BACKGROUND

Mechanical ventilation with high tidal volumes (V(T)) in contrast to mechanical ventilation with low V(T) has been shown to increase plasma levels of proinflammatory and antiinflammatory mediators in patients with acute lung injury. The authors hypothesized that, in patients without previous lung injury, a conventional potentially injurious ventilatory strategy with high V(T) and zero end-expiratory pressure (ZEEP) will not cause a cytokine release into systemic circulation.

METHODS

A total of 39 patients with American Society of Anesthesiologists physical status I-II and without signs of systemic infection scheduled for elective surgery with general anesthesia were randomized to receive mechanical ventilation with either (1) V(T) = 15 ml/kg ideal body weight on ZEEP, (2) V(T) = 6 ml/kg ideal body weight on ZEEP, or (3) V(T) = 6 ml/kg ideal body weight on positive end-expiratory pressure of 10 cm H2O. Plasma levels of proinflammatory and antiinflammatory mediators tumor necrosis factor, interleukin (IL)-6, IL-10, and IL-1 receptor antagonist were determined before and 1 h after the initiation of mechanical ventilation.

RESULTS

Plasma levels of all cytokines remained low in all settings. IL-6, tumor necrosis factor, and IL-1 receptor antagonist did not change significantly after 1 h of mechanical ventilation. IL-10 was below the detection limit (10 pg/ml) in 35 of 39 patients. There were no differences between groups.

CONCLUSIONS

Initiation of mechanical ventilation for 1 h in patients without previous lung injury caused no consistent changes in plasma levels of studied mediators. Mechanical ventilation with high V(T) on ZEEP did not result in higher cytokine levels compared with lung-protective ventilatory strategies. Previous lunge damage seems to be mandatory to cause an increase in plasma cytokines after 1 h of high V(T) mechanical ventilation.

OBJECTIVE

Leflunomide and methotrexate have proven to be efficacious in reducing joint inflammation and slowing destruction in clinical trials of patients with rheumatoid arthritis (RA). This study was conducted to provide more insight into the mechanism of action of these agents in synovial tissue.

METHODS

In a 2-center, prospective, randomized, double-blind clinical trial, we compared leflunomide (20 mg/day, after a 3-day 100 mg/day loading dose) and methotrexate (increased stepwise to 15 mg/week) treatment in patients with active RA. Paired synovial tissue biopsy samples were obtained by knee arthroscopy at baseline and after 4 months of treatment. Frozen synovial tissue sections were stained for macrophages (CD68), T cells (CD3), adhesion molecules (intercellular adhesion molecule 1 [ICAM-1], vascular cell adhesion molecule 1 [VCAM-1]), cytokines (tumor necrosis factor alpha, interleukin-1beta [IL-1beta]), matrix metalloproteinase 1 (MMP-1), and tissue inhibitor of metalloproteinases 1 (TIMP-1).

RESULTS

Paired synovial tissue sections were available in 35 patients (16 taking leflunomide, 19 taking methotrexate). Both drugs displayed equal clinical efficacy, with 8 leflunomide-treated patients (50%) and 10 methotrexate-treated patients (53%) fulfilling the American College of Rheumatology 20% response criteria. Both compounds showed similar effects on synovial tissue: reduced numbers of macrophages and reduced ICAM-1 and VCAM-1 expression were noted after 4 months of treatment. Both leflunomide- and methotrexate-treated patients exhibited a decreased MMP-1:TIMP-1 ratio in the synovial tissue. In the subset of patients fulfilling the 20% response criteria of the American College of Rheumatology, a more pronounced reduction in the expression of ICAM-1, VCAM-1, IL-1beta, and MMP-1 was found compared with the nonresponders.

CONCLUSIONS

Leflunomide and methotrexate are clinically efficacious drugs that interfere with mechanisms involved in joint inflammation and destruction of joint integrity.

In this study, the hypothesis that the release of <em>interleukin</em> (IL)-6 from human muscle is linked to exercise intensity and muscle glucose uptake was investigated. In the overnight fasted state, seven healthy males performed knee extension exercise, kicking with both legs, each at 25 % of maximal power (W(max)) for 45 min (eliciting 23 +/- 1 % of pulmonary maximal oxygen uptake, V(O2,max)) and then simultaneously with one leg at 65 % and the other leg at 85 % W(max) for <em>35</em> min (40 +/- 1 % of pulmonary V(O2,max)). Blood was sampled from a femoral artery and both femoral veins, and blood flow was determined by thermodilution. Thigh plasma flow (0.15 +/- 0.01, 1.4 +/- 0.2, 2.0 +/- 0.1 and 2.3 +/- 0.2 l min(-1) thigh(-1) at rest and 25 %, 65 % and 85 % W(max), respectively) and thigh oxygen uptake (0.02 +/- 0.01, 0.27 +/- 0.03, 0.48 +/- 0.04 and 0.55 +/- 0.05 l min(-1) thigh(-1) at rest and 25 %, 65 % and 85 % W(max), respectively) increased with increasing exercise intensity (P < 0.05). Also, thigh IL-6 release (0.4 +/- 0.1, 1.3 +/- 0.5, 1.5 +/- 0.6 and 2.5 +/- 0.7 ng min(-1) thigh(-1) at rest and 25 %, 65 % and 85 % W(max), respectively) and thigh glucose uptake (0.05 +/- 0.01, 0.3 +/- 0.05, 0.75 +/- 0.16, 1.07 +/- 0.15 mmol min(-1) thigh(-1) at rest and 25 %, 65 % and 85 % W(max), respectively) increased with increasing exercise intensity (P < 0.05). During the last <em>35</em> min of exercise, arterial catecholamine concentrations were higher (P < 0.05) than at rest and during low-intensity exercise. During exercise, thigh IL-6 release was positively related to both thigh glucose uptake (P < 0.001) and thigh glucose delivery (P < 0.005), but not to thigh glucose extraction. Thigh IL-6 release was also positively related to arterial plasma adrenaline concentration. The pre-exercise muscle glycogen concentration tended to correlate with the arteriovenous IL-6 concentration difference at rest, and the postexercise glycogen concentration was inversely correlated with IL-6 release during the final <em>35</em> min of exercise. In conclusion, the study indicates that IL-6 release from human muscle is positively related to exercise intensity, arterial adrenaline concentration and muscle glucose uptake. This supports the hypothesis that IL-6 may be linked to the regulation of glucose homeostasis during exercise. The observation of a relationship between IL-6 release and muscle glycogen store both at rest and after exercise suggests that IL-6 may act as a carbohydrate sensor.

