BACKGROUND

Adiponectin is an adipose tissue-derived specific protein that has antiinflammatory as well as anti-atherosclerotic effects. In the United States, many patients with COPD are obese and die of cardiovascular diseases. However, in Japan, patients with COPD are frequently cachexic and die of respiratory failure. This study was designed to investigate the role of adiponectin in these differences in characteristics of COPD.

METHODS

We enrolled normal-weight and underweight male patients with COPD (n = <em>31</em>; age, 71 +/- 1 years; body mass index [BMI], 20.1 +/- 0.6 kg/m(2)) and age-matched, healthy, male, control subjects (n = 12). The adiponectin levels were measured by enzyme-linked immunosorbent assay. Correlation of adiponectin levels with pulmonary function and serum levels of proinflammatory cytokines (tumor necrosis factor [TNF]-alpha and <em>interleukin</em>-6) were estimated.

RESULTS

Adiponectin levels in patients with COPD were significantly higher than those in control subjects (p<0.01) and inversely correlated with BMI (r = - 0.55, p<0.01). Even in the normal-weight patients with COPD, adiponectin levels were significantly higher than those in control subjects (p<0.01). Adiponectin levels in patients with COPD significantly correlated with percentage of predicted residual volume (r = 0.40, p<0.05). In patients with TNF-alpha levels>> 5 pg/mL, there was a significant correlation between plasma adiponectin and serum TNF-alpha levels (r = 0.68, p<0.05).

CONCLUSIONS

Plasma adiponectin levels in patients with COPD were elevated and correlated with body weight loss, hyperinflation, and systemic inflammation. Increased adiponectin may reduce cardiovascular events in underweight patients with COPD.

OBJECTIVE

To investigate the T-helper cell cytokine profiles in two well-defined clinical uveitis entities caused by an infectious mechanism.

METHODS

Cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, and interferon [IFN]-gamma) were measured in ocular fluid samples obtained from patients with herpes simplex- or varicella-zoster virus-induced acute retinal necrosis (ARN; n = 17) and toxoplasma chorioretinitis (n = 27) using enzyme-linked immunosorbent assay techniques. The data were compared with data for 51 control samples taken during cataract surgery (n = 10), vitrectomy in diabetic retinopathy (n = 10), eye bank eyes (n = 10) and with samples from patients with "autoimmune" uveitis (n = 21).

RESULTS

Interleukin-6 was detected in 44 of 51 control samples and 43 of 44 eyes of patients with uveitis. The highest levels in the control samples were detected in 9 of 10 vitreous samples from patients with diabetic retinopathy (mean, 648 pg/ml). In 8 of 10 samples taken from patients during cataract surgery and in 7 of 10 eye bank eyes the amount of IL-6 was significantly lower (mean, 10 pg/ml and 136 pg/ml, respectively). Interleukin-6 levels in patients with ARN (mean, 1436 pg/ml) were significantly higher than in those with toxoplasma chorioretinitis (mean, 272 pg/ml). Interleukin-2 was detected in one of the samples from patients with toxoplasma chorioretinitis (1105 pg/ml) and in three samples from the control subjects suffering from Fuchs' heterochromic anterior uveitis (mean, 752 pg/ml). No IL-4 (<2 pg/ml) was detected either in patient or control samples. Interferon-gamma could be detected in 7 of 17 ARN patients (range, 277-3483 pg/ml), in 13 of 27 samples from patients with toxoplasma chorioretinitis (range, 12-250 pg/ml), and in 1 of 21 of the samples from control subjects with uveitis (31 pg/ml) but was absent in nonuveitic control samples. Interleukin-10 was detected in 10 of 17 ARN patients (range, 29-3927 pg/ml), in 13 of 27 samples from patients with toxoplasma chorioretinitis (range, 4-67 pg/ml), and in only 3 of 51 control samples (6 pg/ml, 16 pg/ml, and 20 pg/ml).

CONCLUSIONS

Various immunoregulatory cytokines (IL-6, IL-10, and IFN-gamma) were detected in ocular fluid samples from patients with uveitis. A separate role for either a T-helper type 1 or T-helper type 2 response in the pathogenesis of clinical uveitis could not be proven.

Type 1 diabetes mellitus (T1DM) is associated with increased microvascular complications and is a proinflammatory state. The toll-like receptors (TLRs) are pattern recognition receptors on monocytes and important in atherosclerosis. We have shown increased TLR2 and TLR4 expression on monocytes of T1DM compared with controls. In this report, we tested the surface expression of TLR2 and TLR4 on monocytes of T1DM patients with microvascular complications (T1DM-MV) compared with those without (T1DM) and healthy controls. The study was performed at the University of California Davis. Healthy controls (n = <em>31</em>), T1DM patients (n = <em>31</em>), and T1DM-MV patients (n = 34) were included. The TLR2 and TLR4 surface expression was significantly increased in T1DM-MV monocytes compared with T1DM and controls (P < .01). In addition, nuclear factor κB activity and <em>interleukin</em>-1β release were significantly increased in monocytes from T1DM-MV compared with T1DM (P < .005). Thus, we make the novel observation that TLR2 and TLR4 expression and signaling are increased in T1DM-MV compared with T1DM and may contribute to the accentuated proinflammatory state and complications of T1DM.

