Fish oil supplementation with various lipid emulsions suppresses in vitro cytokine release in home parenteral nutrition patients: a crossover study.

Long-chain n-3 polyunsaturated fatty acids modulate immune cell functions. The primary objective of this study was to evaluate the impact of different lipid emulsions (LEs) with supplemented doses of fish oil (FO) on serum cytokine concentration and in vitro cytokine production in patients with intestinal failure on home parenteral nutrition (HPNPs). We hypothesized that FO supplementation would diminish lipopolysaccharide (LPS)-stimulated cytokine production. Twelve HPNPs receiving Smoflipid for at least 3 months were given FO (Omegaven) for a further 4 weeks. After this cycle, the patients were randomized to subsequently receive 1 cycle with Lipoplus and 1 cycle with ClinOleic for 6 weeks or vice versa plus 4 weeks of added Omegaven after each cycle in a crossover design. Comparison of the baseline LE regimens showed lower LPS-stimulated production of IL-1ß in the HPNPs on Lipoplus than on the Smoflipid and ClinOleic regimens, as well as lower IL-8 compared to the Smoflipid regimen. Omegaven reduced IL-8 concentration in serum under the Lipoplus regimen and diminished LPS-stimulated production of IL-1ß under the Smoflipid and ClinOleic. IL-6 and TNF-α production was depressed only in those on Smoflipid. Irrespective of the LE used, the HPNPs compared to the healthy controls showed higher IL-6, IL-8, and TNF-α concentrations in serum and LPS-stimulated production of IL-6 as well as lower n-6/n-3 long-chain polyunsaturated fatty acids in the erythrocyte phospholipids. LPS-stimulated production of IL-6 correlated negatively with the parenteral dose of eicosapentaenoic acid + docosahexaenoic acid. In conclusion, FO-supplemented parenteral nutrition suppresses in vitro cytokine production.

The evaluation of survival and proliferation of lymphocytes in autologous mixed leukocyte reaction with dendritic cells. The comparison of incorporation of (3)H-thymidine and differential gating method.

Dendritic cells (DCs) play the key role in T-lymphocyte proliferation and induction of antitumour response. The mixed leukocyte reaction (MLR) of T-lymphocytes and DCs is essential instrument for immunological mechanisms studies. Conventionally used method for determination of T-lymphocytes proliferation, (3)H-thymidine incorporation, provides only general information. The method of flow cytometry and differential gating seems to be more suitable for quantitative and qualitative analysis of T-lymphocyte proliferation. It is based on time limited acquisition of events and on its distribution according to forward and side scatter values. We decided to compare these two methods and determine mutual correlation and compatibility. Eleven patients were studied and in all cases DCs promoted the survival and proliferation of both CD4 and CD8 lymphocytes. Both methods retained consistency with regard to survival and proliferation of CD4/CD8 lymphocytes. However, the correlation of these methods was not convincing. Therefore, both these methods might be used for evaluation of MLR, but each of them gives specific and complementary information.

Defective cytotoxicity of T lymphocytes in myelodysplastic syndrome.

OBJECTIVE: The capacity of mononuclear blood cells to form autoreactive cytotoxic T lymphocytes was investigated in order to elucidate the mechanism of successful immunosuppressive therapy in some myelodysplastic syndrome (MDS) patients (autoreactivity studies). The failure in autoreactivity studies raised the question of alloreactive cytotoxic T lymphocyte formation in MDS (alloreactivity studies). MATERIALS AND METHODS: Sixteen MDS patients and relevant controls were examined. Autoreactive lymphocytes directed against autologous bone marrow mononuclear cells and alloreactive lymphocytes directed against unrelated third-party cells were tested using cytotoxicity assay. In addition, we used one-way mixed lymphocyte reaction, human androgen receptor test for clonality detection, and enzyme-linked immunosorbent assay kits for tumor necrosis factor and interferon-gamma testing. RESULTS: We did not confirm the presence of autoreactive T cells in eight of nine MDS patients tested. The response to allogeneic cells was impaired in 11 of 16 MDS patients, more often in refractory anemia (RA; 80%) than in RA with ring sideroblasts (40%). Interestingly, the response to allogeneic cells in mixed lymphocyte reaction was normal in all MDS patients. T lymphocytes were polyclonal in all but one patient. Tumor necrosis factor and interferon-gamma level in supernatants of mononuclear cells was significantly reduced in RA. CONCLUSION: The presumed autoaggressive T cells were not confirmed in MDS in our experimental arrangement. Alloreactivity studies demonstrated the impairment of effector cytotoxic phase of cell-mediated immunological reaction in MDS, namely in RA. The significance of our finding of defective cytotoxicity for pathogenesis, clinical course, and even for therapy is discussed together with other immunological defects reported so far.

