Maria Concetta Renda

Intestinal permeability and genetic determinants in patients, first-degree relatives, and controls in a high-incidence area of Crohn's disease in Southern Italy.

OBJECTIVE:A defect of gastrointestinal barrier function is considered to represent an important step in the pathogenesis of Crohns disease (CD) but the mechanisms leading to an increased intestinal permeability (IP) are poorly understood. Since IP is influenced by pro-inflammatory mediators, it seems likely that a genetically determined abnormal immune response may lead to a loss of barrier function.METHODS:In a geographic area in Southern Italy with high incidence of CD we investigated IP (lactulose/mannitol testing) together with the three main mutations of the NOD2/CARD15 and the D299G polymorphism of the toll-like receptor (TLR)-4 gene in 23 families of CD patients (patients and first-degree relatives).RESULTS:Forty-eight percent of CD patients and 40% of their healthy relatives were found to have an abnormal IP compared to 5% of an appropriate control population (p < 0.0001). IP, however, was not associated with the L1007finsC mutation of the NOD2/CARD15 or the D299G variant of the TLR-4 gene. Allele frequency of the only L1007finsC mutation of CARD15 was significantly increased in patients (8.7%, p < 0.003) and in relatives (8.3%, p < 0.024) compared with controls (2.4%), whereas the D299G variant of the TLR-4 gene was found to be increased only in relatives (8.3%, p < 0.022), but not in patients (4.3%) compared with the control population (1.7%).CONCLUSIONS:There was no association between IP and genetic markers. Our findings showed a very high proportion of healthy first-degree relatives to bare alterations suggested to constitute determinants of CD. Mutations of NOD2/CARD15 or TLR-4, however, do not lead to permeability defects emphasizing the importance of additional environmental and/or genetic factors for pathogenesis.

A Prospective Study of Hepatocellular Carcinoma Incidence in Thalassemia

Hepatocellular carcinoma (HCC) is a complication of cirrhosis. Due to blood transfusions, patients with β-thalassemia (thal) are often infected with either hepatitis C virus (HCV) or hepatitis B virus (HBV). In the past, many patients did not survive long enough to develop HCC. The recent improvements in prognosis have helped in the diagnosis of HCC that has developed. The aim of this study was to evaluate HCC incidence in β-thal. We performed liver ultrasound (US) on all adults without a previous diagnosis of HCC. Risk factors (iron overload, HCV infection, HBV infection, cirrhosis) were evaluated. One hundred and eight thalassemia patients have been evaluated; of whom three were excluded (two patients as they were under the age of 18 years and one patient because he had a previous history of HCC). Seventy-two patients [31 had thalassemia major (TM), 41 had thalassemia intermedia (TI)] with risk factors (iron overload in 72, HCV infection in 46, HBV infection in two, cirrhosis in 10) and 33 (four with TM and 29 with TI) without risk factors underwent liver US. Overall, two patients were found to have a newly developed HCC. Of these two patients, one was treated with surgery and the other with percutaneous radiofrequency. Further follow-up did not show any evidence of recurrence after 23 and 15 months, respectively. Ultrasound screening can allow early detection and treatment of HCC in thalassemia patients.

IL28B polymorphisms influence stage of fibrosis and spontaneous or interferon-induced viral clearance in thalassemia patients with hepatitis C virus infection

