Airway hyperresponsiveness and airway inflammation are hallmarks of allergic asthma, the etiology of which is crucially linked to the presence of Th2 cytokines. A role for the complement anaphylatoxins C3a and C5a in allergic asthma was suggested, as deficiencies of the C3a receptor (C3aR) and of complement factor C5 modulate airway hyperresponsiveness, airway inflammation, and Th2 cytokine levels. However, such models do not allow differentiation of effects on the sensitization phase and the effector phase of the allergic response, respectively. In this study, we determined the role of the anaphylatoxins on the effector phase of asthma by pharmacological targeting of the anaphylatoxin receptors. C3aR and C5a receptor (C5aR) signaling was blocked using the nonpeptidic C3aR antagonist SB290157 and the neutralizing C5aR mAb <em>20</em>/70 in a murine model of Aspergillus fumigatus extract induced pulmonary allergy. Airway hyperresponsiveness was substantially improved after C5aR blockade but not after C3aR blockade. Airway inflammation was significantly reduced in mice treated with the C3aR antagonist or the anti-C5aR mAb, as demonstrated by reduced numbers of neutrophils and eosinophils in bronchoalveolar lavage fluid. Of note, C5aR but not C3aR inhibition reduced lymphocyte numbers in bronchoalveolar lavage fluid. Cytokine levels of <em>IL</em>-5 and <em>IL</em>-13 in bronchoalveolar lavage fluid were not altered by C3aR or C5aR blockade. However, blockade of both anaphylatoxin receptors markedly reduced <em>IL</em>-4 levels. These data suggest an important and exclusive role for C5aR signaling on the development of airway hyperresponsiveness during pulmonary allergen challenge, whereas both anaphylatoxins contribute to airway inflammation and <em>IL</em>-4 production.

OBJECTIVE

Preclinical studies showed that immunization with peripheral blood mononuclear cells (PBMC) loaded with tumor antigen peptides plus interleukin-12 (<em>IL</em>-12) induced CD8+ T-cell responses and tumor rejection. We recently determined that recombinant human (rh) <em>IL</em>-12 at 30 to 100 ng/kg is effective as a vaccine adjuvant in patients. A phase II study of immunization with Melan-A peptide-pulsed PBMC + rh<em>IL</em>-12 was conducted in <em>20</em> patients with advanced melanoma.

METHODS

Patients were HLA-A2-positive and had documented Melan-A expression. Immunization was performed every 3 weeks with clinical re-evaluation every three cycles. Immune responses were measured by ELISpot assay before and after treatment and through the first three cycles, and were correlated with clinical outcome.

RESULTS

Most patients had received prior therapy and had visceral metastases. Nonetheless, two patients achieved a complete response, five patients achieved a minor or mixed response, and four patients had stable disease. The median survival was 12.25 months for all patients and was not yet reached for those with a normal lactate dehydrogenase. There were no grade 3 or 4 toxicities. Measurement of specific CD8+ T-cell responses by direct ex vivo ELISpot revealed a significant increase in interferon gamma-producing T cells against Melan-A (P =.015) after vaccination, but not against an Epstein-Barr virus control peptide (P =.86). There was a correlation between the magnitude of the increase in Melan-A-specific cells and clinical response (P =.046).

CONCLUSIONS

This immunization approach may be more straightforward than dendritic cell strategies and seems to have clinical activity that can be correlated to a biologic end point.

Interleukin-1 (<em>IL</em>-1) induces <em>IL</em>-1, tumor necrosis factor alpha (TNF alpha), and <em>IL</em>-6 gene expression and synthesis in a variety of cells. In this study, we investigated the ability of human recombinant <em>IL</em>-1 receptor antagonist (<em>IL</em>-1ra) to inhibit <em>IL</em>-1-induced cytokine production in human peripheral blood mononuclear cells (PBMC) and isolated monocytes. <em>IL</em>-1ra alone at concentrations as high as 1 microgram/mL did not induce <em>IL</em>-1 alpha, <em>IL</em>-1 beta, TNF alpha, or <em>IL</em>-6 synthesis. Suppression of <em>IL</em>-1-induced <em>IL</em>-1, TNF alpha, or <em>IL</em>-6 synthesis was dose-dependent (P less than or equal to .0001). At a twofold molar excess, <em>IL</em>-1ra inhibited <em>IL</em>-1-induced <em>IL</em>-1 or TNF alpha synthesis by 50% (P less than .01); an equimolar concentration of <em>IL</em>-1ra inhibited synthesis of these two cytokines by over <em>20</em>% (P less than .05). A 10-fold molar excess of <em>IL</em>-1ra over <em>IL</em>-1 beta reduced <em>IL</em>-1 beta-induced <em>IL</em>-1 alpha by 95% (P = .01) and <em>IL</em>-1 alpha-induced <em>IL</em>-1 beta by 73% (P less than .01). <em>IL</em>-1ra added to PBMC 8 hours after stimulation with <em>IL</em>-1 beta was still able to inhibit <em>IL</em>-1 alpha, TNF alpha, and <em>IL</em>-6 synthesis (P less than or equal to .01). A similar reduction in <em>IL</em>-1 beta-induced <em>IL</em>-1 alpha was observed when <em>IL</em>-1 beta was removed from the cultures after 8 hours of stimulation (P less than .05), suggesting a prolonged presence of <em>IL</em>-1 or restimulation of <em>IL</em>-1 receptors on monocytes is required for the induction of cytokines. In elutriated monocytes, a 10-fold molar excess of <em>IL</em>-1ra reduced <em>IL</em>-1 beta-induced <em>IL</em>-1 alpha by 82% (P less than .05), TNF alpha by 64% (P = .05), and <em>IL</em>-6 by 47% (P less than .05). 125I-<em>IL</em>-1 beta was bound to purified monocytes, cross-linked, and immunoprecipitated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a band at 85 Kd corresponding to the 68-Kd <em>IL</em>-1 receptor type II (<em>IL</em>-1RtII). Excess unlabeled <em>IL</em>-1 beta or <em>IL</em>-1ra blocked the binding of 125I-<em>IL</em>-1 beta to the <em>IL</em>-1RtII. We conclude that <em>IL</em>-1ra inhibits <em>IL</em>-1-induced cytokine synthesis and competes with <em>IL</em>-1 for the <em>IL</em>-1RtII on human monocytes.

