We previously reported that the human CD4 molecule is capable of transducing a positive signal when activated by an anti-CD4 mAb B66. This antibody, in contrast to many other anti-CD4 mAb, induced IL2 production and proliferation of resting CD4+ peripheral blood T lymphocytes in the absence of any other signal. We further reported that anti-CD4 mAb B66 was able to induce IL2 production in murine T-cell hybridoma cells transfected with full-length human CD4 cDNA. In the present study, we extend these findings by demonstrating that anti-CD4 mAb B66 was able to induce Ca2+ mobilization and IL2 production in a CD3/TcR- variant 31-13, of the CD3/TcR+ Jurkat cell line. We further showed that anti-CD4 mAb B66 was able to activate CD4+ cells from the promonocytic cell line U937. In these cells, mAb B66 induced Ca2+ mobilization when cross-linked with a second antibody and, in addition, the production of large quantities of IL1 beta was measured. In essence, our findings provide direct evidence that cross-linking of CD4 may cause T-cell activation in the absence of the coexpression of the CD3/TcR molecular complex and that, in addition, CD4 might transduce a positive signal in CD4+ cells of the myeloid lineage.

OBJECTIVE

To carry out an immunoassay analysis of biomarkers expressed in gingival crevicular fluid (GCF) with the main goal of finding a useful diagnostic pattern to distinguish between resorbing deciduous teeth and nonresorbing controls.

METHODS

A split-mouth design was used in this study with a total of 22 GCF samples collected from 11 patients in the mixed dentition. For each child, one deciduous molar with radiographic evidence of root resorption was used as the test tooth whereas the contralateral first permanent molar with formed roots was used as the control tooth. Samples were processed with immunoassays using a panel of selected biomarkers including interleukin-1 beta (IL-1b), interleukin-1 receptor antagonist (IL-1RA), nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase-9 (MMP-9), and dentin sialoprotein (DSP).

RESULTS

There were no statistically significant differences in levels of IL-1b, OPG, and MMP-9 between test and control sites (P>> .05). IL-1RA was the only biomarker to show a significant down-regulation (P = .04) in GCF samples collected from resorbing teeth. RANKL data showed a heavily skewed distribution and was deemed unreliable. Only one deciduous GCF sample had detectable levels of DSP; therefore, no further statistical calculation was applicable because of the limited amount of data for this biomarker.

CONCLUSIONS

This study indicated that IL1-RA is down-regulated in GCF from resorbing primary molars, thus suggesting this cytokine as a potential analyte to be included in a panel that can discriminate between resorbing and nonresorbing teeth.

This review shows that numerous neuropeptides and hormones are involved in the regulation of intestinal transit. Many gastrointestinal hormones known to act on smooth muscle to influence muscle contractility also play a role in the genesis of abrupt changes associated with alimentary behaviour. In many monogastrics and ruminants, the cyclic occurrence of the migrating motor complex (MMC) is linked to peripheral hormonal factors only slightly influenced by the nature of food. Motilin is the major hormone involved in triggering the gastric migrating motor complex while somatostatine and enkephalins are implicated in the propagation along the small intestine. Other hormones, like CCK8, insulin, gastrin, and neurotensin, trigger the development of an intestinal feed pattern but CCK released at the central nervous system ventromedial hypothalamus is involved in maintaining the postprandial type of activity. Gastrointestinal transit may be altered in physiopathological situations in which CRF, TRH and some cytokines (IL1 beta, TNF alpha) play an important role.

A computer-designed hydropathically complementary peptide to human interleukin 1 beta (IL1 beta) precursor sequence 204-215 recognized the 204-215 peptide as well the entire IL1 beta protein with binding affinities in the micromolar range. Interaction between the complementary pair was characterized by analytical high-performance liquid affinity chromatography on columns derivatized with the computer-generated peptide. Recognition selectivity was clearly shown by the ability of the computer-generated complementary peptide columns to purify the IL1 beta-(204-215)-peptide from complex synthetic mixtures with high yields, independently of the type of solid support used. Recognition specificity was demonstrated by the inability of the IL1 beta-(204-215)-peptide and IL1 beta molecules to interact with blank columns or columns derivatized with other non-related peptides. Furthermore, scrambling the sequence of the computer-generated peptide or the IL1 beta-(204-215)-peptide in such a way as to alter their hydropathic profiles had the effect of abolishing binding. The complementary pair failed to interact in the presence of competing peptide, thus providing further evidence of specificity. Computer-generated complementary peptide affinity columns also proved useful for purification of recombinant human IL1 beta protein directly from crude Escherichia coli lysates.