Background Polymorphisms in the interleukin-28B are important determinants in the spontaneous and drug-induced control of hepatitis C virus infection. Design and Methods We assessed the association of rs8099917 and rs12979860 polymorphisms with spontaneous viral clearance, severity of liver fibrosis, and response to interferon-monotherapy in 245 thalassemia major patients with hepatitis C virus infection. Results Ninety-eight patients (40%) had a spontaneous viral clearance while 147 patients (60%) developed a chronic infection. Spontaneous viral clearance was more frequent among patients with the T/T genotype of rs8099917 polymorphism (OR 2.130; P=0.008) or C/C genotype of rs12979860 polymorphism (OR 2.425; P=0.001). During observation, 131 patients with chronic infection underwent a liver biopsy; age (OR 1.058; P=0.01) G/T or G/G genotypes of rs8099917 polymorphism (OR 3.962; P=0.001), and C/T or T/T genotypes of rs12979860 polymorphism (OR 3.494; P=0.005) were associated with severe liver fibrosis, independent of liver iron concentration. Finally, T/T genotype of rs8099917 polymorphism (OR 3.014; P=0.03) or C/C genotype of rs12979860 polymorphism (OR 3.285; P=0.01), age (OR 0.902; P=0.001), female gender (OR 3.418; P=0.01) and 2 or 3 virus C genotypes (OR 4.700; P=0.007) were independently associated with sustained virological response in 114 patients treated with alpha-interferon. Conclusions Polymorphisms in the interleukin-28B are associated with the control of hepatitis C virus infection in thalassemia major patients, and understanding allelic patterns has an important role in determining prognosis and therapeutic management.

CLINICAL AND HEMATOLOGICAL RESPONSES TO HYDROXYUREA IN SICILIAN PATIENTS WITH Hb S/β-THALASSEMIA

Although, several reports have detailed that hydroxyurea can ameliorate the clinical course of adult and pediatric patients with sickle cell anemia (Hb S or βS), few clinical studies have been carried out in patients with βS/β-thalassemia. In a two-year clinical study, we evaluated the efficacy of hydroxyurea in a group of 22 adult Sicilian patients with βS/β-thalassemia with severe phenotypes. Among the 20 patients evaluated during 2 years of treatment, we observed a very good clinical response with a 93% reduction of the annual number of crises (median 7 versus 0.5 crises per year; P ≤ 0.001) and of days in hospital (mean 22±21.9 versus 1.2±2.3; P < 0.001), a significant increase in Hb F (7.5±5.3% versus 25.2±5.2%; P < 0.001) and in MCV (73.1±4.8 fL versus 96.4±7.2 fL; P < 0.001), and no significant modifications in Hb (9.6±1.3 g/dL versus 10.0±1.5 g/dL; P > 0.05) and in WBC (11.4±3.9 × 109/L versus 10.2±3.9 × 109/L; P > 0.05). Twelve patients had no crises from the first month of treatment; 16 patients showed a 2–3-fold increase over baseline in Hb F. During the study no severe complications and no important side effects of hydroxyurea were observed. Our data suggest that hydroxyurea efficacy in patients with βS/β-thalassemial may be greater than that described in patients with sickle cell disease. This pattern and durability of response will need to be confirmed in a larger, randomized, clinical trial.

The Role of CARD15 Mutations and Smoking in the Course of Crohn's Disease in a Mediterranean Area

AIM:To evaluate the role of CARD15 mutations and smoking in the main events of Crohns disease (CD).PATIENTS AND METHODS:A total of 182 patients with CD were included in a prospective study in order to evaluate the role of CARD15 mutations and smoking in the main outcomes of disease course: first operation and surgical recurrence. The following variables were evaluated in a univariable and multivariable analysis: age, sex, site of disease, pattern, smoking habit, extraintestinal manifestations, duration of disease, and CARD15 mutation. The Kaplan–Meier method for survival curves and Cox model for multivariable analysis were, respectively, used.RESULTS:A total of 110 patients were operated on and 32 were reoperated on. The 7-yr cumulative free rate of surgery was 42% (95% CI 34–51%). At multivariate analysis only stricturing and penetrating pattern were predictors of surgery (HR 1.7, 95% CI 1–2.8; HR 3.2, CI 1.8–5.5, respectively). The 7-yr cumulative free rate of reoperation was 75% (95% CI 0.52–0.88). At multivariable analysis in the model with any CARD15 mutation, only smoking habit at diagnosis (HR 3.6, 95% CI 1.4–9.1) was predictive of surgical recurrence. When single mutations were considered in the model smoking (HR 4.2, 95% CI 1.8–10.1) and L1007fs mutation (HR 2.9, 95% CI 1.1–7.3) were predictive of reoperation.CONCLUSIONS:In CD, smoking predicts recurrence after surgery. The role of CARD15 mutations in the clinical course of CD remains undefined.