TRAIL-induced apoptosis of HL60 leukemia cells: two distinct phenotypes of acquired TRAIL resistance that are accompanied with resistance to TNFalpha but not to idarubicin and cytarabine.

TNF-related apoptosis-inducing ligand (TRAIL) is a proapoptotic cytokine implicated in cancer cell surveillance. A potential of TRAIL as a cancer-specific therapeutic agent has been proposed, either as a single agent or in combination with chemotherapy. Prolonged exposure of TRAIL-sensitive leukemia cell line, wild-type (WT) HL60 cells to recombinant soluble TRAIL or to cytostatic agents, cytarabine and idarubicin, resulted in the establishment of resistant subclones with distinct phenotypic features. The TRAIL resistant HL60 subclones were characterized by decreased expression of TRAIL and TNFalpha death receptors. These resistant subclones had impaired activation of caspases 8 and 10 in response to TRAIL and TNFalpha, decreased TRAIL-induced nuclear translocation of NFkappaB RelA/p65, and dysregulation of the expression of several apoptosis regulators. Among the TRAIL resistant HL60 subclones we identified two separate phenotypes that differed in the expression of CD14, osteoprotegerin, and several apoptosis regulators. Both these TRAIL resistant HL60 subclones were resistant to TNFalpha, suggesting disruption of the extrinsic apoptotic pathway, but not to cytostatic agents, cytarabine and idarubicin. The concurrently derived HL60 subclones were cytarabine and idarubicin-resistant but remained sensitive to TRAIL-induced apoptosis. We identified distinct pathways for the development of HL60 leukemia cell resistance to apoptosis induction. These findings are relevant for the design of more effective strategies for leukemia therapy.

Extracorporeal photochemotherapy (ECP) in treatment of patients with c-GVHD and CTCL.

We evaluated the immunomodulatory and clinical effect of 279 extracorporeal photochemotherapy (ECP) procedures which were performed in six patients with chronic extensive GVHD and in two patients with CTCL (cutaneous T-cell lymphoma)/Mycosis fungoides. ECP was performed using the off line regimen. In five of six patients with c-GVHD the improvement of sclerodermatous skin changes, joint mobility, and the reduction of joint pain was observed. Two patients with CTCL responded to ECP with rapid improvement of the skin changes. In patients with c-GVHD and CTCL who responded to ECP efficiently, we found the similar tendency to increase in the number of CD 3/8+ T-lymphocytes and the decrease of CD 4/8 IRI. In patients with CTCL we observed also the decrease in levels of CD 3/4+ T-lymphocytes and in the number of leukocytes. The influence of ECP on T-cell subsets and on the dendritic cells function, which we observed in our previous study, leads to the suggestion that interactions between T-cell subsets and dendritic cells may participate in the process of ECP. ECP did not cause any significant changes in levels of IgG, parameters of liver and renal functions in patients with c-GVHD and with CTCL. No increased incidence of infections and no serious adverse reactions in patients have been observed so far.

Translational efficiency in patients with Diamond-Blackfan anemia.

BACKGROUND AND OBJECTIVES: Diamond-Blackfan anemia (DBA) is a rare congenital pure red cell aplasia characterized by normochromic macrocytic anemia, reticulocytopenia, and normocellular bone marrow with a selective deficiency of erythroid precursors. Ribosomal protein S19 (RPS19), currently the only gene associated with DBA, is mutated in 25% of DBA patients, but its role in erythropoiesis is unknown. We attempted to elucidate the importance of RPS19 in translation in relation to the pathogenesis of DBA. DESIGN AND METHODS: We measured translation and proliferation rates in unstimulated and phytohemagglutinin (PHA)-stimulated lymphocytes isolated from DBA patients, as well as in K562 cells expressing several RPS19 mutants to directly test the effect of RPS19 mutations on translation. The effect of leucine on overall translation was also studied. RESULTS: We found that the level of translation was on average 48-73% of controls in both unstimulated and PHA-activated DBA lymphocytes irrespective of mutations in RPS19. The addition of leucine increased the translational level in RPS19-non-mutated DBA cells, but not in cells with an RPS19 mutation. In unstimulated DBA cells, proliferation was significantly impaired in both RPS19-mutated and non-mutated cells, but in both groups could be efficiently activated by PHA. Studies on K562 cells showed that RPS19 mutations affecting RPS19 conserved arginines R56Q and R62Q could significantly inhibit the rate of protein synthesis, indicating the importance of RPS19 in translation. INTERPRETATION AND CONCLUSIONS: Our results indicate that inefficient translation may be the main cause of DBA, and administration of leucine may be beneficial for at least some DBA patients.