Evidence of survival benefit of extended (D2) lymphadenectomy in western patients with gastric cancer based on a new concept: a prospective long-term follow-up study

Human basophils have recently been shown to rapidly produce and release interleukin (IL-)4 and IL-13 as well as histamine and eicosanoids. Since both IL-4 and IL-13 can initiate and maintain late phase allergic reactions we addressed whether some widely used anti-allergic drugs can inhibit the anti-IgE induced release of these cytokines from enriched human basophils. Basophils were enriched (47-92% purity) by Ficoll density centrifugation followed by elutriation and negative selection of contaminating cells using immunomagnetic beads. Basophils were stimulated with sub-optimal dilutions of anti-IgE in the presence or absence of various drugs and the release of histamine and cytokines were measured after 30 min and 4 h, respectively. The beta-2 agonist salmeterol, the H1-receptor antagonist terfenadine and the phosphodiesterase inhibitor theophylline inhibited the release of IL-4 and IL-13 by more than 50% following 4 h of basophil stimulation with anti-IgE. These drugs also inhibited the release of histamine following 30 min stimulation, although with less efficacy than for IL-4 and IL-13. Short preincubation of basophils with salmeterol or terfenadine before stimulation gave rise to significantly greater inhibition of histamine release but had less effect on the inhibition of cytokine release. The effects of theophylline, however, were not significantly affected by preincubation of the cells with the drug. In contrast to the aforementioned drugs, salbutamol and cetirizine were ineffective at inhibiting both histamine and cytokine release from basophils. These results suggest that a number of anti-allergic drugs may mediate their effects, in part, in reducing late phase allergic responses due to their actions on IL-4 and IL-13 secretion from basophils.     

Endogenous superallergen protein Fv induces IL-4 secretion from human Fc epsilon RI+ cells through interaction with the VH3 region of IgE

We investigated the mechanism whereby protein Fv (pFv), a human sialoprotein found in normal liver and largely released in the intestinal tract in patients with viral hepatitis, induces mediator release from basophils and mast cells and evaluated whether it also induces IL-4 synthesis and secretion in basophils. pFv is a potent stimulus for histamine and IL-4 release from purified basophils. Histamine and IL-4 secretion from basophils activated by pFv was significantly correlated (rs = 0.70; p < 0.001). There was also a correlation (rs = 0.58; p < 0.01) between the maximum pFv- and anti-IgE-induced IL-4 release from basophils. The average t1/2 for pFv-induced histamine release was lower (3.5+/-1.5 min) than for IL-4 release (79.5+/-8.5 min; p < 0.01). IL-4 mRNA, constitutively present in basophils, was increased after stimulation by pFv and was inhibited by cyclosporin A and tacrolimus. Basophils from which IgE had been dissociated by brief exposure to lactic acid no longer released IL-4 in response to pFv and anti-IgE. The response to an mAb cross-linking the alpha-chain of Fc epsilon RI was unaffected by this treatment. Three human VH3+ monoclonal IgM concentration-dependently inhibited pFv-induced secretion of IL-4 and histamine from basophils and of histamine from human lung mast cells. In contrast, VH6+ monoclonal IgM did not inhibit the release of IL-4 and histamine induced by pFv. These results indicate that pFv, which acts as an endogenous superallergen, interacts with the VH3 domain of IgE to induce the synthesis and release of IL-4 from human Fc epsilon RI+ cells.     

Expression of protein kinase C isozymes in human basophils: regulation by physiological and nonphysiological stimuli