Type II collagen is composed of alpha1(II) chains encoded by the COL2A1 gene. Alteration of this cartilage marker is a common feature of osteoarthritis. <em>Interleukin</em>-6 (IL-6) is a pro-inflammatory cytokine that needs a soluble form of receptor called sIL-6R to exert its effects in some cellular models. In that case, sIL-6R exerts agonistic action. This mechanism can make up for the partial or total absence of membrane-anchored IL-6 receptors in some cell types, such as chondrocytes. Our study shows that IL-6, sIL-6R, or both inhibit type II collagen production by rabbit articular chondrocytes through a transcriptional control. The cytokine and/or sIL-6R repress COL2A1 transcription by a -63/-<em>35</em> sequence that binds Sp1.Sp3. Indeed, IL-6 and/or sIL-6R inhibit Sp1 and Sp3 expression and their binding activity to the 63-bp promoter. In chromatin immunoprecipitation experiments, IL-6.sIL-6R induced an increase in Sp3 recruitment to the detriment of Sp1. Knockdown of Sp1.Sp3 by small interference RNA and decoy strategies were found to prevent the IL-6- and/or sIL-6R-induced inhibition of COL2A1 transcription, indicating that each of these Sp proteins is required for down-regulation of the target gene and that a heterotypic Sp1.Sp3 complex is involved. Additionally, Sp1 was shown to interact with Sp3 and HDAC1. Indeed, overexpression of a full-length Sp3 cDNA blocked the Sp1 up-regulation of the 63-bp COL2A1 promoter activity, and by itself, inhibits COL2A1 transcription. We can conclude that IL-6, sIL-6R, or both in combination decrease both the Sp1.Sp3 ratio and DNA-binding activities, thus inhibiting COL2A1 transcription.

Bcl2's antiapoptotic function is regulated by phosphorylation. Bcl2 also regulates cell cycle progression, but the molecular mechanism is unclear. Bcl2 is functionally expressed in mitochondria where it can act as an antioxidant that may regulate intracellular reactive oxygen species (ROS). Since ROS have been reported to act as second messengers in cell signaling, we tested whether Bcl2 phosphorylation regulates ROS and cell cycle progression. G1 ->> S transition and ROS levels were measured in cells expressing either the gain of function phosphomimetic Bcl2 mutants S70E and T69E/S70E/S87E (EEE) or the nonphosphorylatable and survival-deficient mutants S70A and T69A/S70A/S87A (AAA). Expression of S70E and EEE but not the A-containing Bcl2 mutants retards G1 ->> S transition by <em>35</em>% to 50% and significantly slows cell growth in association with reduced levels of intracellular ROS. In addition to expression of the phosphomimetic Bcl2 mutants, either <em>interleukin</em>-3 withdrawal or treatment of cells with the antioxidant pyrrolidine dithiocarbamate (PDTC) also reduces intracellular ROS levels in association with up-regulation of p27 and accumulation of cells in G0/G1. Retardation of G1 ->> S transition can be overridden by directly adding H2O2 to the cells in a mechanism that involves down-regulation of p27 and activation of Cdk2. Thus, Bcl2 may regulate G1 ->> S transition by a novel signaling mechanism that couples regulation of intracellular ROS with p27 and Cdk2. Furthermore, phosphorylation of Bcl2 may functionally link its antiapoptotic, cell cycle retardation, and antioxidant properties.

Regulatory T (Treg) cells pose a major barrier to effective anti-tumor immunity. Although Treg cell depletion enhances tumor rejection, the ensuing autoimmune sequelae limits its utility in the clinic and highlights the need for limiting Treg cell activity within the tumor microenvironment. <em>Interleukin</em>-<em>35</em> (IL-<em>35</em>) is a Treg cell-secreted cytokine that inhibits T cell proliferation and function. Using an IL-<em>35</em> reporter mouse, we observed substantial enrichment of IL-<em>35</em>(+) Treg cells in tumors. Neutralization with an IL-<em>35</em>-specific antibody or Treg cell-restricted deletion of IL-<em>35</em> production limited tumor growth in multiple murine models of human cancer. Limiting intratumoral IL-<em>35</em> enhanced T cell proliferation, effector function, antigen-specific responses, and long-term T cell memory. Treg cell-derived IL-<em>35</em> promoted the expression of multiple inhibitory receptors (PD1, TIM3, LAG3), thereby facilitating intratumoral T cell exhaustion. These findings reveal previously unappreciated roles for IL-<em>35</em> in limiting anti-tumor immunity and contributing to T cell dysfunction in the tumor microenvironment.

