Relationship between the fatty acid composition of rat lymphocytes and immune functions.

The effects of dietary lipids on the fatty acid composition, activation and proliferation of lymphocytes were investigated. Weanling male Wistar rats were fed for 8 weeks on one of two low-fat diets which contained 50 g lipid/kg, or one of two high-fat diets containing 200 g lipid/kg, from either coconut oil or soyabean oil. The fatty acid composition of phospholipids from splenocyte membranes was affected by dietary lipid manipulation, and these differences influenced lymphocyte functions. Increased levels of linoleic acid in spleen lymphocytes correlated negatively with interleukin-2 receptor alpha-chain expression determined either by measuring the mean fluorescence or by the proportion of cells staining positive for CD25, and with the cell proliferation index. However, we found a positive correlation between interleukin-2 receptor alpha-chain expression determined by measuring the mean fluorescence and the cell proliferation index with the oleic acid concentration of spleen lymphocytes. Since phospholipid hydrolysis occurs early in lymphocyte activation, immunosuppressive effects induced by polyunsaturated fatty acids, described in the literature, could be due to an increase of linoleic acid or a decrease of oleic acid affecting many components of plasma-membrane-associated events involved in lymphocyte activation.

Adrenal medullary explants as an efficient tool for pain control: adhesive biomolecular components are involved in graft function ex vivo.

Adrenal medullary (AM) tissue transplantation into the central nervous system has been reported as a potential source of opioid peptides and catecholamines, which have analgesic effects useful in the control of chronic pain. Clinical trials, involving allogeneic graft of whole tissue explants into the subarachnoid space of the lumbar spinal cord, have already been reported. The aim of the present study was to determine whether adhesion and function of AM explants were related in some extent and how this relationship could account for improvement of AM tissue in terms of analgesic activity before grafting. Our experiments demonstrated a significant correlation between the adherent state of AM organoids during culture and a sustained secretion of Met-enkephalin and catecholamines by chromaffin cells (CC). These findings suggest that optimal culture condition for AM organoid adhesion can be defined for maintenance of tissue, prior to transplantation. Using immunocytochemistry, flow cytometry, and ELISA assays we showed that different cell adhesion molecules (CAMs) and extracellular matrix ECM proteins were expressed and released by AM cells during culture. Adherent AM organoids expressed increased levels of specific neural CAMs (NCAM and HNK-1 epitope) and integrin chains (beta1, alpha1, alpha2, alpha4, alpha5) and deposited markedly higher levels of fibronectin, but also laminin and collagen IV. Those molecules and probably adhesion processes they control might be involved in the maintenance of the CC-secreting neuroendocrine phenotype through cellular signaling pathways.

Intrathecal grafting of unencapsulated adrenal medullary tissue can bring CD4 T lymphocytes into CSF: a potentially deleterious event for the graft.

Adrenal medullary tissue including chromaffin cells was grafted intrathecally in cancer patients to relieve intractable pain. The central nervous system (CNS) is considered an immune privileged site. Therefore, non-HLA-matched and unencapsulated tissue was grafted in 15 patients and 1 sham control in a series of at least 20 grafts. We observed an increase in CSF lymphocyte counts in 15/20 allografts (75%). In contrast to peripheral blood, CD4 T cells predominated in the CSF, but failed to exhibit an activated phenotype (CD25+ CD45RO+ HLA-DR+). The positive effect of graft on pain, the high met-enkephalin levels, the absence of any increase in CSF cytokine levels particularly for IFN-gamma or IL-2 (but not IL-10 and IL-6), indirectly indicated that the graft was tolerated despite the presence of CSF lymphocytes. The single treatment failure and three of four cases of partial efficacy occurred in grafts where CSF lymphocytes were present. Moreover, when assayed (n = 7), the CD4+ CSF lymphocytes still retained the capacity to exhibit ex vivo a normal or enhanced frequency of T CD4 cells producing IFN-gamma and IL-2. Taken together, our observations indicate that impairment of the local immunosuppressive balance can lead to activation of those CSF CD4 T cells and drive a rejection process. This study suggests further work on the purification and/or the immunoisolation of tissues grafted in the CNS will be necessary, particularly when the possibility of long-term and repeated grafting is considered.