The expression of protein kinase C (PKC) isozymes in human basophils and the regulation of PKC isozymes during basophil activation by phorbol 12-myristate 13-acetate (PMA) +/- ionomycin, f-met-leu-phe (FMLP), and anti-IgE antibody were examined. In human basophils (> 98% purity), PKCbetaI, betaII, delta, and were expressed, PKCalpha was difficult to detect, and PKCgamma and eta were undetectable. In unstimulated basophils, PKCbetaI and betaII were found primarily in the cytosol fraction (95% +/- 3% of total and 98% +/- 1%, respectively). Within 5 minutes of stimulation with PMA (100 ng/mL), both PKCbetaI and betaII were translocated to the membrane fraction (85% +/- 4% and 83% +/- 6%, respectively). In resting cells, 48% +/- 3% and 61% +/- 10% of PKCdelta and , respectively, existed in the membrane fraction. Within 1 minute of stimulation with PMA, 90% +/- 6% of PKC was found in the membrane fraction, however, no translocation of PKCdelta was apparent. Stimulation with FMLP caused modest translocation ( approximately 20%) of all PKC isozymes by 1 minute, whereas stimulation with anti-IgE antibody led to no detectable changes in PKC location throughout a 15-minute period of measurement. However, concentrations of PMA and ionomycin that alone caused no PKC translocation and little histamine release, together caused significant histamine release but no apparent PKC translocation. Studies with bis-indolylmaleimide analogs showed inhibition of PMA-induced, but not anti-IgE-induced, histamine release. These pharmacological studies suggest that PKC does not play a prodegranulatory role in human basophil IgE-mediated secretion.     

The effect of IL-4 on human nasal mucosal responses

Interleukin (IL)-4 causes the dose limiting sensation of nasal congestion when administered systematically at doses of 3 micrograms/kg or higher thrice daily to humans. This side effect was observed in a group of patients treated as part of an immunotherapy protocol for cancer management. To determine the source of this congestion, nasal secretions were collected prospectively in a group of patients at baseline and after provocation with normal saline, methacholine (which stimulates glandular secretion), and histamine (which causes increased vascular permeability). Nasal lavages obtained at baseline and after provocation were analyzed for the presence of these glandular and vascular proteins and inflammatory mediators. Washings and provocations were performed before IL-4 administration, after 24 hours of IL-4 treatment, and after 3 days of treatment, at a time when nasal congestion was maximal. Compared with histamine challenge before IL-4 treatment, the secretion of the plasma proteins albumin and IgG were significantly decreased after 3 days of IL-4 treatment. IL-4 treatment had no apparent effect on methacholine-induced responses. Thus systemically administered IL-4 causes the subjective sensation of nasal congestion, increased histamine in nasal lavages, and the development of vascular unresponsiveness to histamine, without affecting parasympathetic responses to histamine. The relationships among increases in nasal lavage histamine, vascular unresponsiveness to histamine, and the sensation of nasal congestion are unclear.     

Molecular and biochemical characterization of the protein template controlling biosynthesis of the lipopeptide lichenysin

To assess the duration of immunosuppression in FK506-dosed pigs, an undiluted whole blood assay was established to measure reactivities of T cells in their physiological milieu. PMA and ionomycin were shown to induce IL-2 production in swine blood. The IC50 of FK506 in inhibiting IL-2 production in whole blood and isolated PBMC stimulated with PMA and ionomycin measured 1.2 and 0.04 nM, respectively. These data underscore the influence of red blood cells and plasma proteins on drug potency. IL-2 levels were determined in blood drawn immediately before and 1, 24, 48, and 72 h after iv dosing. For pigs dosed with 0.05 mg/kg, 50% recovery of IL-2 production was observed at 16 h and 100% at 35 h after dosing. For pigs dosed with 0.15 mg/kg, 50% recovery was observed at 38 h and 100% at 72 h. Blood concentrations of FK506 at 50 and 100% recovery of IL-2 production measured 10.8 and 2.2 nM for pigs dosed with 0.05 mg/kg and 6.1 and 1.1 nM for pigs dosed with 0.15 mg/kg, respectively. These concentrations are severalfold higher than predicted from the IC50 of FK506 for inhibiting IL-2 production in the whole blood assay. These data suggest that the true potency of FK506 in blood after dosing is influenced by additional factors, which could include plasma protein binding, the presence of active or interfering metabolites, serum interfering factors, and sequestration of drug in blood cells. Our results demonstrate the utility of an undiluted whole blood assay for assessing the duration of immunosuppression in drug-dosed animals and emphasize the importance of assessing drug potency in the whole blood environment ex vivo.     

