Interleukin 16 expression and phenotype of interleukin 16 producing cells in Crohn's disease.

BACKGROUND: The mechanisms involved in the initiation and maintenance of Crohn's disease are poorly understood. Previous studies have demonstrated an increased number of infiltrating CD4+ T cells within the inflammatory affected bowel wall in Crohn's disease. Novel therapy approaches using anti-CD4 antibodies are thought to be effective in Crohn's disease. AIMS: Interleukin 16 (IL-16) has been characterised as a chemokine with selective chemoattraction for CD4+ inflammatory T cells. In this study, cellular expression of IL-16 in Crohn's disease and ulcerative colitis was investigated. METHODS: Expression of IL-16 was analysed in tissue samples of Crohn's disease, ulcerative colitis, and normal controls by applying reverse transcription-polymerase chain reaction, non-radioactive in situ hybridisation, and immunohistochemistry. Double staining methods were used to characterise cells expressing IL-16. The amount of infiltrating CD4+ cells was determined by immunohistochemistry and correlated with the corresponding IL-16+ cell number by step sections. RESULTS: An increased number of IL-16+ cells in Crohn's disease in comparison with ulcerative colitis and control probes was demonstrated. IL-16 was expressed by CD4 and CD8 positive T cells. In addition, in active Crohn's disease there was a substantial number of IL-16 positive mast cells. The increased number of CD4+ lymphocytes correlated positively with the increased number of IL-16 positive cells in Crohn's disease. CONCLUSION: Our results demonstrate that increased expression of IL-16 in T cells and mast cells in active Crohn's disease is associated with increased numbers of CD4+ lymphocytes. Local expression of IL-16 seems to play a significant role in the initiation and persistence of the inflammatory process in Crohn's disease, presumably by IL-16 mediated recruitment of CD4+ cells, mostly lymphocytes, into the bowel wall.

Expression of the T-cell chemoattractant chemokine lymphotactin in Crohn's disease.

Recruitment of lymphocytes is a prominent feature of the inflammatory process in Crohn's disease (CD). The present study was undertaken to investigate the expression of the novel lymphocyte-specific chemoattractant lymphotactin (Lptn) as a potential regulatory factor for the recruitment of T cells in CD. The expression of Lptn mRNA was quantified in resection specimens of patients with CD in comparison to normal controls without signs of inflammation by real-time quantitative reverse transcriptase-polymerase chain reaction and localized by nonradioactive in situ hybridization. Furthermore, the phenotype of cells expressing Lptn mRNA was characterized. In contrast to normal controls Lptn mRNA was significantly increased in tissue samples affected by CD. Cells expressing Lptn were identified as T cells, mast cells, and unexpectedly dendritic cells. Lptn mRNA was found to be up-regulated on stimulation with phorbol-12-myristate-13-acetate and concanavalin A in T cells isolated from peripheral blood, which could be prevented by dexamethasone, cyclosporine A, and FK506. A similar regulation mechanism could be identified for the Lptn receptor GPR-5 in peripheral T cells. In addition, Lptn mRNA expression could be induced in mature monocyte-derived dendritic cells. The results indicate that local expression of Lptn by activated T cells and to a lesser extent by mast cells and dendritic cells represents a key regulator for lymphocyte trafficking and maintenance of the inflammatory process observed in CD, which might be partly mediated through an autocrine/paracrine pathway of activated T cells.

In vitro reactivity of cryoglobulin IgM and IgG in hepatitis C virus-associated mixed cryoglobulinemia.