OBJECTIVE

Interleukin (IL)-10 has recently been shown to promote survival of neurons and glia. The purpose of this report is to investigate whether IL-10 has any role in protecting retinal ganglion cells (RGCs) from death under conditions in which growth factors are removed, or in which oxidative stress is present. Signal transduction pathways that activate IL-10 signaling in RGCs were studied in both stress conditions.

METHODS

Effects of various interleukins on the viability of the RGC cell line was determined, and apoptotic cells were quantified. Immunoblot analysis was preformed to identify the IL-10 receptor (IL-10R) and phosphorylated or nonphosphorylated Akt and STAT-3 proteins in RGC extracts. Immunohistochemistry was performed on the rat retinal sections to identify native IL-10R.

RESULTS

Apoptosis of RGCs in the absence of growth factors with or without dexamethasone (1 microM) occurred in 68.5% +/- 3.4% and 53.4% +/- 2.6% of cells, respectively, after 96 hours. Addition of IL-10 at a concentration of 50 ng/mL significantly reduced the apoptotic population of RGCs to 28.2% +/- 2.3% in the absence of growth factors with dexamethasone and to 31% +/- 2.7% in the absence of growth factors alone. RGCs as well as native retina expressed functional IL-10R as determined by immunoblot analysis and by the ability of IL-10 to phosphorylate Stat-3. However, IL-10 failed to phosphorylate Akt in these cells.

CONCLUSIONS

IL-10 caused a 59% and 42% reduction in the apoptotic population of serum-deprived cells with and without dexamethasone treatment, respectively. These observations establish that activation of IL-10R promotes survival of RGCs and this survival-promoting activity is due to IL-10 signaling through the Stat-3 pathway, which inhibits the cell death and not through the Akt cell survival pathway.

BACKGROUND

There is growing evidence that interleukin 17 (IL-17) producing T cells are involved in the pathogenesis of systemic lupus erythematosus (SLE). Previous studies showed that increased percentages of T-cell subsets expressing the costimulatory molecules CD80 and CD134 are associated with disease activity and renal involvement in SLE. The aim of this study was to investigate the distribution and phenotypical characteristics of IL-17 producing T-cells in SLE, in particular in patients with lupus nephritis, with emphasis on the expression of CD80 and CD134.

METHODS

Thirty-four patients (3 male, 31 female, mean age 41 ± 15 years) fulfilling at least four of the American College of Rheumatology (ACR) revised criteria for the diagnosis of SLE and 24 healthy controls were enrolled. T-cells from the peripheral blood were analysed by fluorescence activated cell sorting (FACS) for their expression levels of CD80, CD134 and CCR6. In vitro stimulated CD3+IL17+ cells were also investigated for the expression of these costimulatory markers. Finally, renal biopsies from SLE patients were evaluated for the presence of CD134 expressing T-cells.

RESULTS

Percentages of IL-17 expressing T-cells were significantly increased in patients with active disease as compared to healthy controls (1.46 ± 0.58% versus 0.93 ± 0.30%, P = 0.007). The percentage of IL-17 producing T-cells was correlated with disease activity as assessed by systemic lupus erythematosus disease activity index (SLEDAI) (r = 0.53, P = 0.003). In patients, most of the IL-17 producing T-cells were confined to the CCR6+ T-cell subset (80 ± 13%). Expression of CD80 and CD134 on the IL-17 producing T-cell subset was higher in SLE than in healthy controls (HC) (CD134: 71.78 ± 14.51% versus 51.45 ± 16.58%, P = 0.002; CD80: 25.5 ± 14.99% versus 14.99 ± 5.74%, P = 0.02). Also, patients with lupus nephritis expressed higher levels of CD134+ on CD3+IL-17+ cells as compared to HC (72.69 ± 11.54% versus 51.45 ± 16.58%, P = 0.006). Furthermore, renal biopsies of lupus nephritis patients showed infiltration of CD134+ T cells.

CONCLUSIONS

Percentages of IL-17 expressing T-cells correlate with disease activity. Further, these cells show increased expression of costimulatory markers such as CD134 and CD80. The presence of CD134+ T-cells in renal biopsies of lupus nephritis patients suggest that these cells migrate to the kidney and might contribute to inflammatory processes through IL-17 secretion.