The polymorphisms in the interleukin (<em>IL</em>)-28B (interferon-lambda [IFN]-λ3) gene are strongly associated with the efficacy of hepatitis C virus (HCV) clearance. Dendritic cells (DCs) sense HCV and produce IFNs, thereby playing some cooperative roles with HCV-infected hepatocytes in the induction of interferon-stimulated genes (ISGs). Blood dendritic cell antigen 3 (BDCA3)(+) DCs were discovered as a producer of IFN-λ upon Toll-like receptor 3 (TLR3) stimulation. We thus aimed to clarify the roles of BDCA3(+) DCs in anti-HCV innate immunity. Seventy healthy subjects and <em>20</em> patients with liver tumors were enrolled. BDCA3(+) DCs, in comparison with plasmacytoid DCs and myeloid DCs, were stimulated with TLR agonists, cell-cultured HCV (HCVcc), or Huh7.5.1 cells transfected with HCV/JFH-1. BDCA3(+) DCs were treated with anti-CD81 antibody, inhibitors of endosome acidification, TIR-domain-containing adapter-inducing interferon-β (TRIF)-specific inhibitor, or ultraviolet-irradiated HCVcc. The amounts of <em>IL</em>-29/IFN-λ1, <em>IL</em>-28A/IFN-λ2, and <em>IL</em>-28B were quantified by subtype-specific enzyme-linked immunosorbent assay (ELISA). The frequency of BDCA3(+) DCs in peripheral blood mononuclear cell (PBMC) was extremely low but higher in the liver. BDCA3(+) DCs recovered from PBMC or the liver released large amounts of IFN-λs, when stimulated with HCVcc or HCV-transfected Huh7.5.1. BDCA3(+) DCs were able to induce ISGs in the coexisting JFH-1-positive Huh7.5.1 cells. The treatments of BDCA3(+) DCs with anti-CD81 antibody, cloroquine, or bafilomycin A1 reduced HCVcc-induced <em>IL</em>-28B release, whereas BDCA3(+) DCs comparably produced <em>IL</em>-28B upon replication-defective HCVcc. The TRIF-specific inhibitor reduced <em>IL</em>-28B release from HCVcc-stimulated BDCA3(+) DCs. In response to HCVcc or JFH-1-Huh7.5.1, BDCA3(+) DCs in healthy subjects with <em>IL</em>-28B major (rs8099917, TT) released more <em>IL</em>-28B than those with <em>IL</em>-28B minor genotype (TG).

CONCLUSIONS

Human BDCA3(+) DCs, having a tendency to accumulate in the liver, recognize HCV in a CD81-, endosome-, and TRIF-dependent manner and produce substantial amounts of IL-28B/IFN-λ3, the ability of which is superior in subjects with IL-28B major genotype.

A series of experiments was performed to determine the role of interleukin (<em>IL</em>)-1 in the induction of tolerance to global ischemia in Mongolian gerbils. In Group I, a 2-min "preconditioning" ischemia protected CA1 hippocampal neurons in gerbils subjected to 3.5 min ischemia 3 days later. CA1 neuronal density was: sham, 171 +/- 3/mm; 3.5 min ischemia, 30 +/- 30/mm; 2 and 3.5 min ischemia 162 +/- 6/mm. Experiments in Group II addressed the role of <em>IL</em>-1 in the induction of tolerance by sublethal ischemia. Arterial <em>IL</em>-1 alpha and <em>IL</em>-1 beta became elevated between 1 and 3 days after a 2-min ischemic exposure. <em>IL</em>-1 alpha was: sham, 6.4 +/- 0.6 ng/ml; and 2-day, 10.2 +/- 1.2 ng/ml. <em>IL</em>-1 beta was: sham, 6.4 +/- 0.5 ng/ml; and 2-day, 17.3 +/- 2 ng/ml. Recombinant human <em>IL</em>-1 receptor antagonist (<em>IL</em>-1ra) i.p. blocked ischemic tolerance induction by 2-min preconditioning ischemia: 2-min ischemia + vehicle, 162 +/- 6/mm; and 2-min ischemia + <em>IL</em>-1ra, 67 +/- 17/mm. Experiments in Group III assessed the capacity of <em>IL</em>-1 to induce tolerance to brain ischemia. <em>IL</em>-1 alpha i.p. (0, 10, <em>20</em> micrograms/kg) for 3 days prior to 3.5-min forebrain ischemia provided significant CA1 neuroprotection in a dose-dependent manner: 2 +/- 2, 68 +/- 83, and 129 +/- 42/mm, respectively. <em>IL</em>-1 beta (15 micrograms/kg) in combination with either <em>IL</em>-1ra (100 mg/kg) or <em>IL</em>-1ra vehicle i.p. on the same schedule demonstrated a significant CA1 neuroprotection that could be nullified by <em>IL</em>-1ra: <em>IL</em>-1 beta + <em>IL</em>-1ra vehicle, 153 +/- 16/mm; and <em>IL</em>-1 beta + <em>IL</em>-1ra, 67 +/- 36/mm. Recognition that tolerance arises from stimulation of a known receptor (<em>IL</em>-1RI) permits molecular analysis of the intracellular signaling that is critical for production of that state.