Histamine increases anti-CD3 induced IL-5 production of TH2-type T cells via histamine H2-receptors

Besides its proinflammatory functions histamine released from basophils and mast cells during immediate-type hypersensitivity reactions is known to inhibit several lymphocyte functions like IL-2 and gamma-IFN production. Recently, it has been shown that T helper cells of type 2 phenotype (TH2) represent the T cell fraction which may play a pivotal role in the promotion of the allergic inflammatory eosinophilic late-phase reaction by secretion of cytokines, especially IL-4 and IL-5. We have investigated the effect of histamine on anti-CD3 induced IL-4 and IL-5 production by TH2 cells. Histamine in concentrations between 10(-7) and 10(-5) mol/l concentration-dependently increased anti-CD3 induced IL-5 production up to 120%, whereas IL-4 production was not affected. The activity of histamine in increasing IL-5 production was mimicked by the H2-receptor agonist dimaprit. Histamine induced increase in IL-5 production was inhibited by histamine H2-receptor antagonists, but remained unaffected by H1- or H3-receptor antagonists. Administration of forskolin which directly stimulates the production of cAMP, the second messenger of the H2-receptor, also resulted in an increase in anti-CD3 induced IL-5 production. These results indicate that the histamine-mediated increase in anti-CD3 induced IL-5 production is mediated via H2-receptors. Consequently, histamine released from mast cells and basophils during the early-phase allergic reaction may act as an important stimulatory signal for the initiation of the allergic inflammatory late-phase reaction by increasing local IL-5 production of allergen triggered TH2 cells.     

Low level of interleukin 10 synthesis during liver allograft rejection

Interleukin 10 (IL-10) is a macrophage and T-cell-derived cytokine with potent immunosuppressive properties. To assess its role in liver allograft rejection, we evaluated the plasma level and in situ production of IL-10 after liver transplantation and designed in vitro studies to asses the effects of IL-10 on the allogeneic response. Normal controls and liver transplant recipients with acute rejection, chronic rejection, other complications (recurrent hepatitis C, biliary complications), or no complications were evaluated. The plasma IL-10 level was measured by an immunoenzymatic technique. IL-10 expression in the liver was detected on frozen liver biopsies by in situ hybridization and immunohistochemistry. Plasma IL-10 levels were not elevated during acute or chronic rejection, when compared with liver recipients with uncomplicated transplants. IL-10 mRNA and protein expressions in the liver graft were restricted to rare scattered sinusoidal cells of transplant recipients with acute or chronic rejection, as well as in those with no complications. In mixed lymphocyte cultures performed with peripheral blood mononuclear cells (PBMC) from normal subjects, IL-10 decreased the cell proliferation in a dose-dependent manner, and this immunosuppression was synergistic with that of cyclosporine or FK506. These findings indicate that IL-10 production is low during allograft rejection. Thus, IL-10 therapy in association with cyclosporine or FK506 might be proposed after liver transplantation.     

Human T cells require IL-2 but not G1/S transition to acquire susceptibility to Fas-mediated apoptosis

The interaction between Fas ligand and Fas, both expressed on activated T cells, is the major pathway in the regulation of activation-induced cell death. However, activated T cells that express membrane Fas are initially resistant to anti-Fas-induced apoptosis and become susceptible only after proliferation in vitro. Since IL-2 is known to regulate activation-induced cell death, we studied the effect of IL-2 on anti-Fas-mediated apoptosis. Interference with the IL-2 pathway was achieved by 1) inhibition of cytokine synthesis using cyclosporin A or FK506, 2) neutralization of IL-2 by anti-IL-2 Ab, 3) inhibition of binding to IL-2R by CD25 mAb, and 4) blocking of IL-2R signaling by rapamycin. We show that Fas expression is independent of the IL-2 pathway, whereas Fas-mediated apoptosis does not develop in the presence of inhibitors of IL-2 production or signaling. While the addition of rIL-2 reversed the inhibitory effect of cyclosporin A and FK506, the addition of rIL-4, rIL-7, or rIFN-gamma did not, although these cytokines induced progression into the S phase of the cell cycle. Aphidicolin-treated activated T cells that do not progress into the S phase were susceptible to Fas-mediated apoptosis. Therefore, Fas-mediated apoptosis is controlled by signals generated by IL-2 in agreement with the reported alteration of apoptosis in mice deficient in IL-2 or IL-2R.     

