Altered T cell receptor beta repertoire patterns in pediatric ulcerative colitis.

The antigenic specificity of T cells occurs via generation and rearrangement of different gene segments producing a functional T cell receptor (TCR). High-throughput sequencing (HTS) allows in-depth assessment of TCR repertoire patterns. There are limited data concerning whether TCR repertoires are altered in inflammatory bowel disease. We hypothesized that pediatric ulcerative colitis (UC) patients possess unique TCR repertoires, resulting from clonotypical expansions in the gut. Paired blood and rectal samples were collected from nine newly diagnosed treatment-naive pediatric UC patients and four healthy controls. DNA was isolated to determine the TCR-ß repertoire by HTS. Significant clonal expansion was demonstrated in UC patients, with inverse correlation between clinical disease severity and repertoire diversity in the gut. Using different repertoire variables in rectal biopsies, a clear segregation was observed between patients with severe UC, those with mild-moderate disease and healthy controls. Moreover, the overlap between autologous blood-rectal samples in UC patients was significantly higher compared with overlap among controls. Finally, we identified several clonotypes that were shared in either all or the majority of UC patients in the colon. Clonal expansion of TCR-ß-expressing T cells among UC patients correlates with disease severity and highlights their involvement in mediating intestinal inflammation.

The state of hepatitis B and C in the Mediterranean and Balkan countries: report from a summit conference.

The burden of disease due to chronic viral hepatitis constitutes a global threat. In many Balkan and Mediterranean countries, the disease burden due to viral hepatitis remains largely unrecognized, including in high-risk groups and migrants, because of a lack of reliable epidemiological data, suggesting the need for better and targeted surveillance for public health gains. In many countries, the burden of chronic liver disease due to hepatitis B and C is increasing due to ageing of unvaccinated populations and migration, and a probable increase in drug injecting. Targeted vaccination strategies for hepatitis B virus (HBV) among risk groups and harm reduction interventions at adequate scale and coverage for injecting drug users are needed. Transmission of HBV and hepatitis C virus (HCV) in healthcare settings and a higher prevalence of HBV and HCV among recipients of blood and blood products in the Balkan and North African countries highlight the need to implement and monitor universal precautions in these settings and use voluntary, nonremunerated, repeat donors. Progress in drug discovery has improved outcomes of treatment for both HBV and HCV, although access is limited by the high costs of these drugs and resources available for health care. Egypt, with the highest burden of hepatitis C in the world, provides treatment through its National Control Strategy. Addressing the burden of viral hepatitis in the Balkan and Mediterranean regions will require national commitments in the form of strategic plans, financial and human resources, normative guidance and technical support from regional agencies and research.

Has the time come to control hepatitis A globally? Matching prevention to the changing epidemiology.

For the first time a global meeting on hepatitis A virus (HAV) infection as vaccine preventable disease was organized at the end of 2007. More than 200 experts from 46 countries gathered to investigate the changing global HAV epidemiology reflecting the increasing numbers of persons at risk for severe clinical disease and mortality from HAV infection. The benefits of childhood and adult hepatitis A (HepA) vaccination strategies and the data needed by individual countries and international health organizations to assess current HepA prevention strategies were discussed. New approaches in preventing HAV infection including universal HepA vaccination were considered. This introductory paper summarizes the major findings of the meeting and describes the changing epidemiology of HAV infections and the impact of HepA vaccination strategies in various countries. Implementation of HepA vaccination strategies should take into account the level of endemicity, the level of the socio-economic development and sanitation, and the risk of outbreaks. A stepwise strategy for introduction of HepA universal immunisation of children was recommended. This strategy should be based on accurate surveillance of cases and qualitative documentation of outbreaks and their control, secure political support on the basis of high-quality results, and comprehensive cost-effectiveness studies. The recognition of the need for increased global attention towards HepA prevention is an important outcome of this meeting.

Iloprost treatment for refractory Raynaud's phenomenon in two infants.

