M. Alvarez-Mon

Mesenteric Th1 polarization and monocyte TNF-α production: First steps to systemic inflammation in rats with cirrhosis†

A systemic inflammatory state with increased circulating tumor necrosis factor alpha (TNF‐α) has been related to the bacterial infection susceptibility and hemodynamic derangement of patients with cirrhosis. We compared the activation status of immune cell subpopulations defined by 4‐color cytometry in mesenteric and peripheral lymph nodes and blood of rats with CCl4‐cirrhosis to define the immune response initiation site, the T‐cell and monocyte contribution to pro‐inflammatory cytokine production, as well as the pathogenic role of enteric bacteria in the cirrhosis immune response. Th1 cells and monocytes were expanded in the mesenteric nodes (P < .001) and blood (P < .001) of rats with cirrhosis, and activated to produce interferon gamma (P < .0001) and TNF‐α (P < .0001), respectively. The greater numbers of recently activated CD134+ Th cells in mesenteric nodes compared with blood, the correlation between their numbers in mesenteric nodes and blood (r = 0.66, P < .001), and the expansion of activated CD45RC− Th cells, which are unable to re‐enter lymph nodes, in mesenteric nodes but not in blood or axillary nodes points to mesenteric nodes as the origin site of activated Th cells. Abrogation of bacterial translocation by bowel decontamination reduced the number of activated Th cells and monocytes, and normalized interferon gamma production by Th cells and TNF‐α production by monocytes in mesenteric nodes and blood, respectively. In conclusion, in cirrhosis, enteric bacteria start off an orchestrated immune response cascade in mesenteric nodes involving Th1 polarization and monocyte activation to TNF‐α production. Later, the recirculation of these activated effector immune cells into blood promotes systemic inflammation. (HEPATOLOGY 2005;42:411–419.)

Treatment With the Immunomodulator AM3 Improves the Health-Related Quality of Life of Patients With COPD

BACKGROUNDnCOPD has a severe impact on patient quality of life. AM3 is an orally effective immunomodulator that can normalize the defective antimicrobial functions of the immune system effector cells of COPD patients.nnnOBJECTIVESnWe analyzed the effect of AM3 on exacerbation frequency and health-related quality of life (HRQL) of COPD patients with moderate disease.nnnDESIGNnA randomized, double-blind, placebo-controlled trial.nnnSETTINGnOutpatient departments of 21 hospitals.nnnMETHODSnA total of 253 COPD patients with a mean age of 67.7 years (SD, 8.1 years) and mean FEV(1) percentage of predicted of 49.6% (SD, 10.2%) were evaluated. Patients received (orally) either 3 g/d AM3 or a matched placebo for 180 consecutive days. Patient quality of life was measured using the St. Georges Respiratory Questionnaire (SGRQ).nnnRESULTSnThere were no differences in the exacerbation frequency of the two groups (0.82 episodes per patient in the AM3 arm vs 0.84 in the placebo arm), and 55.3% of patients were exacerbation free in the AM3 arm compared to 48.8% in the placebo arm (p = 0.11). At the end of treatment, quality of life was significantly better in the AM3 arm than in the placebo arm (SGRQ total score, 32.9; SD, 16.4, compared to 37.5; SD, 17.5 [p < 0.05]: activity score, 47.5; SD, 22.4, compared to 54.6; SD, 20.5 [p < 0.05]). The improvements in total SGRQ scores were 8.9 U (SD, 13.4 U) in the AM3 arm and 5.6 U (SD, 15.9 U) in the placebo arm (p = 0.076). Improvements on the symptoms subscale were 15.9 U (SD, 20.7 U) for the AM3 arm and 10.2 U (SD, 21.3 U) for the placebo arm (p < 0.05). Both AM3 and the placebo were clinically, biochemically, and hematologically well tolerated.nnnCONCLUSIONSnAM3 is a safe, easily tolerated, effective treatment that improves the quality of life of COPD patients as measured by SGRQ scores. This effect was observed with no significant reduction in the frequency of exacerbations.

