BACKGROUND

To investigate whether immunologic factors in breast milk change in response to nursing infants' infection.

RESULTS

Total CD45 leukocyte count dropped from 5,655 (median and interquartile range: 1,911; 16,871) in the acute phase to 2,122 (672; 6,819) cells/ml milk after recovery with macrophage count decreasing from 1,220 (236; 3,973) to 300 (122; 945) cells/ml. Tumor necrosis factor-α (TNFα) levels decreased from 3.66 ± 1.68 to 2.91 ± 1.51 pg/ml. The decrease in lactoferrin levels was of borderline statistical significance. Such differences were not recorded in samples of the controls. Interleukin-10 levels decreased in the sick infants' breast milk after recovery, but also in the healthy controls, requiring further investigation. Secretory immunoglobulin A levels did not change significantly in the study or control group.

CONCLUSIONS

During active infection in nursing infants, the total number of white blood cells, specifically the number of macrophages, and TNFα levels increase in their mothers' breast milk. These results may support the dynamic nature of the immune defense provided by breastfeeding sick infants.

METHODS

Breast milk from mothers of 31 infants, up to 3 months of age, who were hospitalized with fever, was sampled during active illness and recovery. Milk from mothers of 20 healthy infants served as controls.

Previous studies indicated that inorganic pyrophosphatase of Ascaris suum (AsPPase) plays an important role in larval survival in the host. Here we describe a precise role for AsPPase in larval molting and development and also describe the potential role of recombinant AsPPase (rAsPPase) in protective immunity to A. suum infection. Using reverse transcriptase PCR analysis, we found that disruption of AsPPase gene function by RNA interference resulted in suppression of AsPPase mRNA levels. RNA interference also caused inhibition of molting of third-stage larvae (<em>31</em>%) and suppression of native protein expression, as demonstrated by a 56% reduction in enzyme activity and quantified by immunoblot and immunofluorescence analyses, suggesting that AsPPase has a role in the molting process. The anatomic location of the AsPPase native enzyme in the hypodermis of larvae along with its elevated expression prior to and during the molting process supports such a role. Anti-rAsPPase immunoglobulin G (IgG) also resulted in 57% inhibition of molting of A. suum lung-stage third-stage larvae to fourth-stage larvae in vitro with developmental arrest. Antigenic epitopes of AsPPase overlapped the enzyme active sites. Mice immunized with rAsPPase exhibited high antigen-specific IgG antibody responses and were protected (>70%) against a challenge A. suum migratory-phase infection. Splenic T cells from rAsPPase-immunized mice produced low levels of T helper 1-type cytokines (gamma interferon and <em>interleukin</em>-2) in vitro but exhibited an elevated <em>interleukin</em>-10 response. A significantly high level of IgG1 subclass antibodies was found in immunized mice. Our results establish that AsPPase has a critical role in the molting and development of Ascaris roundworms and suggest the potential of AsPPase for use as a candidate vaccine against ascariasis.

The characteristics of soluble <em>interleukin</em>-6 receptor (sIL-6R) in murine sera were examined. To investigate a relationship between serum sIL-6R level and autoimmune diseases, quantitative analysis of serum sIL-6R in MRL/lpr mice was performed by an enzyme-linked immunosorbent assay. The serum sIL-6R level in MRL/lpr mice of both sexes was below the detection limit (< 1.0 ng/ml) at 8 weeks of age, but it increased in accordance with age and reached 42 +/- 9.3 ng/ml in female and <em>31</em> +/- 13 ng/ml in male mice at 30 weeks of age. In MRL/+ mice, although an age-associated increase in serum sIL-6R level was observed, it was much less extensive than that in MRL/lpr mice. Elevated serum sIL-6R level at the age of 30 weeks was observed in female and male (NZB x NZW)F1 mice (32 +/- 10 ng/ml and 17 +/- 5.0 ng/ml, respectively), and male BXSB/Mpj Yaa mice (42 +/- 18 ng/ml), suggesting that elevated serum sIL-6R in aged mice is one of the characteristics of autoimmune-prone mice. Quantitative analysis of serum IL-6 in MRL/lpr revealed that the serum sIL-6R level correlated well with the serum IL-6 level. We also showed that sIL-6R in the sera from MRL/lpr mice could mediate the IL-6 functions through the IL-6 signal-transducing receptor component gp130, suggesting that elevated production of sIL-6R may partly contribute to development of autoimmune disease in MRL/lpr mice.

BACKGROUND

Preoperative alteration of T cell-mediated immunity as well as an altered immune response to surgical stress were found in long-term alcoholic patients. The aim of this study was to evaluate perioperative T cell-mediated immune parameters as well as cytokine release from whole blood cells after lipopolysaccharide stimulation and its association with postoperative infections.

