Effects of Cytokines, Without and with Helicobacter pylori Components, on Mucus Secretion by Cultured Gastric Epithelial Cells

Cytokines are suspected to play a crucial rolein the pathogenesis of Helicobacter pylori associatedgastric diseases. Hence, considerable attention has beenpaid to the actions of cytokines on gastric cells. We examined the effects of cytokines onmucus secretion by gastric epithelial cells, without orwith H. pylori components. Mucus secretion by culturedgastric epithelial cells was assessed as secretion of [³H]glucosamine-prelabeledhigh-molecularweight glycoproteins. Interleukin(IL)-1 and IL-6 significantly stimulated mucussecretion, but other cytokines such as IL-7, IL-8,IL-10, interferon (IFN)- and tumor necrosis factor (TNF)- had noeffect. H. pylori lysate caused a decrease in both basaland stimulated secretion of mucus. In addition,IFN- significantly potentiated the lysate-induced reduction of basal and stimulated secretion.Cell viability was not affected by any of treatments.These results indicate that IL-1 and IL-6stimulate mucus secretion, while IFN- potentiatesH. pylori-decreased secretion by gastric epithelialcells.     

Differences Between Interferon-α and-β Treatment for Patients with Chronic Hepatitis C Virus Infection

To compare virological, biochemical, and immuneresponses to human lymphoblastoid interferon(IFN-) and human fibroblast interferon(IFN-) in patients with chronic hepatitis C virus(HCV) infection, 120 patients were randomly assigned to threegroups (group A, 60 patients receiving IFN-, 6million units (MU) once a day, daily for one month andthrice weekly for five months; group B, 40 patients receiving 6 MU IFN- once a day daily fortwo months; and group C, 20 patients receiving 3 MUIFN- twice a day (6 MU/day) daily for two months).Serum soluble interleukin-2 receptor (sIL-2R) and interleukin-6 (IL-6) levels were measured byenzyme-linked immunosorbent assay. Patients withsustained clearance of serum HCV RNA detected bypolymerase chain reaction (PCR) at six months after IFNtreatment were defined as having complete response to IFNtreatment. A low level of HCV RNA (10^-4copies/50 mul, measured by competitive PCR) and HCV RNAof genotype 2a were favorable factors for a completeresponse to both IFNs. Complete response in group Atreatment was strongly associated with early HCV RNAclearance, in contrast with group B. A significantlyhigher HCV RNA negativity at the second week from start of treatment was noted in group C (80.0%),compared with groups A (41.6%) and B (27.5%). sIL-2Rlevels rose in each group during IFN administration. Ingroup C, alanine aminotransferase (ALT) and IL-6 levels were remarkably elevated. These findingsindicate that timing of serum HCV RNA negativity insustained response differs between IFN- andIFN- administrations and that early HCV RNAclearance was induced by twice-a-day IFN-treatment.     

Amino Acid Polymorphism at Residue 71 in HLA-DR Beta Chain Plays a Critical Role in Susceptibility to Ulcerative Colitis

The association of ulcerative colitis withdistinct HLA-DRB1 alleles has not been easy toascertain. Recent studies show that among HLA-DR2alleles, DRB1*15 but not DRB1*16, is associated with thedisease. Similarly, in the HLA-DR1 group, only DRB1*0103is increased in ulcerative colitis patients. The aim ofour study was to identify critical DRB1 residues thatmight account for these differences. We typed 121 patients with ulcerative colitis and 275controls using gene amplification and sequence-specificoligonucleotide probing for HLA-DRB1 and DRB3. Weobserved a strong negative association between HLA-DRB1 alleles that encode lysine at position 71 intheir -chain and susceptibility to ulcerativecolitis. Differences in the prevalence among otheralleles differing only in the third hypervariable region suggested a hierarchy of susceptible andprotective class II alleles related to the presence ofan acidic, neutral, or basic amino acid residue atposition 71. These data implicate most strongly theamino acid residues in the third hypervariable regionof the DR chain, especially DR71, in theassociation between ulcerative colitis and HLA. However,this does not exclude the contribution of other parts of the molecule and otherimmunoregulatory genes.     