OBJECTIVE

To investigate the effects of physiological doses of hydrocortisone on synthesis and turnover of cell associated matrix (CAM) by human chondrocytes obtained from normal articular cartilage.

METHODS

Human articular cartilage cells were obtained from visually intact cartilage of the femoral condyles of five donors and maintained in culture for one week to reach equilibrium in accumulated CAM compounds. 0, 0.05, 0.20, and 1.0 micro g/ml hydrocortisone was added to the nutrient media during the entire culture period. Cells were liberated and levels of CAM aggrecan, type II collagen, and fibronectin, of intracellular IGF-1, IL1alpha and beta, and of their respective plasma membrane bound receptors IGFR1, IL1RI, and the decoy receptor IL1RII, were assayed by flow cytometry.

RESULTS

In comparison with controls, hydrocortisone treated chondrocytes, at all concentrations, expressed significantly higher plasma membrane bound IGFR1. Intracellular IGF-1 levels remained unchanged. Together with these changes, reflecting an increased ability to synthesise extracellular matrix (ECM) macromolecules, hydrocortisone treated cells expressed significantly higher amounts of the plasma membrane bound decoy IL1RII. Concurrently, intracellular IL1alpha and beta levels and membrane bound IL1RI were down regulated. Levels of CAM aggrecan, type II collagen, and fibronectin were significantly up regulated in the chondrocytes treated with hydrocortisone.

CONCLUSIONS

0.05 micro g/ml hydrocortisone treated chondrocytes had decreased catabolic signalling pathways and showed an enhanced ability to synthesise ECM macromolecules. Because IL1 activity was decreased and the expression of IL1RII decoy receptor enhanced, more of the ECM macromolecules produced remained accumulated in the CAM of the chondrocytes. The effects were obtained at doses comparable with physiological plasma levels of hydrocortisone in humans.

In autoimmune diseases striking abnormalities of T and B cell activation and of cytokine production are present. In 14 patients with autoimmune hemolytic anemia (AIHA), idiopathic or in the course of: lymphoma, B hepatitis, carcinoma, drug therapy (alpha-methyldopa), systemic lupus erythematosus (SLE), and not yet submitted to immunosuppressive therapy, the PBL proliferative response to PHA and the IL1 alpha, IL2, IL4 and IL2R serum levels have been valued. While the stimulation index of PBL was strongly reduced in 10 cases (64 +/- 56 vs 138 +/- 45 in the control group), IL1 alpha, IL2 and IL2R were greatly increased in all the patients, and IL4 in 5 (IL1 alpha :199 +/- 268 pg/ml in patients vs 0.30 +/- 0.2 in controls; IL2:716 +/- 311 pg/ml vs 16 +/- 4; IL4:29 +/- 13 pg/ml vs 13 +/- 7; IL2R:1233 +/- 471 U/ml vs 256 +/- 114). Cytokine serum levels were not related with the associated disease, with the CD4+ and CD8+ cells absolute number or with PBL blastogenic in vitro response. The high serum levels of cytokines and IL2R suggest that in AIHA there exist a CD4+ lymphocyte hyperactivation (the low proliferative response of PBL might imply a temporary functional exhaustion of T lymphocytes) as in the other autoimmune diseases.

In order to test the hypothesis that stratification of Mexican Modification of the Systemic Lupus Erythematosus Disease Activity Index (MEX-SLEDAI) simplifies the genetic study of SLE, we evaluated the genetic susceptibility to inflammation and defects in clearance of immune complexes among SLE patients in Taiwan. SLE phenotypes were stratified according to the MEX-SLEDAI scores into two subgroups (<or=10 and >10), and then according to renal disorder and neurological disorder, aiming to minimize any loss of power associated with disease heterogeneity. Upon stratification, <em>IL1</em>-<em>beta</em> polymorphism and LTA were significantly associated with SLE within the MEX-SLEDAI <or=10 subgroup. When SLE patients were classified into two subgroups with or without renal disorder to stratify the genetic study, we could find that the stratification with renal disorder could partially confirm the hypothesis that stratification of MEX-SLEDAI score simplifies the genetic study of complex diseases such as SLE. So we concluded that in the mild disease state of SLE, stratification of disease phenotypes, especially <em>IL1</em>-<em>beta</em> and LTA, according to MEX-SLEDAI scores could reveal new associations between candidate genes and disease activity index of SLE.