METHODS

Fifty-four patients undergoing elective surgery of the aerodigestive tract were included in this prospective observational study. Long-term alcoholic patients (n = <em>31</em>) were defined as having a daily ethanol consumption of at least 60 g and fulfilling the Diagnostic and Statistical Manual of Mental Disorders for either alcohol abuse or alcohol dependence. The nonalcoholic patients (n = 23) were defined as drinking less than 60 g ethanol/day. Blood samples to analyze the immune status were obtained on morning before surgery and on the morning of days 1, 3, and 5 after surgery.

RESULTS

Basic patient characteristics did not differ between groups. Before surgery, the T helper 1:T helper 2 ratio (Th1: Th2) was significantly lower (P < 0.01), whereas plasma interleukin 1beta and lipopolysaccharide-stimulated interleukin 1ra from whole blood cells were increased in long-term alcoholic patients. After surgery, a significant suppression of the cytotoxic lymphocyte ratio (Tc1:Tc2), the interferon gamma:interleukin 10 ratio from lipopolysaccharide-stimulated whole blood cells, and a significant increase of plasma interleukin 10 was observed. Long-term alcoholics had more frequent postoperative infections compared with nonalcoholic patients (54%vs. 26%; P = 0.03).

CONCLUSIONS

T helper cell-mediated immunity was significantly suppressed before surgery and possibly led to inadequate cytotoxic lymphocyte and whole blood cell response in long-term alcoholic patients after surgery. This altered cell-mediated immunity might have accounted for the increased infection rate in long-term alcoholic patients after surgery.

Bacterial sepsis is a frequent complication in patients with cancer who are receiving high doses of <em>interleukin</em>-2. We evaluated the function of neutrophils from such patients to determine whether there was any abnormality in this form of host defense. Before <em>interleukin</em>-2 therapy, neutrophils from <em>31</em> patients with metastatic cancer were normal in assays of random migration and chemotaxis. Superoxide production, phagocytosis, secretion of granule proteins, and bactericidal activity were also normal. Neutrophils from the patients near the end of the first course of <em>interleukin</em>-2 had severely impaired chemotaxis in response to a formylated peptide stimulus (mean [+/- SEM], 49.6 +/- 7.4 percent of base line; P less than 0.001). The detect in chemotaxis improved 5 to 10 days after patients completed the first course of <em>interleukin</em>-2 therapy but recurred toward the end of the second course of such therapy (35.3 +/- 6.9 percent of base line; P less than 0.001). The chemotactic response to a second stimulus (zymosan-activated serum) was also abnormal, but random migration, superoxide production, bactericidal activity, and the secretion of neutrophil granule constituents remained normal or increased throughout treatment with <em>interleukin</em>-2. We conclude that patients who receive <em>interleukin</em>-2 immunotherapy acquire an acute, profound, and reversible defect in neutrophil chemotaxis that may contribute to the high morbidity resulting from bacterial infections in these patients.

Cytokines produced by tumor cells may have various effects on antitumor immune responses and tumor growth. In the present study, the cytokine production of <em>31</em> lung cancer cell lines was evaluated, while any correlation with the histological type, the induction of tumor-specific cytotoxic T lymphocytes (CTL) in vitro, and angiogenesis and the infiltration of inflammatory cells in tumor tissues were also examined. Production of <em>interleukin</em> (IL)-1alpha, IL-1beta, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-alpha, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor, transforming growth factor (TGF)-beta and vascular endothelial growth factor (VEGF) in the culture supernatant was measured using enzyme-linked immunosorbent assay. Each cytokine was produced in a substantial number of the tumor cell lines. In particular, IL-6, IL-8, TGF-beta and VEGF were produced in 18 (55%), 29 (94%), <em>31</em> (100%) and 28 (90%) of <em>31</em> cell lines, respectively. However, neither IL-4 nor TNF-alpha was produced at all by any tumor cell line. TGF-beta production was significantly higher in adenocarcinoma than in squamous cell carcinoma (P = 0.03). Immunohistochemical staining revealed the magnitude of macrophage infiltration, and angiogenesis in surgically resected tumor tissue specimens correlated well with GM-CSF and IL-8 production from the corresponding cell lines. Among six lung cancer cell lines, CTL were induced in the three lung cancer cell lines that produced a lower amount of TGF-beta (<100 pg/mL). These findings suggested that TGF-beta produced by tumor cells could inhibit the induction of CTL in vitro. The present results suggest that the production of various cytokines from tumor cells might exert various paracrine effects both in vivo and in vitro.

<em>Interleukin</em>-10 (IL-10), originally described as a product of TH2 cell clones, has been recognized as a potential immunosuppressive cytokine. To investigate the relevance of IL-10 in melanoma patients in vivo, we studied IL-10 serum levels in 104 untreated patients in different stages of the disease; 20 healthy subjects and 22 patients with inflammatory dermatoses served as controls. Serum levels were measured by ELISA. Only one of <em>31</em> patients with stage I melanoma (3%) and one of 16 stage II patients (6%) showed detectable IL-10 levels. Interestingly, six of 17 patients with lymph node metastases (stage III, 35%) and 29 of 40 patients with widespread disease (stage IV, 73%) revealed IL-10 levels of 15-480 pg/ml. No healthy person and only one control patient had a detectable IL-10 serum level. The data suggest that IL-10 in melanoma patients may contribute to down-modulation of anti-tumour responses in vivo.