Cytomegalovirus Disappearance After Treatment for Refractory Ulcerative Colitis in 2 Patients Treated with Infliximab and 1 Patient with Leukapheresis

To the Editor: Human cytomegalovirus (HCMV) is a herpesvirus widely spread in the general population. In immunocompetent hosts it rarely causes serious illness but the infection can result in Epstein– Barr virus-like mononucleosis syndrome. In immunologically impaired patients the virus may cause serious multiorgan involvement and complications due to the cytomegalovirus-associated disease. The role of HCMV in patients with inflammatory bowel disease (IBD) is not clearly known. Data from the literature have shown a possible role in exacerbating a colitis flare,1,2 while some authors have reported a role as a bystander and innocent observer.3 Virus interaction with the host’s immune system involves both innate and adaptive immunity. T cells play an important role in controlling viral replication and disease development, but do not eliminate the virus completely; all herpesviruses possess the key property of latency and reactivation.4 The virus is maintained in latency in a non-replicative state in myeloid lineage progenitor cells and it reactivates, in response to inflammatory cytokines and chemokines, when peripheral blood monocytes differentiate into macrophages. The virus produces molecules designed to disrupt or manipulate host inflammatory/ immune responses: interleukin 1 and tumor necrosis factor alpha (TNF) support leukocyte activation and recruitment.5 An important clinical question is what to do when, in severe colitis, there is an indication to use infliximab and HCMV is identified in biopsy specimens or in peripheral blood. Is the presence of HCMV a contraindication to infliximab? We reviewed 3 cases of patients with steroid-dependent moderate-to-severe ulcerative colitis (UC) treated with infliximab and/or leukapheresis for active disease in whom a clinical response with virus disappearance was obtained. Three UC patients were admitted to our department for a severe disease relapse (8 bloody bowel movements per day, abdominal pain and tenderness). Therapy with intravenous steroid (methylprednisolone 1 mg/kg/day) was started, but after 5 days the symptoms were stable. Screening for treatment with infliximab was negative. At sigmoidoscopy, colitis activity was severe. In 1 patient antigenemia pp65 for HCMV detection in peripheral blood was positive, but negative in the other 2. Also, in all patients rectal biopsies (morphologic, immunohistochemical, and polymerase chain reaction [PCR] analysis) were positive for HCMV detection. On the basis of this evidence infliximab treatment was avoided in the first patient, who was treated with leukapheresis (8 sessions, blood volume of 1800 mL in 1 hour). The other 2 patients were treated with 3 infusions (0, 2, and 6 weeks) of infliximab at a dose of 5 mg/kg. All the patients obtained a clinical response with symptoms improvement. At the end on the treatments the endoscopic and histological activity was mild and the morphological and immunohistochemical analysis did not detect HCMV, while PCR proved to be positive. In the first patient antigenemia pp65 for HCMV detection in peripheral blood became negative. Previous data have shown that anti-TNF therapy may cause widespread HCMV infection in refractory Crohn’s disease (CD) through vasculitis, which allows the virus to circulate within the shed endothelial cells.6 An interruption of the replicative sequence may induce an HCMV latent state in infected cells determining immunologic tolerance by the host immune system. Fietze et al7 hypothesized that TNFplays a role in the reversal of HCMV latency and that inhibition of TNFrelease or action may be an alternative strategy for preventing HCMV-associated morbidity in allograft recipients. The replication of HCMV could also increase TNFexpression, allowing it to maintain a steady viral load. According to this evidence, some authors have shown a possible role of TNFin the reactivation of HCMV in septic patients.8 The mechanisms of action of leukapheresis devices have been extensively reviewed and are believed to exert an immunomodulatory and/or antiinflammatory effect on patients with systemic inflammatory disease. Dumont et al9 have shown a substantial reduction in the viral genome load in subjects positive for anti-CMV antibodies and HCMV-DNA, evaluated by qualitative PCR, after leukocyte reduction methods. According to this experience, treatment with anti-TNFantibodies and leukapheresis may be carried out without risk of colitis relapse associated with superimposed colonic HCMV infection. Low TNFactivity after infliximab treatment may interfere both with the viral replication and the inflammation maintenance avoiding colitis, while the decrease in circulating HCMV-infected cells after leukapheresis reduces the risk of colitis flare due to superimposed HCMV action. The improvement of colonic inflammation due to these treatments reduces viral replication, and the detection of HCMV in rectal biopsies only by PCR assay may correspond to a latent and non-pathogenic state of the virus. Although the available evidence arises from anecdotal or experimental observations, this report may be an interesting and useful proposal for further Copyright