Soluble cytokine receptors (SCR) can either act as inhibitors, by competitively inhibiting cytokines from binding to their membrane-bound receptors, or as enhancers, by serving as cytokine carriers. We have previously found that the levels of the Th2 cytokines interleukin (IL)-4, IL-5, IL-6, and IL-10 were positively correlated to eosinophils and IgE in nasal fluids from 60 children with seasonal allergic rhinitis. In this study, nasal fluids were reexamined to analyze IL-4sR, IL-6sR, IL-1 beta, TNF-alpha, IL-1sR2, TNF-sR1, and TNFsR2 in relation to eosinophils, neutrophils, ECP, and IgE. In allergic patients IL-4sR increased significantly during the pollen season, and weak, but positive correlations with IgE and eosinophils were found (r = 0.45, P < 0.001 and r = 0.4, P < 0.001 respectively). By contrast, none of the other SCR showed increases or correlations with IgE. However, positive correlations between IL1 beta, TNF-alpha, IL-6sR, IL-1sR2, TNF-sR1, TNF-sR2, and either neutrophils or ECP were found. Also, in healthy controls, these cytokines and their receptors were positively correlated to neutrophils or ECP. Thus, increased levels of the soluble IL-4 receptor, as well as IgE, were specifically associated with allergic rhinitis, whereas all other SCR correlated with either inflammatory cells or their products, in both allergic and healthy subjects. These results may suggest that SCR in vivo act as cytokine enhancers, rather than inhibitors.

Cytokine gene loci are one of the potential susceptibility factors in immuno-inflammatory diseases. Interleukin1 (IL-1) is a major monocyte derived proinflammatory cytokine that mediates tissue damage and cartilage loss in chronic arthritis. IL-1 locus has been linked to various autoimmune diseases, but data on juvenile idiopathic arthritis (JIA) is limited. Ninety-four patients with enthesitis related arthritis (ERA) category of JIA (ILAR criteria) were included in the study. One hundred eighty-five healthy blood donors were used as controls. IL-1Ra VNTR polymorphism was done by PCR using specific primers, and allele assignment was done on product size obtained on 2% agarose gel. IL-1α-889 and IL-β-511 polymorphism was done using PCR-RFLP method. Among 94 patients, 87 were males, and the mean age at onset of disease was 11.8 + 1.8 years; 17 had history of uveitis, and 13 had positive family history. Forty-two had enthesitis, and 51 had inflammatory back pain; all had arthritis. Seventy-six were HLA B27 positive. All three loci genotypes were in the Hardy-Weinberg equilibrium in controls. There was no difference in genotype frequency among patients and controls for IL-1Ra, IL-1α-889 and IL-β-511. IL1*RN2 homozygosity was more common in patients, whereas carriage rate for IL1*RN1 was less frequent in patients. Haplotype frequencies were also similar in both groups. IL-1 gene cluster is not a susceptibility locus for ERA.

BACKGROUND

We report cloning and characterization of a novel Leishmania infantum protein which we termed Lepp12, and we examine its possible implication in the interference with intramacrophage signaling pathways.

RESULTS

The protein Lepp12 contains 87 amino acid sequence and exhibits 5 potential phosphorylation sites by protein kinase C (PKC). Recombinant GST-Lepp12 is phosphorylated in vitro by exogenous PKC and by PKC-like activities present in promastigote and in the myelomonocytic THP-1 cell line, indicating that at least one phosphorylation site is functional on the recombinant Lepp12. The natural Lepp12 protein is present in L. infantum promastigotes, as evidenced using specific anti-Lepp12 antibodies produced by immunopurification from acute phase VL patient sera. Interestingly, human patient sera are strongly reactive with GST-Lepp12, demonstrating immunogenic properties of Lepp12 in man, but no immune response to Lepp12 is detectable in experimentally infected animals. When isolated from promastigotes, Lepp12 migrates as two species of apparent MW of 18.3 kDa (major) and 14 kDa (minor), localizes in the nuclear fraction and appears constitutively phosphorylated. Natural Lepp12 is phosphorylable in vitro by both exogenous PKC and PKC-like activity present in THP-1 extracts. The intracellular Lepp12 transfected into THP-1 cells activates these cells to produce IL-1beta and induces an enhancing effect on PMA stimulated IL-1beta synthesis, as demonstrated using GST-Lepp12 transfectants.