Raynaud's phenomenon (RP) is rare in young children. We describe two infants with severe RP, manifesting as fingertip necrosis, who were resistant to conventional vasodilators and were treated successfully with iloprost, a prostacyclin analogue. The application of iloprost is safe and should be considered in children with threatening ischemic digits.

Hepatitis B virus, hepatitis C virus and other blood-borne infections in healthcare workers: guidelines for prevention and management in industrialised countries.

The Viral Hepatitis Prevention Board (VHPB) convened a meeting of international experts from the public and private sectors in order to review and evaluate the epidemiology of blood-borne infections in healthcare workers, to evaluate the transmission of hepatitis B and C viruses as an occupational risk, to discuss primary and secondary prevention measures and to review recommendations for infected healthcare workers and (para)medical students. This VHPB meeting outlined a number of recommendations for the prevention and control of viral hepatitis in the following domains: application of standard precautions, panels for counselling infected healthcare workers and patients, hepatitis B vaccination, restrictions on the practice of exposure-prone procedures by infected healthcare workers, ethical and legal issues, assessment of risk and costs, priority setting by individual countries and the role of the VHPB. Participants also identified a number of terms that need harmonization or standardisation in order to facilitate communication between experts.

Role in nude mice of interferon and natural killer cells in inhibiting the tumorigenicity of human hepatocellular carcinoma cells infected with hepatitis B virus.

The human hepatoma cell line, PLC/PRF/5, which is persistently infected with hepatitis B virus (HBV), has integrated HBV-DNA, secretes HBV surface antigen (HBsAg), and does not grow readily in congenitally athymic (nu/nu) mice. The present investigation was undertaken to ascertain whether the low tumorigenicity of this cell line was governed by a host immune response and/or was related to expression of HBsAg. Subcutaneous injection of 4-5 X 10(6) cells into BALB/c nude mice produced localized encapsulated tumors with morphologic features of primary hepatocellular carcinoma in 25% of the animals within 29-40 d. No tumor growth was observed at lower cell inocula. In contrast, SK-HEP-1, an HBV-negative human hepatoma cell line, produced tumors at 1-5 X 10(6) cells inocula in 66% of the animals. Immunosuppression of mice with antilymphocyte serum (ALS) or irradiation increased tumor incidence in mice inoculated with 1 X 10(6) PLC/PRF/5 cells to almost 100% and produced local invasiveness. Immunosuppression also reduced the latency, i.e., time to tumor appearance, and increased mean tumor weight. These results suggest that tumorigenicity was limited by the host immune response. The nature of the response was delineated by treating nude mice challenged with tumor cells with sheep anti-mouse interferon globulin (anti-IFN). When 2 X 10(6) cells were injected, tumor growth occurred in 75% of anti-IFN-treated mice, whereas controls injected with the same number of cells, but not receiving anti-IFN, failed to develop tumors. The tumors in the anti-IFN-treated mice were highly invasive and the latency period until tumor appearance was reduced to 3-5 d. An inverse correlation was found between susceptibility of the hepatoma cells to natural killer (NK) activity in vitro and resistance to tumor growth in vivo. In vitro cytotoxicity for PLC/PRF/5 cells was eliminated by anti-NK 1.1 and complement, establishing the effector cell as an NK cell. NK cell activity 14 d after inoculation of mice with PLC/PRF/5 cells was augmented against PLC/PRF/5 target cells but not against SK-HEP-1 cells. Treatment of mice with ALS, irradiation, or anti-IFN abolished NK activity against PLC/PRF/5 cells. Co-cultivation of nude mouse spleen cells with PLC/PRF/5 but not with HBsAg or SK-HEP-1 cells induced secretion of murine IFNalpha. These results suggest that the IFN/NK cell system may play a role in limiting tumorigenicity and invasiveness of HBV-infected human hepatocellular carcinoma cells by a mechanism similar to that found for other cells persistently infected with viruses.

Antigenic characterization of human hepatocellular carcinoma. Development of in vitro and in vivo immunoassays that use monoclonal antibodies.