Identification of a basophil leukocyte interleukin-3-regulated protein that is identical to IgE-dependent histamine-releasing factor

This study aimed to identify basophil leukocyte proteins associated with interleukin (IL)-3 and/or anti-IgE activation by two-dimensional (2-D) gel electrophoresis. We noticed one particular protein showing increased synthesis after recombinant human (rh)IL-3 and, to a lesser extent, anti-IgE stimulation. The protein was also present in the culture medium in increased amounts after rhIL-3 stimulation. On the basis of comigration with proteins in published 2-D gel electrophoresis databases and immunoblotting with a specific monoclonal antibody, we identified this protein as translationally controlled tumor protein (TCTP), also known as p23 or IgE-dependent histamine-releasing factor. The antibody was shown to be specific for TCTP/IgE-dependent histamine-releasing factor by blotting on 2-D gels of proteins from human lymphocytes and the human basophilic cell line KU812, followed by N-terminal amino-acid sequencing of the bound protein. Densitometric analysis of the gels showed that the synthesis of IgE-dependent histamine-releasing factor in human basophil leukocytes was dose dependent upon rhIL-3 stimulation with an optimum of 100 ng/ml. The level of the protein in the medium was also highest at an optimal rhIL-3 concentration of 100 ng/ml. Supernatants from cultured basophils were able to stimulate histamine release from other basophils. This histamine release was decreased by precipitation of TCTP/IgE-dependent histamine-releasing factor from these supernatants.     

Role of serum amyloid A as an intermediate in the IL-1 and PMA-stimulated signaling pathways regulating expression of rabbit fibroblast collagenase

The matrix metalloproteinase collagenase is expressed by resident tissue cells only when needed for biological remodeling. Exogenous addition of inflammatory and growth-promoting cytokines stimulates collagenase expression in early passage fibroblast cultures. In addition, the signal for collagenase expression in response to phorbol-12 myristate-13 acetate (PMA) or to agents which alter cell shape in early passage fibroblast cultures is routed extracellularly to an autocrine cytokine intermediate, IL-1 alpha. Importantly, fibroblasts, when freshly isolated from the tissue, are not competent for IL-1 alpha gene expression and, therefore, cannot produce collagenase in response to shape change agents. However, they do make a small amount of collagenase in response to PMA via an IL-1-independent pathway that has not been further characterized. In this paper, we investigate the role of a second autocrine, serum amyloid A3 (SAA3), in IL-1-dependent and -independent collagenase gene expression. We demonstrate that SAA3 is required for effective stimulation of collagenase expression by either exogenous or endogenous IL-1. Furthermore, while freshly isolated fibroblasts cannot express IL-1 alpha they can express SAA3, and this autocrine mediator acts independently of IL-1 alpha to control the low level of collagenase expression that can be stimulated by PMA. These results provide further evidence for a newly emerging paradigm of collagenase regulation which emphasizes the requirement for extracellular routing of signals. They also suggest that SAA3 might be utilized independently of IL-1 alpha to control tissue remodeling in vivo.     

Topical FK506 suppresses cytokine and costimulatory molecule expression in epidermal and local draining lymph node cells during primary skin immune responses

Recently, it has been shown that the immunosuppressive macrolide lactone, FK506, exerts good therapeutic efficacy in inflammatory skin diseases. The aim of this study was to analyze the influence of topical FK506 on molecular (IL-1alpha, IL-1beta, IL-2, IL-4, IL-12 p35, IL-12 p40, macrophage inflammatory protein-2 (MIP-2), granulocyte-macrophage CSF (GM-CSF), TNF-alpha, and IFN-gamma) and cellular (I-A+/CD80+, I-A+/CD54+, I-A+/CD69+, I-A+/B220+, and CD4+/CD25+) events in epidermal (EC) and local draining lymph node (LNC) cells during primary contact hypersensitivity responses. Cytokine mRNA levels for IL-1alpha, IL-1beta, GM-CSF, TNF-alpha, MIP-2, and IFN-gamma in EC and for IL-2, IL-4, IL-12 p35, IL-12 p40, and IFN-gamma in LNC were increased and resulted in significant LNC proliferation during oxazolone-induced contact hypersensitivity. Topical FK506 treatment dose-dependently suppressed oxazolone-induced LNC proliferation. This effect was correlated with decreased IL-1alpha, IL-1beta, GM-CSF, TNF-alpha, MIP-2, and IFN-gamma mRNA expression within the epidermis and decreased IL-12 p35 and p40 mRNA expression in LNC. Further analysis of the LNC cytokine pattern revealed that the production of both Thl (IFN-gamma and IL-2) and Th2 (IL-4) cytokines was dramatically impaired after topical FK506 treatment. Flow cytometric analysis showed that topical FK506 decreased the population of epidermis-infiltrating CD4+ T cells and suppressed the expression of CD54 and CD80 on I-A+ EC and LNC during hapten-induced contact hypersensitivity. Furthermore, topical FK506 profoundly impaired oxazolone-induced up-regulation of CD25 expression on CD4+ LNC and dramatically decreased hapten-induced expansion of I-A+/B220+ and I-A+/CD69+ LNC subsets. In conclusion, these results give new insights into the mechanisms of action of topical FK506 treatment.     