The present study tests the hypotheses that local bioavailability of insulin-like growth factor I (IGF-I) is capable of regulating muscle protein balance and that muscle-directed IGF-I can selectively maintain muscle mass during bacterial infection. Initial studies in C57BL/6 mice demonstrated that increasing or decreasing bioavailable IGF-I within muscle by local administration of either Leu(24) Ala(<em>31</em>) IGF-I or IGF binding protein 1, respectively, produced proportional changes in surrogate markers (eg, phosphorylation of 4E-BP1 and S6K1) of protein synthesis. We next examined the ability of a sustained local administration of IGF-I to prevent sepsis-induced muscle atrophy over a 5-day period. At the time of cecal ligation and puncture or sham surgery, mice had a time-release pellet containing IGF-I implanted next to the gastrocnemius and a placebo pellet placed in the contralateral limb. Data indicated that IGF-I released locally only affected the adjacent muscle and was not released into the circulation. Gastrocnemius from septic mice containing the placebo pellet was atrophied and had a reduced IGF-I protein content. In contrast, locally directed IGF-I increased IGF-I protein within adjacent muscle to basal control levels. This change was associated with a proportional increase in muscle weight and protein, as well as increased phosphorylation of 4E-BP1 and the redistribution of eIF4E from the inactive eIF4E4EBP1 complex to the active eIF4EeIF4G complex. Local IGF-I also prevented the sepsis-induced increase in atrogin-1 messenger RNA in the exposed muscle. Finally, local IGF-I prevented the sepsis-induced increase in muscle <em>interleukin</em>-6 messenger RNA. Thus, muscle-directed IGF-I attenuates the sepsis-induced atrophic response apparently by increasing muscle protein synthesis and potentially decreasing proteolysis. Collectively, our data suggest that agents that increase the bioavailability of IGF-I within muscle per se might be effective in ameliorating the sepsis-induced loss of muscle mass without having undesirable effects on metabolic processes in distant organs.

OBJECTIVE

The administration of interleukin-2 (IL-2) may increase the frequency of peripherally circulating FOXP3-positive regulatory immune cells, thus potentially compromising this treatment option for patients with metastatic renal cell carcinoma. The impact of IL-2-based therapy on the accumulation of FOXP3-positive immune cells in the tumor microenvironment in metastatic renal cell carcinoma is unknown.

METHODS

Baseline (n = 58) and on-treatment (n = 42) tumor core biopsies were prospectively obtained from patients with clear cell metastatic renal cell carcinoma before and during IL-2-based immunotherapy. Immunohistochemical expression of FOXP3 was estimated by stereological counting technique and correlated with other immune cell subsets and overall survival.

RESULTS

A significant increase in absolute intratumoral FOXP3-positive immune cells was observed comparing baseline (median 23 cells/mm2; range, 0-183) and on-treatment biopsies (median, 89 cells/mm2; range, 11-388; P < 0.001). The relative increase in individual patients was median 4.7-fold, range 0.3 to 230. FOXP3-positive cells were positively correlated with CD3-positive, CD4-positive, and CD8-positive tumor-infiltrating immune cells at baseline and during treatment (P < 0.05 in all comparisons). All patients achieving high numbers (>180 cells/mm2) of on-treatment FOXP3-positive intratumoral immune cells were dead within 22 months (n = 11), whereas patients with low numbers (<180 cells/mm2) of on-treatment FOXP3-positive cells (n = 31) had a 5-year survival rate of 19% (hazard ratio, 2.2; confidence interval, 1.03-4.5; P = 0.043). All long-term survivors were characterized by low-baseline FOXP3-positive cells and a modest absolute rise in FOXP3-positive cells.

CONCLUSIONS

Intratumoral FOXP3-positive regulatory immune cells significantly increased during IL-2-based immunotherapy, and high numbers of on-treatment FOXP3-positive cells were correlated with poor prognosis in patients with metastatic renal cell carcinoma.

OBJECTIVE

The purpose of this study was to evaluate the clinical activity and toxicity of recombinant human Interleukin (IL)-12 in patients with relapsed and refractory non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD).

METHODS

Forty-two previously treated patients (32 patients with NHL and 10 patients with HD) were enrolled on the study. Patients were treated with either intravenous (n = 11) or subcutaneous (n = 31) administration of IL-12. The patients had received a median of three prior treatment regimens, and 16 patients had undergone prior autologous stem cell transplantation.

RESULTS

All patients were assessable for toxicity, and 39 of 42 (93%) patients were assessable for response. Six of 29 (21%) patients with NHL had a partial or complete response, whereas none of the 10 patients with HD responded. Furthermore, 15 patients had stable disease that lasted for up to 54 months. Progression-free survival in patients with indolent NHL, aggressive NHL, and HD was 6, 2, and 2.5 months, respectively. Treatment was well tolerated, and the most common toxicity was flu-like symptoms. Reversible grade 3 hepatic toxicity was observed in three patients requiring dose reduction. IL-12 therapy increased the median number of peripheral blood CD8 T lymphocytes from 423/microl to 576/microl (P = 0.0019). Furthermore, IL-12 therapy decreased serum vascular endothelial growth factor and basic fibroblast growth factor concentrations in 37% of the patients.