The CD59 Ag is a <em>20</em>-kDa protein that is widely expressed on most leukocytes and RBC, is coupled to the membrane by a phosphatidylinositol-glycan anchoring structure, plays a role in cell interaction between monocytes and T cells, and also functions as an inhibitor of cytolysis by the terminal C components C5b-9. Because this molecule is structurally related to the murine Ly-6 family of Ag, we have investigated whether anti-CD59 mAb might be capable of activating human T lymphocytes in a manner similar to that described for antibodies to the murine Ly-6 Ag. In the presence of the appropriate co-stimulators, mAb to one of the two epitopes on CD59 were capable of inducing both a rise in intracytoplasmic free Ca2+, inositol phosphate production, <em>IL</em>-2 production, and T cell proliferation. Anti-CD59-induced inositol phosphate turnover and <em>IL</em>-2 production were dependent on co-expression of the CD3/TCR complex. CD59-loss mutants of the Jurkat cell line were completely responsive to stimulation by anti-CD3 thereby demonstrating that CD59 does not play a role as a signal transducer downstream from the TCR. Taken together, these results demonstrate that the CD59 Ag can play multiple distinct roles in the regulation of the immune response.

Experimental allergic encephalomyelitis (EAE) is a Th1 cell-mediated autoimmune disease model of multiple sclerosis (MS). Vitamin D deficiency is commonly observed in MS patients and vitamin D supplements reduce the clinical symptoms of EAE and MS. Earlier studies have shown that in vivo treatment with vitamin D analogs ameliorates EAE in association with the inhibition of <em>IL</em>-12 production and Th1 differentiation. The mechanisms in the regulation of Th1 response by vitamin D in EAE/MS are, however, not known. We show that in vivo treatment of C57BL/6 and SJL/J mice (i.p.) with 100 ng of 1,25 dihydroxyvitamin D3, on every other day from Day 0-30, ameliorates EAE in association with the inhibition of <em>IL</em>-12 production and neural antigen-specific Th1 response. In vitro treatment with 1,25(OH)2D3 inhibited IFNgamma-induced tyrosine phosphorylation of STAT1, without affecting JAK2, in EOC-<em>20</em> microglial cells. Treatment of activated T cells with 1,25(OH)2D3 also inhibited the <em>IL</em>-12-induced tyrosine phosphorylation of JAK2, TYK2, STAT3, and STAT4 in association with a decrease in T cell proliferation in vitro. These findings highlight the fact that vitamin D modulates JAK-STAT signaling pathway in <em>IL</em>-12/IFNgamma axis leading to Th1 differentiation and further suggest its use in the treatment of MS and other Th1 cell-mediated autoimmune diseases.

Chemical modification of proteins with polyethylene glycol (PEGylation) can increase plasma half-lives, stability, and therapeutic potency. To make a PEGylated recombinant immunotoxin with improved therapeutic properties, we prepared a mutant of anti-Tac(Fv)-PE38 (LMB-2), a recombinant immunotoxin composed of a single-chain Fv fragment of the anti-human Tac monoclonal antibody to the <em>IL</em>-2 receptor alpha subunit fused to a 38-kDa fragment of Pseudomonas exotoxin. For site-specific PEGylation of LMB-2, one cysteine residue was introduced into the peptide connector (ASGCGPE) between the Fv and the toxin. This mutant LMB-2 (cys1-LMB-2), which retained full cytotoxic activity, was then site-specifically conjugated with 5 or <em>20</em> kDa of polyethylene glycol-maleimide. When compared with unmodified LMB-2, both PEGylated immunotoxins showed similar cytotoxic activities in vitro but superior stability at 37 degrees C in mouse serum, a 5- to 8-fold increase in plasma half-lives in mice, and a 3- to 4-fold increase in antitumor activity. This was accompanied by a substantial decrease in animal toxicity and immunogenicity. Site-specific PEGylation of recombinant immunotoxins may increase their therapeutic potency in humans.