CONCLUSIONS

Together these results indicate that Lepp12 represents a substrate for PKC or other PKC-like activities present in the promastigote form and the host cell and therefore may interfere with signal transduction pathways involving PKC.

The stimulation of activated T cells with soluble peptides or peptide-pulsed T-APC in the absence of professional APC can anergize peptide-specific T cells. Here, we studied human T cell clones (TCCs) that either proliferate (T-responder) or do not proliferate (T-nonresponder) to activated T cells as antigen-presenting cells (APC) and investigated the efficacy of anergy induction in these two types of TCCs. The TCCs were specific to the p30 peptide from tetanus toxoid and secreted either a Th0- or a Th1-like cytokine pattern. To induce anergy, the TCCs were first stimulated by addition of the peptides directly to the cell cultures without additional APC (T-APC). Anergy was detected by restimulating these TCCs on professional B-APC. The proliferation, production of cytokines (IL2, IFN-gamma, IL4, IL5, IL1B-APC, the restimulation of TCCs primed by T-APC lead to a drastic reduction of proliferation and cytokine production for both T-responder and T-nonresponder TCCs. The functional down-regulation of TCCs mediated by soluble peptides could be overcome by addition of IL2, but not by IL1 or IL4. We concluded that the induction of T-cell anergy does not require cell proliferation.

Chest-wall invading malignancies usually necessitate the resection of the respective part of the thoracic wall. Gore-Tex® is the material of choice that is traditionally used to repair thoracic defects. This material is well accepted by the recipient; however, though not rejected, it is an inert material and behaves like a 'foreign body' within the thoracic wall. By contrast, there are materials that have the potential to physiologically integrate into the host, and these materials are currently under in vitro and also in vivo investigation. These materials offer a gradual but complete biodegradation over time, and severe adverse inflammatory responses can be avoided. Here, we present a novel material that is a biodegradable nanocomposite based on poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles in comparison to the traditionally employed Gore-Tex® being the standard for chest-wall replacement. On a mouse model of thoracic wall resection, that resembles the technique and localization applied in humans, poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles and Gore-Tex® were implanted subcutaneously and additionally tested in a separate series as a chest-wall graft. After 1, 2, 4 and 8 weeks cell infiltration into the respective materials, inflammatory reactions as well as neo-vascularization (endothelial cells) were determined in six different zones. While Gore-Tex® allowed for cell infiltration only at the outer surface, electrospun poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles were completely penetrated by infiltrating cells. These cells were composed mainly by macrophages, with only 4% of giant cells and lymphocytes. Total macrophage count increased by time while the number of IL1-β-expressing macrophages decreased, indicating a protective state towards the graft. As such, poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles seem to develop ideal characteristics as a material for chest-wall replacement by (a) having the advantage of full biodegradation, (b) displaying stable chest-wall structures and (c) adapting a physiological and integrating graft compared to Gore-Tex®.

In type 1 diabetes, beta cells are attacked and destroyed by auto reactive T cells causing major impairment of blood glucose metabolism and, ultimately, the development of life-threatening complications. Currently, the treatment of this chronic disease is based on the use of endogenous insulin and no curative therapies are available. Treatment approaches in this respect need to be directed toward the primary causes of the disease tackling beta cells' auto reactive T cells. The goal of any curative intervention in type 1 diabetes is the preservation of insulin-secreting cells. This may be achieved by the abrogation of the pathogenic reactivity to beta cell auto antigens while preserving full capacity to generate a normal immune response against foreign antigens. In this review, some of the most promising drugs for immune intervention in type 1 diabetes are presented and discussed including phase 3 clinical trials that involve: DiaPep277, Anti-CD3 Otelixizumab, Glutamic Acid Decarboxylase (GAD) and anti-IL1 receptor antagonist. These approaches are currently being tested in international multicenter trials and all of them have a very similar outcome in terms of a beneficial effect on beta cells.