CONCLUSIONS

The ability of recombinant human IL-12 therapy to increase the number of circulating CD8+ cells and induce clinical remissions in patients with relapsed NHL warrants further investigation of the drug.

<em>Interleukin</em> (IL) <em>31</em>Ralpha (glycoprotein 130-like monocyte receptor and glycoprotein 130-like receptor) heterodimerizes with oncostatin M receptor beta to bind IL-<em>31</em>, a cytokine expressed preferentially by CD4(+) T helper type 2 (Th2) cells. However, the functions of IL-<em>31</em>-IL-<em>31</em>R signaling in immune regulation remain unknown. Here, we identify a novel role for IL-<em>31</em>R in limiting type 2 inflammation in the lung. After intravenous injection of Schistosoma mansoni eggs, IL-<em>31</em>Ralpha(-/-) mice developed severe pulmonary inflammation, characterized by an increase in the area of granulomatous inflammation, increased numbers of resistin-like molecule alpha(+) cells, and enhanced collagen deposition compared to WT counterparts. In vitro, macrophages generated from IL-<em>31</em>Ralpha(-/-) mice promoted enhanced ovalbumin-specific CD4(+) T cell proliferation and purified naive CD4(+) T cells from IL-<em>31</em>Ralpha(-/-) mice exhibited enhanced proliferation and expression of Th2 cytokines, identifying a T cell- and macrophage-intrinsic regulatory function for IL-<em>31</em>R signaling. In contrast, the generation of CD4(+) T cell-mediated Th1 responses were normal in IL-<em>31</em>Ralpha(-/-) mice, suggesting that the regulatory role of IL-<em>31</em>R signaling is limited to type 2 responses. Together, these data implicate IL-<em>31</em>R signaling as a novel negative regulatory pathway that specifically limits type 2 inflammation.

Nerve injury and the consequent release of <em>interleukins</em> (ILs) are processes implicated in pain transmission. To study the potential role of IL-1 in the pathogenesis of allodynia and hyperalgesia, IL-1alpha and comparative IL-1beta, IL-6, and IL-10 mRNA levels were quantified using competitive RT-PCR of the lumbar spinal cord and dorsal root ganglia (DRG; L5-L6) three and seven days after chronic constriction injury (CCI) in rats. Microglial and astroglial activation in the ipsilateral spinal cord and DRG were observed after injury. In naive and CCI-exposed rats, IL-1alpha mRNA and protein were not detected in the spinal cord. IL-1beta and IL-6 mRNAs were strongly ipsilaterally elevated on day seven after CCI. In the ipsilateral DRG, IL-1alpha, IL-6, and IL-10 mRNA levels were increased on days three and seven; IL-1beta was elevated only on day seven. Western blot analysis revealed both the presence of IL-1alpha proteins (45 and <em>31</em> kDa) in the DRG and the down-regulation of these proteins after CCI. Intrathecal administration of IL-1alpha (50-500 ng) in naive rats did not influence nociceptive transmission, but IL-1beta (50-500 ng) induced hyperalgesia. In rats exposed to CCI, an IL-1alpha or IL-1 receptor antagonist dose-dependently attenuated symptoms of neuropathic pain; however, no effect of IL-1beta was observed. In sum, the first days after CCI showed a high abundance of IL-1alpha in the DRG. Together with the antiallodynic and antihyperalgesic effects observed after IL-1alpha administration, this finding indicates an important role for IL-1alpha in the development of neuropathic pain symptoms.

Associations between Helicobacter pylori (HP) infection and lifestyle factors have been reported by several authors, but little is known about the host factors associated with the infection. This study aims to examine the infection rate of HP according to gene polymorphisms of <em>interleukin</em> (IL)-1A, IL-1B, and IL-1RN, and to investigate the interactions with lifestyle factors. Subjects were 241 non-cancer outpatients who had participated in a HP eradication program. Polymorphisms at - 889 (T to C) of IL-1A, at - <em>31</em> (C to T; T allele makes a TATA box) and - 511 (C to T) of IL-1B, and at intron 2 (86-bp VNTR (variable number of tandem repeats)) of IL-1RN were genotyped by PCR (polymerase chain reaction), PCR-RFLP (restriction fragment length polymorphism) and PCR-CTPP (PCR with confronting two-pair primers). It was found that IL-1B polymorphisms at - <em>31</em> and - 511 were near-completely linked, but in the opposite way to that in Caucasians; - <em>31</em>C / - 511T and - <em>31</em>T / - 511C alleles were dominant in the present subjects. The HP infection rate was substantially different among the genotypes of IL-1B C - <em>31</em>T; 45.2% (19 / 42) for the C / C, 67.7% (90 / 133) for the C / T, and 63.6% (42 / 66) for the T / T. The age-sex adjusted odds ratio (OR) relative to the C / C genotype was 2.32 (95%CI (confidence interval), 1.10 - 4.92) for the T / C genotype and 2.46 (1.06 - 5.74) for the T / T genotype. The OR for the T / T genotype was significantly modified by smoking status; interaction term = 14.6 (1.12 - 190). The polymorphisms of IL-1A and IL-1RN were not associated with the infection rate. The results suggested that the T allele of IL-1B C - <em>31</em>T is associated with vulnerability to persistent HP infection, and that the vulnerability is modified by smoking.