Several libraries of monoclonal antibodies have been produced by immunization of Balb/c mice with single cell suspensions of nontrypsin-treated human hepatocellular carcinoma cell (HCC) lines in order to study the antigenic properties of transformed hepatocytes. The antibodies were characterized with regards to specificity for hepatoma-associated antigens and their capability for use as reagents in radioimmunoassays (RIAs) and tumor localization in vivo. Three such antibodies namely, P215457, PM4E9917, P232524 of the IgG2a, IgG2a, and IgG1 isotypes, respectively, not only recognized separate and distinct antigenic determinants on four human hepatoma cell lines but also reacted with epitopes present on chemically induced rat hepatoma cell lines. In contrast, only 1 of 38 other human malignant and transformed cell lines demonstrated reactivity with the three antibodies; normal human tissues were also found to be unreactive. Monoclonal antibody P215457 densely stained the plasma membrane by indirect immunofluorescence, showed rapid binding activity to HCC cells in suspension, and precipitated a 50,000-mol wt cell surface protein; antibody PM4E9917 also stained the plasma membrane and precipitated a 65,000-mol wt protein, whereas P232534 recognized cytoplasmic antigenic determinants. With these antibodies "simultaneous sandwich" RIAs were established that detect soluble hepatoma-associated antigens in culture supernatants. Finally, the Fab fragment of P215457 was found to be useful in tumor localization in vivo. This antibody fragment when labeled with 131I was shown to localize by radionuclide-imaging studies in human hepatoma grown in nude mice. Thus, these investigations demonstrate that monoclonal antibodies may be produced against epitopes that reside almost exclusively on transformed hepatocytes and such antibodies may be successfully employed in the development of in vitro and in vivo immunoassays.

Erythropoietin-induced polycythemia in athymic mice following transplantation of a human renal carcinoma cell line.

An established cloned human renal carcinoma line RC-1, which has been continuously maintained in culture for several years and which produces erythropoietin, was injected s.c. into BALB/c athymic mice and produced tumors. Tumorigenicity was directly correlated with the number of RC-1 cells inoculated. Tumor cell histology resembled the original patient-derived tumor. Tumor-bearing mice developed hepatosplenomegaly and significant reticulocytosis with elevated hemoglobin and hematocrit values that were proportional to tumor mass. In addition, red cell mass and blood volume of nude mice increased over 100% as compared to control mice or to animals bearing nonrelevant neoplasms. Large amounts of immunoreactive erythropoietin could be extracted from the nude mouse RC-1 tumors. These results indicate that the RC-1 cell line is tumorigenic and produces biologically active erythropoietin in vivo in athymic mouse hosts, thus providing a reproducible model to study ectopic erythropoietin production and its regulation in vivo.

Fluidity of membrane lipids and lateral mobility of concanavalin A receptors in the cell surface of normal lymphocytes and lymphocytes from patients with malignant lymphomas and leukemias.

Lymphocytes isolated from the peripheral blood of patients with nonmalignant and malignant disorders were studied for fluidity of membrane lipids and lateral mobility of concanavalin A (Con A) receptors. The degree of fluidity of the surface membrane lipid core was monitored quantitatively by fluorescence polarization analysis using the probe 1,6-diphenyl-1,3,5-hexatriene embedded in lipid regions of the surface membrane of intact cells. Mobility of Con A surface receptors was determined by the cap-forming ability after binding of fluorescent Con A. The present studies were performed on lymphocytes from 28 patients with malignant lymphomas, 22 patients with leukemia, 28 individuals who either were healthy or had nonmalignant disorders, and 5 patients with carcinoma. The results showed that lymphocytes and mononuclear cells from patients with malignant lymphomas and leukemias have a more fluid lipid layer in their surface membrane than do lymphocytes obtained from healthy individuals or from patients with other malignant and nonmalignant disorders. This increase in membrane fluidity was less pronounced in lymphocytes isolated from leukemic patients in clinical remission and from leukemic patients receiving treatment with steroids. The results also show a marked difference in the cap-forming ability of lymphocytes from patients with malignant lymphomas or leukemia as compared with lymphocytes from patients with non-malignant disorders or carcinoma. Lymphocytes isolated from lymphoma and chronic lymphatic leukemia patients during remission stages of the disease exhibited a higher cap-forming ability. The cap-forming ability of cells from patients with chronic lymphocytic leukemia was unaffected by treatment with steroids. The present results, which are in line with previous observations, have shown that normal lymphocytes can be characterized by a low degree of lipid fluidity but a high degree of mobility of Con A receptors, whereas leukemic lymphocytes are characterized by a high degree of lipid fluidity but a low degree of mobility of Con A receptors. These results confirmed our general hypothesis on the dynamic interrelation between membrane lipids and membrane protein receptors, and they indicate that the widely accepted term "membrane fluidity" requires better consideration for different membrane components.