Cigarette smoke is a powerful inducer of inflammatory responses resulting in disruption of major cellular pathways with transcriptional and genomic alterations driving the cells towards carcinogenesis. Cell culture and animal model studies indicate that (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea, possesses potent anti-inflammatory and antiproliferative activity capable of selectively inhibiting cell growth and inducing apoptosis in cancer cells without adversely affecting normal cells. Here, we demonstrate that EGCG pretreatment (<em>20</em>-80 microM) of normal human bronchial epithelial cells (NHBE) resulted in significant inhibition of cigarette smoke condensate (CSC)-induced cell proliferation. Nuclear factor-kappaB (NF-kappaB) controls the transcription of genes involved in immune and inflammatory responses. In most cells, NF-kappaB prevents apoptosis by mediating cell survival signals. Pretreatment of NHBE cells with EGCG suppressed CSC-induced phosphorylation of IkappaBalpha, and activation and nuclear translocation of NF-kappaB/p65. NHBE cells transfected with a luciferase reporter plasmid containing an NF-kappaB-inducible promoter sequence showed an increased reporter activity after CSC exposure that was specifically inhibited by EGCG pretreatment. Immunoblot analysis showed that pretreatment of NHBE cells with EGCG resulted in a significant downregulation of NF-kappaB-regulated proteins cyclin D1, MMP-9, <em>IL</em>-8 and iNOS. EGCG pretreatment further inhibited CSC-induced phosphorylation of ERK1/2, JNK and p38 MAPKs and resulted in a decreased expression of PI3K, AKT and mTOR signaling molecules. Taken together, our data indicate that EGCG can suppress NF-kappaB activation as well as other pro-survival pathways such as PI3K/AKT/mTOR and MAPKs in NHBE cells, which may contribute to its ability to suppress inflammation, proliferation and angiogenesis induced by cigarette smoke.

To study the effects of the cytokines <em>IL</em>-1 and TNF-alpha on the transendothelial migration of neutrophils, human umbilical vein endothelial cells (HUVEC) were grown to confluence on connective tissue prepared from human amniotic membrane. Pretreatment of HUVEC-amnion cultures with r<em>IL</em>-1 beta (7.5 ng/ml) or rTNF-alpha (5 ng/ml) for 4 h resulted in rapid migration of from <em>20</em> to 50% of subsequently added neutrophils across the endothelial monolayer. In contrast, only 3 +/- 3% of added neutrophils penetrated the HUVEC monolayer in the absence of any stimulus. The number of neutrophils that migrated across cytokine-treated HUVEC was similar to the number that traversed untreated monolayers in response to gradients of FMLP; in addition, it was only 35% less than the number of neutrophils that migrated in response to leukotriene B4. No consistent additive effect was seen when migration was induced by both cytokine pretreatment of the HUVEC and a chemotactic gradient. The number of neutrophils that migrated across <em>IL</em>-1-treated cultures was proportional to the number added over the range of 2.5 x 10(5) to 4 x 10(6) neutrophils. When used at optimal concentrations, <em>IL</em>-1 and TNF-alpha were equally effective in stimulating neutrophil migration; no additive effect was seen when HUVEC were pretreated with optimal doses of both cytokines together. Direct addition of <em>IL</em>-1 or TNF-alpha to a 1-h migration assay had no effect on neutrophil adhesion to or migration across HUVEC, either in the presence or absence of a chemotactic gradient. Stimulation of neutrophil transendothelial migration in this system did not appear to be caused by adsorption of cytokine by the amniotic tissue, nor was it due to contamination of the cytokine preparations by LPS. These results suggest that <em>IL</em>-1 and TNF-alpha, generated at sites of inflammation, may act upon the endothelium to promote emigration of neutrophils from the vasculature.

Bronchopulmonary dysplasia (BPD) is a common lung disease of premature infants, with devastating short- and long-term consequences. The pathogenesis of BPD is multifactorial, but all triggers cause pulmonary inflammation. No therapy exists; therefore, we investigated whether the anti-inflammatory interleukin-1 receptor antagonist (<em>IL</em>-1Ra) prevents murine BPD. We precipitated BPD by perinatal inflammation (lipopolysaccharide injection to pregnant dams) and rearing pups in hyperoxia (65% or 85% O2). Pups were treated daily with <em>IL</em>-1Ra or vehicle for up to 28 d. Vehicle-injected animals in both levels of hyperoxia developed a severe BPD-like lung disease (alveolar number and gas exchange area decreased by up to 60%, alveolar size increased up to fourfold). <em>IL</em>-1Ra prevented this structural disintegration at 65%, but not 85% O2. Hyperoxia depleted pulmonary immune cells by 67%; however, extant macrophages and dendritic cells were hyperactivated, with CD11b and GR1 (Ly6G/C) highly expressed. <em>IL</em>-1Ra partially rescued the immune cell population in hyperoxia (doubling the viable cells), reduced the percentage that were activated by up to 63%, and abolished the unexpected persistence of <em>IL</em>-1α and <em>IL</em>-1β on day 28 in hyperoxia/vehicle-treated lungs. On day 3, perinatal inflammation and hyperoxia each triggered a distinct pulmonary immune response, with some proinflammatory mediators increasing up to <em>20</em>-fold and some amenable to partial or complete reversal with <em>IL</em>-1Ra. In summary, our analysis reveals a pivotal role for <em>IL</em>-1α/β in murine BPD and an involvement for MIP (macrophage inflammatory protein)-1α and TREM (triggering receptor expressed on myeloid cells)-1. Because it effectively shields newborn mice from BPD, <em>IL</em>-1Ra emerges as a promising treatment for a currently irremediable disease that may potentially brighten the prognosis of the tiny preterm patients.