The human "26-kd protein' is a secreted glycoprotein expressed, for example, in (blood) leukocytes, in epithelial cells treated with various inducers, but most strongly in <em>interleukin</em>-1 (IL-1)-treated fibroblasts. After finding it has antiviral and 2-5A synthetase-inducing activity, one group of authors called this protein IFN-beta 2. However, recently the full-length 26-kd cDNA sequence was shown to be identical with that of a B-cell-differentiating lymphokine called BSF-2, and another report suggested that the 26-kd protein could support the growth of some transformed murine B cell lines. To define its biological activities, we expressed the recombinant 26-kd protein by translating in Xenopus laevis oocytes a pure, synthetic chimeric mRNA containing the 26-kd protein coding region surrounded by Xenopus laevis beta-globin untranslated regions. A similar construction, but containing the HuIFN-beta cDNA coding region, was used to produce HuIFN-beta by the same procedure. Both recombinant glycoproteins were secreted, glycosylated, and their amounts were measured by [<em>35S</em>]methionine incorporation by the oocyte. Here we show that the recombinant 26-kd protein exhibits a high growth factor activity when assayed on an IL-HP1-dependent murine B cell hybridoma (sp. act. approximately 2 X 10(8) U/mg) as well as a potent differentiating activity on human CESS cells (sp. act. approximately 5 X 10(7) U/mg). While rHuIFN-beta was inactive in the latter two assays, it had the expected antiviral activity of 1-5 X 10(8) U/mg. The parallel recombinant 26-kd protein preparations had no detectable antiviral activity (i.e. a maximal specific activity of 1-3 X 10(2) U/mg, if any). The 26-kd protein is thus clearly an <em>interleukin</em>, and considering the confusing nomenclature now in use, this factor may better be renamed "<em>interleukin</em> 6'.

OBJECTIVE

Progression from the acute to chronic phase of inflammatory bowel disease cannot be easily evaluated in patients and has not been characterized in animal models. We report a longitudinal study investigating changes in the mucosal immune response in an experimental model of colitis.

METHODS

Severity of colitis, body mass, stool consistency and blood content, serum amyloid A, and tissue histology were examined in <em>interleukin</em> (IL)-10-deficient mice over <em>35</em> weeks. The corresponding production of IL-12, IL-18, interferon gamma, tumor necrosis factor alpha, IL-4, and IL-13 by lamina propria mononuclear cells in the inflamed intestine was measured. Administration of neutralizing antibody to IL-12 at distinct times during disease progression permitted evaluation of its therapeutic potential.

RESULTS

The clinical manifestations and intestinal inflammation delineated an early phase of colitis (10-24 weeks), characterized by a progressive increase in disease severity, followed by a late phase (>25 weeks), in which chronic inflammation persisted indefinitely. Lamina propria mononuclear cells from mice with early disease synthesized progressively greater quantities of IL-12 and interferon gamma, whereas production of both cytokines dramatically declined and returned to pre-disease levels in the late phase of colitis. Consistent with this pattern, neutralizing antibody to IL-12 reversed early, but not late, disease. In contrast, IL-4 and IL-13 production increased progressively from pre- to early to late disease.

CONCLUSIONS

Colitis that develops in IL-10-deficient mice evolves into 2 distinct phases. IL-12 plays a pivotal role in early colitis, whereas its absence and the synthesis of IL-4 and IL-13 in late disease indicate that other immune mechanisms sustain chronic inflammation.

It is now <em>35</em> years since Brandtzaeg and Kraus (1965) published their seminal work entitled "Autoimmunity and periodontal disease". Initially, this work led to the concept that destructive periodontitis was a localized hypersensitivity reaction involving immune complex formation within the tissues. In 1970, Ivanyi and Lehner highlighted a possible role for cell-mediated immunity, which stimulated a flurry of activity centered on the role of lymphokines such as osteoclast-activating factor (OAF), macrophage-activating factor (MAF), macrophage migration inhibition factor (MIF), and myriad others. In the late 1970s and early 1980s, attention focused on the role of polymorphonuclear neutrophils, and it was thought that periodontal destruction occurred as a series of acute exacerbations. As well, at this stage doubt was being cast on the concept that there was a neutrophil chemotactic defect in periodontitis patients. Once it was realized that neutrophils were primarily protective and that severe periodontal destruction occurred in the absence of these cells, attention swung back to the role of lymphocytes and in particular the regulatory role of T-cells. By this time in the early 1990s, while the roles of <em>interleukin</em> (IL)-1, prostaglandin (PG) E(2), and metalloproteinases as the destructive mediators in periodontal disease were largely understood, the control and regulation of these cytokines remained controversial. With the widespread acceptance of the Th1/Th2 paradigm, the regulatory role of T-cells became the main focus of attention. Two apparently conflicting theories have emerged. One is based on direct observations of human lesions, while the other is based on animal model experiments and the inability to demonstrate IL-4 mRNA in gingival extracts. As part of the "Controversy" series, this review is intended to stimulate debate and hence may appear in some places provocative. In this context, this review will present the case that destructive periodontitis is due to the nature of the lymphocytic infiltrate and is not due to periodic acute exacerbations, nor is it due to the so-called virulence factors of putative periodontal pathogens.