Majority of Gliadin-Specific T-Cell Clones from Celiac Small Intestinal Mucosa Produce Interferon-γ and Interleukin-4

An abnormal mucosal cell-mediated immune response plays a fundamental role in the pathogenesis of celiac disease. To characterize locally infiltrating T cells, gliadin-specific T-cell clones were isolated from two treated celiac patients. Mucosal biopsies were cultured in vitro for 24 hr with a peptic-tryptic digest (PT) of gliadin. T-cell clones (TCC) were then isolated by limiting dilution. The production of interferon- (IFN-) and interleukin-4 (IL-4) was evaluated by ELISA in culture supernatants obtained after a short incubation with anti-CD3 and PMA, or with antigen. Twenty-two TCC were specific for gliadin and/or PT. All were CD3⁺, CD4⁺, CD8⁺, TCR ⁺. In one such clone the PT-specific response was inhibited by an anti-DQ, but not by an anti-DR antibody. Of the five gliadin-specific TCC examined, four produced IL-4 and high levels of IFN-; the remaining one initially produced only IL-4, but subsequently also IFN-. All clones obtained from the celiac mucosa, including the gliadin-specific ones, produced high levels of IFN-, in most cases with IL-4. This cytokine profile could explain most of the immunological features of the celiac mucosa.     

Effects of Cytokines on the Binding of Leukocytes to Cultured Rat Hepatocytes and on the Expression of ICAM-1 by Hepatocytes

Adhesions of leukocytes to hepatocytes andsinusoidal endothelial cells mediates the induction andprogression of hepatic injury. However, in contrast toendothelial cells, information regarding the regulation of interactions between leukocytes andhepatocytes is limited. In the present study, weinvestigated the effect of inflammatory mediatorsincluding lipopolysaccharide (LPS), staphylococcalenterotoxin B (SEB), interferon- (IFN-), tumornecrosis factor- (TNF-), andinterleukin-1 (IL-1) on the adhesion ofpolymorphonuclear leukocytes or lymphocytes to primarycultured rat hepatocytes, and on the expression of intercellular adhesionmolecule-1 (ICAM-1) gene in hepatocytes. Bothpolymorphonuclear leukocyte and lymphocyte adhesion tohepatocytes were enhanced after exposure of hepatocytes to IFN- and TNF-, but not afterexposure to LPS, SEB or IL-1. The adhesion inducedby either IFN- or TNF- was inhibited bymonoclonal antibodies against ICAM-1 or lymphocytefunction-associated antigen-1 (LFA-1). Nonstimulated hepatocytesexpressed faintly ICAM-1 mRNA, which increased slightlyduring the culture period. ICAM-1 mRNA expression wasup-regulated to a greater extent by incubating hepatocytes with IFN- or TNF-,and peaked after 12 hr of incubation with TNF-and after 24 hr with IFN-. These results indicatethat IFN- and TNF- induce the expressionof ICAM-1 on parenchymal hepatocytes and that theLFA-1-ICAM-1 pathway plays an important role in theinteraction between hepatocytes and neutrophils orlymphocytes.     

Cytokines activate genes of the endocytotic pathway in insulin-producing RINm5F cells