This study investigated the influence of age, body composition, physical fitness, training volume and intensity, and underlying systemic inflammation on exercise-induced inflammation and innate immune function in a heterogeneous group of cyclists. Subjects included <em>31</em> male cyclists (mean ± standard deviation, age 38.8 ± 10.6 years, body fat 17.8%± 5.6%, VO(2max) 55.8 ± 8.4 mL kg(-1) min(-1)) who cycled for 1.75 h at 60% watts(max) followed by a 10-km time trial (18.3 ± 0.3 min). Blood samples were collected pre-, post-, and 1-h-postexercise, and analyzed for WBCs, 9 cytokines [<em>interleukin</em> (IL)-6, tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, interferon-γ, IL-1β, IL-2, IL-8, IL-10, and IL-12p70], and granulocyte and monocyte phagocytosis (GR-PHAG and MO-PHAG) and oxidative burst activity (GR-OBA and MO-OBA). Exercise-induced changes varied widely, with significant increases measured for 8 of 9 cytokines, GR-PHAG (mean change 99%) (95% confidence limits, 69%, 128%) and MO-PHAG (43%) (28%, 58%), and WBC (160%) (133%, 187%), and decreases for GR-OBA (-30%) (-43%,-16%) and MO-OBA (-23%) (-36%,-10%). Correlation and stepwise regression analysis revealed that changes in these variables were not related to age, body fat percentage, VO(2max), training volume, or pre-exercise C-reactive protein. Performance measures, specifically the average heart rate and rating of perceived exertion, were correlated with changes in several variables, including IL-8 (r=0.68 and 0.67, respectively, P<0.001) and IL-6 (r=0.51, P=0.004, and r=0.46, P=0.011, respectively). In summary, variance in exercise-induced inflammation and innate immunity in male cyclists in response to 2 h of endurance exercise is best explained by exercise intensity.

Seventy renal allograft biopsies were done in <em>31</em> patients, routinely at 1, 2, and 3 months posttransplant, and as clinically indicated, using an automated biopsy "gun." The histological diagnosis was made according to the Banff schema, which emphasizes tubulitis and vascular inflammation over mononuclear cell infiltration. Fifty-three biopsies satisfied histological inclusion criteria. Twenty-nine biopsies were obtained from stable patients, defined as those in whom serum creatinine had changed < 10% in 2 weeks, and in whom immunosuppression (cyclosporine, azathioprine, and prednisone) had not been increased in that interval. Of these biopsies, 30% (9/29) showed rejection, which could not have been predicted from pretransplant (HLA mismatch, panel-reactive antibody titer) or posttransplant (cyclosporine and serum <em>interleukin</em> 2 receptor levels) variables. The significance of these early subclinical rejection episodes is unknown, and their effects on long-term graft histology and function are being examined in a controlled study.

BACKGROUND

Clinical trials in cystic fibrosis (CF) have been hindered by the paucity of well characterised and clinically relevant outcome measures.

OBJECTIVE

To evaluate a range of conventional and novel biomarkers of CF lung disease in a multicentre setting as a contributing study in selecting outcome assays for a clinical trial of CFTR gene therapy.

METHODS

A multicentre observational study of adult and paediatric patients with CF (>10 years) treated for a physician-defined exacerbation of CF pulmonary symptoms. Measurements were performed at commencement and immediately after a course of intravenous antibiotics. Disease activity was assessed using 46 assays across five key domains: symptoms, lung physiology, structural changes on CT, pulmonary and systemic inflammatory markers.

RESULTS

Statistically significant improvements were seen in forced expiratory volume in 1 s (p<0.001, n=32), lung clearance index (p<0.01, n=32), symptoms (p<0.0001, n=37), CT scores for airway wall thickness (p<0.01, n=<em>31</em>), air trapping (p<0.01, n=30) and large mucus plugs (p=0.0001, n=<em>31</em>), serum C-reactive protein (p<0.0001, n=34), serum <em>interleukin</em>-6 (p<0.0001, n=33) and serum calprotectin (p<0.0001, n=<em>31</em>).

CONCLUSIONS

We identify the key biomarkers of inflammation, imaging and physiology that alter alongside symptomatic improvement following treatment of an acute CF exacerbation. These data, in parallel with our study of biomarkers in patients with stable CF, provide important guidance in choosing optimal biomarkers for novel therapies. Further, they highlight that such acute therapy predominantly improves large airway parameters and systemic inflammation, but has less effect on airway inflammation.

