During infection, vertebrates develop "sickness syndrome," characterized by fever, anorexia, behavioral withdrawal, acute-phase protein responses, and inflammation. These pathophysiological responses are mediated by cytokines, including TNF and <em>IL</em>-1, released during the innate immune response to invasion. Even in the absence of infection, qualitatively similar physiological syndromes occur following sterile injury, ischemia reperfusion, crush injury, and autoimmune-mediated tissue damage. Recent advances implicate high-mobility group box 1 (HMGB1), a nuclear protein with inflammatory cytokine activities, in stimulating cytokine release. HMGB1 is passively released during cell injury and necrosis, or actively secreted during immune cell activation, positioning it at the intersection of sterile and infection-associated inflammation. To date, eight candidate receptors have been implicated in mediating the biological responses to HMGB1, but the mechanism of HMGB1-dependent cytokine release is unknown. Here we show that Toll-like receptor 4 (TLR4), a pivotal receptor for activation of innate immunity and cytokine release, is required for HMGB1-dependent activation of macrophage TNF release. Surface plasmon resonance studies indicate that HMGB1 binds specifically to TLR4, and that this binding requires a cysteine in position 106. A wholly synthetic <em>20</em>-mer peptide containing cysteine 106 from within the cytokine-stimulating B box mediates TLR4-dependent activation of macrophage TNF release. Inhibition of TLR4 binding with neutralizing anti-HMGB1 mAb or by mutating cysteine 106 prevents HMGB1 activation of cytokine release. These results have implications for rationale, design, and development of experimental therapeutics for use in sterile and infectious inflammation.

Autoimmune diseases often result from an imbalance between regulatory T (Treg) cells and interleukin-17 (<em>IL</em>-17)-producing T helper (TH17) cells; the origin of the latter cells remains largely unknown. Foxp3 is indispensable for the suppressive function of Treg cells, but the stability of Foxp3 has been under debate. Here we show that TH17 cells originating from Foxp3(+) T cells have a key role in the pathogenesis of autoimmune arthritis. Under arthritic conditions, CD25(lo)Foxp3(+)CD4(+) T cells lose Foxp3 expression (herein called exFoxp3 cells) and undergo transdifferentiation into TH17 cells. Fate mapping analysis showed that <em>IL</em>-17-expressing exFoxp3 T (exFoxp3 TH17) cells accumulated in inflamed joints. The conversion of Foxp3(+)CD4(+) T cells to TH17 cells was mediated by synovial fibroblast-derived <em>IL</em>-6. These exFoxp3 TH17 cells were more potent osteoclastogenic T cells than were naive CD4(+) T cell-derived TH17 cells. Notably, exFoxp3 TH17 cells were characterized by the expression of Sox4, chemokine (C-C motif) receptor 6 (CCR6), chemokine (C-C motif) ligand <em>20</em> (CCL<em>20</em>), <em>IL</em>-23 receptor (<em>IL</em>-23R) and receptor activator of NF-κB ligand (RANKL, also called TNFSF11). Adoptive transfer of autoreactive, antigen-experienced CD25(lo)Foxp3(+)CD4(+) T cells into mice followed by secondary immunization with collagen accelerated the onset and increased the severity of arthritis and was associated with the loss of Foxp3 expression in the majority of transferred T cells. We observed <em>IL</em>-17(+)Foxp3(+) T cells in the synovium of subjects with active rheumatoid arthritis (RA), which suggests that plastic Foxp3(+) T cells contribute to the pathogenesis of RA. These findings establish the pathological importance of Foxp3 instability in the generation of pathogenic TH17 cells in autoimmunity.