Cyclooxygenase (Cox) exists in two forms in human endothelial cells (HUVEC). We have raised antibodies that recognize the sequence of the carboxyl-terminal portion of the human Cox-2 (C)-NASSSRSGLD-DINPTVLLK. Cyclooxygenase activity of HUVEC challenged with <em>interleukin</em> 1 alpha or a phorbol ester increased in parallel with the mass of a protein doublet analyzed by Western blot using antibodies directed against the Cox-2 peptide; a monoclonal antibody directed against Cox-1 showed a small change in protein mass. A <em>35S</em>-labeled protein doublet with a molecular mass of approximately 70,000 daltons was immunoprecipitated with the anti-Cox-2 antiserum in L-[<em>35S</em>] methionine-labeled cells stimulated with <em>interleukin</em> 1 alpha. This protein was not recovered by pretreating the antiserum with the Cox-2 peptide before immunoprecipitation. A minor variation in <em>35S</em>-immunoprecipitated protein was obtained with the polyclonal anti-Cox-1 antibody. Both immunoprecipitated Cox-1 and Cox-2 possessed cyclooxygenase activity that was inhibited by flurbiprofen. Endoglycosidase H treatment of immunoprecipitated Cox-2 proteins caused a decline in the apparent molecular size similar to that observed with immunoprecipitated Cox-1 or sheep cyclooxygenase but did not suppress the doublet. These results show by direct protein measurement that HUVEC synthesize the novel Cox-2 under appropriate stimulation, with little changes of Cox-1.

OBJECTIVE

Pancreatic beta-cell apoptosis is a common feature of Type 1 and Type 2 diabetes and leptin exerts an anti-apoptotic function in these cells. The beta-cell line INS-1 was used to test the hypothesis that the adipocyte hormone adiponectin might mediate an anti-apoptotic effect comparable to leptin.

METHODS

Apoptosis was induced by culturing cells with a cytokine combination (interleukin-1beta/interferon-gamma) or palmitic acid in absence or presence of leptin or the globular domain of adiponectin (gAcrp30), respectively.

RESULTS

INS-1 cells had a prominent sensitivity towards cytokine- and fatty acid-induced apoptosis, resulting in about three- and six-fold increases in caspase 3 activation and DNA fragmentation, respectively. gAcrp30 strongly (50-60%) inhibited palmitic acid-induced apoptosis, with a weaker effect against cytokine-induced apoptosis (35%). The same result was observed for leptin with both adipokines being non-additive. Reduction of apoptosis by an inhibitor of IkappaB-kinase (IKK) indicated the involvement of the nuclear factor (NF)-kappaB pathway in both cytokine- and fatty acid-induced apoptosis, however, leptin and gAcrp30 were unable to block NF-kappaB activation. Cytokine- and fatty-acid-induced suppression of glucose/forskolin-stimulated insulin secretion was completely prevented through the action of gAcrp30, whereas leptin was only effective against lipotoxicity-mediated beta-cell dysfunction.

CONCLUSIONS

Our data show that gAcrp30 partially rescues beta cells from cytokine- and fatty-acid-induced apoptosis and completely restores autoimmune- and lipotoxicity-induced dysfunction of insulin-producing cells. We suggest that gAcrp30 exerts its anti-apoptotic function without modulating NF-kappaB activation. This novel beta cell protective function of gAcrp30 might serve to counteract autoimmune- and lipotoxicity-induced beta-cell destruction.

OBJECTIVE

Anti-tumor necrosis factor a (anti-TNFalpha) therapy has shown efficacy in the treatment of rheumatoid arthritis (RA). Since interleukin-1 (IL-1), TNFalpha, and IL-17 have many additive and/or synergistic effects in vitro, we tested whether their combined inhibition by soluble receptors would lead to an enhanced effect on ex vivo models of synovial inflammation and bone destruction.

METHODS

RA synovium and bone explants were cultured for 7 days in the presence of 1 microg/ml soluble TNFalpha receptor (STNFR; as in current therapy), type II soluble IL-1 receptor (sIL-1RII), or sIL-17R either alone or in combination. Their effects on the production of IL-6 and the release of C-telopeptide of type I collagen (CTX), a marker of type I collagen destruction, were measured by enzyme-linked immunosorbent assay.

RESULTS

In synovium, each soluble receptor alone decreased IL-6 production and CTX release by approximately 35% and approximately 55%, respectively. The combination of all 3 receptors was more effective, inhibiting IL-6 production and collagen degradation by up to 70%. Neither sIL-17R, sIL-1RII, or sTNFR alone had no effect (or an effect of <20% inhibition) on IL-6 production in 18%, 33%, and 22%, respectively, of the samples. In bone, sIL-17R, sIL-1RII, and sTNFR decreased IL-6 production by 23%, 50%, and 37%, respectively, while the combination decreased IL-6 production by 75%. A 50% inhibition of CTX release was obtained with sIL-1RII for 63% of the samples versus 38% of the samples with either sTNFR or sIL-17R. However, the combination of all 3 receptors was not more potent than sIL-1RII alone.

CONCLUSIONS

The inhibitory effect of sTNFR on IL-6 production and collagen degradation in RA synovium and bone was increased in combination with sIL-17R and sIL-1RII. These results support the concept of combination therapy, which may increase the percentage of responding patients as well as the degree of individual patient response.

OBJECTIVE

Tofacitinib, an orally administered Janus kinase inhibitor, blocks signaling through γ-chain-containing cytokines (interleukins 2, 4, 7, 9, 15, and 21). We performed a phase 2 trial to measure its efficacy in patients with moderate-to-severe active Crohn's disease.

METHODS

Patients (N = 139; age, ≥18 y) with moderate-to-severe active Crohn's disease were assigned randomly to groups given 1 mg (n = 36), 5 mg (n = 34), or 15 mg (n = 35) tofacitinib or placebo (n = 34), twice daily for 4 weeks, at 48 centers in 12 countries. The primary end point was the proportion of clinical responders at week 4 (decrease from baseline in the Crohn's Disease Activity Index score of ≥70 points [Response-70]). Secondary end points included clinical remission (Crohn's Disease Activity Index score of <150 points) at week 4.