There was a significant correlation between microvessel counts and interleukin (<em>IL</em>)-8 levels and between infiltrated macrophage counts and <em>IL</em>-8 levels in uterine cervical cancers. Immunohistochemical staining revealed that the localization of <em>IL</em>-8 was similar to that of CD68 for macrophages. The prognosis of the <em>20</em> patients with high <em>IL</em>-8 (>1000 pg/mg protein) in uterine cervical cancers was extremely poor, whereas the 24-month survival rate of the other 60 patients with low <em>IL</em>-8 (<1000 pg/mg protein) was 67%. Therefore, this indicates that <em>IL</em>-8 might be a prognostic indicator as an angiogenic factor supplied from macrophages within and around the tumor.

Estrogen's action on bone may be mediated by cytokines produced by monocytes. We have reported a decreased ratio of interleukin-1beta (<em>IL</em>-1beta) to interleukin-1 receptor antagonist (<em>IL</em>-1ra) produced by whole blood cultures in vivo in women taking hormone replacement therapy (HRT). Also, one study has shown an effect of estradiol on tumor necrosis factor-alpha (TNF-alpha) secretion by separated monocytes in vitro. The aim of this study was to evaluate the effect of estrogen in vitro on the secretion of cytokines using whole blood cultures. Subjects consisted of 12 healthy postmenopausal women, ages 57-69 years, 4-<em>20</em> years since menopause. Cytokines <em>IL</em>-1beta, interleukin-1alpha (<em>IL</em>-1alpha), <em>IL</em>-1ra, interleukin-6 (<em>IL</em>-6), TNF-alpha, and granulocyte macrophage-colony stimulating factor (GM-CSF) were measured in unstimulated and in stimulated (500 ng/mL lipopolysaccharide [LPS]) whole blood cultures treated with 17beta-estradiol (E(2)) at concentrations of 10(-12)--10(-6) mol/L. We found significant decreases in the spontaneous secretion of <em>IL</em>-6, TNF-alpha, <em>IL</em>-1ra, <em>IL</em>-1beta, and ratio of <em>IL</em>-1beta/<em>IL</em>-1ra compared with control, at physiological concentrations of E(2). The action of E(2) was blocked by the use of the antiestrogen ICI 182780 in coculture. A decrease in cytokine secretion was not observed when the inactive form of estrogen, 17alpha-estradiol, was used in place of 17beta-estradiol. GM-CSF and <em>IL</em>-1alpha were not detectable in unstimulated cultures. Cytokine levels measured in stimulated cultures were not attenuated by treatment with E(2). We conclude that E(2) inhibits the spontaneous secretion of cytokines measured in whole blood cultures at physiological concentrations, and that the powerful stimulatory effect of LPS prevents any significant inhibition by E(2) in stimulated cultures.

In the standard model of cytokine-induced signal transducer and activator of transcription (STAT) protein family signaling to the cell nucleus, it is assumed that STAT3 is recruited to the cytoplasmic side of the cell surface receptor complex from within a cytosolic monomer pool. By using Superose-6 gel-filtration chromatography, we have discovered that there is little monomeric STAT3 (91 kDa) in the cytosol of liver cells (human hepatoma Hep3B cell line and rat liver). The bulk of STAT3 (and STAT1, STAT5a, and -b) was present in the cytosol as high molecular mass complexes in two broad distributions in the size range <em>20</em>0-400 kDa ("statosome I") and 1-2 MDa ("statosome II"). Upon treatment of Hep3B cells with interleukin-6 (<em>IL</em>-6) for 30 min (i) cytosolic tyrosine-phosphorylated STAT3 was found to be in complexes of size ranging from <em>20</em>0-400 kDa to 1-2 MDa; (ii) a small pool of monomeric STAT3 and tyrosine-phosphorylated STAT3 eluting at 80-100 kDa was observed, and (iii) most of the cytoplasmic DNA-binding competent STAT3 (the so-called SIF-A "homodimer") co-eluted with catalase at 230 kDa. In order to identify the protein components of the <em>20</em>0-400-kDa statosome I cytosolic complexes, we used the novel technique of antibody-subtracted differential protein display using anti-STAT3 antibody. Eight polypeptides in the size range from <em>20</em> to 114 kDa co-shifted with STAT3; three of these (p60, p<em>20</em>a, and p<em>20</em>b) were co-shifted in an <em>IL</em>-6-dependent manner. In-gel tryptic fragmentation and mass spectroscopy identified the major <em>IL</em>-6-dependent STAT3-co-shifted p60 protein as the chaperone GRP58/ER-60/ERp57. Taken together, these data (i) emphasize the absence of a detectable STAT3 monomer pool in the cytosol of cytokine-free liver cells as posited by the standard model, and (ii) suggest an alternative model for STAT signaling in which STAT3 proteins function in the cytoplasm as heteromeric complexes with accessory scaffolding proteins, including the chaperone GRP58.