Inflammation is a major contributor to atherosclerosis by its effects on arterial wall biology and lipoprotein metabolism. <em>Interleukin</em>-10 (IL-10) is an anti-inflammatory cytokine that may modulate the atherosclerotic disease process. We investigated the effects of adeno-associated virus (AAV) vector-mediated gene transfer of IL-10 on atherogenesis in apolipoprotein E (ApoE)-deficient mice. A murine myoblast cell line, C2C12, transduced with AAV encoding murine IL-10 (AAV2-mIL10) secreted substantial amounts of IL-10 into conditioned medium. The production of monocyte chemoattractant protein-1 (MCP-1) by the murine macrophage cell line, J774, was significantly inhibited by conditioned medium from AAV2-mIL10-transduced C2C12 cells. ApoE-deficient mice were injected with AAV5-mIL10 into their anterior tibial muscle at 8 weeks of age. The expression of MCP-1 in the vascular wall of the ascending aorta and serum MCP-1 concentration were decreased in AAV5-mIL10-transduced mice compared with AAV5-LacZ-transduced mice. Oil red-O staining of the ascending aorta revealed that IL-10 gene transfer resulted in a <em>31</em>% reduction in plaque surface area. Serum cholesterol concentrations were also significantly reduced in AAV5-mIL10-transduced mice. To understand the cholesterol-lowering mechanism of IL-10, we measured the cellular cholesterol level in HepG2 cells, resulting in its significant decrease by the addition of IL-10 in a dose-dependent manner. Furthermore, IL-10 suppressed HMG-CoA reductase expression in the HepG2 cells. These observations suggest that intramuscular injection of AAV5-mIL10 into ApoE-deficient mice inhibits atherogenesis through anti-inflammatory and cholesterol-lowering effects.

The autosomal recessive lpr (lymphoproliferation) gene is responsible for a thymus-dependent massive lymphoproliferation associated with the development of lupus-like autoimmune disease. Phenotypic analysis of adult lpr/lpr lymph nodes has demonstrated accumulation of a dull Lyt-1+, Thy-1+ population that expresses neither Lyt-2 nor L3T4 antigens. With the use of a depletion method based on complement-mediated lysis with an anti-Lyt-2 monoclonal antibody (<em>31</em> M) and a new anti-L3T4 monoclonal antibody (RL 172.4), we have purified the Lyt-2- L3T4- subset from lymph nodes or spleens of C57BL/6-lpr/lpr mice and determined whether they are immunologically functional in vitro. Production of neither <em>interleukin</em> 2 nor interferon-gamma was detected by the double-negative subset after stimulation with concanavalin A and/or phorbol myristate acetate. The frequencies of allospecific cytotoxic T lymphocyte (CTL) precursors and lectin-induced antigen-nonspecific CTL precursors were diminished to almost undetectable levels, whereas the Lyt-2+ population from lpr/lpr mice had CTL-precursor frequencies comparable with that of +/+ mice. These results show that the major cell subset of adult lpr/lpr lymph nodes or spleens is composed of lymphocytes with markedly limited potential for lymphokine production or antigenic stimulation.

BACKGROUND

IL-<em>31</em> is a cytokine expressed by T cells following activation with cytokines or staphylococcal exotoxins. A major function of IL-<em>31</em> in atopic dermatitis (AD) is the induction of pruritus in the skin via the IL-<em>31</em> receptor on sensory nerve cells. However, the regulation of the IL-<em>31</em> receptor and pro-inflammatory functions of IL-<em>31</em> in human monocytes and monocyte-derived cells are yet to be studied in detail.

OBJECTIVE

To investigate the regulation and function of IL-<em>31</em> receptors in resting and activated human monocytes, macrophages and dendritic cells.

METHODS

Human monocytes, macrophages and dendritic cells were stimulated with staphylococcal exotoxins (SEB, alpha-toxin) or cytokines (IFN-gamma, IL-13). IL-<em>31</em>RA expression and regulation were then investigated at both the mRNA and the protein level. Subsequently, functional effects of IL-<em>31</em> stimulation on cytokine secretion were measured at the protein level.

RESULTS

Staphylococcal exotoxins significantly up-regulated IL-<em>31</em>RA expression on monocytes and macrophages but not on dendritic cells at both the mRNA and the protein level. IL-<em>31</em> enhanced the secretion of IL-1beta, IL-6 and IL-18 and up-regulated CD86 expression. In patients with AD, functional IL-<em>31</em>RA was also detected following stimulation of PBMC with IFN-gamma. However, this was not observed in healthy individuals.