RESULTS

A clinical response was observed in 36% (P = .467), 58% (P = .466), and 46% (P ≥ .999) of patients given the 1-, 5-, and 15-mg doses of tofacitinib, compared with 47% of patients given placebo. Clinical remission was observed in 31% (P = .417), 24% (P = .776), and 14% (P = .540) of patients given the 1-, 5-, and 15-mg doses of tofacitinib, compared with 21% of patients given placebo. The 15-mg dose of tofacitinib reduced levels of C-reactive protein and fecal calprotectin from baseline. Adverse and serious adverse events were similar among groups. Dose-dependent increases in low- and high-density lipoprotein cholesterol were observed in patients given the 5- or 15-mg doses of tofacitinib.

CONCLUSIONS

There were no significant differences in the percentage of patients with moderate-to-severe active Crohn's disease who achieved clinical responses (Response-70) or clinical remission after 4 weeks' administration of tofacitinib (1, 5, or 15 mg) or placebo twice daily. However, a large percentage of patients given placebo achieved Response-70 or remission. Reductions in C-reactive protein and fecal calprotectin levels among patients given the 15-mg dose of tofacitinib indicate its biologic activity. ClinicalTrials.gov number: NCT00615199.

We aimed to study the effect of iron deficiency anemia (IDA) on immunity. In 32 children with IDA and 29 normal children, the percentage of T-lymphocyte subgroups, the level of serum <em>interleukin</em>-6 (IL-6); and the phagocytic activity, the oxidative burst activity of neutrophils and monocytes and the levels of immunoglobulins were compared. There was no difference in the distribution of T-lymphocyte subgroups. The mean IL-6 levels was 5.6+/-3.9 pg/ml in children with IDA and 10.3+/-5.3 pg/ml in the control group (P<0.001). The percentage of neutrophils with oxidative burst activity when stimulated with pma was 53.4+/-32.7% in children with IDA and 81.7+/-14.3% in the control group (P=0.005). The percentage of monocytes with oxidative burst activity was 13.8+/-11.7% in children with IDA and <em>35</em>+/-20.0% in the control group (P<0.001) when stimulated with pma. and 4.3+/-3.1 versus 9.7+/-6.0% (P=0.008) when stimulated with fMLP. The ratio of neutrophils with phagocytic activity was 58.6+/-23.3% in the anemic group; and 74.2+/-17.7% in the control group (P=0.057). The ratio of monocytes with phagocytic activity was 24.3+/-12.0% in the anemic group; and 42.9+/-13.4% in the control group (P=0.001). IgG4 level was 16.7+/-16.6 mg/dl in children with IDA and 51.8+/-40.7 mg/dl in healthy children (P<0.05). These results suggest that humoral, cell-mediated and nonspecific immunity and the activity of cytokines which have an important role in various steps of immunogenic mechanisms are influenced by iron deficiency anemia.

OBJECTIVE

To assess the presence of Toll-like receptors (TLRs) 1-9 in human articular cartilage, and to investigate the effects of lipopolysaccharide (LPS)-induced activation of TLR-4 on biosynthetic activity and matrix production by human articular chondrocytes.

METHODS

TLRs 1-9 were assessed in human articular cartilage by reverse transcription-polymerase chain reaction (RT-PCR); TLR-4 was also analyzed by Western blotting and immunohistochemistry. Articular chondrocytes were isolated from human donors and from wild-type or TLR-4(-/-) mice. Chondrocyte monolayer cultures were incubated with <em>interleukin</em>-1beta (IL-1beta) and LPS in the absence or presence of bone morphogenetic protein 7 (BMP-7) and IL-1 receptor antagonist (IL-1Ra). Neosynthesis of sulfated glycosaminoglycans (sGAG) was measured by (<em>35</em>)S-sulfate incorporation. Endogenous gene expression of cartilage markers as well as IL-1beta was examined using RT-PCR. The involvement of p38 kinase or p44/42 kinase (ERK-1/2) in LPS-mediated TLR-4 signaling was investigated by immunoblotting, RT-PCR, and sGAG synthesis.

RESULTS

TLRs 1-9 were found on the messenger RNA (mRNA) level in human articular chondrocytes. The presence of TLR-4 was also observed on the protein level. In murine and human articular chondrocytes, but not in chondrocytes derived from TLR-4(-/-) mice, stimulation with LPS resulted in a decrease in total proteoglycan synthesis. IL-1beta mRNA expression was increased by TLR-4 activation, whereas expression of aggrecan and type II collagen was significantly decreased. The presence of BMP-7 and IL-1Ra antagonized the anti-anabolic effects of LPS. Blocking of p38, but not ERK-1/2, resulted in inhibition of both LPS-mediated IL-1beta gene expression and the negative effects of LPS on matrix biosynthesis.

CONCLUSIONS

These data demonstrate the presence of TLRs in human articular cartilage. The suppressive effects of LPS on cartilage biosynthetic activity are dependent on the presence of TLR-4, are governed, at least in part, by an up-regulation of IL-1beta, and are mediated by p38 kinase. These in vitro data indicate an anti-anabolic effect of TLR-4 in articular chondrocytes that may hamper cartilage repair in various joint diseases.

BACKGROUND

Myeloid differentiation factor (MyD)-88 is a key adaptor protein that plays a major role in the innate immune pathway. How MyD88 may regulate host response in inflammatory heart disease is unknown.