In vivo effects of olive oil-based lipid emulsion on lymphocyte activation in rats.

Numerous studies suggest that immune function may be compromised by lipid emulsions rich in polyunsaturated fatty acids, especially linoleic acid. In our study, we compared the effect of a new olive oil-based lipid emulsion (ClinOleic(R)) containing 18% linoleic acid, and an emulsion based on soybean oil (Ivelip(R); 52% linoleic acid) on lymphocyte functions. Weaning Wistar rats (n= 24) were fed for 4 weeks on an oral diet that contained 12% of total energy as lipids from soybean oil. Then they received, during 6 days, a total parenteral nutrition (260 kcal/kg/d) in which 12% of total energy was brought by one of the two lipid emulsions. The fatty acid profile of spleen lymphocyte phospholipids reflected lipid intakes, with a higher content of oleic acid in ClinOleic(R) group and linoleic acid in Ivelip(R) group. A greater proportion of cells expressed the interleukin-2 receptor a-chain (CD25) after administration of ClinOleic(R) when compared to Ivelip(R) (55.43 +/- 3.47 vs 45.48 +/- 3.26%, P<< 0.05). Moreover, the CD25 expression was positively correlated with oleic acid content of spleen lymphocyte phospholipids (r= 0.500, P<< 0.018). These results show that ClinOleic(R) is able to induce, in vivo, a greater proportion of cells expressing CD25, and suggest that oleic acid could have a role in the observed effects.

Ex vivo flow cytometry determination of intracytoplasmic expression of IL-2, IL-6, IFN-gamma, and TNF-alpha in monocytes and T lymphocytes, in chronic hemodialysis patients.

AIMS: To determine the intracytoplasmic expression of TNF-alpha, IL-2, IL-6 and IFN-gamma, ex vivo and in vitro, in both monocytes and T lymphocytes by flow cytometry after appropriate stimulation using phorbol myristate acetate (PMA)/ionomycin or lipopolysaccharides (LPS) in the presence of monensin, in order to assess the bio(in)compatibility of different dialysis membranes. METHODS: We examined monocytes and T lymphocytes taken from chronic hemodialysis patients (using either cuprophane (CUP), n = 6; polyacrylonitrile (AN 69), n = 6; or polysulfone (PS), n = 6 membranes), before and after a dialysis session. We compared the results with those obtained from end-stage chronic renal failure patients (n = 3) and healthy volunteers (n = 11). RESULTS: Before any stimulation there was a statistically significant difference in the percentages of TNF-alpha, IL-6, and IFN-gamma- expressing monocytes with respect to the dialysis membrane used. The highest percentages were observed for CUP and AN69 patients with figures of around 30% for each cytokine; the lowest percentages were found in PS patients and healthy volunteers. One hour after LPS stimulation the patterns remained unchanged for TNF-alpha and IFN-gamma, whereas the percentages of IL-6-expressing cells in PS patients and in healthy volunteers reached the figures obtained in the other groups. When we examined the percentage of IFN-gamma-, TNF-alpha- and IL-6-expressing monocytes in patients before and after a dialysis session, before any stimulation, we found that the results were significantly different for the three membranes (p = 0.01). Thus, a dialysis session with polysulfone membranes had no significant effect on the precentages of IFN-gamma-, TNF-alpha-, and IL-6-expressing monocytes, whereas percentages were significantly lower after the dialysis session when using cuprophane or AN69 membranes, suggesting a release of these cytokines by the monocytes during dialysis. A significant number of IFN-gamma- and IL-2-expressing T lymphocytes were only detected after 18 hours of PMA/ionomycin stimulation. The percentages of IFN-gamma-expressing T cells recorded for the different membranes were not statistically different from those recorded for healthy subjects or pre-dialysis patients, i.e., they were between 11.5 and 20%. However, the percentages of IL-2-expressing T lymphocytes were significantly different between the 5 groups, i.e., 31.3, 30.5, 18.6, 13.9 and 7. 6%, respectively, for CUP patients, pre-dialysis patients, healthy volunteers, PS and AN69 patients. This suggests that pre-dialysis and CUP patients have, at baseline, a stimulation of their T lymphocytes. Finally, a 4-hour dialysis session had no impact on the percentages of IL-2-expressing T lymphocytes, whereas it was associated with a significant decrease in the percentage of IFN-gamma-expressing cells, but only when cuprophane membranes were used. CONCLUSION: Cytokine flow cytometry enables one to study, ex vivo, i.e., without any stimulation of the cells, and in vitro after appropriate stimulation, the bio(in)compatibility of dialysis membranes assessed by the intracytoplasmic cytokine profiles of TNF-alpha, IFN-gamma, IL-6 and IL-2, evaluated at the single cell level.