Loss of lineage antigens is a common feature of apoptotic lymphocytes

The analysis of apoptosis in cell populations involves the detection of their specific lineage antigen (LAg) expression. This experimental approach relies on their assumed constant expression, but it is unclear whether such expression is actually maintained during cell death. We examined whether the loss of LAgs is a common feature of apoptotic lymphocytes and whether some might completely lose their LAgs. The changes in the expression of CD3, CD5, CD8, CD4, CD28, CD56, and CD19 were monitored in highly purified lymphocyte populations obtained by negative selection in a fluorescence‐activated cell sorter. These were cultured for 24 h with or without phytohemagglutinin or staurosporin. For each LAg‐positive subset studied, apoptosis was consistently more common among cells showing partial or total loss of LAg expression compared with cells maintaining their initial LAg levels. The kinetics of expression loss was rapid for CD8, CD56, and CD28, and more than 80% of initial expression was lost in the early stages of apoptosis but was slower for CD3, CD5, and CD4. For CD3 and CD5, expression was dependent on the apoptotic stimulus used. It is interesting that loss of antigen expression was independent of cell size. This phenomenon was also found in nonmanipulated, highly pure CD19 B lymphocytes of peripheral blood mononuclear cells from B chronic lymphocytic leukemia patients. Loss of LAg expression appeared to be a common feature of apoptotic lymphocytes under all the conditions assayed. The different kinetic patterns of LAg loss suggest apoptotic cells might actively regulate this process.

Systemic effects of TNF‐α secreted by circulating monocytes and fatigue in cirrhosis

We read with interest the article by Kerfoot et al. describing a 1.8-fold expansion of circulating monocytes activated to tumor necrosis factor (TNF) production 10 days after bile-duct resection in mice, and the accompanying editorial.1,2 Notably, activated monocytes are recruited into brain tissue by abnormally activated endothelial cells with subsequent activation of resident macrophages to TNFproduction. The authors hypothesize that the TNFdelivered to the brain might contribute to the sickness behavior of cholestasis. We would like to point out that the key finding of Kerfoot’s study, i.e., an expansion in peripheral blood of TNF-secreting monocytes capable of infiltrating somatic tissues,3 is a characteristic shared by human and experimental cirrhosis,4,5 a disease in which fatigue is a prominent feature.6 In rats with CCl4 cirrhosis and patients with cirrhosis, numbers of TNFsecreting monocytes in peripheral blood increase by 45and 26fold, respectively. The marked increase in TNF-secreting monocytes is accompanied by polarization of the T-helper cell compartment toward a Th1 pattern of activation with increased interferonproduction.5 Expansion in the peripheral blood of these abnormally activated immune cells leads to increased serum levels of proinflammatory cytokines, including TNF, interferon, interleukin (IL)-1 , and IL-6.5,7,8 Experimental and clinical data have shown that immune cell activation initiates in the mesenteric lymph nodes and is promoted by gut bacterial translocation.4,5,8 Thereafter, the activated monocytes and T lymphocytes gain access to the peripheral blood by recirculation. Thus, the notion proposed in Kerfoot’s article and the accompanying editorial would need to be explored in cirrhosis to establish the infiltration of brain tissue by activated monocyte/macrophages and their potential role in the pathogenesis of associated fatigue. In addition, we would like to draw attention to the possibility that the marked systemic inflammation demonstrated in cirrhosis may also be involved in the pathogenesis of fatigue, a proposal based on a combination of several known mechanisms including: (1) the combined direct effects on the brain of circulating pro-inflammatory cytokines elevated in cirrhosis, such as TNF, interferon, IL-1 , and IL-6; and (2) the direct skeletal muscle disturbance with increased proteolysis of muscle fibers provoked by TNFalone, or in combination with other cytokines such as interferon.9 Immunological mechanisms underlie the pathogenesis of cholestasis and liver cirrhosis, and their complications including fatigue.1-5,8,10 Their unsuspected role in liver diseases, that were not previously considered immune-mediated, points to immune components as novel targets in translational medicine efforts directed to broaden therapeutic options.

Spontaneously increased B cell growth factor and B cell differentiation factor activities in the synovial fluid of patients with rheumatoid arthritis.

The presence of factors implicated in B cell proliferation and differentiation was studied in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and from patients with ankylosing spondylitis (AS) and traumatic joint injury. Culture with Staphylococcus aureus Cowan I B cell blasts showed strong B cell growth factor (BCGF) activity in the SF from patients with RA. This BCGF activity was significantly greater than that found in SF from patients with traumatic joint injury and similar to that of patients with AS. The presence of B cell differentiation factor (BCDF) for IgM(mu) in the SF from patients with RA was also demonstrated and was significantly greater than that found in SF from patients with AS and traumatic joint injury. Moreover, a significantly increased BCDF for IgG(gamma) was also found in the SF from patients with RA compared with that observed in those patients with traumatic joint injury, which, however, was similar to that of patients with AS.