Histamine potently suppresses human IL-12 and stimulates IL-10 production via H2 receptors

IL-12 and IL-10, respectively, stimulate Th1 and Th2 immune responses. The development of some allergic reactions, infections, and tumors are associated with excessive histamine production and a shift toward Th2 responses. Here we address the possibility that this association is causally linked, at least in part, to modulation of IL-12 and IL-10 production by histamine. We report that histamine dose-dependently inhibited the secretion of human IL-12 (p70) and increased the production of IL-10 in LPS-stimulated whole blood cultures. These effects of histamine were antagonized by cimetidine, an H2 receptor antagonist, but not by selective H1 and H3 receptor blockers, and were mimicked by an H2 receptor agonist. The effects of histamine on IL-12 and IL-10 secretion were independent of endogenous secretion of IL-10 or exogenous addition of IL-12, while Ro 20-1724, a phosphodiesterase inhibitor, potentiated the effects of histamine on IL-12 and IL-10 production, implicating cAMP in its actions. Similar modulatory effects of histamine on IL-12 and IL-10 production, which were reversed by the H2 antagonist cimetidine, were observed in PBMC and isolated monocytes stimulated by Staphylococcus aureus Cowan strain 1 and LPS, respectively. Thus, histamine, via stimulation of H2 receptors on peripheral monocytes and subsequent elevation of cAMP, suppresses IL-12 and stimulates IL-10 secretion, changes that may result in a shift of Th1/Th2 balance toward Th2-dominance. This may represent a novel mechanism by which excessive secretion of histamine potentiates Th2-mediated allergic reactions and contributes to the development of certain infections and tumors normally eliminated by Th1-dependent immune mechanisms.     

Fc gammaRIII-mediated regulation of hematopoiesis in murine bone marrow cells by interleukin-3 and CD95 (Fas/Apo-1)

The interleukin-3 (IL-3)-dependent murine bone marrow-derived cell line FDC-P2/185-4 (185-4) undergoes apoptosis when IL-3 is withdrawn from culture medium. Previous results from our studies indicated that a high concentration of aggregated mouse IgG prevented apoptosis of 185-4 cells through Fc gammaRIII by an autocrine mechanism, producing IL-3. But after 24 hours, 185-4 cells expressed CD95 (Fas/Apo-1) on their surfaces on stimulation via Fc gammaRIII. In addition, this CD95 was functional and apoptosis was induced by anti-CD95 monoclonal antibody (MoAb). We investigated how these conflicting effects were induced by Fc gammaRIII stimulation within the context of cell survival and death. The results showed that IL-3 was induced by calcium ionophore and that the IL-3 induced by Fc gammaRIII stimulation was blocked by EGTA or FK506, but not by staurosporine (protein kinase C [PKC] inhibitor), indicating the important role of calcium-calcineurin in this system. On the other hand, the CD95 expression induced by Fc gammaRIII stimulation was blocked by staurosporine, but not by EGTA or FK506, and phorbol myristate acetate (PMA) induced CD95 expression in the same manner as Fc gammaRIII, indicating the involvement of PKC in the CD95 expression induced by Fc gammaRIII stimulation. Thus, Fc gammaRIII-mediated stimulation even while promoting immediate survival of the bone marrow cells, also triggers mechanisms that will facilitate their eventual deletion at the end of the response. These results suggest that a balance between cell survival and death is maintained to avoid unlimited cell growth caused by Fc gammaRIII-ligand interaction in hematopoiesis during inflammation.     