Differences in Type 1 and Type 2 intracytoplasmic cytokines, detected by flow cytometry, according to immunosuppression (cyclosporine A vs. tacrolimus) in stable renal allograft recipients.

Recent multicenter, randomized clinical trials have shown that in renal transplant patients tacrolimus (FK506) was more efficient than cyclosporine A (CsA) at preventing acute rejection. In order to try and evaluate whether this difference was related to a different in vivo T-cell suppression we assessed, in a prospective study, the frequencies of interleukin (IL)-2-, IL-4-, IL-5-, IL-6-, IL-10-, interferon-gamma (IFN-gamma)- and double-positive IL-2/IFN-gamma-producing whole T cells, CD4 + and CD8 + T-cell subsets by means of cytokine flow cytometry. This was performed after in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with phorbol myristate acetate (PMA) and ionomycin, in the presence of monensin, in 14 healthy volunteers (controls) and in 14 renal transplant patients. The immunosuppression of the latter was based either on CsA (n = 7) or on FK506 (n = 7). Cytokine-expressing T-cell frequencies were assessed immediately pretransplantation (DO), and subsequently 3 months (M3) and 6 months (M6) afterwards in fasting patients prior to the morning intake of the immunosuppressive drug. We found that at DO the frequencies of IL-2-(22 +/- 2% vs. 22.2 +/- 2%), IFN-gamma-(26 +/- 3% vs. 29 + 3.4%) and IL-4-(0.8 +/- 0.2% vs. 1.4 +/- 0.2%)-expressing T lymphocytes were not significantly different between the controls and the patients, respectively. Conversely, the frequency of IL-2/IFN-gamma double positive cells was higher in the latter (9.3 +/- 1.6%) than in the controls (5.6 +/- 0.8); p = 0.06. Finally, on D0 the frequencies of IL-5-, IL-6-, and IL-10-producing T lymphocytes were lower than 1%, in both groups, as well as after grafting, i.e. on M3 and M6. As compared to baseline (DO): (a) chronic immunosuppression significantly decreased the frequencies of IL-2-, IL-4- and IL-2/IFN-gamma-expressing T cells, whereas those of IFN-gamma, IL-5, IL-6, and IL-10 were not significantly affected; (b) the frequencies of cytokine-expressing T cells were not statistically different between M3 and M6; (c) the decrease in the frequencies of IL-2- and IL-2/IFN-gamma-expressing T cells affected CD4 + and CD8 + cells equally; (d) there was a marginal decrease in the frequency of IFN-gamma-expressing cells only in the CD4 + subset but not in the CD8 population; and (e) for CsA, but not for FK506, the frequency of the IL-2-expressing T cells was negatively correlated with the whole blood trough levels. When we compared the frequencies of cytokine-expressing cells in FK506- and CsA-treated patients, we found that the frequency of IL-2-expressing T cells was significantly lower with FK506 (10.9+/-1.61%) than with CsA (16.3 +/- 1.8%; p = 0.03), whereas the frequencies of the other cytokine-expressing cells were not statistically different between the two groups. In conclusion, our study clearly demonstrated that studied ex vivo, FK506 and CsA decrease the frequencies of cells expressing IL-2, IL-4 and IL-2/IFN-gamma in vivo but do not affect those expressing IFN-gamma. Meanwhile, the frequency of IL-2-producing T cells was more affected with FK506 than with CsA and was negatively correlated with the CsA trough level. Finally, our results regarding IL-2 might explain to some extent the higher efficiency of FK506 in vivo than CsA.

