Nitric oxide affects immune cells bioenergetics: long-term effects of nitric-oxide derivatives on leukaemic Jurkat cell metabolism.

Major advances in dissecting mechanisms of NO-induced down-regulation of the anti-tumour specific T-cell function have been accomplished during the last decade. In this work, we studied the effects of a NO donor (AT38) on leukaemic Jurkat cell bioenergetics. Culturing Jurkat cells in the presence of AT38 triggered irreversible inhibition of cell respiration, led to the depletion of 50% of the intracellular ATP content and induced the arrest of cell proliferation and the loss of cell viability. Although a deterioration of the overall metabolic activity has been observed, glycolysis was stimulated, as revealed by the increase of glucose uptake and lactate accumulation rates as well as by the up-regulation of GLUT-1 and PFK-1 mRNA levels. In the presence of NO, cell ATP was rapidly consumed by energy-requiring apoptosis mechanisms; under a glucose concentration of about 12.7mM, cell death was switched from apoptosis into necrosis. Exposure of Jurkat cells to DMSO (1%, v/v), SA and AT55, the non-NO releasing moiety of AT38, failed to modulate neither cell proliferation nor bioenergetics. Thus, as for all NSAIDs, beneficial effects of AT38 on tumour regression are accompanied by the suppression of the immune system. We then showed that pre-treating Jurkat cells with low concentration of cyclosporine A, a blocker of the mitochondrial transition pore, attenuates AT38-induced inhibition of cell proliferation and suppresses cell death. Finally, we have studied and compared the effects of nitrite and nitrate on Jurkat cells to those of NO and we are providing evidence that nitrate, which is considered as a biologically inert anion, has a concentration and time-dependent immunosuppressive potential.

The tissue response to photopolymerized PEG-p(HPMAm-lactate)-based hydrogels.

Hydrogels are three-dimensional networks of crosslinked hydrophilic polymers widely used for protein delivery and tissue engineering. To be eligible for in vivo applications, the hydrogels should not evoke an adverse tissue response. In this study the angiogenic and inflammatory responses in vivo after implantation of photopolymerized thermosensitive poly(hydroxypropyl methacrylamide lactate)-poly(ethyl copolymer hydrogels are investigated. Hydrogels consisting of polymers with different crosslink densities were subcutaneously implanted in Balb/c mice and histological evaluation of the tissue response was performed. The implants showed an acute and localized inflammatory reaction upon implantation, mainly characterized by a strong infiltration of granulocytes. The acute inflammatory reaction was followed by a milder chronic inflammation which was characterized by infiltration of macrophages and persistent but decreasing levels of granulocytes. The number of macrophages and blood vessels was associated with the biodegradation and resorption of the biomaterial and increased in time as the degradation of the materials progressed. The observed degradation rates in vivo correlated well with previously observed in vitro degradation rates, which suggests that hydrolysis is the main mechanism governing the degradation.

Immune responses during and after exercise of constant and alternating intensity above the lactate threshold.

AIM: Intense and prolonged exercise greatly affects circulating cytokine levels. The purpose of this study was to investigate the possible changes in tumour necrosis factor -a (TNF-a), interleukin 6 (IL-6) and cortisol concentrations during and after prolonged exercise of constant and alternating intensity of the same duration and total work performed. METHODS: Ten male subjects underwent two main cycling exercise trials lasting one hour each. On one occasion, exercise intensity was alternated between 46.5±1.9% of maximal oxygen uptake (VO2max ) for 40 s and 120% of VO2max for 20 s, so that the mean intensity corresponded to 105% of the lactate threshold. On the other occasion, exercise intensity was constant at 105% of the lactate threshold. Levels of TNF-a, IL-6 after lipo polysaccharide (LPS) stimulation as well as cortisol were measured at rest, 30 and 60 minutes of exercise and 1 hour after. RESULTS: No significant differences were observed in TNF-a concentrations between the two exercise protocols (P= 0.75), but there was a significant time effect (P<0.01). TNF-a was increased in both groups from a resting value of 436.1±102.5 to 649.5±187.7 pg/mL (P<0.05) at the end of exercise and was subsequently decreased 1 hour post exercise to 305.9±78.8 pg/mL (P<0.01). No significant difference in IL-6 and cortisol concentrations was observed between the two exercise protocols (P=0.13, P=0.10 accordingly). CONCLUSION: In conclusion, prolonged constant and alternating intensity exercise of the same mean intensity and duration seemed to provoke similar changes in aspects of immune response in healthy subjects.

Monocarboxylate transporter MCT1 is a target for immunosuppression.

Current immunosuppressive therapies act on T lymphocytes by modulation of cytokine production, modulation of signaling pathways or by inhibition of the enzymes of nucleotide biosynthesis. We have identified a previously unknown series of immunomodulatory compounds that potently inhibit human and rat T lymphocyte proliferation in vitro and in vivo in immune-mediated animal models of disease, acting by a novel mechanism. Here we identify the target of these compounds, the monocarboxylate transporter MCT1 (SLC16A1), using a strategy of photoaffinity labeling and proteomic characterization. We show that inhibition of MCT1 during T lymphocyte activation results in selective and profound inhibition of the extremely rapid phase of T cell division essential for an effective immune response. MCT1 activity, however, is not required for many stages of lymphocyte activation, such as cytokine production, or for most normal physiological functions. By pursuing a chemistry-led target identification strategy, we have discovered that MCT1 is a previously unknown target for immunosuppressive therapy and have uncovered an unsuspected role for MCT1 in immune biology.