The production of macrophage inflammatory protein-1 alpha by human basophils

Macrophage inflammatory protein-1alpha (MIP-1alpha) has previously been shown to be produced by mononuclear cells, eosinophils, and neutrophils. Its production by basophils has not been investigated. The objective of this study was to investigate the production of MIP-1alpha by basophils. Peripheral blood basophils were separated by Percoll gradient centrifugation, cultured overnight, and processed for double immunocytochemistry using Abs against MIP-1alpha and FcepsilonRIalpha (alpha subunit of IgE receptor type 1). We demonstrated that basophils expressed immunoreactive MIP-1alpha upon stimulation with anti-IgE. Less than 5% of the basophils stained for MIP-1alpha without stimulation. The secretion of MIP-1alpha by basophils was studied by ELISA. In these experiments, basophils were further enriched to 65 to 99% (median, 86%) by a negative selection method. Basophils released MIP-1alpha when stimulated by Abs against IgE and FCepsilonRIalpha as well as IL-3 and the calcium ionophore, A23187. In parallel experiments, PBMC, eosinophils, and neutrophils did not produce MIP-1alpha in response to anti-IgE, but they did so in response to A23187. No MIP-1alpha release was detected in platelet preparations. Preincubation with IL-3 (15 min or 18 h) augmented anti-IgE-included basophil MIP-1alpha production. The secretion of MIP-1alpha by basophils was detectable shortly after stimulation and gradually increased over 24 h. Since MIP-1alpha has potent inflammatory and histamine-releasing activities, its production by basophils may indicate a positive feedback mechanism for allergic inflammation.     

IL-4 regulation of perforin gene expression and BLT-esterase production in alpha CD3-induced activated killer cells

This present study examines Il-4 regulation of perforin gene expression and cytolytic granule production in alpha CD3-induced activated killer cells CD3-AK. After stimulation of resting T cells with alpha CD3, proliferative response could be detected at 1 day after activation. The expression of perforin mRNA and production of cytolytic granules (using BLT-E as indicator) was detected on days 2-4, and this time course correlated with the generation of lytic CD3-AK cells. These findings indicate that killer cells generation is a late event during the course of alpha CD3 activation. Generation of CD3-AK cells is primarily PKC dependent and is blocked by the depletion or inhibition of PKC by PMA or SSP. These changes are accompanied by the suppression of perforin gene expression (mRNA) and BLT-E production. However, adding IL-4 into the cultures restored the perforin mRNA expression and BLT-E production, and also the cytolytic activity of the CD3-AK cells. Furthermore, for preactivated CD3-AK cells cultured in IL-2, SSP also suppressed the perforin mRNA and BLT-E with the concomitant reduction of cytolytic activity. Similar to the resting T cells, in the SSP-maintained preactivated CD3-AK cells, switching the cytokine from IL-2 to IL-4/IL-2 restored perforin mRNA expression and BLT-E production, with concomitant restoration of the cytolytic activity. In contrast, switching from IL-4/IL-2 gave the opposite effect. These results could be reproduced by using amiloride which also inhibited PKC activity but did not affect the growth of preactivated CD3-AK cells. These findings indicate that IL-4 may play a role in the late stage of alpha CD3 activation to regulate the expression of perforin gene and probably the translation process during the generation of activated killer cells.     

Generation, persistence, and modulation of Th0 effector cells: role of autocrine IL-4 and IFN-gamma

Many studies have classified CD4 responses into either Th1-like or Th2-like, based on cytokine secretion profiles, but little significance has been placed on Th0 cells. This has largely resulted from studies that suggested that Th0 populations primarily comprise individual Th1 and Th2 cells. Here, we show that priming of Ag-specific naive CD4 cells with moderate dose IL-4 generates a Th0 population that is evident after 3 days in vitro and becomes prevalent after successive encounters with Ag over a 9-day period. By intracellular cytokine staining, the majority (>60%) of effector cells generated in this way produce either IL-4, IFN-gamma and IL-2, or IL-4 and IFN-gamma without IL-2. Endogenous IFN-gamma secreted over the initial 3 days of culture was critical for generating Th0 cells, since neutralization allowed IL-4 to induce differentiation into Th2-like cells. Successive encounters with Ag were required for generating Th0 cells, and their stability and persistence were governed by the balance of endogenous IL-4 and IFN-gamma secreted during the later stages of differentiation. Studies blocking Fas-induced cell death showed that this process played no role in Th0 cell generation, and differential death of committed Th1 or Th2 cells was not required for Th0 persistence. These data suggest that Th0 cells can be as prevalent as Th1- or Th2-like cells after naive CD4 activation, that the relative levels of autocrine IL-4 and IFN-gamma are important to the lack of commitment, and that not all cells are predestined to the Th1 or Th2 phenotypes early in the response.     