The CBF.78 monoclonal antibody to human sialophorin has distinct properties giving new insights into the CD43 marker and its activation pathway.

We confirm here the CD43 specificity of the CBF.78 monoclonal antibody (mAb) and compare its phenotypic and functional capacities to classical group-A mAbs (DFT1, MEM-59) and to 2 other CD43 mAbs (RDP/AD9, 161-46). It reacts with stable human CD43 transfectants in a sialic acid independent way and blocks completely cell binding of RDP/AD9 or 161-46 more or less but not DFT1 and MEM-59. Its distribution differs from all other CD43. B lymphocytes, but surprisingly the majority of granulocytes or monocytes are CBF.78 negative. CBF.78 is expressed on all T lymphocytes, but the number of CBF.78 molecules/cell is low and equally represented on resting T CD4 and CD8 cells. In comparison to naive T lymphocytes, CD45RO cells increase their CBF.78 epitopes much more than other CD43 epitopes. At a single cell level, confocal microscopy shows that CBF.78 can exist independently of other epitopes. CBF.78 is able to induce homotypic adhesion in different cell lines but not in peripheral blood lymphocytes and is unable to relocalise the targeted molecules. U937 cell line that is not agglutinated by CBF.78 (or RDP/AD9) undergoes a stronger adhesion with PMA, when this reagent is combined with this mAb. By itself CBF.78 is unable to activate T lymphocytes and to costimulate CD3 mAbs but partially blocks PMA. The phosphorylation of the tyrosine kinase p59fyn and p56lck, driven by CBF.78, is weak and almost blocked by PMA. Altogether these data support the hypothesis that there are at least 3 modes of interaction between PKC and CD43 pathways: each pathway is inhibitory towards the other but the CD43 one can also be synergistic.

A new rejection criteria in the heterotopically placed rat heart by non-invasive measurement of Dp/Dtmax.

BACKGROUND: The heterotopic heart of rats has been a useful model in the evaluation of immunomodulatory protocols. Graft palpation usually determines the day of rejection. We present in this paper an original method of graft monitoring in allograft rejection. METHODS: Heterotopic cardiac abdominal transplantation was performed in Lewis isografts (n = 15) and in ACI to Lewis allograft (n = 15). A balloon connected to a measurement device was inserted in the left ventricle, and calculation of Dp/Dtmax was possible by recording the intra-left ventricular pressure. A ten-day follow-up was achieved with a daily comparison of palaption, ECG, and Dp/Dtmax. RESULTS: In transplanted hearts, Dp/Dtmax did not change in isografts but significantly decreased in allograft on posttransplantation Day 5 (PTD 5) vs PTD 0.1 and 3 (p < .01). Dp/Dtmax values on PTD 5 and 6 were also statistically significant in allograft vs isograft group (p < .01). Histological analysis at this time showed the occurrence of acute rejection in the allograft group. Graft palpation, and ECG remained normal until PTD 10 and no difference was observed between iso and allo groups. CONCLUSION: This study shows that daily measurement of Dp/Dtmax in heterotopic heart is made possible by our implantable system without interrupting the graft, and gives a more accurate definition of graft rejection than a combination of palpation and ECG. In addition, this method would seem desirable when differences in survival may be expected to be of lesser magnitude.

Kinetics of intracytoplasmic Th1 and Th2 cytokine production assessed by flow cytometry following in vitro activation of peripheral blood mononuclear cells.