Strength, workload, anaerobic intensity and the immune response to resistance exercise in women.

AIM: The mechanism linking exercise intensity to the magnitude of the immune response is not completely understood. The purpose of this investigation was to determine whether the immune response to resistance exercise was associated with (1) changes in workload or (2) anaerobic exercise intensity. METHODS: Previously untrained women underwent 6 months of resistance training for lower and upper body (TOTAL, n = 34) or for upper body alone (UPPER, n = 30). Lymphocyte subsets [T (CD3+), CD4+, CD8+, NK and B], functional markers (CD45RA+ and CD45RO+), and mitogen (phytohemagglutinin-M, concanavalin A and pokeweed mitogen) and superantigen (staphylococcus a. cowans)-stimulated proliferation were measured from blood samples collected pre- and post-exercise for a squat resistance exercise consisting of six sets of 10 repetitions at 75% of one repetition maximum. This protocol was performed before (T0) and after 3 (T3) and 6 months (T6) of training. RESULTS: Lymphocyte recruitment to the circulation and proliferation following resistance exercise did not differ between training groups at any time, although the TOTAL group performed at a higher workload as training progressed. With respect to anaerobic intensity, exercise-induced increases in NK, CD4+, CD8+ and B lymphocyte concentrations were 42 (P = 0.07), 76 (P < 0.05), 72 (P < 0.05) and 242% (P < 0.01) greater in women in the highest compared with the lowest post-exercise lactate quartiles. Lymphocyte proliferation did not differ between lactate quartiles. CONCLUSIONS: Anaerobic intensity, rather than increased strength and workload, is associated with the number of lymphocytes recruited to the circulation, but not T and B cell proliferation responses.

Host metabolites that phenotypically increase the resistance of Haemophilus influenzae type b to clearance mechanisms.

Unidentified low-molecular-weight factor(s) in serum or nasopharyngeal secretions were known to phenotypically increase the resistance of Haemophilus influenzae type b (Hib) to bactericidal and opsonic antibodies, and resistance was attributed to two hypothetical mechanisms. Serum components generating resistance were studied. Mechanism 1, present in some Hib strains and their capsule-deficient mutants and accompanied by apparent increases in lipopolysaccharide content, was reproduced with a mixture of glucose, lactate, urea, and bicarbonate. Mechanism 2, present only in capsulated Hib and accompanied by increased capsulation, was reported with a mixture of Ca++ and lactate. Hib incubated with these compounds in buffer or grown in serum filtrate was resistant, but Hib grown in conventional media containing the metabolites in serum filtrate was resistant, but Hib grown in conventional media containing the metabolites was not. The resistant phenotype, which resembles Hib in vivo, may depend on nutrient balance as well as the specific factors. Lactate apparently is an important energy source for Hib.

Serum lactate dehydrogenase in muine leprosy: source and isozymes

The bulk of the lactate dehydrogenase (LDH) that increases in the serum of mice infected with Mycobacterium lepraemurium (MLM) derives from the liver and corresponds to the isozyme V. MLM-induced granulomas continuously arise in the liver and steadily increase in size until the animal's death. Growing granulomas push the adjacent hepatocytes away and cause them to disrupt and to release their cytoplasmic contents, including LDH. The LDH is then picked up by the infiltrating phagocytes and/or admixed with the circulating blood. Other LDH-containing organs (including the testis with its additional isozyme LDH-X) in the infected or normal animals do not seem to significantly contribute to the serum levels of LDH. The study of the liver-associated histochemical and biochemical changes in this controlled model of murine leprosy allows us to gain insight into the overall pathology of this mycobacteriosis. In some respects this sheds light on the liver involvement in human leprosy; a subject on which results of all sorts have been published

Locomotion of human lymphoblasts towards glucose and other sugars. Effects of pH, temperature and oxygen.

The effect of basic physical and chemical changes in the environment on the locomotion of human lymphoblasts was studied. The lymphoblasts were obtained by culture of human blood lymphocytes either with mitogens or in mixed lymphocyte culture. Lymphoblasts required glucose for optimal locomotion, and, if placed in glucose free media, they migrated towards glucose sources. A filter checkerboard assay showed chemokinesis and was suggestive of chemotaxis to glucose. Among other sugars tested only mannose enhanced locomotion, and products of glycolysis such as lactate and pyruvate inhibited locomotion. Lymphoblast locomotion was inhibited by azide, fluoride and 2-deoxyglucose but the effect of 2-deoxyglucose could be reversed by the addition of glucose. Lymphoblasts migrated faster at 40 degrees C than at 37 degrees C and their pH optimum was between 7.1 and 7.4. They migrated well in anaerobic conditions.