Tumorigenicity in nude mice of a human hepatoma cell line containing hepatitis B virus DNA.

The human hepatocellular carcinoma cell line PLC/PRF/5, which synthesizes and secretes hepatitis B surface antigen, was grown under optimal conditions in tissue culture, using Eagle's minimal essential medium supplemented with 10% fetal bovine serum and 10(-11) M triiodothyronine on collagen rafts. Injection s.c. of the PLC/PRF/5 cell line into athymic BALB/c nude mice resulted in the growth of a well-circumscribed, moderately differentiated hepatocellular carcinoma. The intervals until tumor appearance and tumor "take" rates were dependent on inoculum dose. Four to 5 x 10(6) cells induced tumor growth in 29% of 14 injected mice within 29 to 40 days, while 7 to 13 X 10(6) cells induced tumors in all 15 mice within 10 to 12 days after inoculation. Hepatitis B surface antigen was detected in the nude mouse serum and tumor tissue, and its concentration roughly correlated with tumor weight. A low level of antibody against hepatitis B surface antigen was detected in five tumor-bearing animals, as well as in one mouse which did not produce a tumor. Hepatitis B core antigen and its antibody and hepatitis B e antigen and its antibody were not detected in 26 mice, using immunohistochemical and radioimmunoassay methods. alpha-Fetoprotein, carcinoembryonic antigen, and alpha-antitrypsin were detected in nude mice tumors, using the peroxidase-antiperoxidase technique. Finally, hepatitis B virus DNA, identified in the nude mouse tumor by molecular hybridization techniques, was compared to PLC/PRF/5 cell line hepatitis B virus DNA.

Hepatitis A booster vaccination: is there a need?

Hepatitis A is one of the most common vaccine-preventable infectious diseases in the world. Effective vaccines against hepatitis A have been available since 1992, and they provide long-term immunity against the infection. However, there is no worldwide consensus on how long protection will last or whether there will be a need for hepatitis A virus (HAV) booster vaccinations in the future. In most countries, booster-vaccination policy is guided by manufacturers' recommendations, national authorities, or both. In June, 2002, a panel of international experts met to review the long-term immunogenicity and protection conferred by HAV vaccine in different population groups. Data have shown that after a full primary vaccination course, protective antibody amounts persist beyond 10 years in healthy individuals, and underlying immune memory provides protection far beyond the duration of anti-HAV antibodies. The group concluded that there is no evidence to lend support to HAV booster vaccination after a full primary vaccination course in a healthy individual. However, further investigations are needed before deciding if boosters can be omitted in special patient-groups.

IgA deficiency associated with chronic hepatitis C virus infection. A cause or an effect?

OBJECTIVE: To evaluate the role of IgA in hepatitis C virus (HCV) infection. We have tested serum IgA levels in patients with antibodies to HCV. DESIGN: A retrospective study. PATIENTS: The IgA levels were tested in serum samples from 94 patients with antibodies to HCV examined during 1989-1990. RESULTS: Low IgA levels were found in 16/94 (17%) patients. In three of these 16 patients (3.2% of the original 94), no IgA was detected by radial immunodiffusion. In nine of 16 patients, previous pre-HCV infection serum samples with undetectable anti-HCV antibodies were available. In four of these nine patients, IgA deficiency was found in the preinfection serum, while in the remaining five patients, previous IgA levels were normal and the occurrence of anti-HCV was associated with the recent development of IgA deficiency. CONCLUSIONS: The results of this study indicate that IgA deficiency is a risk factor for HCV infection in some patients, whereas in others it might be caused by the viral disease.