BACKGROUND: Standard cytokine detection methods are unable to determine which cells are the producing cells. We report on the extent and under which conditions the multilabeling capability of flow cytometry (FCM) can bring new advances into the field. METHODS: Five different cytokines, interleukin-2 (IL-2), -4, -5, -10 and interferon-gamma (IFN-gamma), were assessed simultaneously under five ex vivo stimulation conditions in peripheral blood mononuclear cells from five healthy volunteers in a 5-day kinetic study. A second group of 35 volunteers was assessed for IFN-gamma and IL-2 production. RESULTS: This study showed that (a) intracytoplasmic cytokines were almost undetectable within unstimulated cells, (b) intracytoplasmic cytokines were detected only in CD69(+) T lymphocytes, and (c) intracytoplasmic IL-2 and IFN-gamma were dramatically upregulated after stimulation with phorbol myristate acetate (PMA)-ionomycin in a biphasic response or with PMA-phytohemagglutinin (one major peak only at 18 h) but to a lesser extent with other stimuli such as monoclonal antibodies. Th2 cytokines were detected at a later time point and at lower levels. PMA/ionomycin stimulation after 4 h and 18 h of culture in 35 other volunteers individualized several subgroups according to the frequency of IFN-gamma- or IL-2-producing cells--IFN-gamma delayed producers (n = 10/35), IFN-gamma low producers (n = 8/35), and IL-2 delayed producers (n = 16/35)--as opposed to IFN-gamma or IL-2 normal producers. CONCLUSIONS: FCM appears to be a good tool to examine cell cytokine status in pathology (allergy, autoimmune disease, etc.) provided that optimal stimulation conditions and multiple time-point cultures are used. It also seems to be a relevant method to define new Th subsets further.

Rheumatoid factor and antikeratin antibody are independent from presence of DR4 or DR1 in rheumatoid arthritis.

BACKGROUND: Presence of HLA-DR4, rheumatoid factor, and antikeratin antibody may predict severe rheumatoid arthritis. OBJECTIVE: To investigate potential associations between HLA DR4, rheumatoid factor and antikeratin antibody in rheumatoid arthritis patients. PATIENTS AND METHODS: Retrospective review of 169 patients followed at the Rangueil Teaching Hospital rheumatology department for rheumatoid arthritis. RESULTS: No differences in prevalences of DR4 and DR1 were found between patients with and without rheumatoid factor or between patients with and without antikeratin antibody, suggesting that the production of rheumatoid factor and/or antikeratin antibody is not dependent on genetic factors. Substantial overlap was seen between rheumatoid factor and antikeratin antibody. All three parameters can be identified at first evaluation of a patient with joint disease. CONCLUSION: It would be of interest to evaluate the cumulative predictive value of DR4 or DR1, rheumatoid factor and antikeratin antibody at disease onset.

HLA class II allele and haplotype frequencies in Ethiopian Amhara and Oromo populations.

HLA class II alleles were identified in 181 healthy unrelated Ethiopian children of both sexes and in 350 European controls from the South of France. The Ethiopian individuals belonged to the two major ethnic groups of the country: Oromo (N=83) and Amhara (N=98). In both panels, genetic polymorphism of HLA class II alleles was analysed for the first time by molecular typing of DRB1, DQA1 and DQB1 loci. Allelic and phenotypic frequencies were compared with those of European controls and other African populations. Construction of HLA class II three-locus haplotypes was also performed. The study revealed some differences between the two groups. Characteristic features of Central and North African populations appeared on the Ethiopian HLA genotypes. Surprisingly, DRB1*11 presented one of the lowest gene frequencies in both Ethiopian ethnic groups in contrast to Europeans and West Africans. Furthermore, this decrease was more marked than those observed using serological techniques in other geographically close East African countries. Oromo and Amhara only showed minor differences in spite of their different origins and histories. One significant difference consisted of a lower DRB1*01 gene frequency in Oromo as reported in most West African people. Some new or rare haplotypes were also observed in the Oromo group. Our results underline the distinctive features of the Ethiopian populations among the few HLA genotyping data available for East African groups and emphasise the major interest of such investigations in this region of Africa.

Absence of imprinting of HLA class Ia genes leads to co-expression of biparental alleles on term human trophoblast cells upon IFN-gamma induction.