RESULTS

We found that the cardiac protein level of MyD88 was significantly increased in the hearts of wild-type mice after exposure to Coxsackievirus B3 (CVB3). MyD88(-/-) mice showed a dramatic higher survival rate (86%) in contrast to the low survival (<em>35</em>%) in the MyD88(+/+) mice after CVB3 infection (P<0.0001). Pathological examination showed a significant decrease of cardiac and pancreatic inflammation in the MyD88(-/-) mice. Viral concentrations in the hearts were significantly decreased in the MyD88(-/-) mice. Cardiac mRNA levels for <em>interleukin</em> (IL)-1beta, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and IL-18 were significantly decreased in the MyD88(-/-) mice. Similarly, serum levels of T-helper 1 cytokines were significantly decreased in the MyD88(-/-) mice. In contrast, cardiac protein levels of the activated interferon regulatory factor (IRF)-3 and IFN-beta were significantly increased in the MyD88(-/-) mice but not other usual upstream signals to IRF-3. The cardiac expression of coxsackie-adenoviral receptor and p56(lck) were also significantly decreased.

CONCLUSIONS

MyD88 appears to be a key contributor to cardiac inflammation, mediating cytokine production and T-helper-1/2 cytokine balance, increasing coxsackie-adenoviral receptor and p56(lck) expression and viral titers after CVB3 exposure. Absence of MyD88 confers host protection possibly through novel direct activation of IRF-3 and IFN-beta.

The cDNA for human beta 2-interferon (IFN-beta 2)/B-cell differentiation factor 2/hepatocyte-stimulating factor was expressed in Escherichia coli to yield a fusion protein which contains the 182 carboxyl-terminal amino acids of IFN-beta 2 fused to a 34-amino acid prokaryotic leader peptide (rIFN-beta 2). When added to cultures of human hepatoma cell line Hep3B2, rIFN-beta 2 as well as preparations of natural IFN-beta 2 enhance secretion of positive acute phase reactants such as alpha 1-antichymotrypsin, complement C3, fibrinogen, and alpha 1-acid glycoprotein and inhibit secretion of albumin, confirming that a protein derived from the IFN-beta 2 gene can have hepatocyte-stimulating factor activity. We have prepared a rabbit polyclonal antiserum to the E. coli-derived human IFN-beta 2 fusion protein. This polyclonal antiserum inhibits the hepatocyte-stimulating and B-cell differentiation activities of appropriate IFN-beta 2 preparations. The anti-rIFN-beta 2 antiserum has been used in immunoprecipitation experiments and in Western blots to help define the secretory proteins derived from the IFN-beta 2 gene in fibroblasts and monocytes. "Uninduced" human FS-4 fibroblasts as well as those induced with <em>interleukin</em>-1 alpha, tumor necrosis factor, or bacterial lipopolysaccharide secrete at least five forms of IFN-beta 2 of apparent molecular mass in the range from 23 to 30 kDa which can be resolved by polyacrylamide gel electrophoresis under denaturing and reducing conditions. The three higher molecular mass forms are not observed when FS-4 cells are induced in the presence of tunicamycin, suggesting that these forms are N-glycosylated (gp28, gp29, and gp30). Although secretion of the two lower molecular mass forms is resistant to tunicamycin, they are labeled by [3H]glucosamine (gp23-gp25). The inclusion of cycloheximide during the [<em>35S</em>]methionine labeling of induced FS-4 cells results in the preferential synthesis and secretion of the 29-kDa triplet. Human monocytes induced with bacterial lipopolysaccharide also secrete several distinct forms of IFN-beta 2 in the size range from 23 to 30 kDa which co-migrate in polyacrylamide gels with those obtained from FS-4 cells. Our observations help relate previous descriptions of multiple forms of hepatocyte-stimulating factor to specific proteins derived from the IFN-beta 2 gene.

We investigated the phenotype of cells expressing messenger RNA encoding <em>interleukin</em> 4 (IL-4), IL-5, IL-2, and interferon gamma (IFN-gamma) in bronchoalveolar lavage (BAL) and bronchial biopsies (BX) from seven mild atopic asthmatic patients and nine nonasthmatic controls. Immunocytochemistry followed by in situ hybridization using either <em>35S</em>- or digoxigenin-labeled riboprobes was performed on cytospins from BAL and BX, respectively. With BAL or BX, in situ hybridization alone showed significant increases in percentages of IL-2, IL-4, and IL-5 mRNA+ cells when asthmatics were compared to nonasthmatic controls. Double immunocytochemistry-in situ hybridization revealed that>> 70% of IL-4 and IL-5 mRNA+ cells were activated T cells (CD3+). The remaining IL-4 and IL-5 mRNA+ signals were colocalized to tryptase+ mast cells, and activated eosinophils (EG2+). Rare IL-4 and IL-5 mRNA+ cells were observed in nonasthmatic controls, the majority being CD3+ cells, as were IL-2 and IFN-gamma mRNA+ cells (in both asthmatics and controls). A few IL-4 (< 8%) and IL-5 (< 5%) mRNA+ signals did not colocalize with any of the cells identified by immunocytochemistry. Thus, we provide further evidence that CD3+ T cells are the most abundant cells expressing IL-4 and IL-5 mRNA in BAL and BX from allergic asthma. Fewer, but detectable, numbers of tryptase+ mast cells and EG2+ eosinophils also expressed these transcripts.

OBJECTIVE

To determine if the serum level of interleukin-6 (IL-6) was elevated in patients with hepatic malignancies or correlated with radiologic tumor burden.

BACKGROUND

High serum levels of IL-6 signify an adverse prognosis in many patients with cancer. IL-6 is a growth factor for bile duct epithelium.