Aims/hypothesis Cytokines are important humoral mediators of beta cell destruction in autoimmune diabetes. The aim of this study was to identify novel cytokine-induced genes in insulin-producing RINm5F cells, which may contribute to beta cell death or survival.Methods A global gene expression profile in cytokine-exposed insulin-producing RINm5F cells was achieved by automated restriction fragment differential display PCR. The expression of selected candidate genes was confirmed by real-time RT-PCR analysis.Results Exposure of RINm5F cells to IL-1 or to a cytokine mixture (IL-1, TNF-, IFN-) for 6 h resulted in the differential expression of a functional gene cluster. Apart from the well-known up-regulation of the cytokine-responsive genes iNOS, NF-B, MnSOD and Hsp70, several genes that belong to the functional cluster of the endocytotic pathway were identified. These endocytotic genes comprised: clathrin, megalin, synaptotagmin and calcineurin, which were up-regulated by IL-1 or the cytokine mixture. In contrast, the expression of the calcineurin inhibitor CAIN and of the GDP/GTP exchange protein Rab3 was down-regulated by cytokines. Other up-regulated cytokine-responsive genes were: agrin, murine adherent macrophage protein mRNA (MAMA) and transport-associated protein (TAP1/MTP), whereas the plasma membrane calcium ATPase (PMCA) 2 and PMCA 3 genes were down-regulated by cytokines.Conclusions/interpretation Our results indicate that genes of the endocytotic pathway are regulated by pro-inflammatory cytokines. This might affect the density of cytokine receptors at the beta cell surface and concomitantly the sensitivity of the cells to cytokine toxicity. A better understanding of the functional cross-talk between endocytotic and cytokine signalling pathways could further the development of novel strategies to protect pancreatic beta cells against toxic effects of pro-inflammatory cytokines.Electronic Supplementary Material Electronic supplementary material is available in the online version of this article at Abbreviations CAIN calcineurin inhibitor - EP extension-protected adaptor - Hsp70 heat shock protein 70 - iNOS inducible nitric oxide synthase - MAMA murine adherent macrophage protein - MnSOD manganese superoxide dismutase - NF-B nuclear factor kappa B - NO nitric oxide - PMCA plasma membrane calcium ATPase - Rab3 rab3 GDP/GTP exchange protein - RFDD-PCR restriction fragment differential display PCR - SD standard adaptor - Syt synaptotagmin V - TAP1 transport-associated protein 1     

On the pathogenesis of preleukemic myelodysplastic syndromes

Summary After a single pulse dose of DMBA, rats develop bone-marrow hypoplasia, which is almost compensated for by regeneration after 16 weeks. Subsequently, dysplastic signs of hemopoiesis appear in all experimental animals as massive extrusion of normoblasts into the peripheral blood, red-cell anisoand poikilocytosis, nuclear deformities, atypical mitoses, and PAS-positivity, as well as megaloblastoid maturation dissociation of erythroblasts and nuclear and granulation anomalies of neutrophilic granulocytes and monocytes, comparable to human pseudo-Pelger cells and paraneutrophils. At the time of death (112-497 days after DMBA pulse) experimental animals showed hyperplastic bone marrow with increased granulopoietic/erythropoietic ratios and an augmented, mainly erythropoietic, hemopoiesis in the spleen, with splenomegaly in six rats. Splenic hemopoiesis is accompanied by white pulp atrophia. The cause of death was septicopyemia in three rats, anemia in three, and bleeding in one rat. None of the animals developed a leukemic blast phase. Myelodysplastic changes in this experiment are the same as have been shown to precede leukemia in rats treated with five DMBA pulses (Fohlmeister et al. 1981). Possible relations of myelodysplasia and leukemia are discussed.Supported by Deutsche Forschungsgemeinschaft (DFG)     

Differences of cellular composition and adhesion molecule expression in “leukemic” as compared with “normal” human long-term bone marrow cultures

Summary Human long-term bone marrow cultures (HLTBMCs) were established with bone marrow samples collected from 15 patients with acute myeloid leukemia (AML) and compared with HLTBMCs from eight healthy volunteers. During 6 weeks of culture, the cellular composition of HLTBMCs was quantitatively studied. The cells of the HLTBMCs were divided into three main categories: fibroblasts, macrophages, and other cells (endothelial cells, hematopoietic cells and undefined cells). HLTBMCs derived from healthy volunteers demonstrated a very consistent development. The number of fibroblasts increased during culture and the number of macrophages decreased, resulting in a steady state after 3 weeks of culture. In contrast, HLTBMCs derived from patients with AML showed a strikingly different pattern of irregular development and a steady state was not reached under our conditions. The APAAP technique was used to demonstrate expression of adhesion molecules. VLA2, VLA5, VLA6, LFA1, Mac1, p150/95, ₂-chain, HCAM, ICAM1, NCAM, and VCAM1 were more expressed on normal as compared with leukemic bone marrow stromal cells, although this reached significance only for ₂-chain and NCAM. VLA1, 3, and 4 were expressed in a higher percentage on leukemic stroma (not significant). More expression was seen on normal as opposed to leukemic macrophages for the adhesion molecules tested, except for VLA5. The differences reached significance for the majority of molecules tested. It is concluded that striking differences exist in cellular composition and adhesion molecule expression between HLTBMCs from healthy individuals and those from patients with AML. This may have an impact on the pathogenesis of AML.     