OBJECTIVE

Recent studies point to the clinical and research utility of saliva as a valuable diagnostic aid for monitoring periodontal health. The objectives of this study were to detect novel biomarkers attributed to chronic inflammation in saliva and to determine if the levels of these markers correlate with severity of periodontitis and with standard obesity measures in participants in a periodontal maintenance program.

METHODS

In this cross-sectional assessment of 63 participants, unstimulated whole saliva was collected after recording anthropometric and clinical parameters of obesity and periodontitis, respectively. The levels of interleukin-1 receptor antagonist (IL-1ra), sCD40L, granzyme B and alpha-fetoprotein (AFP) in saliva were determined using multiplex proteomic immunoassays. The correlation between the four tested biomarker concentrations and obesity/periodontal measures was determined.

RESULTS

Positive correlation between fat% and granzyme B levels (r=0.292; p=0.020) and negative correlation between BMI and sCD40L (r=0.256; p=0.043) was observed. In addition, positive correlation between severity of periodontal disease and levels of IL1-ra (r=0.253; p=0.046) and negative correlation between periodontitis severity and sCD40L salivary levels (r=0.272; p=0.031) was noted. None of the above correlations remained statistically significant after multiple comparisons adjustment. After adjustment for clinical covariates, the relationship between sCD40L and periodontal severity remained suggestive (p=0.081).

CONCLUSIONS

Levels of four novel biomarkers of periodontitis were detectable in saliva of subjects enrolled in a periodontal maintenance program. Prospective studies with larger sample sizes and other populations are warranted to explore the diagnostic applicability of these markers.

The in vitro effect of the dextroisomer r-verapamil on blast cells derived from patients with acute myelogenous leukemia (AML) was studied. R-verapamil caused a dose-dependent inhibition of AML blast proliferation in the presence of stem-cell factor, leukemia inhibitory factor, interleukin 4, interleukin 6, and interleukin 10 when these cytokines were tested both alone and in different combinations. R-verapamil also inhibited the growth of clonogenic AML blast cells. The antiproliferative effect was not specific for AML blast cells, because r-verapamil also inhibited cytokine-dependent proliferation of blast cells derived from patients with acute lymphoblastic leukemia. The inhibitory effects of r-verapamil and anti-IL1 serum were additive, suggesting that the antiproliferative effect of r-verapamil does not depend solely on inhibition of IL1-mediated effects. Although r-verapamil inhibited spontaneous AML blast proliferation, for a majority of patients it caused only minimal, if any, inhibition of spontaneous cytokine secretion (IL1 alpha, IL1 beta, TNF alpha, IL6) by AML blast cells. Thus, although inhibition of IL1 effects may contribute in certain patients to the antiproliferative effect of r-verapamil, mechanisms other than IL1 inhibition seem to be more important in mediating the effects of r-verapamil.

FSTL3 as adipokine takes part in dyslipidemia and inflammatory response, but the association of FSTL3 with atherosclerosis is unclear. This study indicated that FSTL3 showed significantly higher level (control: 7.68 ± 3.10 vs. AS: 9.29 ± 2.37 ng/mL; P < 0.001) in atherosclerosis, and FSTL3 expressed higher in plaque of ApoE knockout mice and located in macrophages. Oxidized low-density lipoproteins induced expression and secretion of FSTL3, meanwhile FSTL3 promoted lipid accumulation in macrophages. The advanced study found that FSTL3 upregulated CD36 and LOX-1 expression in a dose-dependent manner; however, FSTL3 also evoked interleukin 1-β (IL1-β), monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor-α, and matrix metalloproteinase-9 (MMP-9) secretion in macrophages. On the contrary, that downregulated FSTL3 attenuated expression of oxidized low-density lipoproteins induced CD36, LOX-1, and inflammatory cytokines expressing. All of these results demonstrated that FSTL3 as a novelty cytokine takes part in the process of atherosclerosis through increasing lipid accumulation and inflammation through regulating CD36 and LOX-1 expression.