The immunogenicity and protective efficacy of the recombinant <em>31</em>-kDa outer membrane protein from Brucella melitensis (rOmp<em>31</em>), administered with incomplete Freund's adjuvant, were evaluated in mice. Immunization of BALB/c mice with rOmp<em>31</em> conferred protection against B. ovis and B. melitensis infection. rOmp<em>31</em> induced a vigorous immunoglobulin G (IgG) response, with higher IgG1 than IgG2 titers. In addition, spleen cells from rOmp<em>31</em>-immunized mice produced <em>interleukin</em> 2 (IL-2) and gamma interferon, but not IL-10 or IL-4, after in vitro stimulation with rOmp<em>31</em>, suggesting the induction of a T helper 1 (Th1) response. Splenocytes from rOmp<em>31</em>-vaccinated animals also induced a specific cytotoxic-T-lymphocyte activity, which led to the in vitro lysis of Brucella-infected macrophages. In vitro T-cell subset depletion indicated that rOmp<em>31</em> immunization elicited specific CD4+ T cells that secrete IL-2 and gamma interferon, while CD8+ T cells induced cytotoxic-T-lymphocyte activity. In vivo depletion of T-cell subsets showed that the rOmp<em>31</em>-elicited protection against B. melitensis infection is mediated by CD4+ T cells while the contribution of CD8+ T cells may be limited. We then evaluated the immunogenicity and protective efficacy of a known exposed region from Omp<em>31</em> on the Brucella membrane, a peptide that contains amino acids 48 to 74 of Omp<em>31</em>. Immunization with the synthetic peptide in adjuvant did not elicit a specific humoral response but elicited a Th1 response mediated by CD4+ T cells. The peptide in adjuvant induced levels of protection similar to those induced by rOmp<em>31</em> against B. melitensis but less protection than was induced by rOmp<em>31</em> against B. ovis. Our results indicate that rOmp<em>31</em> could be a useful candidate for the development of subunit vaccines against B. melitensis and B. ovis.

OBJECTIVE

The death of chondrocytes by apoptosis is characteristic of degenerative joint diseases, such as osteoarthritis (OA). Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been shown to play an important role in the development of OA. In this study we analyzed the effects of TNF-alpha and IL-1beta on cell death in normal human chondrocytes.

METHODS

Normal human chondrocytes were isolated from knee cartilage obtained at autopsy from 30 adult cadaveric donors. The cells were stimulated with TNF-alpha (10 ng/ml) or IL-1beta (5 ng/ml) in the presence or absence of Ro 31-8220 (Ro: a structurally related analog of bisindolylmaleimide that inhibits mitogen-activated protein kinase phosphatase 1 [MKP-1]) (Ro; 10 microM), an MKP-1 inhibitor, which induces apoptosis in chondrocytes. Apoptosis was evaluated by flow cytometry (propidium iodide) and nuclear morphology was evaluated with 4',6'-dianidino-2-phenylindole dihydrochloride. The expressions of caspase-8, -7 and -3 and Bcl-2 were analyzed by Western blot and the activation of caspase-3 and -8 was measured by flow cytometry. Prostaglandin E2 (PGE2) was evaluated by enzyme-linked immunosorbent assay.

RESULTS

At 24 h the percentage of apoptotic (hypodiploid) nuclei induced by TNF-alpha+Ro was higher than the level induced by Ro alone. The combination of IL-1beta (5 ng/ml) with Ro did not show a synergistic effect. A morphological analysis demonstrated that treatment with TNF-alpha+Ro resulted in a large number of cells with condensed nuclei and DNA fragmentation. Western blot studies indicated that IL-1beta+Ro did not induce the time-dependent activation of caspase-8, -7 and -3 as seen with TNF-alpha+Ro. As quantified by flow cytometry, TNF-alpha+Ro induced a higher level of caspase-3 and -8 activation than that seen with IL-1beta+Ro. Pre-incubation for 2h with caspase inhibitors for caspase-3, -7, -8 and pan-caspase significantly decreased the hypodiploid DNA peak induced by treatment with TNF-alpha+Ro at 24 h. Indomethacin increased the cell death induced by IL-1beta+Ro; however, apoptosis induced by TNF-alpha+Ro was not modified by indomethacin.

CONCLUSIONS

These results confirm that TNF-alpha and IL-1beta regulate apoptosis differently in this human chondrocyte model and that the differing effects of these cytokines are PGE2-independent. Indomethacin potentiates the effect of IL-1 on cell death and this may explain the reported effect of indomethacin on the progression of joint destruction.

OBJECTIVE

The rs1143679 variant of ITGAM, encoding the R77H variant of CD11b (part of complement receptor 3; CR3), is among the strongest genetic susceptibility effects in human systemic lupus erythematosus (SLE). The authors aimed to demonstrate R77H function in ex-vivo human cells.

METHODS

Monocytes/monocyte-derived macrophages from healthy volunteers homozygous for either wild type (WT) or 77H CD11b were studied. The genotype-specific expression of CD11b, and CD11b activation using conformation-specific antibodies were measured. Genotype-specific differences in iC3b-mediated phagocytosis, adhesion to a range of ligands and the secretion of cytokines following CR3 ligation were studied. The functionality of R77H was confirmed by replicating findings in COS7 cells expressing variant-specific CD11b.