Bacterial infection stimulates the host to mount a rapid inflammatory response. A 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines was shown to contribute to this response by inducing polygonal B-cell activation. This stimulatory motif is <em>20</em> times more common in the DNA of bacteria than higher vertebrates. The current work shows that the same motif induces the rapid and coordinated secretion of interleukin (<em>IL</em>) 6, <em>IL</em>-12, and interferon gamma (but not <em>IL</em>-2, <em>IL</em>-3, <em>IL</em>-4, <em>IL</em>-5, or <em>IL</em>-10) in vivo and in vitro. Stimulatory CpG DNA motifs induced B, T, and natural killer cells to secrete cytokine more effectively than did lipopolysaccharide. Thus, immune recognition of bacterial DNA may contribute to the cytokine, as well as the antibody production characteristic of an innate inflammatory response.

Understanding the factors that influence T helper 1 (T(H)1)- and T(H)2-cell responses has been one of the main focuses of immunology for almost <em>20</em> years. Whereas the central role of interleukin-12 (<em>IL</em>-12) in the generation of T(H)1 cells has long been appreciated, subsequent studies indicated that <em>IL</em>-23 and <em>IL</em>-27, two cytokines that are closely related to <em>IL</em>-12, also regulate T(H)1-cell responses. However, as discussed in this article, it is now recognized that the ability of <em>IL</em>-23 to stimulate a unique T-cell subset to produce <em>IL</em>-17 has a dominant role in autoimmune inflammation. By contrast, <em>IL</em>-27 has a role in limiting the intensity and duration of adaptive immune responses.

Mature circulating polymorphonuclear cells (PMN) have the shortest half-life among leukocytes and undergo rapid programmed cell death in vitro. In this study, we have examined the possibility that inflammatory signals (cytokines and bacterial products) can regulate PMN survival. PMN in culture were found to rapidly die, with percentages of survival at 24, 48, 72, and 96 hours of 97.3% +/- 1.9%, 36.8% +/- 5.3%, 14.5% +/- 3.1%, and 4.2% +/- 2.9%, respectively (mean +/- SE of <em>20</em> different donors). PMN incubated with interleukin-1 beta (<em>IL</em>-1 beta), tumor necrosis factor, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, and interferon-gamma (IFN-gamma), but not with prototypic chemoattractants (fMLP, recombinant C5a, and <em>IL</em>-8), showed a marked increase in survival, with values ranging at 72 hours of incubation from 89.5% +/- 5.8% for <em>IL</em>-1 beta to 47.6% +/- 6.4% for IFN-gamma. The calculated half-life was 35 hours for untreated and 115 hours for <em>IL</em>-1-treated PMN. PMN activated with lipopolysaccharide (LPS) or inactivated streptococci also showed a longer survival compared with untreated cells (94.4% +/- 3.2% and 95.5% +/- 2.4%, respectively, at 72 hours). PMN surviving in response to LPS or <em>IL</em>-1 beta retained the capacity to produce superoxide anion when treated with phorbol esters or fMLP. All inducers of PMN survival protect these cells from programmed cell death because they reduced cells with morphologic features of apoptosis and the fragmentation of DNA in multiples of 180 bp. Thus, certain cytokines and bacterial products can prolong PMN survival by interfering with the physiologic process of apoptosis. Prolongation of survival may be important for the regulation of host resistance and inflammation, and may represent a crucial permissive step for certain cytokines and microbial products that activate gene expression and function in PMN.