Participation of the actin cytoskeleton in the transduction of proliferative signals has been established through the use of compounds that disrupt the cytoskeleton. To address the possibility that actin also participates in the transduction of an apoptotic signal, we have studied the response of the murine interleukin 2 (<em>IL</em>-2)-dependent T cell line CTLL-<em>20</em> to treatment with the actin-binding compound jasplakinolide upon <em>IL</em>-2 deprivation. Like phalloidin, jasplakinolide stabilizes F-actin and promotes actin polymerization. Treatment of CTLL-<em>20</em> cells with jasplakinolide, in the presence or absence of recombinant human <em>IL</em>-2, altered actin morphology as assessed by confocal fluorescence microscopy. Jasplakinolide was not toxic to CTLL-<em>20</em> cells, nor was apoptosis induced in the presence of exogenous recombinant human <em>IL</em>-2. However, actin stabilization at the time of <em>IL</em>-2 deprivation enhanced apoptosis by changing the time at which CTLL-<em>20</em> cells committed to the apoptotic pathway. This effect of jasplakinolide correlated with its ability to stabilize polymerized actin, as treatment with a synthetic analog of jasplakinolide with a greatly reduced ability to bind actin, jasplakinolide B, did not enhance apoptosis. The enhancement occurred upstream of the induction of caspase-3-like activity and could be inhibited by the overexpression of the anti-apoptotic protein Bcl-xL. These data suggest that the actin cytoskeleton plays an active role in modulating lymphocyte apoptosis induced by cytokine deprivation.

The receptor for <em>IL</em>-26 (AK155), a cytokine of the <em>IL</em>-10 family, has not previously been defined. We demonstrate that the active receptor complex for <em>IL</em>-26 is a heterodimer composed of two receptor proteins: <em>IL</em>-<em>20</em>R1 and <em>IL</em>-10R2. Signaling through the <em>IL</em>-26R results in activation of STAT1 and STAT3 which can be blocked by neutralizing Abs against <em>IL</em>-<em>20</em>R1 or <em>IL</em>-10R2. <em>IL</em>-10R2 is broadly expressed on a wide variety of tissues, whereas only a limited number of tissues express <em>IL</em>-<em>20</em>R1. Therefore, the ability to respond to <em>IL</em>-26 is restricted by the expression of <em>IL</em>-<em>20</em>R1. <em>IL</em>-10, <em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22, and <em>IL</em>-24 fail to signal through the combination of <em>IL</em>-10R2 and <em>IL</em>-<em>20</em>R1 proteins, demonstrating that this receptor combination is unique and specific for <em>IL</em>-26.

OBJECTIVE

The present study aimed at investigating the effects of Lactobacillus casei 01 supplementation on symptoms and inflammatory biomarkers of rheumatoid arthritis (RA) in women.

METHODS

In this randomized double-blind clinical trial, female patients with established RA for more than 1 year, <em>20</em>-80 years of age and body mass index (BMI) lower than 40, who followed stable medication for 3 months prior to the supplementation, were randomly allocated to receive either one capsule containing 10(8) colony forming units (CFU) of L. casei 01, or a placebo for 8 weeks; allocation was stratified by BMI and menopausal status. Disease activity score-28 (DAS28) was calculated, European League Against Rheumatism (EULAR) response was evaluated and the cytokines, interleukin (<em>IL</em>)-1β, <em>IL</em>-6, <em>IL</em>-10, <em>IL</em>-12 and tumor necrosis factor (TNF)-α were measured.

RESULTS

Thirty patients were recruited in each group; 22 and 24 patients were analyzed in the probiotic and placebo groups, respectively. L. casei 01 supplementation decreased serum high-sensitivity C-reactive protein (hs-CRP) levels, tender and swollen joint counts, global health (GH) score and DAS28 (P < 0.05). More patients in the L. casei 01 group had moderate response to the treatment, based on the EULAR criteria, at the end of the study (P < 0.01). At the end of the study, a significant difference was observed between the two groups for IL-10, IL-12 and TNF-α changes through the study course (P < 0.05), in favor of the probiotic group. No adverse effects were reported for the intervention.

CONCLUSIONS

Probiotic supplementation may be an appropriate adjunct therapy for RA patients and help alleviate symptoms and improve inflammatory cytokines.

Psoriatic arthritis (PsA) is an inflammatory joint disease, characterized by extensive bone resorption, whose mechanisms have not been fully elucidated. Thus, in the present study we investigated the involvement of RANKL, TNFalpha, and <em>IL</em>-7 in the osteoclastogenesis of PsA patients. In vitro osteoclastogenesis models, consisting of unfractionated and T-cell-depleted mononuclear cells from peripheral blood (PBMCs) and synovial fluid (SFMCs) of <em>20</em> PsA patients as well as from healthy donors were studied. Freshly isolated T and B cells from PBMCs and T cells and fibroblasts from SFMCs of PsA patients were subjected to RT-PCR to detect the levels of RANKL, TNFalpha, and <em>IL</em>-7. Osteoclastogenesis was studied in the presence of RANK-Fc, anti-TNFalpha, and anti <em>IL</em>-7 functional antibodies. We demonstrate that lymphocytes and fibroblasts support osteoclast (OC) formation in PsA patients through the production of osteoclastogenic cytokines. In particular, OC formation was completely abolished in unstimulated T cell-depleted PBMC cultures, and reduced by approximately 70% in unstimulated T cell-depleted SFMC cultures. Freshly isolated T cells from PBMCs and SFMCs of PsA patients overexpressed RANKL and TNFalpha, while fibroblasts from synovial fluid produced only RANKL. We show that the presence of RANK-Fc and/or anti-TNFalpha functional antibodies reduced OC formation. Moreover, T and B cells from PBMCs as well as T cells and fibroblasts from SFMCs expressed <em>IL</em>-7 mRNA. Finally, the anti-<em>IL</em>-7 functional antibody significantly reduced osteoclastogenesis. Our results suggest that fibroblasts, B and T lymphocytes support OC formation by producing RANKL, TNFalpha, and <em>IL</em>-7, contributing to the aggressive bone resorption in PsA patients.