METHODS

Using bioactive and enzyme-linked immunosorbent assays, serum level of IL-6 was measured in 35 healthy adults and in 60 patients presenting for definitive management of cholangiocarcinoma (CC) (15 patients), hepatocellular carcinoma (HCC) (14), metastatic colorectal cancer (MCRC) (26), and benign biliary disease (BBD) (5). Patients with clinical conditions known to raise the serum level of IL-6 were excluded. Tumor burden was calculated from concurrent computed tomography scans. IL-6 levels were measured 2 weeks after resection in 3 CC patients. Secretion of IL-6 was examined in 3 human CC cell lines.

RESULTS

An elevated level of bioactive IL-6 was detected in every patient with CC and in 13 of 14 patients with HCC, 14 of 26 patients with MCRC, 2 of 5 patients with BBD, and 3 of 35 healthy adults. Median and mean levels of bioactive IL-6 were higher in CC than in other neoplasms (p < 0.026) and for all tumor groups differed from healthy adults (p < or = 0.026). IL-6 level was elevated more often in primary than in secondary liver neoplasms (p = 0.02), distinguished patients with CC or MCRC from BBD (p = 0.014 and 0.031, respectively), correlated with tumor burden in CC (p < 0.001), and dropped sharply after CC resection. CC line SG231 secreted bioactive IL-6.

CONCLUSIONS

In selected patients, a high serum level of IL-6 marks patients with CC and correlates with tumor burden both before and after resection. IL-6 levels are elevated in patients with other liver neoplasms and may distinguish patients with hepatic malignancies from those with benign disease.

BACKGROUND

Adherence to the Mediterranean diet (MD) is associated with reduced morbidity and mortality due to cardiovascular disease. However, how the MD exerts its effects is not fully known.

OBJECTIVE

To assess the 12-month effects of two enhanced MDs compared to a low-fat diet on inflammatory biomarkers related to atherosclerosis and plaque vulnerability in a subcohort of the PREDIMED (Prevención con Dieta Mediterránea) study.

METHODS

A total of 164 participants at high risk for cardiovascular disease were randomized into three diet groups: MD supplemented with 50mL/d of extra virgin olive oil (MD+EVOO) or 30 g/d of nuts (MD+Nuts) and a low-fat diet. Changes in classical cardiovascular risk factors, inflammatory biomarkers of atherosclerosis and plaque vulnerability were measured after 12 months of intervention.

RESULTS

Compared to participants in the low-fat diet group, those receiving MD+EVOO and MD+Nuts showed a higher decrease in systolic (6mmHg) and diastolic (3mmHg) blood pressure (P = 0.02; both), as well as a reduction of 10% and 8% in LDL-cholesterol (P = 0.04), respectively. Patients in the MD+Nuts group showed a significant reduction of 34% in CD40 expression on monocyte surface compared to low-fat diet patients (P = 0.03). In addition, inflammatory biomarkers related to plaque instability such as C-reactive protein and <em>interleukin</em>-6 were reduced by 45% and <em>35</em>% and 95% and 90% in the MD+EVOO and MD+Nuts groups, respectively (P<0.05; all) compared to the low-fat diet group. Likewise, sICAM and P-selectin were also reduced by 50% and 27%, respectively in the MD+EVOO group (P = 0.04) and P-selectin by 19% in MD+Nuts group (P = 0.04) compared to the low-fat diet group.

CONCLUSIONS

Adherence to the MD is associated with an increase in serum markers of atheroma plaque stability which may explain, at least in part, the protective role of MD against ischemic heart disease.

BACKGROUND

www.controlled-trials.com ISRCTN<em>35</em>739639.

BACKGROUND

Type 1 diabetes results from T-cell-mediated destruction of β cells. Findings from preclinical studies and pilot clinical trials suggest that antithymocyte globulin (ATG) might be effective for reducing this autoimmune response. We assessed the safety and efficacy of rabbit ATG in preserving islet function in participants with recent-onset type 1 diabetes, and report here our 12-month results.

METHODS

For this phase 2, randomised, placebo-controlled, clinical trial, we enrolled patients with recent-onset type 1 diabetes, aged 12-<em>35</em> years, and with a peak C-peptide of 0.4 nM or greater on mixed meal tolerance test from 11 sites in the USA. We used a computer generated randomisation sequence to randomly assign patients (2:1, with permuted-blocks of size three or six and stratified by study site) to receive either 6.5 mg/kg ATG or placebo over a course of four days. All participants were masked and initially managed by an unmasked drug management team, which managed all aspects of the study until month 3. Thereafter, to maintain masking for diabetes management throughout the remainder of the study, participants received diabetes management from an independent, masked study physician and nurse educator. The primary endpoint was the baseline-adjusted change in 2-h area under the curve C-peptide response to mixed meal tolerance test from baseline to 12 months. Analyses were by intention to treat. This is a planned interim analysis of an on-going trial that will run for 24 months of follow-up. This study is registered with ClinicalTrials.gov, number NCT00515099.

RESULTS

Between Sept 10, 2007, and June 1, 2011, we screened 154 individuals, randomly allocating 38 to ATG and 20 to placebo. We recorded no between-group difference in the primary endpoint: participants in the ATG group had a mean change in C-peptide area under the curve of -0.195 pmol/mL (95% CI -0.292 to -0.098) and those in the placebo group had a mean change of -0.239 pmol/mL (-0.361 to -0.118) in the placebo group (p=0.591). All except one participant in the ATG group had both cytokine release syndrome and serum sickness, which was associated with a transient rise in interleukin-6 and acute-phase proteins. Acute T cell depletion occurred in the ATG group, with slow reconstitution over 12 months. However, effector memory T cells were not depleted, and the ratio of regulatory to effector memory T cells declined in the first 6 months and stabilised thereafter. ATG-treated patients had 159 grade 3-4 adverse events, many associated with T-cell depletion, compared with 13 in the placebo group, but we detected no between-group difference in incidence of infectious diseases.