We have shown that thalidomide (Thal) and its immunomodulatory derivatives (IMiDs), proteasome inhibitor PS-341, and As(2)O(3) act directly on multiple myeloma (MM) cells and in the bone marrow (BM) milieu to overcome drug resistance. Although Thal/IMiDs, PS-341, and As(2)O(3) inhibit nuclear factor (NF)-kappaB activation, they also have multiple and varied other actions. In this study, we therefore specifically address the role of NF-kappaB blockade in mediating anti-MM activity. To characterize the effect of specific NF-kappaB blockade on MM cell growth and survival in vitro, we used an IkappaB kinase (IKK) inhibitor (PS-1145). Our studies demonstrate that PS-1145 and PS-341 block TNFalpha-induced NF-kappaB activation in a dose- and time-dependent fashion in MM cells through inhibition of IkappaBalpha phosphorylation and degradation of IkappaBalpha, respectively. Dexamethasone (Dex), which up-regulates IkappaBalpha protein, enhances blockade of NF-kappaB activation by PS-1145. Moreover, PS-1145 blocks the protective effect of <em>IL</em>-6 against Dex-induced apotosis. TNFalpha-induced intracellular adhesion molecule (ICAM)-1 expression on both RPMI8226 and MM.1S cells is also inhibited by PS-1145. Moreover, PS-1145 inhibits both <em>IL</em>-6 secretion from BMSCs triggered by MM cell adhesion and proliferation of MM cells adherent to BMSCs. However, in contrast to PS-341, PS-1145 only partially (<em>20</em>-50%) inhibits MM cell proliferation, suggesting that NF-kappaB blockade cannot account for all of the anti-MM activity of PS-341. Importantly, however, TNFalpha induces MM cell toxicity in the presence of PS-1145. These studies demonstrate that specific targeting of NF-kappaB can overcome the growth and survival advantage conferred both by tumor cell binding to BMSCs and cytokine secretion in the BM milieu. Furthermore, they provide the framework for clinical evaluation of novel MM therapies based upon targeting NF-kappaB.

The pandemic outbreak of coronavirus disease <em>20</em>19 (COVID-19) is rapidly spreading all over the world. Reports from China showed that about <em>20</em>% of patients developed severe disease, resulting in a fatality of 4%. In the past two months, we clinical immunologists participated in multi-rounds of MDT (multidiscipline team) discussion on the anti-inflammation management of critical ill COVID-19 patients, with our colleagues dispatched from Chinese leading PUMC Hospital to Wuhan to admit and treat the most severe patients. Here, from the perspective of clinical immunologists, we will discuss the clinical and immunological characteristics of severe patients, and summarize the current evidence and share our experience in anti-inflammation treatment, including glucocorticoids, <em>IL</em>-6 antagonist, JAK inhibitors and choloroquine/hydrocholoroquine, of patients with severe COVID-19 that may have an impaired immune system.

We hypothesized that the rising levels of inflammatory markers with aging is explained by cardiovascular risk factors and morbidity becoming progressively more prevalent in older persons. Information on inflammatory markers, cardiovascular risk factors, and diseases was collected in 595 men and 748 women sampled from the general population (age, <em>20</em>-102 years). In both men and women, older age was associated with higher levels of interleukin-6 (<em>IL</em>-6), <em>IL</em>-1 receptor antagonist (<em>IL</em>-1ra), <em>IL</em>-18, C-reactive protein (CRP), and fibrinogen, while soluble <em>IL</em>-6 receptor (s<em>IL</em>-6r) increased significantly with age only in men. Adjusting for cardiovascular risk factors and morbidity, the age regression coefficients became substantially smaller in models predicting <em>IL</em>-6, <em>IL</em>-1ra, <em>IL</em>-18, and fibrinogen and larger in the model predicting s<em>IL</em>6r. Adjustment for cardiovascular morbidity substantially reduced the effect of age on CRP in men but not in women. Findings were confirmed in a subgroup of 51 men and 45 women with low risk profile and no cardiovascular morbidity. Part of the "proinflammatory state" in older persons is related to the high prevalence of cardiovascular risk factor and morbidity.