TCR1+ large granular lymphocyte proliferation in rheumatoid arthritis

The T-lymphoproliferative syndrome is characterized by a proliferation of large granular lymphocytes (LGL). It is often associated with neutropenia, and in 30% of cases with rheumatoid arthritis (RA). Phenotypic analysis has demonstrated that in most cases of RA with T-proliferative disease, the LGL represent T cells with a clonal rearrangement of the / T cell receptor (TCR2). Here, three patients with / TCR1⁺ LGL proliferation suffering from long-standing arthritis and neutropenia are described. The first patient with RA showed an expansion of a heterogeneous CD2⁺ CD16⁺ CD56^- LGL population, of which 30% coexpressed TCR1 with V1 rearrangement. The second patient with ankylosing spondylitis and RA was suffering from proliferation of TCR1⁺ (V9^-, V1^-), CD2⁺ CD16^- CD56^- LGL with low coexpression of CD8. The third patient with RA was suffering from a proliferation of TCR1⁺ (V1⁺, V9^-) CD4^- CE8^- CD16^- CD56^- lymphocytes. On the basis of these unusual findings, the pathogenetic role of TCR1⁺ T cells in RA is discussed.     

Temperature and Serum Proinflammatory Cytokine Changes in Patients with NSCLC after BAL

We examined the effects of bronchoalveolar lavage (BAL) and BAL fluid characteristics on the systemic proinflammatory cytokine expression and their relation to clinical and laboratory findings. Thirty patients suspected to have lung cancer were subjected to fiber-optic bronchoscopy (FOB) and BAL. Clinical and laboratory findings were determined at baseline, 4 h, and 24 h, including lung auscultation, temperature, chest X-ray, WBC, neutrophils, and serum IL-1, IL-6, and TNF-. BAL fluid characteristics were determined including cytokine levels. Fifteen volunteers served as controls to determine serum variation of the same cytokines. Significant temperature elevation was defined as 1°C increase compared to baseline. BAL was associated with temperature and serum TNF- and IL-6 but not IL-1 increase at 4 h. Four patients (13.3%) developed temperature over 38°C. In controls there were no significant changes between baseline and 24 h measurements for the same cytokines. Eleven patients (36.6%) developed a significant temperature elevation 4 h after BAL. These patients had a statistically significant (p < 0.05) increase in serum IL-6 at 4 h and in TNF- at both 4 and 24 h after BAL compared with the nonsignificant temperature increase group. BAL characteristics were not different between the two groups. On the other hand, BAL fluid IL-6 and TNF- levels were significantly higher (p < 0.05) in the nonfever group. Significant temperature increase was observed in 36.6% of the patients undergoing BAL and associated with significant serum TNF- and IL-6 increase at 4 h. Lung cytokines levels, alveolar macrophages, and BAL fluid characteristics are not related to temperature and serum proinflammatory cytokine increase. The hypothesis of alveolar macrophages derive from cytokine production and shift to the systemic circulation cannot be supported by our data. Abbreviations: NSCLC = non-small-cell lung carcinoma, BAL = bronchoalveolar lavage, FOB = fiber-optic bronchoscopy, IL-6 = interleukin 6, IL-1 = interleukin 1-beta, TNF- = tumor necrosis factor alpha, WBC = white blood cells, G-CSF = granulocyte colony stimulating factor, IM = intramuscular, ECG = electrocardiogram.     

Plasma immunoreactive glucagon fractions in four cases of glucagonoma: Increased “Large glucagon — immunoreactivity”

Summary Immunoreactive glucagon (IRG) fractions from plasma of 8 normal subjects and 4 patients with glucagon secreting tumors were studied by gel filtration techniques on Bio Gel P-30 and Sephadex G-50 columns. The pancreatic glucagon specific anti serum (30K) of Unger was utilized to measure IRG. Columns were calibrated with labelled albumin, proinsulin, insulin and glucagon. Four peaks were defined in normal and tumor bearing patients: peak I (>20000 mol. wt.), peak II (primarily 9000 mol. wt.), peak III pancreatic glucagon (3500 mol. wt.) and peak IV small glucagon (<3500 mol. wt.). Glucagonoma patients differed from our normal and reported normal subjects in that peak II contained most of the circulating IRG. The percent of IRG associated with peak II was 9.5–31.5% in normals and 39.1–61.2% in glucagonomas. Glucagon-like biological activity in an isolated hepatocyte system was demonstrated for all peaks. However, relative to immunoreactivity, peak II showed reduced activity (25–33%). Immunoassay of dilutions of all peaks revealed the probability of immuno determinants identical with porcine pancreatic glucagon. The presence of heterogeneous IRG peaks with biological glucagon-like activity suggest that the larger molecules may be prohormones. Further, it is possible that specific elevation of peak II may be a diagnostic feature of glucagonomas.Supported in part by NIH Grant #AM 17431-02.     