Although reperfusion of an ischemic organ is essential to prevent irreversible tissue damage, it may amplify tissue injury. This study investigates the role of endogenous testosterone in myocardial ischemia reperfusion and apoptosis in male rats. Material and method. Twenty four male rats were randomized into 4 equal groups: Group (1), sham group, rats underwent the same anesthetic and surgical procedure as the control group except for LAD ligation; Group (2), Active control group, rats underwent LAD ligation; Group (3), castrated, rats underwent surgical castration, left 3wks for recovery, and then underwent LAD ligation; and Group (4), Goserelin acetate treated, rats received 3.6 mg of Goserelin 3 wks before surgery and then underwent LAD ligation. At the end of experiment, plasma cTn I, cardiac TNF- α , IL1- β , ICAM-1, and Apoptosis level were measured and histological examination was made. Results. Compared to sham group, the levels of myocardial TNF- α , IL-1 β , ICAM-1, apoptosis, and plasma cTn I were significantly increased (P < 0.05) in control group and all rats showed significant myocardial injury (P < 0.05). Castration and Goserelin acetates significantly counteract the increase in myocardial levels of TNF- α , IL-1 β , ICAM-1, plasma cTn I, and apoptosis (P < 0.05) and significantly reduce (P < 0.05) the severity of myocardial injury. We conclude that castration and Goserelin acetates ameliorate myocardial I/R injury and apoptosis in rats via interfering with inflammatory reactions.

We have studied the in vitro effects of gold sodium thiomalate (GST) and auranofin (Auf) on the production of interleukin 1 (IL1) expressed as thymocyte co-stimulatory activity (TCSA), and interleukin 1 beta (IL1 beta) as modulated by interferon gamma (IFN gamma). Adherent cells (ADC), of which 80% were monocytes, were obtained from human peripheral blood, and stimulated with lipoprotein polysaccharide (LPS) for 24-48 h. TCSA and IL1 beta production by fresh ADC (0-24 h) was significantly higher than that of aged ADC (24-48 h). The addition of IFN gamma to ADC cultures, however, maintained the capacity of aging ADC to respond optimally to LPS. The addition of GST or Auf inhibited this modulatory effect of IFN gamma, resulting in a marked reduction of TCSA and IL1 beta production. The effects of IFN gamma on the production of IL1 may be important in the pathogenesis of rheumatoid arthritis (RA). The inhibition by GST and Auf of IFN gamma modulation may contribute to the therapeutic efficacy of these drugs in RA.

We report the generation of macrophage-hybridomas, obtained by somatic cell fusion between macrophage-enriched C3H.eB spleen cell population, and a drug-resistant MPC-11 myeloma cell line, designated as 4T00.1L1 clone. Screening for hybridomas possessing macrophage properties was carried out by assaying the presence of two macrophage-specific enzymes: lysozyme and nonspecific esterase. Two hybridomas, E2-7 and E2-10, were selected for further studies. We found that clones of E2-7 (E2-7.7) did not express Fc receptors but possessed cell-surface Ia molecules. In contrast, clones of E2-10 (E2-10.20) possessed Fc receptors but were devoid of Ia molecules. E2-7.7 did, however, express Fc receptors after mitomycin treatment, whereas E2-10.20 eliminated the expression of Fc receptors after treatment with mitomycin C. Opsonized erythrocytes were phagocytized by E2-10.20 cells, but not by E2-7.7. Phagocytosis was thus correlated with the possession of Fc receptors. Testing the response of KLH-primed lymph node cells to KLH-pulsed hybridoma cells, we found that E2-7.7 cells caused antigen-specific lymphoproliferative response, whereas E2-10.20 did not. Thus, antigens could be presented by E2-7.7 but not by E2-10.20 cells. The response was shown to be mediated by T but not by B lymphocytes. The difference in antigen-presenting capacity could not be attributed to differences in antigen uptake by the different hybridomas, because the two hybridomas manifested the same level of pinocytosis. Both hybridomas produced IL1. The differences in the properties of the two hybridomas may indicate that the normal partners represent two distinct subpopulations of macrophages. The segregation of functional properties among the hybridoma clones may lead to a clarification of the dependence of distinct functions on defined molecular structures.