Feasibility of DNA diagnosis of haemoglobinopathies on coelocentesis

At 5–12 weeks of gestation the amniotic sac is surrounded by celomic fluid, which contains cells of fetal origin. This fluid can be sampled by celocentesis, which involves the ultrasound‐guided insertion of a needle through the vagina. The aim of this study was to examine the feasibility of prenatal diagnosis of haemoglobinopathies from the celomic fluid using a specific protocol. Celocentesis was performed at 7–9 weeks gestation in 26 singleton pregnancies at risk for haemoglobinopathies. In 25 cases more than 30 fetal cells were recovered from the celomic fluid and in all these cases molecular analysis for haemoglobinopathies was possible and the results were confirmed by subsequent chorionic villus sampling or amniocentesis. The results of this study suggest that reliable diagnosis of thalassemia syndromes can be performed from 7 weeks gestation by celocentesis. Further work is necessary to demonstrate the safety of celocentesis before widespread use.

Marked impact of IL28B genotype in the natural clearance of hepatitis C virus in patients with haemoglobinopathies

Driss, F., Tertian, G., Becquemont, L., Haddad, B., Cynober, T., Raphael, M. & Tchernia, G. (2007) Management of high risk pregnancy in sickle cell disease by a strategy of prophylactic red cell transfusion or automated red cell exchange. Transfusion Clinique et Biologique, 14, 386–392. Kalff, A., Dowsing, C. & Grigg, A. (2010) The impact of a regular erythrocytapheresis programme on the acute and chronic complications of sickle cell disease in adults. British Journal of Haematology, 149, 768–774.

Embryo-fetal erythroid megaloblasts in the human coelomic cavity†

The coelomic cavity is part of the extraembryonic mesoderm, surrounding amniotic cavity, embryo, and yolk sac in the early gestation. It is now believed to represent an important transfer interface and a reservoir of nutrients for the embryo. Coelocentesis by ultrasound‐guided transvaginal puncture offers an easier access to the early human embryo, from 28 days post‐fertilization. However, despite some studies about its biochemical composition being reported, our knowledge about the presence of cellular elements and their quality in this compartment are still limited. Here we studied human coelomic fluids sampled from 6.6 (48 days) to 10 weeks of gestation, demonstrating the presence of functional embryonic erythroid precursors, that is, megaloblasts in the coelomic cavity. The ease of access of the coelomic cavity could allow the development of novel strategies for diagnostic or therapeutic purposes by ultrasound imaging and ultrasound‐guided puncture. J. Cell. Physiol. 225: 385–389, 2010.

Typing of the immunological system in human embryos by coelocentesis

Coelocentesis offers a new opportunity for gaining access to the human embryos from 28 d postfertilization. However, while some studies about its biochemical composition have been reported, our knowledge about immunological pattern of this compartment is still limited. For this reason, we studied the human coelomic fluids sampled from 6.6 to 10 wk of gestation. The majority of cellular population consisted in mesenchymal/epithelial cells. In fluids sampled before 10 wk we found only a preT Cell Receptor expression and an absence or a very low frequency of B lymphocytes, T lymphocytes and NK (natural killer) antigens. These preliminary data suggest that the immunological system in human embryos could be in the ideal conditions to start a process of tolerance induction.