without peritoneal signs and scattered red macules on bilateral lower extremities. Admission laboratory test results were significant for leukocytosis (13,300/cmm), with left shift and bandemia, hemoglobin of 11 g/dL, and creatinine of 1.8 mg/dL. Urinalysis revealed 3 red blood cells (50–60 per high-powered field) and proteinuria (300 mg/dL). Abdominopelvic computed tomography was performed with findings suggestive of diffuse colitis and terminal ileitis and surrounding fat stranding. In view of her recent normal colonoscopy and antibiotic use before symptomatology, the patient was admitted, stool studies were sent to rule out infections, she was started on intravenous fluids and empiric metronidazole. The stool studies were negative for cultures, leukocytes, and Clostridium difficile toxin. She continued to have abdominal pain, nausea, vomiting, and episodes of hematochezia. The patient underwent an upper endoscopy and a colonoscopy for further diagnostic workup. The upper endoscopy was significant for diffuse mucosal edema, hemorrhage and ulceration starting from the postbulbar duodenum (Figure A, B, C). Colonoscopy revealed patchy erythema throughout the colon without gross ulceration. Biopsies ere obtained and sent to pathology and special staining. The day after endoscopy, the scant macular rash that was noted on initial valuation of the patient was noted to have evolved into a diffuse purplish, purpuric rash extending over bilateral lower extremities (Figure ). Scattered purpuric rash was also noted over abdomen and lower back. Biopsies of the skin rash were obtained and sent for ermatopathologic evaluation. Her renal function worsened further and serum creatinine increased to 3 mg/dL. What is the likely diagnosis based on the clinical presentation, skin findings, and upper endoscopic images? What specific stains ust be requested from the biopsy specimens to confirm the diagnosis? See the GASTROENTEROLOGY web site (www.gastrojournal.org) for more information on submitting your favorite image to Clinical Challenges and Images in GI. Conflicts of interest: The authors disclose no conflicts.