Human trophoblast cells have developed various efficient regulatory mechanisms to prevent cell surface expression of the classical HLA-A, -B, and (but not always) -C class I molecules. This allows them to escape maternal alloimmune attack during pregnancy. However, recent results have demonstrated that such a lack of expression could be reversed in villous cytotrophoblast cells purified from term placenta by in vitro IFN-gamma treatment. In this context, we investigated whether both maternal and paternal HLA class Ia antigens were co-dominantly expressed in such trophoblast cells. Using polymerase chain reaction sequence-specific primers for HLA-A and HLA-C alleles, we detected transcripts of both paternal and maternal origins, showing that these genes were not affected by genomic imprinting, at least in term placenta. After in vitro IFN-gamma treatment, the polymorphic HLA-A and HLA-B antigens of both parental origins become detectable at the cell surface, as assessed by flow cytometry and/or complement-dependent microtoxicity test. Appearance of paternal antigens on trophoblast cells upon IFN-gamma induction raises the question of the in vivo biological consequences of this phenomena, in term of materno-fetal tolerance and in particular of a potential allogeneic cytotoxic immune response.

Clinical implications from studies of HLA antigens in idiopathic nephrotic syndrome in children.

HLA class I and II antigen frequencies were determined in two large cohorts of children with idiopathic nephrotic syndrome (NS) from Southwest France (n = 199) and Southwest Germany (n = 152) and compared with unrelated healthy individuals from the same geographical areas. Strength of HLA association was expressed by the relative risk (RR) estimated by Odd's ratio. We examined 105 steroid-resistant and 242 steroid-sensitive NS patients who were subdivided in non-relapsers, infrequent relapsers and frequent relapsers or steroid-dependent patients. In steroid-sensitive patients significant associations were found with HLA-DR7 (RR 5.1 in French, 3.2 in Germans), -DQ2 (RR 4.7/2.3) and with the phenotypic combination HLA-DR3/DR7 (RR 5.6/7.7). Significant negative associations were encountered with HLA-DR2, -DR6 and -DQ1. The associations were stronger in frequent relapsers/steroid-dependent patients than in infrequent relapsers and were not significant in non-relapsers. In steroid-resistant patients the only significant association found was with the combined occurrence of HLA-DR3/DR7. We propose that in childhood NS tissue typing for selected HLA class II antigens is helpful in prediciting the clinical course.

Immune responses in humans after 60 days of confinement.

A confinement experiment in a normobaric diving chamber was undertaken to better understand the effect of confinement and isolation on human psychology and physiology. Pre- and postconfinement blood samples were obtained from four test subjects and control donors to analyze immune responses. No modification in the levels of CD2+, CD3+, CD4+, CD8+, CD19+, and CD56+ cells was observed after confinement. Mitogen-induced T-lymphocyte proliferation and interleukin-2 receptor expression were not altered significantly. Whole blood interferon-alpha and gamma-induction and plasma cortisol levels were also unchanged, as was natural killer cell activity. These data suggest that in humans, no specific components of the immune response are affected by a 2-month isolation and confinement of a small group.

Transplantation of human chromaffin cells for control of intractable cancer pain.

Adrenal medullary chromaffin cells produce high levels of endogenous opioid peptides. Recent data suggest that transplantation injected locally into the spinal subarachnoid space reduced intractable malignant pain. In order to determine the feasibility, the efficacy and the risks of using adrenal medullary tissue for control of irreducible pain, we have developed a transplantation protocol on cancer pain patients selected when they required chronic intrathecal injection of morphine and progressively increasing doses to maintain the level of analgesic effects. At the present time, our clinical trial involves 8 patients. We report here our initial results (mean follow-up: 5 months). The various data collected before and after the intrathecal administration of chromaffin cells included: 1) Pain evaluation over time, with concomitant narcotic intake, 2) CSF sampling through an implanted access port to determine the following biological parameters: biochemical assay for opioid peptides, cell count and phenotyping of lymphocytes, 3) peripheral blood samples for lymphocyte typing. The results confirm the efficacy of adrenal medullary transplantation into spinal CSF for controlling irreducible cancer pain. Complementary intrathecal and oral morphine were totally stopped in 2 cases and stabilized in 5 others. It seems essential to have an important volume of grafted tissue to achieve analgesia with high levels of metenkephalin in CSF. A progressive decrease in metenkephalin release was observed from 2 to 4 months after the transplantation. Two patients with a long-term follow-up (8 and 12 months) needed another intrathecal chromaffin cell graft.