BACKGROUND/AIMS: Mixed cryoglobulinemia is frequently associated with chronic hepatitis C virus infection. We aimed to clarify the mechanism, kinetics and participating proteins in cryoprecipitate formation, which are still being debated. METHODS: Eighteen patients with cryoglobulinemia were studied. Isolated serum cryoprecipitates and purified cryoglobulin IgM and IgG fractions were analyzed in vitro by turbidimetry for temperature-dependent complex formation. Immunoglobulin reactivity, i.e. in cryoprecipitates and in cryoglobulin-free sera, was studied using immunoblot and enzyme immunoassays. HCV RNA was detected by reverse transcriptase/polymerase chain reaction. RESULTS: By turbidimetry, purified cryo-IgM precipitated (in the absence of HCV RNA) with cryo-IgG as well as with non-cryoglobulin IgG and with IgG Fc or F(ab')2 fragments. In contrast, purified cryo-IgG did not precipitate with non-cryoglobulin IgM. Anti-HCV IgG reactivity was found in cryoglobulin-free sera, in cryoprecipitates and in purified cryoglobulin IgG fractions. The respective titers were similar. Purified cryo-IgM did not react to HCV-encoded proteins. Binding of cryo-IgM to heterologous IgG was inhibited by intact IgG (up to a mean of about 52%) as well as by IgG Fc (33%) and F(ab')2 fragments (17%). Binding of cryo-IgM to IgG was enhanced at low temperature (4 degrees C vs. 37 degrees C), particularly for type III cryoglobulin IgM. CONCLUSIONS: In hepatitis C virus-associated cryoglobulinemia the in vitro precipitate formation depended on cryo-IgM, while IgG appeared to act as an unspecific antigenic partner. Hepatitis C viral particles were probably not required. Cryo-IgM binding occurred primarily to intact IgG. Anti-HCV reactivity of either cryo-IgM or cryo-IgG was not necessary for precipitate formation. Regarding the pathogenesis, a direct hepatitis C virus protein-dependent stimulation of B-cells producing cryo-IgM seems to be unlikely.

Interferon-alpha treatment of hepatitis C virus-associated mixed cryoglobulinemia.

BACKGROUND/AIMS: Chronic hepatitis C virus infection is frequently associated with mixed cryoglobulinemia. The efficacy of interferon-alpha treatment in the presence of cryoglobulinemia, particularly the rate of sustained responders, has not yet been well defined. METHODS: Fifty-nine consecutive patients with chronic HCV infection were studied prospectively with regard to the presence of cryoglobulinemia and their biochemical and virological response to interferon-alpha2a therapy. RESULTS: Cryoglobulins were detected in sera of 23 patients. For this latter group of patients, significant differences were found compared to the 36 patients without cryoglobulinemia, i.e. the prevalence of female sex was higher, the duration of liver disease was longer and distinctive laboratory abnormalities, e.g. higher rheumatoid factor activity, were noted as well as a higher prevalence of cirrhosis. The distribution of HCV genotypes and serum HCV RNA titers was similar in the two groups. Interferon-alpha treatment regimens were not different regarding mean cumulative dose and mean duration of therapy. The response to therapy was almost identical, i.e. 35% of patients with cryoglobulinemia showed a sustained response compared to 22% of patients without cryoglobulinemia. The percentages of patients showing a relapse or breakthrough were similar in both groups. Pre-treatment viremia levels were higher in non-responders compared to sustained responders. Non-responders appeared to be more frequent among patients infected with genotypes 1a and 1b, especially among male patients without cryoglobulinemia. CONCLUSIONS: The presence of cryoglobulinemia per se in chronic HCV-infected patients does not adversely affect the outcome of interferon-alpha therapy, including the rate of sustained response.

Increased intercellular adhesion molecule-1 serum concentration in cholestasis.