Characterization of an acute T lymphoblastic leukemia expressing the γ/δ T-cell receptor

Summary Leukemic cells of a 20 year old patient, suffering from acute lymphoblastic leukemia, were characterized by surface marker and functional analysis. A significant cell population within this type of leukemia expresses concomitantly the CD4 and CD8 antigen on the same cell and might represent a new differentiation stage of T-cells with the / receptor. The leukemic cells show a distinct pattern of growth response to mitogens and lymphokines, which might correlate to their differentiation stage. Moreover, a natural killer-like activity can be induced in these cells by IL-2.Abbreviations FITC fluorescein isothiocyanate - PE phycoerythrin - IL-2 interleukin 2; - / TCR gamma/delta T cell receptor - NK natural killer - PBL peripheral blood lymphocytes - T-ALL acute T lymphoblastic leukemia - ConA concanavalin A - PMA phorbol myristate acetate - BM bone marrow - IL-2R IL-2 receptor - TdT terminal deoxynucleotidyl transferase Supported by the Deutsche Forschungsgemeinschaft (DFG Wi-728/3-1)     

Inhibition by 16,16-Dimethyl Prostaglandin E2 of Tumor Necrosis Factor-α and Interleukin-1β Production and Messenger RNA Expression in Human Monocytes Stimulated by Helicobacter pylori

We investigated the effects of 16,16-dimethylprostaglandin E₂ on the production of tumornecrosis factor- and interleukin-1 in humanmonocytes stimulated with Helicobacter pylori. Monocytes isolated from human peripheral blood wereincubated for 24 hr with the extract of H. pyloridiluted 1:100 to 1:100,000 by volume, a combination ofthe extract and 16,16-dimethyl prostaglandinE₂, or a vehicle alone. The extract stimulated theproduction of tumor necrosis factor- andinterleukin-1 and the expression of theirmessenger RNA in a dose-dependent manner. 16,16-Dimethylprostaglandin E₂ inhibited the production of thesecytokines and their messenger RNA in the presence of H.pylori at doses higher than 10^-6 M,predominantly with tumor necrosis factor-. These data suggest that antiinflammatory effects ofprostaglandins on gastric mucosa are in part related totheir effects on inhibition of production ofproinflammatory cytokines by monocytes.     

Impairment of monocyte “lectin-like” receptor activity in Type 1 (insulin-dependent) diabetic patients

Summary In order to investigate whether the ability of peripheral blood monocytes to bind bacteria is impaired in diabetes, we studied carbohydrate-binding (lectin-like) receptors and the receptor for the Fc portion of immunoglobulin on monocytes from 25 male Type 1 (insulin-dependent) diabetic patients and 10 age-matched healthy control subjects. Peripheral blood monocytes from the diabetic patients expressed lower levels of lectin-like receptors compared to the control subjects, whereas the expression of the receptor for the Fc portion of immunoglobulin was similar in both populations. There was no correlation between the degree of lectin-like binding activity and plasma glucose concentration or glycaemic control. Recognition of unopsonized bacteria by the lectin-like receptor is impaired in Type 1 diabetes; this may affect the efficient elimination of potential pathogens.     