609 SMALL INTESTINE INFLAMMATION IN RATS WITH CCL4 CIRRHOSIS: ROLE OF ENTERIC BACTERIA

macrophages during conditions of excess iron by blocking the iron exporter ferroportin. In addition, IL-6 mediated upregulation of hepcidin during inflammation is the major mechanism of the socalled ‘anemia of chronic disease’. We here identify H2O2 which is co-released by inflammatory cells as potent transcriptional activator of hepcidin independent of upstream regulators IL-6 and BMP6. Material andmethods: H2O2 release (1–8mM) by immune cells was mimicked using purified enzymes glucose oxidase and catalase as recently described (GOX/CAT system). Huh7 hepatoma cells were treated with either IL-6 or BMP6 alone, or in combination with steady state H2O2 over 24h. Hepcidin regulation was assessed by quantitative real time PCR. Members of the intracellular signaling cascade such as STAT3 were assessed by Western blotting. Promotor studies were performed using hepcidin-promoter constructs with various deletions fused to luciferase as reporter gene with Renilla as control reporter gene. Results: Steady state non-toxic H2O2 concentrations comparable to those by inflammatory cells rapidly and drastically upregulate hepcidin in a dose-dependent way in Huh7 cells by a factor of 10. In addition, H2O2 further potentiates IL6 and BMP6 mediated upregulation of hepcidin 5-fold. Interestingly, induction was also not blunted by confounding hypoxia (2%). The H2O2mediated hepcidin response is an early response reaching the maximum mRNA-concentration already after 6h. Promoter studies identified the STAT3 element as major promoter region of H2O2 mediated hepcidin induction. Indeed, phosphorylation of STAT3 was confirmed under these conditions using Western blotting. Conclusions: Our studies establish H2O2 as as an important regulatory link between systemic iron regulation and oxidative stress. Impaired H2O2-hepcidin signaling in chronic liver diseases could contribute to hepatic accumulation of iron in cirrhotic patients.

528 THE DEFECTIVE FUNCTION OF GUT DENDRITIC CELLS OF RATS WITH CIRRHOSIS AND BACTERIAL TRANSLOCATION IS CORRECTED BY BOWEL DECONTAMINATION WITH ANTIBIOTICS

528 THE DEFECTIVE FUNCTION OF GUT DENDRITIC CELLS OF RATS WITH CIRRHOSIS AND BACTERIAL TRANSLOCATION IS CORRECTED BY BOWEL DECONTAMINATION WITH ANTIBIOTICS L. Munoz, M. Ubeda, M.J. Borrero, M. Lario, R. Frances, D. Diaz, J. Monserrat, L. Lledo, J. Such, M. Alvarez-Mon, A. Albillos. University of Alcala, Ciberehd, Alcala de Henares, Madrid, Hospital General Universitario de Alicante, Ciberhed, Alicante, Hospital Universitario Pŕincipe de Asturias, University of Alcala, Ciberehd, Alcala de Henares, Hospital Universitario Ramon y Cajal, University of Alcala, Ciberehd, Madrid, Spain E-mail: leticia.munoz@uah.es

Pattern of restoration of the immune alterations by the treatment with high doses of Interferon alpha in high risk melanoma patients

7534 Background: IFNa is currently use in the adjuvant treatment of patients with high risk of melanoma. The mechanism of action of IFNa is complex. Since it, IFNa is able to modulate different regulatory and effectors cells of the immune system. Therefore, the optimization of the clinical use of IFNa requires the analysis of the biological effects of the biotherapy. Herein, we have investigated the effects of high doses of IFNa in the expression of chemokine receptors by peripheral blood (PB) lymphocytes and the spontaneous cytotoxic (NK) activity of PB mononuclear cells (PBMC) in patients with high risk melanoma.nnnMETHODSn15 patients with melanoma stage III were treated with intravenous IFNa 20MU/m2 5 days per week for 4 weeks follow by intramuscular IFNa 10 MU/m2 3 days per weeks for 48 weeks. PBMC were obtained by venous puncture before the first dose of IFNa, every last day of the first 4 weeks of treatment, and at 3, 6, 9 and 12 months of the maintenance treatment. The expression of differentiation antigens and chemokine receptors were measured by direct surface immunofluorescence and flowcytometry. NK activity was measured by 51Cr release assay using as targets K562 cells. 15 age and sex matched healthy donors were use as controls.nnnRESULTSnBefore the biotherapy, melanoma patients showed significantly reduced expression of CXCR4 and CCR2 on the surface of CD19+ B cells (P<0.05), as well as reduced CXCR4+CD3+ T cells and CD16+CD3- NK cells (P<0.05). The expression of CXCR4 by B, T and NK cells was normalized, as compared to controls, after 4 weeks of treatment. The normalization of the expression of CCR2 in the latter lymphocyte subsets was achieved only after 3 months of treatment. NK cytotoxic activity in PBMC remained significant decreased during the first 9 months but normalization occurred after 12 months of treatment.nnnCONCLUSIONSnThis study demonstrates that high doses of IFNa promote clear immunomodulatory effects in high risk melanoma patients. Notably, the normalization of immunological parameters is sequential and beneficial accumulative effects are evident after 9 months which provides a rational to recommend maintenance therapy. No significant financial relationships to disclose.