Chronic mucocutaneous candidiasis disease (CMCD) may be caused by autosomal dominant (AD) <em>IL</em>-17F deficiency or autosomal recessive (AR) <em>IL</em>-17RA deficiency. Here, using whole-exome sequencing, we identified heterozygous germline mutations in STAT1 in 47 patients from <em>20</em> kindreds with AD CMCD. Previously described heterozygous STAT1 mutant alleles are loss-of-function and cause AD predisposition to mycobacterial disease caused by impaired STAT1-dependent cellular responses to IFN-γ. Other loss-of-function STAT1 alleles cause AR predisposition to intracellular bacterial and viral diseases, caused by impaired STAT1-dependent responses to IFN-α/β, IFN-γ, IFN-λ, and <em>IL</em>-27. In contrast, the 12 AD CMCD-inducing STAT1 mutant alleles described here are gain-of-function and increase STAT1-dependent cellular responses to these cytokines, and to cytokines that predominantly activate STAT3, such as <em>IL</em>-6 and <em>IL</em>-21. All of these mutations affect the coiled-coil domain and impair the nuclear dephosphorylation of activated STAT1, accounting for their gain-of-function and dominance. Stronger cellular responses to the STAT1-dependent <em>IL</em>-17 inhibitors IFN-α/β, IFN-γ, and <em>IL</em>-27, and stronger STAT1 activation in response to the STAT3-dependent <em>IL</em>-17 inducers <em>IL</em>-6 and <em>IL</em>-21, hinder the development of T cells producing <em>IL</em>-17A, <em>IL</em>-17F, and <em>IL</em>-22. Gain-of-function STAT1 alleles therefore cause AD CMCD by impairing <em>IL</em>-17 immunity.

Adoptive immunotherapy with T cells expressing a tumor-specific chimeric T-cell receptor is a promising approach to cancer therapy that has not previously been explored for the treatment of lymphoma in human subjects. We report the results of a proof-of-concept clinical trial in which patients with relapsed or refractory indolent B-cell lymphoma or mantle cell lymphoma were treated with autologous T cells genetically modified by electroporation with a vector plasmid encoding a CD<em>20</em>-specific chimeric T-cell receptor and neomycin resistance gene. Transfected cells were immunophenotypically similar to CD8(+) effector cells and showed CD<em>20</em>-specific cytotoxicity in vitro. Seven patients received a total of <em>20</em> T-cell infusions, with minimal toxicities. Modified T cells persisted in vivo 1 to 3 weeks in the first 3 patients, who received T cells produced by limiting dilution methods, but persisted 5 to 9 weeks in the next 4 patients who received T cells produced in bulk cultures followed by 14 days of low-dose subcutaneous interleukin-2 (<em>IL</em>-2) injections. Of the 7 treated patients, 2 maintained a previous complete response, 1 achieved a partial response, and 4 had stable disease. These results show the safety, feasibility, and potential antitumor activity of adoptive T-cell therapy using this approach. This trial was registered at www.clinicaltrials.gov as #NCT00012<em>20</em>7.

<em>IL</em>-22 belongs to a family of cytokines structurally related to <em>IL</em>-10, including <em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-24, and <em>IL</em>-26. In contrast to <em>IL</em>-10, <em>IL</em>-22 has proinflammatory activities. <em>IL</em>-22 signals through a class II cytokine receptor composed of an <em>IL</em>-22-binding chain, <em>IL</em>-22RA1, and the <em>IL</em>-10RB subunit, which is shared with the <em>IL</em>-10R. In the present study, we show that short-term cultured human epidermal keratinocytes express a functional <em>IL</em>-22R but no <em>IL</em>-10R. Accordingly, <em>IL</em>-22 but not <em>IL</em>-10 induces STAT3 activation in keratinocytes. Using a cDNA array screening approach, real-time RT-PCR, and Western blot analysis, we demonstrate that <em>IL</em>-22 up-regulates, in a dose-dependent manner, the expression of S100A7, S100A8, S100A9, a group of proinflammatory molecules belonging to the S100 family of calcium-binding proteins, as well as the matrix metalloproteinase 3, the platelet-derived growth factor A, and the CXCL5 chemokine. In addition, <em>IL</em>-22 induces keratinocyte migration in an in vitro injury model and down-regulates the expression of at least seven genes associated with keratinocyte differentiation. Finally, we show that <em>IL</em>-22 strongly induces hyperplasia of reconstituted human epidermis. Taken together, these results suggest that <em>IL</em>-22 plays an important role in skin inflammatory processes and wound healing.