BACKGROUND/AIMS: Increase of serum levels of the soluble intercellular adhesion molecules in patients with the cholestatic liver diseases primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are known and have been thought to indicate activation of the immune system and the grade of the inflammatory process. In hepatitis and cholestatic diseases, expression of adhesion molecules was found on the surface of bile duct epithelia and hepatocytes. MATERIALS AND METHODS: Serum levels of sICAM-1 in patients with intrahepatic cholestasis in PBC (n = 42) and extrahepatic cholestasis (n = 18) due to choledocholithiasis were investigated. sICAM-1 levels and "classical" cholestasis parameters as alkaline phosphatase (ALP), gamma-glutamyl-transpeptidase (gamma-GTP) and bilirubin levels were compared. Furthermore, sICAM-1 concentrations and "classical" cholestasis parameters were analysed before and after therapy with ursodeoxycholic acid (UDCA). In addition, sICAM-1 was detected in serum and bile fluid of four patients with cholestasis due to choledocholithiasis. Soluble ICAM-1 levels in sera and, if accessible, in bile fluids were determined using a commercially available ELISA system. Statistics were done by Wilcoxon's signed rank exact test and Spearman's rank correlation test. Sensitivity and specificity of cholestasis parameters and sICAM-1 concentrations was analysed by receiver operating characteristic (ROC) curves. RESULTS: Increased sICAM-1 serum concentrations in a similar range were found in patients with PBC (range 251-2620 micrograms/l; median 966 micrograms/l) as well as in patients with extrahepatic cholestasis (257-2961 micrograms/l; median 760 micrograms/l) compared to healthy controls (n = 12; 220-500 micrograms/l; median 318 micrograms/l). sICAM-1 levels correlated significantly to histological stage I to IV (p < 0.001), ALP (range 107-1877 U/l; median 545 U/l; r = 0.496, p = 0.0008), bilirubin (range 0.3-26 mg/dl; median 0.8 mg/dl; r = 0.52; p < 0.0004) and gamma-GTP levels (range 43-705 U/l; median 221 U/l; r = 0.36; p = 0.02) in PBC patients. In PBC patients a histological stage III or IV (n = 21) could be predicted with high sensitivity (95%) and specificity (85%) if sICAM-1 levels were above 840 micrograms/l. After treatment of PBC patients with UDCA, sICAM-1 levels decreased significantly with decline of other "classical" cholestasis parameters. Increased sICAM-1 levels (range 257-2961, median 745 micrograms/l) in extrahepatic cholestasis correlated also significantly with serum concentrations of bilirubin (r = 0.8; p < 0.01; range 0.3-19.7, median 1.6 mg/dl), gamma-GTP (r = 0.55; p = 0.03; range 33-1401, median 179 U/l) and ALP (r = 0.61; p = 0.1; range 110-1378, median 562 U/l). sICAM-1 was detectable in bile fluid (264-919 micrograms/l) of four patients with extrahepatic cholestasis and nose-biliary catheterisation. CONCLUSIONS: sICAM-1 concentrations were found to discriminate between histological stage I/II and stage III/IV of PBC with higher sensitivity and specificity than "classical" cholestasis parameters. Increased serum concentrations for sICAM-1 in intra- and in extrahepatic cholestasis and detection of sICAM-1 in the bile may indicate that sICAM-1 is eliminated through the bile. In other words, not only increased synthesis but also decreased elimination may be responsible for increased sICAM-1 serum levels in patients with cholestatic liver diseases.

Recurrent episodes of acute hepatitis associated with LKM-1 (cytochrome P450 2D6) antibodies in identical twin brothers.

BACKGROUND/AIMS: Liver/kidney microsomal antibodies have been noted in liver disease of different etiology, e.g. in autoimmune hepatitis, chronic hepatitis C and D virus infection and in drug-induced liver disease. Unlike these, acute hepatitis of unknown etiology associated with high-titer liver/kidney microsomal-1 antibodies (cytochrome P450 2D6) is reported in identical twin brothers. METHODS: Patients were studied using clinical, biochemical, serological and immunological methods, as well as liver biopsy. RESULTS: The acute icteric episodes were followed by spontaneous remission with complete normalization of liver function tests and liver histology. During the acute phase, serum titer for liver/kidney microsomal-1 antibodies (detected by indirect immunofluorescence, ELISA and Western blot analysis) was exceedingly high and decreased gradually thereafter. Hepatitis C and D virus infection were excluded by repeated serological testing; exposure to drugs or chemicals was not evident. Concomitant autoimmune disease was not detectable. HLA typing for class 1 and 2 antigens was positive for the HLA haplotype DQ2, but negative for HLA B4, B8, DR3 and DR4. CONCLUSIONS: The present observations might suggest a hitherto unreported form of acute hepatitis of unknown etiology, distinct from other liver diseases in which liver/kidney microsomal antibodies have been described so far.

Cryoglobulinemia in chronic hepatitis C virus infection: prevalence, clinical manifestations, response to interferon treatment and analysis of cryoprecipitates.