Mouse-transformed epidermal cell line (Pam 212) generated the soluble mediators for promoting the growth of a mast cell line (MC9) in the presence of retinoic acid at a concentration of 10(-6)-10(-7) M. The effective molecule of MC9 cell growth promoting factor (MC9-GF) was non-dialyzable and eluted between the molecular weight of 45 K and 68 K on a TSK 2000 G column. Chromatofocusing analysis revealed that this factor had a pI range between 7.0 and 7.5. Anti-c-kit ligand antibody abrogated MC9-GF activity and RT-PCR analysis demonstrated that retinoic acid upregulates c-kit ligand mRNA expression by Pam cells. Several recombinant cytokines including IL1-alpha, IL-1 beta, IL-2, IL-3 or IL-4 did not promote MC9 cell growth at a concentration of 100 U/ml. The presence of anti-IL-1 alpha, -IL-1 beta, -IL-2, -IL-3 or -IL-4 antibodies did not abrogate the MC9-GF activity except for anti-c-kit ligand antibody.

Time-varying magnetic fields (TVMF), especially those of extremely low frequency (below 250 Hz), have been reported to have profound effects on biological systems due to the induced currents since the biological systems consist of electrolyte solution. We have been interested in utilizing TVMF for cellular immunomodulations, and have shown that the TVMF could augment macrophage activation. In this study, the effect of TVMF on lymphocyte activation was studied. Murine spleen lymphocytes were isolated from DDY mice and incubated in the presence of Concanavalin A (ConA) for 72 h. The lymphocytes were exposed to TVMF for various durations, from 20 min to 2 h. The proliferation activities of lymphocytes were assayed by ELISA by use of 5-bromo-2'-deoxy-uridine Labeling and Detection Kit III (Roche Diagnostic Corp. Indianapolis, IN, USA). The IL1beta and IL2 concentrations in the culture medium were measured by ELISA assay. The IL2 receptor expression on the lymphocytes was evaluated by FACS analysis by use of FITC-conjugated monoclonal antibody. The proliferation activities were significantly enhanced by the TVMF for up to 40 min exposure from the initiation of ConA stimulation. The degree of augmentation effects, defined by the ratio of activation index of with and without TVMF, was varied from 1.1 to 2.7, and related to the lymphocyte responsiveness to the ConA. The less responsive cells showed more TVMF augmentation effects. The TVMF exposure after 40 min from ConA addition showed no effect, suggesting that the TVMF effects are most likely related to the Ca ion influx. The prolonged exposure of TVMF depressed the augmentation effects, which was caused by the depressed IL-2 receptor expression although both IL1-beta and IL-2 productions were not affected.

Long-term culture of cells in rIL2-containing medium increases LAK activity on a per cell basis and produces an average 30-100-fold expansion over 14-21 days. The stimulation of PBL with anti-CD3 results in a 300-1000-fold increase in cell number while maintaining LAK activity. Cells stimulated with anti-CD3 and cultured in rIL2 for 12 days) can be further stimulated with beta IL1, or beta IFN, producing a further increase in LAK activity. These findings have allowed us to begin understanding the role of different signals in the activation of cells with LAK activity and together with biotechnological advances will allow for the culture of large numbers of cells for experimental and therapeutic purposes.

Recent findings indicate that interleukin-1 beta (IL1 beta), a monokine secreted by stimulated macrophages and monocytes, modulates neuroendocrine functions in a manner similar to classical hormones. In this study we show that IL1 modulates PRL secretion, assessed by reverse hemolytic plaque assay, and describe the effect of the monokine on adenylate cyclase activity and calcium fluxes in rat normal pituitary cells. In basal and vasoactive intestinal peptide (VIP)-stimulated conditions, low doses of IL1 reduced the mean plaque area, a direct index of PRL secretion without affecting the percentage of PRL-secreting cells. Similarly, low concentrations of IL1 inhibited adenylate cyclase activity in both basal and VIP-stimulated conditions, while higher concentrations restored the enzymatic activity to the control value. IL1 also caused a biphasic effect on the free intracellular calcium increase induced by maitotoxin, a calcium channel activator, being inhibitory at low and stimulatory at high concentrations. The effects of IL1 on adenylate cyclase activity and calcium fluxes were reversed by preincubation of the monokine with its polyclonal antibody, thus confirming the specificity of the effects. In conclusion, our data show that IL1 modulates PRL secretion by acting directly on pituitary cells through interaction with the adenylate cyclase-cAMP system and calcium flux.