RESULTS

No genotype-specific difference in CD11b expression or in the expression of CD11b activation epitopes was observed. A <em>31</em>% reduction was observed in the phagocytosis of iC3b opsonised sheep erythrocytes (sRBC(iC3b)) by 77H cells (p=0.003) and reduced adhesion to a range of ligands: notably a 24% reduction in adhesion to iC3b (p=0.014). In transfected COS7 cells, a 42% reduction was observed in phagocytosis by CD11b (77H)-expressing cells (p=0.004). A significant inhibition was seen in the release of Toll-like receptor 7/8-induced pro-inflammatory cytokines from WT monocytes when CR3 was pre-engaged using sRBC(iC3b), but no inhibition in 77H monocytes resulting in a significant difference between genotypes (<em>interleukin</em> (IL)-1β p=0.030; IL-6 p=0.029; tumour necrosis factor alpha p=0.027).

CONCLUSIONS

The R77H variant impairs a broad range of CR3 effector functions in human monocytes. This study discusses how perturbation of this pathway may predispose to SLE.

Cytokines, important biochemical mediators of inflammation, cause a rapid fall in the plasma concentration of cholesterol in vivo. One mechanism by which cytokines may cause acquired hypocholesterolemia is by decreasing the hepatic synthesis and secretion of apolipoproteins. To test this hypothesis, we incubated Hep G2 cells with human recombinant tumor necrosis factor-alpha, <em>interleukin</em>-1 beta, and <em>interleukin</em>-6. Each of the cytokines resulted in a dose-related reduction in the concentrations of apolipoprotein (apo) A-I, apoB, and lecithin:cholesterol acyltransferase (LCAT) activity in the medium after 24 hours of incubation. The effect of cytokines on apolipoprotein accumulation was not affected by preincubation of Hep G2 cells with fatty acids. Cytokines decreased the concentration of cellular apoA-I mRNA in a dose-related fashion but did not affect cellular concentrations of apoB mRNA. The concentrations of triglyceride and cholesterol were also reduced in the medium of cells incubated with cytokines. Total cell sterol synthesis rates were calculated by [14C]acetate incorporation. Cells incubated with <em>interleukin</em>-6 had a <em>31</em>% increase in sterol synthesis rate but a 41% decrease in sterol secretion. These data suggest that these cytokines can decrease the hepatic synthesis and/or secretion of apolipoproteins and that this may explain, in part, the acquired hypocholesterolemia seen during acute and chronic inflammation.

OBJECTIVE

Although inflammation is increasingly implicated in psychiatric disorders, less is known about its role in anorexia nervosa (AN), an illness with low body mass index (BMI).

METHODS

We performed a systematic PubMed literature search until 12/<em>31</em>/2013 and meta-analyzed cross-sectional and longitudinal studies comparing circulating pro- and anti-inflammatory cytokines between patients with anorexia nervosa (AN) and healthy controls (HCs) (1) before and (2) after weight gain, and (3) within AN patients before and after weight gain. Standardized mean differences (SMDs)± 95% confidence intervals (CIs) for results from ≥ 2 studies were calculated.

RESULTS

Of 999 initial hits, 22 studies with 924 participants (AN=512, HCs=412) were eligible. Compared to HCs, tumor necrosis factor (TNF)-alpha (SMD=0.35, 95%CI=0.09-0.61, p=0.008), interleukin (IL)1-beta (SMD=0.51, 95%CI=0.18-0.84, p=0.003), IL-6 (SMD=0.43, 95%CI=0.11-0.76, p=0.009), and TNF-receptor-II (SMD=0.42, 95%CI:0.07-0.78, p=0.02) were significantly elevated in AN, while C-reactive protein (SMD=-0.53, 95%CI=-.77, -0.28, p<0.0001) and IL-6 receptor (SMD=-0.85, 95%CI=-1.33, -0.36, p=0.0006) were significantly decreased. No differences were found for TNF-receptor I and TGF-β. Across a subset of eight longitudinal studies (AN=152, HCs=129), significant weight gain (baseline BMI=15.4 ± 1.5, endpoint BMI=18.2 ± 1.6, p<0.0001) was not associated with significant changes in TNF-α, IL-6 and IL1-β. However, after weight gain, IL-6 was not different anymore compared to HCs (SMD=0.06, 95%CI=-0.32, 0.45, p=0.75). In meta-regression, shorter illness duration (p=0.0008), but not younger age (p=0.71) significantly moderated greater IL-6 levels.

CONCLUSIONS

Despite abnormally low BMI, AN seems to be associated with increased inflammatory cytokines. Whether specific elevated cytokines represent trait or state markers of AN, and whether they could be treatment targets requires further study.