Conservation from rat to human of cytosolic phosphoenolpyruvate carboxykinase and the control of its gene expression.

Structural conservation of cytosolic phosphoenolpyruvate carboxykinase protein and mRNA sequence was found in all species examined from rodents to human. The mitochondrial isoenzyme, in all species tested, represents a distinct protein. Moreover, irrespective of the ratio of cytosolic to mitochondrial isoenzyme, cytosolic phosphoenolpyruvate carboxykinase activity in the human as in the rat is controlled at the level of gene expression and through the same multiple hormonal stimulation. This evolutionary conservation of the cytosolic phosphoenolpyruvate carboxykinase structure and mode of regulation supports the enzymes' physiological importance in mammals.

Relapsing hepatitis A. Review of 14 cases and literature survey.

We have reviewed our experience with 14 cases of relapsing hepatitis A (RH-A), as well as 68 cases reported in the literature. Relapse occurs in 3 to 20% of patients with acute hepatitis A, and rarely takes the form of a polyphasic disease (multiple relapses). After a stage of typical hepatitis A, remission phase ensues, with partial or complete resolution of clinical and biochemical manifestations. Relapse usually occurs after a short period (usually less than 3 weeks). Relapse is usually clinically milder than the first phase, with variable liver function abnormalities and a tendency toward more marked cholestatic features. Not uncommonly, immune manifestations occur during this phase, including purpura, nephritis, and arthralgia, with common laboratory findings of rheumatoid factor as well as false-positive reaction to HCV-EIA tests. The clinical course in relapsing hepatitis A is almost always benign, and uneventful recovery is the rule with few exceptions. Steroid treatment, first reported in the present series, resulted in marked clinical improvement. Preliminary results suggest that R-HA is associated with a continuing viremia as well as shedding of virus in stools during the relapse phase. The pathogenesis of R-HA probably involves an interaction between persistent viral infection and immune mechanisms responding to the continuing antigenic stimulation.

Hepatitis C-associated cryoglobulinemia after liver transplantation.

Mixed cryoglobulinemia is well known to be associated with hepatitis C virus (HCV) infection. We report two cases in which cryoglobulinemia appeared or became grossly exacerbated after orthotopic liver transplantation. In both cases, there was co-appearance of cryoglobulinemia with the reinfection of the grafted liver with HCV. It is postulated that the cryoglobulinemia might be related to secondary HCV infection in these patients.

Sublines of the Burkitt line Daudi, selected on the basis of reactivity with soybean agglutinin, differ in tumorigenicity in athymic mice.

Two subpopulations were isolated on the basis of soybean agglutinin (SBA) binding, from the human Burkitt lymphoma line Daudi. The low- and high-binder sublines maintained this characteristic in continuous passages. Their surface marker profiles, antibodies, scanning electron microscope (SEM), cytochemical reactions and binding of other lectins (concanavalin A and wheatgerm agglutinin) were not different. They differed, however, in growth potential in athymic mice. The low-binder subline had lower frequency of takes, tumor weight and volume, and did not metastasize as compared to the high-binder subline. However, the reaction with F-SBA of all the tumor cells examined was strong (greater than 70%), indicating in vivo selection and tumor development of high binder cells.

Development of a novel C1q immunoadsorbent for removal of circulating immunecomplexes: quantitative isolation of hepatitis B virus surface antigen and immunecomplexes.