CONCLUSIONS

Our findings suggest that a brief course of ATG does not result in preservation of β-cell function 12 months later in patients with new-onset type 1 diabetes. Generalised T-cell depletion in the absence of specific depletion of effector memory T cells and preservation of regulatory T cells seems to be an ineffective treatment for type 1 diabetes.

OBJECTIVE

The goal of this study was to describe the spectrum of clinical signs of mevalonate kinase deficiency (MKD).

METHODS

This was a retrospective French and Belgian study of patients identified on the basis of MKD gene mutations.

RESULTS

Fifty patients from 38 different families were identified, including 1 asymptomatic patient. Symptoms began during the first 6 months of life in 30 patients (60%) and before the age of 5 years in 46 patients (92%). Symptoms consisted of febrile diarrhea and/or rash in 23 of <em>35</em> patients (66%). Febrile attacks were mostly associated with lymphadenopathy (71%), diarrhea (69%), joint pain (67%), skin lesions (67%), abdominal pain (63%), and splenomegaly (63%). In addition to febrile attacks, 27 patients presented with inflammatory bowel disease, erosive polyarthritis, Sjögren syndrome, and other chronic neurologic, renal, pulmonary, endocrine, cutaneous, hematologic, or ocular symptoms. Recurrent and/or severe infections were observed in 13 patients, hypogammaglobulinemia in 3 patients, and renal angiomyolipoma in 3 patients. Twenty-nine genomic mutations were identified; the p.Val377Ile mutation was the most frequently found (29 of 38 families). Three patients died of causes related to MKD. The disease remained highly active in 17 of the 31 surviving symptomatic patients followed up for >5 years, whereas disease activity decreased over time in the other 14 patients. <em>Interleukin</em> 1 antagonists were the most effective biological agents tested, leading to complete or partial remission in 9 of 11 patients.

CONCLUSIONS

MKD is not only an autoinflammatory syndrome but also a multisystemic inflammatory disorder, a possible immunodeficiency disorder, and a condition that predisposes patients to the development of renal angiomyolipoma.

BACKGROUND

Treatment with interleukin-2 (IL-2) and lymphokine-activated killer cells (LAK) resulted in responses in some patients with advanced renal cell carcinoma (RCC). However, the relative therapeutic benefit of the addition of LAK to IL-2 was unknown.

METHODS

A randomized Phase III trial was conducted in patients with RCC comparing continuous intravenous infusion (CI) IL-2 alone with CI IL-2 plus LAK. Interleukin-2 was administered at 3 x 10(6) U/m2/day on days 1-5, 13-17, 21-24, and 28-31. Patients on the LAK treatment arm underwent leukapheresis on days 8-10 and LAK cell reinfusion on days 13-15. The results are reported with long-term follow-up. The published experience with IL-2 alone or with the addition of LAK was investigated in a quantitative literature survey. The response proportions were studied by schedule (high dose bolus, moderate dose, low dose) and by concomitant administration of LAK.

RESULTS

Seventy-one patients were treated, 36 on the IL-2 arm and 35 on the IL-2 plus LAK arm. Four patients (6%) had major responses (two complete, two partial). The median survival of all patients was 13 months (95% confidence interval [CI], 9-18 months). There were no differences between treatment arms with regard to response (P = 0.61) and survival (P = 0.67). More patients on the LAK arm experienced pulmonary toxicity (P = 0.008). The overall weighted response proportion was 16% (95% CI, 8%-24%) for the 39 published series of 1291 patients treated with IL-2. The 95% confidence intervals for response proportion overlapped when compared by schedule and by administration of LAK.

CONCLUSIONS

The dose and schedule of IL-2 used in this study resulted in a low level of antitumor activity and the addition of LAK did not improve the response rate against RCC. Given the infrequent, but reproducible, responses with IL-2 and interferon-based regimens, continued investigation of these agents is warranted as is the study of new cytokines. Alternative treatment strategies should be studied in RCC and new agents and treatment regimens that appear promising in Phase II studies must be studied in randomized trials.

Previous studies have demonstrated the development of an age-dependent resistance to reinfection after chemotherapeutic cure of the helminthic parasite Schistosoma mansoni. Here we report on a longitudinal investigation of cell-mediated responses in infected individuals before and after treatment which was designed to outline those parameters important in mediating a protective response. A well-defined study group of 89 individuals with an age range of 9 to <em>35</em> years was selected from an area of high S. mansoni transmission in the Machakos district of Kenya. Peripheral blood mononuclear cell proliferation and cytokine production (<em>interleukin</em>-2 [IL-2], gamma interferon IL-5, IL-4, and tumor necrosis factor) in response to different crude life cycle-stage antigens of S. mansoni were assessed longitudinally in vitro before, 3 months after, and 1 year after treatment. Detailed statistical analyses of the results from this study have indicated a clear negative association between the proliferative responses to adult- and schistosomulum-stage antigens and subsequent reinfection intensity in older individuals (14 to <em>35</em> years) which was not present in the younger individuals (9 to 13 years). This association was significant even after the effects of age, sex, and exposure had been accounted for in multiple regression analyses. Cytokines were detected predominantly in response to adult worm and egg antigen extracts. An inverse association between the two cytokines gamma interferon and IL-5 was detected in response to all antigens at the three time points investigated, indicating cross-regulation in the production of these two mediators. Differences in antigen-specific cytokine levels between the two age groups were detected, with significantly higher IL-5 levels detected in the older (more resistant) age group. An inverse correlation between this cytokine and reinfection was detected but could not be dissociated from the effects of age and exposure in multiple regression analysis.