CONCLUSIONS

IL-<em>31</em> induces pro-inflammatory effects in activated human monocytes and macrophages. This may have implications for cutaneous inflammation in eczema where an over-expression of IL-<em>31</em> has been described previously. Moreover, our findings provide a new link between staphylococcal colonization and the worsening of inflammation via IL-<em>31</em>. Further therapeutic considerations may include IL-<em>31</em> as a target in AD.

Protein kinase Czeta (PKCzeta) is an intracellular serine/threonine protein kinase that has been implicated in the signaling pathways for certain inflammatory cytokines, including <em>interleukin</em>-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), in some cell types. A study of gene expression in articular chondrocytes from osteoarthritis (OA) patients revealed that PKCzeta is transcriptionally up-regulated in human OA articular cartilage clinical samples. This finding led to the hypothesis that PKCzeta may be an important signaling component of cytokine-mediated cartilage matrix destruction in articular chondrocytes, believed to be an underlying factor in the pathophysiology of OA. IL-1 treatment of chondrocytes in culture resulted in rapidly increased phosphorylation of PKCzeta, implicating PKCzeta activation in the signaling pathway. Chondrocyte cell-based assays were used to evaluate the contribution of PKCzeta activity in NF-kappaB activation and extracellular matrix degradation mediated by IL-1, TNF, or sphingomyelinase. In primary chondrocytes, IL-1 and TNF-alpha caused an increase in NF-kappaB activity resulting in induction of aggrecanase-1 and aggrecanase-2 expression, with consequent increased proteoglycan degradation. This effect was blocked by the pan-specific PKC inhibitors RO <em>31</em>-8220 and bisindolylmaleimide I, partially blocked by Gö 6976, and was unaffected by the PKCzeta-sparing inhibitor calphostin C. A cell-permeable PKCzeta pseudosubstrate peptide inhibitor was capable of blocking TNFand IL-1-mediated NF-kappaB activation and proteoglycan degradation in chondrocyte pellet cultures. In addition, overexpression of a dominant negative PKCzeta protein effectively prevented cytokine-mediated NF-kappaB activation in primary chondrocytes. These data implicate PKCzeta as a necessary component of the IL-1 and TNF signaling pathways in chondrocytes that result in catabolic destruction of extracellular matrix proteins in osteoarthritic cartilage.

<em>Interleukin</em> (IL)-6, a key player in the inflammatory response, may be a useful biomarker in rheumatoid arthritis (RA). The aim was to determine analytical variability, a reference interval in healthy subjects, and long- and short-term variation in serum and plasma IL-6 in healthy subjects and RA patients. An enzyme-linked immunosorbent assay from R&D was used for determination of serum and plasma IL-6. The IL-6 concentration did not depend on the type of anticoagulant used or the 3-h time delay between sampling and processing or repeated freeze-thaw cycles. The median plasma and serum IL-6 in <em>31</em>8 healthy subjects were 1.3 pg ml(-1) (range 0.33-26) and 1.4 pg ml(-1) (range 0.25-23), respectively. The median coefficient of variation in plasma IL-6 in 27 healthy subjects during 1 month, and repeated after 6 and 12 months were 27%, <em>31</em>% and 26%, respectively. No significant long-term changes were observed in serum IL-6 over a 3-year period (14%, p = 0.33). Exercise (cycling) increased serum IL-6 in healthy subjects but not in RA patients. In conclusion, circulating IL-6 is stable regarding sample handling and shows little variation over time. Changes in IL-6 concentrations>> 60% (2 times the biological variation) are likely to reflect changes in disease activity and not only pre-analytical or normal biological variability.

OBJECTIVE

To compare the prevalence of left ventricular (LV) diastolic dysfunction in subjects with and without rheumatoid arthritis (RA), among those with no history of heart failure (HF), and to determine risk factors for diastolic dysfunction in RA.

METHODS

A cross-sectional, community-based study comparing cohorts of adults with and without RA and without a history of HF was carried out. Standard two-dimensional/Doppler echocardiography was performed in all participants. Diastolic dysfunction was defined as impaired relaxation (with or without increased filling pressures) or advanced reduction in compliance or reversible or fixed restrictive filling.

RESULTS

The study included 244 subjects with RA and 1448 non-RA subjects. Mean age was 60.5 years in the RA cohort (71% female) and 64.9 years (50% female) in the non-RA cohort. The vast majority (>98%) of both cohorts had preserved ejection fraction (EF> or =50%). Diastolic dysfunction was more common in subjects with RA at <em>31</em>% compared with 26% (age and sex adjusted) in non-RA subjects (OR=1.6; 95% CI 1.2 to 2.4). Patients with RA had significantly lower LV mass, higher pulmonary arterial pressure and higher left atrial volume index than non-RA subjects. RA duration and <em>interleukin</em> 6 (IL-6) level were independently associated with diastolic dysfunction in RA even after adjustment for cardiovascular risk factors.