The purpose of these studies was to examine the role of cytokines in the pathogenesis of cisplatin nephrotoxicity. Injection of mice with cisplatin (<em>20</em> mg/kg) led to severe renal failure. The expression of cytokines, chemokines, and ICAM-1 in kidney was measured by ribonuclease protection assays and RT-PCR. We found significant upregulation of TNF-alpha, TGF-beta, RANTES, MIP-2, MCP-1, TCA3, <em>IL</em>-1beta, and ICAM-1 in kidneys from cisplatin-treated animals. In addition, serum, kidney, and urine levels of TNF-alpha measured by ELISA were increased by cisplatin. Inhibitors of TNF-alpha production (GM6001, pentoxifylline) and TNF-alpha Ab's reduced serum and kidney TNF-alpha protein levels and also blunted the cisplatin-induced increases in TNF-alpha, TGF-beta, RANTES, MIP-2, MCP-1, and <em>IL</em>-1beta, but not ICAM-1, mRNA. In addition, the TNF-alpha inhibitors also ameliorated cisplatin-induced renal dysfunction and reduced cisplatin-induced structural damage. Likewise, TNF-alpha-deficient mice were resistant to cisplatin nephrotoxicity. These results indicate cisplatin nephrotoxicity is characterized by activation of proinflammatory cytokines and chemokines. TNF-alpha appears to play a central role in the activation of this cytokine response and also in the pathogenesis of cisplatin renal injury.

OBJECTIVE

A phase I/II trial was performed to evaluate the safety and immunogenicity of a novel vaccination with α-type 1 polarized dendritic cells (αDC1) loaded with synthetic peptides for glioma-associated antigen (GAA) epitopes and administration of polyinosinic-polycytidylic acid [poly(I:C)] stabilized by lysine and carboxymethylcellulose (poly-ICLC) in HLA-A2(+) patients with recurrent malignant gliomas. GAAs for these peptides are EphA2, interleukin (IL)-13 receptor-α2, YKL-40, and gp100.

METHODS

Twenty-two patients (13 with glioblastoma multiforme [GBM], five with anaplastic astrocytoma [AA], three with anaplastic oligodendroglioma [AO], and one with anaplastic oligoastrocytoma [AOA]) received at least one vaccination, and 19 patients received at least four vaccinations at two αDC1 dose levels (1 × or 3 × 10(7)/dose) at 2-week intervals intranodally. Patients also received twice weekly intramuscular injections of 20 μg/kg poly-ICLC. Patients who demonstrated positive radiologic response or stable disease without major adverse events were allowed to receive booster vaccines. T-lymphocyte responses against GAA epitopes were assessed by enzyme-linked immunosorbent spot and HLA-tetramer assays.

RESULTS

The regimen was well-tolerated. The first four vaccines induced positive immune responses against at least one of the vaccination-targeted GAAs in peripheral blood mononuclear cells in 58% of patients. Peripheral blood samples demonstrated significant upregulation of type 1 cytokines and chemokines, including interferon-α and CXCL10. Nine (four GBM, two AA, two AO, and one AOA) achieved progression-free status lasting at least 12 months. One patient with recurrent GBM demonstrated sustained complete response. IL-12 production levels by αDC1 positively correlated with time to progression.

CONCLUSIONS

These data support safety, immunogenicity, and preliminary clinical activity of poly-ICLC-boosted αDC1-based vaccines.

To identify susceptibility loci for ankylosing spondylitis, we undertook a genome-wide association study in 2,053 unrelated ankylosing spondylitis cases among people of European descent and 5,140 ethnically matched controls, with replication in an independent cohort of 898 ankylosing spondylitis cases and 1,518 controls. Cases were genotyped with Illumina HumHap370 genotyping chips. In addition to strong association with the major histocompatibility complex (MHC; P < 10(-800)), we found association with SNPs in two gene deserts at 2p15 (rs10865331; combined P = 1.9 x 10(-19)) and 21q22 (rs2242944; P = 8.3 x 10(-<em>20</em>)), as well as in the genes ANTXR2 (rs4333130; P = 9.3 x 10(-8)) and <em>IL</em>1R2 (rs2310173; P = 4.8 x 10(-7)). We also replicated previously reported associations at <em>IL</em>23R (rs11<em>20</em>9026; P = 9.1 x 10(-14)) and ERAP1 (rs27434; P = 5.3 x 10(-12)). This study reports four genetic loci associated with ankylosing spondylitis risk and identifies a major role for the interleukin (<em>IL</em>)-23 and <em>IL</em>-1 cytokine pathways in disease susceptibility.