Global profiling of coxsackievirus- and cytokine-induced gene expression in human pancreatic islets

Aims/hypothesis It is thought that enterovirus infections initiate or facilitate the pathogenetic processes leading to type 1 diabetes. Exposure of cultured human islets to cytolytic enterovirus strains kills beta cells after a protracted period, suggesting a role for secondary virus-induced factors such as cytokines.Methods To clarify the molecular mechanisms involved in virus-induced beta cell destruction, we analysed the global pattern of gene expression in human islets. After 48 h, RNA was extracted from three independent human islet preparations infected with coxsackievirus B5 or exposed to interleukin 1 (50 U/ml) plus interferon (1,000 U/ml), and gene expression profiles were analysed using Affymetrix HG-U133A gene chips, which enable simultaneous analysis of 22,000 probe sets.Results As many as 13,077 genes were detected in control human islets, and 945 and 1293 single genes were found to be modified by exposure to viral infection and the indicated cytokines, respectively. Four hundred and eighty-four genes were similarly modified by the cytokines and viral infection.Conclusions/interpretation The large number of modified genes observed emphasises the complex responses of human islet cells to agents potentially involved in insulitis. Notably, both cytokines and viral infection significantly (p<0.02) increased the expression of several chemokines, the cytokine IL-15 and the intercellular adhesion molecule ICAM-1, which might contribute to the homing and activation of mononuclear cells in the islets during infection and/or an early autoimmune response. The present results provide novel insights into the molecular mechanisms involved in viral- and cytokine-induced human beta cell dysfunction and death.Electronic Supplementary Material Supplementary material is available for this article at .     

Lazy leukocyte syndrome

Summary A-35-year-old woman with a long-lasting history of neutropenia and recurrent infections was found to have defective neutrophil chemotaxis, random motility, and in vivo migration. Although the bone marrow granulocyte reserve was normal, the patient failed to release an appropriate amount of granulocytes after injection of etiocholanolone. These features are characteristic of the so-called Lazy leukocyte syndrome. The clinical presentation of the five cases of this syndrome so far reported and its pathophysiological aspects are discussed.     

Radiologic appearance of gallstones and its relationship with biliary symptoms and awareness of having gallstones

In the course of two cross-sectional epidemiological surveys carried out by the Rome Group for Epidemiology and Prevention of Cholelithiasis (GREPCO), cholecystography was performed in 82 of 126 subjects identified by means of ultrasonography as having gallstones. In four subjects gallstones were not detected by cholecystography. The x-ray characteristics of the gallbladder and gallstones of the remaining 78 subjects were related to age, sex, presence of biliary symptoms in the five years prior to the study, and awareness of having gallstones. Twenty-three of the 78 gallstone subjects (29.5%) showed a nonvisualized gallbladder. Among the 55 subjects with visualized gallbladder, 16 (29.1%) and 28 (50.9%) showed radiopaque and solitary stones, respectively. The mean diameter of the largest stone was 19.7 mm±11.2 (sd). Age was related inversely to the number of stones. X-ray characteristics of gallstones did not differ between men and women. Presence of biliary symptoms in the five years prior to the study or awareness of having gallstones were not related to any radiologic feature, either in univariate or multivariate statistical analysis which included age, sex, weight, and height as possible confounding variables. Nineteen (24.3%) of the 78 subjects showed gallstones which would have been suitable for medical therapy with bile acids (ie, radiolucent, with a diameter of less than 20 mm, and in a visualized gallbladder).Rome Group for the Epidemiology and Prevention of Cholelithiasis (GREPCO) (For the composition of GREPCO, see appendix).This study was supported by a grant from Ministero della Sanità, No. CS/30/245/3458.     

Serum cytokines in patients with rheumatoid arthritis

Serum cytokines such as interleukin 1 (IL-1), interferon (IFN-), and tumor necrosis factor (TNF) were measured in 40 patients with rheumatoid arthritis (RA). In the 40 patients studied, serum IL-1 was detected in 5 patients, IFN- in 10 patients, and TNF in 20 patients. The IL-1-positive group showed increased values of activity indices compared to the IL-1-negative group. Values of serum IFN- correlated well with the number of peripheral blood lymphocytes and CD3⁺ cells and with the percentage of CD3⁺ CD26⁺ cells. Values of serum TNF correlated positively with the number of peripheral blood monocytes and the percentage of CD3⁺ HLA-DR⁺ and CD3⁺ CD25⁺ cells. These results indicated that serum IL-1 in RA patients reflects the activity of RA, while the serum IFN- and TNF in RA patients may be related to circulating activated lymphocytes and monocytes, respectively.