BACKGROUND

Neonatal onset multisystem inflammatory disease (NOMID), an autoinflammatory disease, is characterized by fever, chronic urticarial rash, CNS manifestations, and arthropathy. Approximately 50% of patients with NOMID have de novo missense mutations in CIAS1, which is associated with modulation of the IL-1b and apoptotic pathways. Approximately 60% of NOMID patients have prominent arthropathy, most commonly involving the knees, the cause of which remains poorly understood.

OBJECTIVE

To more fully describe the findings of NOMID arthropathy on MRI and radiography and to provide a better understanding of the origin of the bony lesions.

METHODS

We imaged 20 patients with NOMID to further investigate NOMID-associated bony lesions.

RESULTS

Bony abnormalities were seen in the knees of 11/20 patients. The knee findings included enlarged, deformed femora and patellae in all and tibiae in the majority, without evidence of synovitis. Some patients had other joint involvement. Most had short stature and valgus or varus knee deformities. No association was noted between bony abnormalities and CIAS1 mutations. The abnormalities appeared to be the result of a mass-producing process. The resulting heterogeneously calcified masses appeared to originate in the physis and deformed the adjacent metaphysis and epiphysis.

CONCLUSIONS

These findings suggest that the arthropathy of NOMID is the result of abnormal endochondral bone growth. Further investigation is needed to determine whether this deformity is triggered by inflammation early in development or by CIAS1 mutations causing abnormal chondrocyte apoptosis.

The majority of human immunodeficiency virus (HIV)-seropositive patients develop bone marrow abnormalities associated with hematopoietic malfunction during the progression of disease. One important manifestation of HIV-associated hematopoietic dysfunction is that after myelosuppression, bone marrow recovery, a process known to be mediated in part by the production of stromal cell-derived hematopoietic growth factors, is impaired. We sought to test the hypothesis that bone marrow stromal cells are infected by HIV-1 in vivo and that production of certain stromal cell-derived hematopoietic growth factors is deficient as a consequence. In this report, we demonstrate that bone marrow microvascular endothelial cells (MVEC), a key element of the stroma, are the predominant cells infected by HIV (5% to <em>20</em>%) in bone marrow stromal cultures obtained from 11 consecutive HIV-seropositive patients. Although HIV-infected stromal cultures enriched for MVEC constitutively express normal levels of interleukin (<em>IL</em>)-4, <em>IL</em>-6, granulocyte (G)-colony-stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and Steel factor, <em>IL</em>-1 alpha-induced release of <em>IL</em>-6 and G-CSF is significantly reduced in these cultures. These observations suggest that HIV infection of bone marrow MVEC reduces the capacity of hematopoietic stroma to respond to regulatory signals that normally augment blood cell production during periods of increased demand.

Daidzein and genistein glucuronides (DG and GG), major isoflavone metabolites, may be partly responsible for biological effects of isoflavones, such as estrogen receptor binding and natural killer cell (NK) activation or inhibition. DG and GG were synthesized using 3-methylcholanthrene-induced rat liver microsomes. The Km and Vmax for daidzein and genistein were 9.0 and 7.7 micromol/L, and 0.7 and 1.6 micromol/(mg protein. min), respectively. The absence of ultraviolet absorbance maxima shifts in the presence of sodium acetate confirmed that the synthesized products were 7-O-glucuronides. DG and GG were further purified by a Sephadex LH-<em>20</em> column. DG and GG competed with the binding of 17beta-(3H) estradiol to estrogen receptors of B6D2F1 mouse uterine cytosol. The concentrations required for 50% displacement of 17beta-(3H) estradiol (CB50) were: 17beta-estradiol, 1.34 nmol/L; diethylstilbestrol, 1.46 nmol/L; daidzein, 1.6 micromol/L; DG, 14.7 micromol/L; genistein, 0.154 micromol/L; GG, 7.27 micromol/L. In human peripheral blood NK cells, genistein at <0.5 micromol/L and DG and GG at 0.1-10 micromol/L enhanced NK cell-mediated K562 cancer cell killing significantly (P < 0.05). At>> 0.5 micromol/L, genistein inhibited NK cytotoxicity significantly (P < 0.05). The glucuronides only inhibited NK cytotoxicity at 50 micromol/L. Isoflavones, and especially the isoflavone glucuronides, enhanced activation of NK cells by interleukin-2 (<em>IL</em>-2), additively. At physiological concentrations, DG and GG were weakly estrogenic, and they activated human NK cells in nutritionally relevant concentrations in vitro, probably at a site different from <em>IL</em>-2 action.

Background: Tocilizumab, a humanized monoclonal antibody, targets IL-6 receptors blocking downstream pro-inflammatory effects of IL-6. In preliminary reports it was suggested to be beneficial in patients with severe COVID-19.