CONCLUSIONS

Subjects with RA have a higher prevalence of diastolic dysfunction than those without RA. RA duration and IL-6 are independently associated with diastolic dysfunction, suggesting the impact of chronic autoimmune inflammation on myocardial function in RA. Clinical implications of these findings require further investigation.

Increased serum <em>interleukin</em>-6 (IL-6) concentrations have been reported in patients with thyroid destructive processes. In the present study we measured IL-6 and soluble IL-6 receptor (sIL-6R) concentrations in the serum of normal subjects and patients with Graves' disease using a high sensitivity sandwich enzyme-linked immunoassay. We found increased serum IL-6 and sIL-6R concentrations (69.3 fmol/L, and 964 pmol/L, respectively) in 49 hyperthyroid patients with Graves' disease (GD) compared to those in controls [55.8 fmol/L (P = 0.019) and 772 pmol/L (P = 0.007), respectively]. In <em>31</em> newly diagnosed GD patients, serum concentrations of IL-6 and sIL-6R during the hyperthyroid phase were elevated, and after therapy with methimazole only, serum sIL-6R concentrations returned to normal (940 vs. 726 pmol/L; P < 0.001) but serum IL-6 did not. Serum sIL-6R concentrations (mean +/- 2 SD) were higher in GD patients with active inflammatory thyroid-associated ophthalmopathy than those in patients with inactive or absent thyroid-associated ophthalmopathy (P < 0.05). In conclusion, we have demonstrated activation of the IL-6 system in GD and, for the first time, have measured and found increased serum sIL-6R concentrations in hyperthyroid GD patients.

Tocilizumab (TCZ) is used for treating moderate-to-severe Covid-19 pneumonia by targeting <em>interleukin</em>-6 receptors (IL-6Rs) and reducing cytokine release. Yet, in spite of this therapy, patients with vs. patients without diabetes have an adverse disease course. In fact, glucose homoeostasis has influenced the outcomes of diabetes patients with infectious diseases. Of the 475 Covid-19-positive patients admitted to infectious disease departments (University of Bologna, University Vanvitelli of Napoli, San Sebastiano Caserta Hospital) in Italy since 1 March 2020, <em>31</em> (39.7%) hyperglycaemic and 47 (60.3%) normoglycaemic patients (blood glucose levels ≥140mg/dL) were retrospectively evaluated at admission and during their hospital stay. Of note, 20 (64%) hyperglycaemic and 11 (23.4%) normoglycaemic patients had diabetes (P<0.01). At admission, hyperglycaemic vs. normoglycaemic patients had fivefold higher IL-6 levels, which persisted even after TCZ administration (P<0.05). Intriguingly, in a risk-adjusted Cox regression analysis, TCZ in hyperglycaemic patients failed to attenuate risk of severe outcomes as it did in normoglycaemic patients (P<0.009). Also, in hyperglycaemic patients, higher IL-6 plasma levels reduced the effects of TCZ, while adding IL-6 levels to the Cox regression model led to loss of significance (P<0.07) of its effects. Moreover, there was evidence that optimal Covid-19 infection management with TCZ is not achieved during hyperglycaemia in both diabetic and non-diabetic patients. These data may be of interest to currently ongoing clinical trials of TCZ effects in Covid-19 patients and of optimal control of glycaemia in this patient subset.

Keywords: Covid-19; Diabetes mellitus; Interleukin-6.

Recent studies have shown that human basophils, like mast cells, generate <em>interleukin</em> (IL)-4 following immunological activation and may thus participate in late-phase allergic and inflammatory processes. Here, we report the capacity of human basophils to release IL-13 within 24 h following stimulation with anti-IgE. Additionally, in 14 out of <em>31</em> experiments, we observed that basophils rapidly release performed IL-4 within 5-10 min, as well as newly generated IL-4, which was released 4 h following stimulation of the cells with anti-IgE. In contrast to the biphasic release of IL-4 from the cells, no preformed IL-13 was detected at earlier times (5-30 min). Preformed IL-4 and IL-4 and IL-13 generated de novo were also released after stimulation of the cells with IL-3; an enhanced production of these cytokines was observed using a combination of IL-3 and anti-IgE. We conclude from these data that, by releasing performed IL-4 and IL-4 and IL-13 generated de novo, human basophils may be centrally involved in the orchestration of allergic inflammation by providing a trigger to IL-4-mediated T helper 2 lymphocyte activation, B cell IgE switching, and increased vascular adhesion molecule expression.

To address the long-term physiological consequences of chronic stressors, 14 continuing or current family caregivers of Alzheimer's disease (AD) patients, 17 former AD caregivers, and <em>31</em> control subjects were compared. Continuing and former caregivers did not differ on depressive symptomatology or perceived stress; both groups were significantly more depressed and stressed than controls. Furthermore, continuing and former caregivers did not differ in the response of NK cells in vitro to recombinant interferon-gamma and recombinant <em>interleukin</em>-2, and both groups had a significantly poorer response to these cytokines than controls. The physiological and psychological consequences of chronic stressors may persist well beyond the cessation of the actual stressor.