OBJECTIVE

MicroRNAs (miRNAs) function to fine-tune the control of immune cell signaling. It is well established that there are abnormalities in the <em>interleukin</em>-2 (IL-2)-related signaling pathways in systemic lupus erythematosus (SLE). The miR-<em>31</em> microRNA has been found to be markedly underexpressed in patients with SLE, and thus the present study was undertaken to investigate the role of miR-<em>31</em> in IL-2 defects in lupus T cells.

METHODS

Expression levels of miR-<em>31</em> were quantitated using TaqMan miRNA assays. Transfection and stimulation of cultured cells followed by TaqMan quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and reporter gene assays were conducted to determine the biologic function of miR-<em>31</em>. NF-AT nuclear translocation and expression were quantitatively measured using an ImageStream cytometer. Bioinformatics analysis, small interfering RNA (siRNA) knockdown, and Western blotting were performed to validate miR-<em>31</em> targets and effects.

RESULTS

The expression of miR-<em>31</em> was significantly decreased in lupus T cells, and this was positively correlated with the expression of IL-2. Overexpression of miR-<em>31</em> in T cells increased the production of IL-2 by altering NF-AT nuclear expression and IL2 promoter activity, while knockdown of endogenous miR-<em>31</em> reduced IL-2 production. RhoA expression was directly repressed by miR-<em>31</em> in T cells. Of note, siRNA-mediated knockdown of RhoA enhanced IL2 promoter activity and, consequently, up-regulated IL-2 production. RhoA expression was consistently up-regulated and negatively correlated with the levels of miR-<em>31</em> in lupus T cells. Manipulation of miR-<em>31</em> expression in lupus T cells restored the expression of IL-2 at both the messenger RNA and protein levels.

CONCLUSIONS

MicroRNA-<em>31</em> is a novel enhancer of IL-2 production during T cell activation. Dysregulation of miR-<em>31</em> and its target, RhoA, could be a novel molecular mechanism underlying the IL-2 deficiency in patients with SLE.

Resistance to recombinant human erythropoietin occurs in a small but important proportion of hemodialysis patients. This may be due to increased immune activation because pro-inflammatory cytokines inhibit erythropoiesis in vitro. Using FACScan flow cytometry, the proportion of PMA/ionomycin-stimulated T cells expressing cytokines ex vivo was compared in 18 poor responders to erythropoietin, 14 good responders to erythropoietin, and 14 normal controls. CD4(+) T cells from poor responders expressed more interferon-gamma (IFN-gamma; 19 +/- 6%) compared with good responders (11 +/- 6%, P < 0.001) and controls (12 +/- 6%, P < 0.01). Similarly, CD4+ T cells from poor responders expressed more tumor necrosis factor-alpha (TNF-alpha; poor responders: 51 +/- 19% versus good responders: 27 +/- 15% [P < 0.01] and controls: 30 +/- 19% [P < 0.01]). CD4+ expression of IL-10 was also enhanced (poor responders: 1.6 +/- 1.1% versus good responders: 0.7 +/- 0.6% [P < 0.05] and controls: 0.5 +/- 0.2% [P < 0.01]). Likewise, CD4+ expression of <em>interleukin</em>-13 (IL-13) was increased (poor responders: 4.4 +/- 4.2% versus good responders: 1.6 +/- 1.7% [P < 0.05] and controls: 1.6 +/- 1.5% [P < 0.05]). CD8+ T cells from poor responders also showed enhanced expression of cytokines. For IFN-gamma, poor responder expression was 48 +/- 20% compared with <em>31</em> +/- 17% (P < 0.05) for good responders and 23 +/- 15% (P < 0.01) for controls. TNF-alpha expression for poor responders was 41 +/- 21% versus 25 +/- 14% for good responders (P < 0.05) and 21 +/- 15% for controls (P < 0.01). IL-10 expression for poor responders was 2.0 +/- 1.2% (good responders: 0.7 +/- 0.6% [P < 0.01]; controls: 0.5 +/- 0.2% [P < 0.001]). These data indicate that T cells from poor responders are in an enhanced activation state possibly as a result of chronic inflammation. In the absence of any other cause (such as iron deficiency), the overproduction of cytokines may account for hyporesponsiveness to erythropoietic therapy in patients with renal failure.

The cytokine <em>interleukin</em>-6, which has been shown to be increased in patients with burn injuries, is produced by activated monocytes and endothelial cells and has many in vitro activities, including stimulation of acute-phase protein synthesis in hepatocytes, immunoglobulin synthesis in B lymphocytes, and stimulation of growth of megakaryocytes. In 13 patients with a mean of <em>31</em>% full-thickness burns, we studied the relation of serum <em>interleukin</em>-6 to clinical parameters and parameters of the acute-phase response and immunoglobulin production. <em>Interleukin</em>-6 was already elevated within hours after the injury was sustained, and it remained elevated for several weeks. All components of the acute-phase response were observed: fever, tachycardia, leukocytosis with an associated left shift, elevation of C-reactive protein and alpha 1-antitrypsin, and a decrease in albumin levels. In the second week after burn injury, immunoglobulin M levels peaked, followed by a prolonged elevation of immunoglobulin G levels. Thrombocyte counts initially decreased and rebounded to supranormal levels after 2 weeks. <em>Interleukin</em>-6 levels were positively correlated with acute-phase responses. We believe that the production of <em>interleukin</em>-6 induces the synthesis of acute-phase proteins. High <em>interleukin</em>-6 levels may also be an etiologic factor in the marked immunoglobulin response observed. Likewise, the relation between the megakaryocyte-promoting activity of <em>interleukin</em>-6 and the rebound thrombocytosis requires further investigation.