Methods: In this open-label prospective study we describe clinical characteristics and outcome of 51 patients hospitalized with confirmed and severe COVID-19 pneumonia treated with tocilizumab intravenously. All patients had elevated IL-6 plasma level (>40 pg/mL) and oxygen saturation <93% in ambient air. Clinical outcomes, oxygen support, laboratory data and adverse events were collected over a follow-up of 30 days.

Results: Forty-five patients (88%) were on high-flow oxygen supplementation, six of whom with invasive ventilation. From baseline to day 7 after tocilizumab we observed a dramatic drop of body temperature and CRP value with a significant increase in lymphocyte count (p<0.001). Over a median follow-up time of 34 days from tocilizumab, 34 patients (67%) showed an improvement in their clinical severity class; 31 were discharged; 17 (33%) showed a worsening of their clinical status, of these 14 died (27%). The mortality rate was significantly associated with mechanical ventilation at baseline (83.3% vs 20% of patients on non-invasive oxygen support; p=0.0001). The most frequent side effects were an increase of hepatic enzymes (29%), thrombocytopenia (14%), and serious bacterial and fungal infections (27%).

Conclusion: Tocilizumab exerts a rapidly beneficial effect on fever and inflammatory markers, although no significant impact on the clinical outcome can be inferred by our results. Critically ill patients seem to have a high risk of serious infections with this drug.

Keywords: COVID-19; IL-6 inhibitors; tocilizumab.

BACKGROUND

Little is known about the virological and inflammatory responses of severe pandemic <em>20</em>09 influenza A(H1N1) virus pneumonia during antiviral treatment.

METHODS

In a prospective observational study, we recruited consecutive adults hospitalized with confirmed pandemic <em>20</em>09 H1N1 infection during a 16-week period. Nasopharyngeal aspirate and non-respiratory samples (blood, stool and urine) were collected at presentation, and serial nasopharyngeal flocked swabs (NPFS) and tracheal aspirates (TA) were collected after initiating oseltamivir treatment for quantitative viral RNA assay, using real-time reverse transcriptase-PCR. Serial plasma samples were collected for cytokine/chemokine assay using cytometric bead array. Patients with severe pneumonia (lung infiltrates and hypoxaemia) were compared to those with milder illnesses.

RESULTS

A total of 66 patients were studied (mean age 43 ±<em>20</em> years); 28 (42%) developed severe pneumonia, of whom 10 (15%) required intubation. Severe pneumonia was associated with older age, dyspnoea, delayed presentation >2 days from onset, extrapulmonary virus detection (13-28%) and higher viral concentration despite late-presentation (multiple linear regression, β=0.94, 95% confidence interval 0.15-1.74; P=0.02). Patients with severe pneumonia exhibited slow viral clearance with oseltamivir treatment, particularly in the lower respiratory tract (median [interquartile range] durations of RNA positivity after antiviral initiation were NPFS 6.0 days [3.0-8.0], TA 11.0 days [7.8-14.3] versus milder illness group NPFS of 2.0 days [1.0-3.0] days; P<0.01). High viral load in lower respiratory tract despite upper-tract RNA negativity and viral rebound after stopping treatment were noted in some patients. H275Y mutation was absent. High plasma levels of interleukin (<em>IL</em>)-6, CXCL-8 (<em>IL</em>-8), CCL2 (monocyte chemoattractant protein-1) and soluble tumour necrosis factor receptor-1 were observed, which correlated with the extent and progression of pneumonia in hospital.

CONCLUSIONS

In severe <em>20</em>09 H1N1 pneumonia, viral clearance is slow with treatment, particularly in the lower respiratory tract. A more sustained antiviral regime appears warranted.

BACKGROUND

Hepatic ischemia-reperfusion (I/R) injury is a pivotal clinical problem occurring in many clinical conditions such as transplantation, trauma, and hepatic failure after hemorrhagic shock. Apoptosis and autophagy have been shown to contribute to cell death in hepatic I/R injury. Ethyl pyruvate, a stable and simple lipophilic ester, has been shown to have anti-inflammatory properties. In this study, the purpose is to explore both the effect of ethyl pyruvate on hepatic I/R injury and regulation of intrinsic pathway of apoptosis and autophagy.

METHODS

Three doses of ethyl pyruvate (<em>20</em> mg/kg, 40 mg/kg, and 80 mg/kg) were administered 1 h before a model of segmental (70%) hepatic warm ischemia was established in Balb/c mice. All serum and liver tissues were obtained at three different time points (4 h, 8 h, and 16 h).

RESULTS

Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and pathological features were significantly ameliorated by ethyl pyruvate (80 mg/kg). The expression of Bcl-2, Bax, Beclin-1, and LC3, which play an important role in the regulation of intrinsic pathway of apoptosis and autophagy, was also obviously decreased by ethyl pyruvate (80 mg/kg). Furthermore, ethyl pyruvate inhibited the HMGB1/TLR4/ NF-κb axis and the release of cytokines (TNF-α and IL-6).

CONCLUSIONS

Our results showed that ethyl pyruvate might attenuate to hepatic I/R injury by inhibiting intrinsic pathway of apoptosis and autophagy, mediated partly through downregulation of HMGB1/TLR4/ NF-κb axis and the competitive interaction with Beclin-1 of HMGB1.