Promoting remyelination as a future therapeutic principle in multiple sclerosis?

Certain factors have been studied that might influence whether interferon-gamma (IFN-gamma) or interleukin-4 (IL-4) will predominate in cultures of peripheral blood mononuclear cells (PBMC). ELISA was used to measure cytokine protein, and PCR was used to quantitate mRNA. It was found that induction with plant lectins produced greater yields of IFN-gamma than induction with ionophores, but ionophores alone produced at least equal yields of IL-4 as did lectins. Phorbol myristate acetate (PMA) greatly enhanced IFN yields in the presence of ionophores but had no significant influence on IL-4 production. Calcium-independent cytokine induction using anti-CD28 and PMA resulted in production of both cytokines, whereas depletion of extracellular calcium and magnesium adversely influenced the yield of both IL-4 and IFN-gamma. Finally, calmidazolium, an inhibitor of calmodulin, had an enhancing effect on IFN-gamma yields when phytohemagglutinin (PHA) was the inducer and an adverse effect when A23187 was the inducer. In contrast, calmidazolium reduced IL-4 yields with both PHA and A23187 induction.     

Myocardial viability. Concept, physiopathology. Methods and diagnostic value]

This study was designed to investigate the effect of tacrolimus (FK506) and of cyclosporine (CsA) on tubular function in renal graft recipients. Patients were randomised after renal transplantation to immunosuppressive treatment with FK506 (n = 8) or CsA (n = 8). Patients had a mean age of 45.7 +/- 3.4 yr; there was no difference in age, sex, HLA status or CMV mismatches. Neither was there any difference in the frequency of episodes of acute kidney failure between the groups, nor was there a significant difference in the frequency of episodes of kidney rejection within the first year. The mean FK506 level at the time lay at 14.7 +/- 14.4 ng/mL whole blood, and the mean CsA level at the time of study was 162 +/- 25 ng/mL whole blood. We performed renal function studies 6 months after transplantation: CIn, CPAH, NaHCO3 loading, and Na2SO4 loading. There was no significant impairment of GFR in patients treated with FK506 with 53.6 +/- 2.5 mL/min as compared to 58 +/- 6 mL in group 2. Plasma renin activity (0.6 +/- 0.4 ng/mL vs 2.3 +/- 3; p < 0.01) and aldosterone (69 +/- 17 vs 157 +/- 28.2 pg/mL; p < 0.05) were significantly decreased during treatment with FK506. Fractional HCO3 excretion was low in both groups, indicating that bicarbonate reabsorption in the proximal nephron was unimpaired. Distal renal tubular acidosis was demonstrated in 4 patients of group 1 but in only 1 of group 2. Potassium levels were slightly increased in patients treated with FK506 (5.4 +/- 0.2 mmoL/L) as compared to cyclosporine (4.9 +/- 0.3 mmoL/L; p < 0.05). Distal hydrogen ion secretion, evaluated by the ability to increase urinary pCO2 in a highly alkaline urine, was impaired in patients treated with FK506 (U-B pCO2: 16.1 +/- 4 vs 36 +/- 5.8; p < 0.05) as compared to patients treated with CsA. The maximum acidification capability (NAE) was slightly lowered during therapy with FK506 (67.5 +/- 11.8 versus 86.6 +/- 16.5 mumoL/min, ns). We conclude that FK506 administration results in a decrease in the rate of hydrogen ion secretion by the collecting tubules. This defect was disclosed by the finding of a subnormal pCO2 in a highly alkaline urine. These results show that FK506 is able to induce distal tubular acidosis. Distal tubular acidosis is part of FK506 induced nephrotoxicity, the pathogenesis of this type of hyperkalemic metabolic acidosis found in patients treated with FK506 after renal transplantation has to be further elucidated.