Individuals exposed to dusts from concentrated animal feeding operations report increased numbers of respiratory tract symptoms, and bronchoalveolar lavage samples from such individuals demonstrate elevated lung inflammatory mediators, including <em>interleukin</em> (IL)-8 and IL-6. We previously found that exposure of bronchial epithelial cells to hog barn dusts resulted in a protein kinase C (PKC)-dependent increase in IL-6 and IL-8 release. We hypothesized that cattle feedlot dusts would also generate bronchial epithelial <em>interleukin</em> release in vitro. To test this, we used <em>interleukin</em> ELISAs and direct PKC isoform assays. We found that a dust extract from cattle feedlots [feedlot dust extract (FLDE)] augments PKC activity of human bronchial epithelial cells in vitro. A 5-10% dilution of FLDE stimulated a significant release of IL-6 and IL-8 at 6-24 h in a PKC-dependent manner vs. control medium-treated cells. An increase in PKCalpha activity was observed with 1 h of FLDE treatment, and PKCepsilon activity was elevated at 6 h of FLDE exposure. The PKCalpha inhibitor, Gö-6976, did not inhibit FLDE-stimulated IL-8 and IL-6 release. However, the PKCepsilon inhibitor, Ro <em>31</em>-8220, effectively inhibited FLDE-stimulated IL-8 and IL-6 release. Inhibition of FLDE-stimulated IL-6 and IL-8 was confirmed in a dominant-negative PKCepsilon-expressing BEAS-2B cell line but not observed in a PKCalpha dominant negative BEAS-2B cell line. These data support the hypothesis that FLDE exposure stimulates bronchial epithelial IL-8 and IL-6 release via a PKCepsilon-dependent pathway.

The migration and proliferation of vascular smooth muscle cells (SMCs) during neointima formation in atherosclerosis and angioplasty restenosis is mediated by certain growth factors and cytokines, one action of which may be to promote basement-membrane degradation. To test this hypothesis further, the effects of such growth factors and cytokines on the synthesis of two basement-membrane-degrading metalloproteinases, namely the 72 kDa gelatinase (MMP-2, gelatinase A) and the 95 kDa gelatinase (MMP-9, gelatinase B) and three tissue inhibitors of metalloproteinases (TIMPs) was studied in primary cultured rabbit aortic SMCs. Expression of the 95 kDa gelatinase was increased by phorbol myristate acetate, foetal calf serum, thrombin and <em>interleukin</em>-1alpha (IL-1alpha); platelet-derived growth factor (PDGF) BB alone had no effect but acted synergistically with IL-1alpha. A selective protein kinase C inhibitor, Ro <em>31</em>-8220, abolished induction of the 95 kDa gelatinase. In contrast, none of the agents tested modulated the synthesis of the 72 kDa gelatinase. We conclude that maximal up-regulation of 95 kDa gelatinase expression requires the concerted action of growth factors and inflammatory cytokines mediated, in part, by a protein kinase C-dependent pathway. TIMP-1 and TIMP-2 were highly expressed, and their synthesis was not affected by growth factors or cytokines. Expression of TIMP-3 mRNAs was, however, increased by PDGF and transforming growth factor beta, especially in combination. Divergent regulation of gelatinase and TIMP expression implies that either net synthesis or net degradation of basement membrane can be mediated by appropriate combinations of growth factors and cytokines.

BACKGROUND

Polymorphisms in the gene for <em>interleukin</em> 1beta (IL-1B) have been found to increase the risks of gastric cancer and its precursors in response to Helicobacter pylori infection in white populations. however, there has been no independent confirmation of the role of IL-1B markers in gastric cancer patients from Asian populations. Moreover, there have been conflicting data regarding the effect of IL-1B-511/-<em>31</em> on the risk of gastric cancer or its precursors in Asian populations. Therefore, we assessed an additional polymorphism in the promoter region of IL-1B at position-1473 with the IL-1B-511/-<em>31</em> polymorphisms in a Korean population.

METHODS

In a case-control study, including 3<em>31</em> gastric cancer cases and 433 controls, we assessed the association between the three polymorphisms and the risk of gastric cancer. All genotyping was performed in duplicate. To assess the DNA-binding activity of IL-1B-1473 in vitro, we performed an electrophoretic mobility shift assay (EMSA).