BACKGROUND

Although the prevalence of allergic asthma increased quickly in the past decade, the diagnostic criteria have not been well established. The aim of the present study was to explore whether stem cell factor (SCF), B cell-activating factor (BAFF), and cytokines <em>interleukin</em> (IL)-17 and IL-<em>31</em> are usable parameters for the diagnosis of allergic asthmatics.

METHODS

Blood samples were collected from patients with allergic asthma, control patients, and healthy control subjects. The serum concentrations of SCF, BAFF, IL-17, and IL-<em>31</em> were measured by enzyme-linked immunosorbent assay. The corresponding mRNA levels in peripheral blood mononuclear cells (PBMCs) were determined by real-time reverse-transcription polymerase chain reaction.

RESULTS

A good correlation existed between protein levels of SCF and IL-<em>31</em> and their mRNA levels (SCF: r = 0.6162; IL-<em>31</em>: r = 0.5463). The serum concentrations of SCF and IL-<em>31</em> in allergic asthmatic patients, but not control patients, were significantly higher than those in normal control subjects (SCF: median 1.83 vs 0.85 ng/ml, P < 0.01; IL-<em>31</em>: 50.15 vs 10.01 pg/ml, P < 0.001). Consistently, the levels of SCF and IL-<em>31</em> mRNAs in allergic asthmatic patients' PBMCs were also significantly higher than those in normal control subjects (P = 0.002 and P < 0.001, respectively).

CONCLUSIONS

These findings suggest that allergic asthma is characterized by an elevation of cytokines SCF and IL-<em>31</em> and the measurement of their expression at either protein level in serum or mRNA level in PBMCs will be a valuable parameter for the diagnosis of allergic asthma.

OBJECTIVE

Endothelial progenitor cells (EPC) may participate in the repair of injured coronary endothelium. We have recently identified EPC co-expressing the osteoblastic marker osteocalcin [OCN (+) EPC] and found that their numbers are increased in patients with early and late coronary atherosclerosis. The current study was designed to test the hypothesis that early coronary atherosclerosis is associated with the retention of osteogenic EPC within the coronary circulation.

RESULTS

Blood samples were taken simultaneously from the proximal aorta and the coronary sinus from <em>31</em> patients undergoing invasive coronary endothelial function testing. Using flow cytometry, peripheral blood mononuclear cells were analysed for EPC markers (CD133, CD34, KDR) and OCN. The net gradient of EPC was calculated by multiplying the coronary blood flow by the arteriovenous EPC gradient (a negative net gradient indicating retention of EPC). Similarly, serum samples were analysed for stromal cell-derived factor-1 alpha (SDF-1 alpha) and <em>interleukin</em>-8 (IL-8) and their net production calculated. Compared with controls (n = 17) patients with endothelial dysfunction (ED, n = 14) had a significant net retention of CD34+/CD133-/KDR+/OCN+ EPC [118.38 (0.00, 267.04) vs. -112.03 (838.36, 0.00), P = 0.004]. The retention of OCN (+) EPC correlated with the degree of ED. Patients with ED also showed a net retention of CD34+/CD133-/KDR+ EPC (P = 0.010). Net production of IL-8 was positive in ED [1540.80 (-300.40, 21744.10)pg/mL] but negative in controls [-3428.50 (-11225.00, 647.48), P = 0.025].

CONCLUSIONS

Our study demonstrates that patients with early coronary atherosclerosis are characterized by retention of OCN (+) EPC within the coronary circulation, potentially leading to progressive coronary calcification rather than normal repair.

The functional characterization and subsequent purification of T cell growth factor/<em>interleukin</em> (IL)-2 in the early 1980s established this secreted protein as a key mediator of immune cell activation and provided the prototype that enabled the discovery of numerous cytokines over the ensuing two decades. While soluble immunoregulatory factors were initially identified functionally as biological activities present in the culture supernatants of activated lymphocytes/monocytes, this methodology shifted radically following the completion of the human genome sequence. Computer-generated structural modeling algorithms have replaced functional assays and biochemical purification as the initial means of discovering new cytokines. To date, a total of <em>31</em> <em>interleukins</em>, as well as over a dozen other related hematopoietic factors, have been identified. These cytokines and their receptors may be grouped on the basis of structural homologies as well as by shared ligand and receptor subunits. The challenge now at hand is to define the biological functions of the newly identified cytokines and to elucidate the common and divergent roles of related family members. This point is well illustrated by the IL-12/IL-23/IL-27 family, whose members share ligand and receptor subunits and play somewhat overlapping roles in innate and adaptive immune responses. These three cytokines are not entirely redundant, as they may preferentially activate naïve or memory T cells, induce discrete T cell cytokine profiles, contribute to distinct stages of host immune responses to infectious agents, and differentially promote autoimmunity. Further elucidation of the unique functions of the IL-12 family members may lead to improved immunodiagnostics and therapies.