Chronic hepatitis C virus infection can be associated with mixed cryoglobulinemia and systemic vasculitis. The pathogenesis remains poorly understood. 55 consecutive patients with chronic HCI infection (anti-HCV- and serum HCV RNA-positive) were studies prospectively. Cryoglobulinemia was detected in 28 patients (51%) with a mean cryocrit level of 2.2%. Clinical symptoms of vasculitis were encountered in six patients. Compared to those HCV-infected patients without cryoglobulinemia the following distinctive features were observed in the presence of cryoglobulinemia: increased age (p<0.02), female preponderance (p<0.002), longer-lasting HCV infection (mean of 10.7 vs. 4.7 yrs), higher prevalence of cirrhosis (42.8 vs. 0%), increased serum concentration of IgM and increased rheumatoid factor activity, decreased concentration of serum C4 (each p<0.05). The response to interferon treatment was similar in patients with and without cryoglobulinemia. When cryoprecipitates were analyzed by immunofixation, type II cryoglobulinemia was present in 1/3 and type III in 2/3 of patients. By SDS-PAGE four different proteins were demonstrable in cryoprecipitates each identified by immunoblotting as IgG and IgM heavy or light chains respectively. Cryoprecipitate IgGs were shown to react with HCV structural as well a non-structural proteins in a recombinant immunoblotting assay (RIBA). In contrast, cryoprecipitate IgMs reacted only to the HCV core protein c22-3. HCV RNA was detected in cryoprecipitates without a significant enrichment when compared to the corresponding serum or supernatant HCV RNA content. Given the monoclonality of some cryoprecipitate IgM and their reactivity to HCV core, a cross-reactivity to IgG was postulated. In fact, when performing a computer-assisted search for sequence homology, a motif within the core protein (EGLGWAGWL, conserved in HCV genotypes) was identified homologous to a sequence of IgG heavy chains. Thus, temperature-dependent affinity changes of IgM anti-HCV core (nonapeptide) and ensuing complex formation with IgG via binding to the homologous IgG sequence could be a mechanism of cryoprecipitate formation.

Influence of gender on the monoethylglycinexylidide test in normal subjects and liver donors.

The objective of this study was to investigate the effect of gender on monoethylglycinexylidide (MEGX) formation in normal subjects and cadaveric liver donors. The study included 92 male and female healthy volunteers < 45 years of age and 98 age- and sex-matched liver donors from a previous study, whose livers were used for transplantation. Women < 45 years not taking contraceptives showed significantly lower MEGX concentrations 30 min after lidocaine administration than men [median (16-84th percentile)]: 59 micrograms/L (41-70 micrograms/L) versus 81 micrograms/L (58-98 micrograms/L)]. The lowest MEGX 30 min values were observed in women taking contraceptives: 39 micrograms/L (25-48 micrograms/L). Intraindividual variability of serial MEGX tests was moderate (median: 17.8%, n = 8) when measured in female subjects taking no contraceptives and males. Cadaveric liver donors showed significantly higher MEGX 15 and 30 min values compared with normal subjects (p < or = 0.0001). There was no statistically significant difference between MEGX values obtained in male and female cadaveric donors. The urinary excretion of MEGX was similar in male and female normal subjects. Our results suggest that sex-related differences in MEGX formation as well as the influence of contraceptives have to be taken into account when test results from living related liver donors and patients with less advanced chronic liver disease are evaluated. In cadaveric liver donors, however, sex-related differences do not affect MEGX formation.

Biochemical and immunological characterization of micellar complexes of the envelope glycoprotein of a simian immunodeficiency virus isolated from an African green monkey.

The external envelope glycoprotein gp130 of a simian immunodeficiency virus isolated from an African green monkey (SIVagmTYO-7) was purified as micellar complexes. The molecular weight of the gp130 micelles was about 700 K. On electron microscopy, the micelles appeared as spherical particles with a diameter of 15 to 20 nm. Such aggregates consisted of about 4 to 5 gp130 monomers. Hyperimmune sera raised in rabbits and rhesus monkeys against these gp130 micelles exhibited titers between 10(5) and 10(6). Such sera inhibit the CD4 binding of gp130 and neutralize SIVagmTYO-7 and SIVmac251 but not HIV-2ben.

CD4-binding of gp130 micelles isolated from SIVagmTYO-7.

The binding and binding inhibition of the SIVagmTYO-7 external glycoprotein gp130 in micellar form to the CD4 molecule on human Molt-4 clone 8 cells was investigated. The best binding of gp130 to Molt-4 clone 8 cells occurred at pH 5.5 to 6.5 at 37 degrees C after 4 h or at room temperature after 10 h. The dissociation constant of this reaction was 0.2-0.4 nM, with both soluble CD4 or CD4 on Molt-4 clone 8 cells. This value is close to 0.15 nM determined for the antihuman CD4 monoclonal antibody 30F16H5. After partial deglycosylation of gp130, a 90 kD product arose which still bound to CD4. Fully deglycosylated gp130 of 60 kD was still immunoprecipitable, but had lost the CD4 binding activity. Lens culinaris agglutinin was able to inhibit the gp130-CD4 interaction very efficiently, while the agglutinin of Phaseolus vulgaris was half as efficient and Canavalia ensiformis was inefficient. CD4 binding of gp130 micelles was also inhibited with several anti CD4 monoclonal antibodies directed against the OKT4a epitope as well as with soluble recombinant CD4.