RESULTS

When cases were divided according to the histologic type of the tumor, a significant difference in genotype frequencies for IL-1B-1473 was observed only between intestinal-type cases and controls (odds ratio [OR], 1.8; 95% confidence interval [CI], 1.0-3.5 and OR 2.1 and 95% CI, 1.1-4.2 in the CG and GG genotypes, respectively). In the cases, there was a deviation from Hardy-Weinberg equilibrium in the IL-1B-511/-<em>31</em> loci confined to the intestinal type, due to the excess of heterozygotes. The IL-1B-1473G allele showed decreased binding to nuclear extract, indicating a wearker promoter activity on EMSA.

CONCLUSIONS

We identified a novel single-nucleotide polymorphism, 1473C->>G, in the IL-1B promoter that was significantly associated with gastric cancer among Koreans. Our results also suggest that the association between IL-1B polymorphism and an increased risk of gastric cancer may depend on the histologic type of gastric cancer.

We have identified and characterized a novel cysteine protease named CMH-1 that is a new member of the <em>interleukin</em> 1 beta converting enzyme (ICE) family of proteases with substrate specificity for Asp-X. CMH-1 has the highest similarity to CPP32 (52% amino acid identity) and MCH2 (<em>31</em>% identical). CMH-1 shares conserved amino acid residues that form the core structure of ICE as well as those residues involved in catalysis and in the P1 aspartate binding. Overexpression of CMH-1 in COS cells resulted in the processing of CMH-1 and the induction of apoptosis of transfected cells. Coexpression of CMH-1 with poly(ADP-ribose) polymerase (PARP) also resulted in a specific cleavage of PARP. Purified recombinant CMH-1 cleaved PARP but not <em>interleukin</em> 1 beta precursor in vitro.

The macrophage-derived product, <em>interleukin</em> 1 (IL 1) is thought to play an important regulatory role in the proliferation of T lymphocytes; however, its mechanism of action is unknown. We describe in this report a variant subline of EL4 thymoma cells (EL4-6.1) that displays a high degree of responsiveness to IL 1. We show that recombinant IL 1 can induce both the secretion of <em>interleukin</em> 2 (IL 2) and the expression of IL 2 receptors (IL 2-R) by these cells. EL4-6.1 cells do not constitutively secrete IL 2, nor do they express IL 2-R; but when cultured in the presence of recombinant IL 1, they secrete detectable amounts of IL 2 (5 to 15 U/ml). In the presence of either suboptimal levels of phorbol ester (PMA) or Ionomycin, the addition of IL 1 resulted in up to an 80-fold enhancement in the amount of IL 2 secreted. Stimulation with IL 1 alone or in combination with Ionomycin was unable to induce detectable IL 2-R expression by EL4-6.1 cells. However, in the presence of suboptimal concentrations of PMA, IL 1 induced expression of about 3000 high affinity (dissociation constant, Kd of <em>31</em> pM) and 50,000 low affinity (Kd of 2800 pM) IL 2-R. These IL 2-R were functional, based on their ability to rapidly internalize IL 2. This model system will allow a detailed analysis of the mechanisms involved in the regulation of the immune response by IL 1 and IL 2.

<em>Interleukin</em>-12 (IL-12) plays an important role in the production of interferon gamma (IFN-gamma) and is essential for protection against intracellular pathogens such as Mycobacterium and Salmonella. A <em>31</em>-year-old man had disseminated Mycobacterium avium complex (MAC) infection. The production of IFN-gamma by peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA-PBMCs) was found severely impaired (40.7 pg/mL compared with 833 +/- 289 pg/mL for the patient's and healthy subjects' (n = 3) PHA- PBMCs, respectively), and the patient's PHA-PBMCs completely lacked surface IL-12 receptor beta1 (IL-12Rbeta1) chain. The IL-12Rbeta1 gene transcript in his PHA-PBMCs had an R213W substitution in each allele. Family history showed that both parents were heterozygotes in the R213W substitution. Transfection of a human embryonal kidney cell line 293 (HEKC293) with wild-type IL-12Rbeta1wt gene led to cell surface IL-12Rbeta1 expression; however, no expression was seen in HEKC293 transfected with the mutated IL-12Rbeta1R213W gene. The IL-12Rbeta1 gene transcript, but no IL-12Rbeta1 protein, was detected in PHA-PBMCs and T cells, suggesting a post-translational event(s), most likely a shortened turnover of the protein. The R213W substitution was not detected in the cells of 32 healthy persons or of 25 patients with tuberculosis or MAC infection. Six amino acid substitutions (Q214R, M365T, G378R, H438Y, A525T, and G594E) were identified, but the incidences of such substitutions were not significantly different between the groups. The Q214R substitution is reportedly linked to IL-12Rbeta1 deficiency; however, the study showed that 19 and 10 of 57 Japanese and 6 and 4 of 33 healthy white persons were heterozygous and homozygous for Arg-214, respectively, suggesting that the Q214R substitution represents a polymorphism and is not related to IL-12Rbeta1 deficiency but that the R213W substitution is responsible for IL-12Rbeta1 deficiency.