A technique for large scale production of human C1q from plasma by affinity chromatography on an anti-C1q column is described. Affinity purified C1q was covalently coupled to a newly developed agarose polyacrolein microsphere beads immunoadsorbent. This immunoadsorbent was utilized for quantitative removal of artificially formed bovine serum albumin (BSA)-anti-BSA immune complexes (IC). The C1q affinity column was then used for the isolation of immunecomplexes containing hepatitis B virus (HBV) surface antigen (HBsAg) from serum of an HBsAg carrier. Identical columns may be utilized for quantitative removal of a variety of IC from blood of patients with infectious and autoimmune diseases, as well as neoplastic diseases. Furthermore, dissociated immunecomplexes will provide an additional source for purification of specific antigens.

Clofibrate does not induce peroxisomal proliferation in human hepatoma cell lines PLC/PRF/5 and SK-HEP-1.

The potential of fibrate drugs to induce peroxisomal proliferation in human liver cells was evaluated in athymic nude mice transplanted with human hepatoma cells and treated by clofibrate in vivo as well as in cultured human hepatoma cells in the presence of fibrate drugs added to the culture medium. Clofibrate did not induce peroxisomal activities and neither acted as a peroxisomal proliferator in human PLC/PRF/5 or SK-HEP-1 heterotransplants under conditions of induction of peroxisomal activities in the host rodent liver. Similarly, clofibric acid or bezafibrate did not induce peroxisomal activities in cultured human PLC/PRF/5 or SK-HEP-1 cells under conditions of induction of peroxisomal activities in cultured primary rat liver cells. The lack of response of the human cells to peroxisomal proliferators of the fibrate type may indicate a species specificity with respect to induction of peroxisomal activities by xenobiotic peroxisomal proliferators.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in health care workers (HCWs): guidelines for prevention of transmission of HBV and HCV from HCW to patients.

The transmission of viral hepatitis from health care workers (HCW) to patients is of worldwide concern. Since the introduction of serologic testing in the 1970s there have been over 45 reports of hepatitis B virus (HBV) transmission from HCW to patients, which have resulted in more than 400 infected patients. In addition there are six published reports of transmissions of hepatitis C virus (HCV) from HCW to patients resulting in the infection of 14 patients. Additional HCV cases are known of in the US and UK, but unpublished. At present the guidelines for preventing HCW to patient transmission of viral hepatitis vary greatly between countries. It was our aim to reach a Europe-wide consensus on this issue. In order to do this, experts in blood-borne infection, from 16 countries, were questioned on their national protocols. The replies given by participating countries formed the basis of a discussion document. This paper was then discussed at a meeting with each of the participating countries in order to reach a Europe-wide consensus on the identification of infected HCWs, protection of susceptible HCWs, management and treatment options for the infected HCW. The results of that process are discussed and recommendations formed. The guidelines produced aim to reduce the risk of transmission from infected HCWs to patients. The document is designed to complement existing guidelines or form the basis for the development of new guidelines. This guidance is applicable to all HCWs who perform EPP, whether newly appointed or already in post.

Successful immunization of autologous bone marrow transplantation recipients against hepatitis B virus by active vaccination.

Patients undergoing autologous bone marrow transplantation (BMT) are severely immunosuppressed. These patients are exposed to various infections agents due to delayed and efficient reconstitution of their immune system. Forty-eight patients with hemato-oncological malignancies were immunized against hepatitis B virus (HBV) following autologous BMT. Twenty one were vaccinated more than 10 days before BMT, 17 on days 1-9 before and 10 day after BMT. Thirty three patients (68.7%) seroconverted within 40 days after autologous BMT after receiving one dose of the vaccine before autologous BMT with a relatively low level of anti-HBs, whereas in 11 no anti-HBV antibodies could be detected. Nineteen patients remained seropositive but in 11 the seroconversion was only transient no correlation was found between permanent or transient seroconversion and basic disease, conditioning regimens, post-transplant therapy, immunotherapy and day of vaccination in relation to autologous and day of vaccination in relation to autologous BMT. Active HBV immunization of patients with malignancy undergoing autologous BMT is feasible and levels of antibodies, although low, are above the conventional protective titers. Therefore active immunization of some patients may reduce hepatitis-related complications in the setting of autologous BMT.