The cellular immune response contributes to clearance of hepatitis C virus (HCV) and persists for decades after recovery from infection. The immunological basis for the inefficiency of the cellular immune response in chronically infected persons is not known. Here, we used four HLA-A2 tetramers, specific for two HCV core and two HCV NS3 epitopes, to investigate at the single-cell level effector function and phenotype of HCV-specific CD8+ T cells in <em>20</em> chronically infected and 12 long-term recovered patients. Overall, HCV-specific, tetramer+ T cells were more frequently found in PBMCs of chronically infected patients than in those of recovered patients. However, when compared with HCV-tetramer+ T cells of recovered patients, they displayed an impaired proliferative capacity. As a result of the impaired proliferative capacity, HCV-specific T cell lines derived from chronically infected patients displayed less peptide-specific cytotoxicity than those from recovered patients. In addition, proliferation and ex vivo IFN-gamma production of HCV-tetramer+ cells, but not influenza-virus-specific T cells, were defective in chronically infected patients and could not be restored by in vitro stimulation with peptide and <em>IL</em>-2. At least three distinct phenotypes of HCV-specific CD8+ T cells were identified and associated with certain functional characteristics. In addition, impairment of proliferative, cytokine, and cytotoxic effector functions of tetramer+ T cells in viremic patients was associated with weak ex vivo HCV-specific CD4+ T cell responses. Thus, the defective functions of HCV-specific CD8+ T cells might contribute to viral persistence in chronically infected patients, and knowledge on their reversibility may facilitate the development of immunotherapeutic vaccines.

OBJECTIVE

To determine the efficacy of treatment using high-dose bolus interleukin 2 (IL-2) in patients with metastatic melanoma or renal cell cancer.

METHODS

Consecutive series of all patients treated with high-dose IL-2 in the Surgery Branch of the National Cancer Institute from September 1985 through December 1992.

METHODS

Two hundred eighty-three patients with metastatic melanoma or metastatic renal cell cancer who had failed standard treatment for their cancers.

METHODS

Patients received IL-2 at a dose of 720,000 IU/kg intravenously every 8 hours for a maximum of 15 doses per cycle. Two cycles constituted a treatment course, and patients with stable or responding disease received additional treatment courses. A total of 447 courses of treatment were administered.

METHODS

Regression of measurable tumor, durability of response to treatment, and survival.

RESULTS

Nine patients (7%) with metastatic melanoma achieved complete regression of all disease and 14 patients (10%) had partial regression. Ten patients (7%) with metastatic renal cell cancer experienced complete regression and 20 patients (13%) had partial regression. Of the 19 patients with complete regression, 15 have remained in complete remission from 7 to 91 months after treatment. Three treatment-related deaths (1.1%) occurred early in this series, but as experience with the administration of this IL-2 regimen increased, no treatment-related deaths occurred in 214 patients treated during the last 5 years of the study.

CONCLUSIONS

Biologic therapy with IL-2 can cause significant antitumor effects in patients with advanced metastatic melanoma or renal cell cancer. Because IL-2 does not have a direct effect on cancer cells but rather mediates its antitumor activity by altering host immune reactions, these data represent the best available evidence that immunologic therapy for cancer can be effective in selected patients.

OBJECTIVE

Treatment options for metastatic melanoma are limited. We conducted this phase II trial to assess the efficacy of JS1/34.5-/47-/granulocyte-macrophage colony-stimulating factor (GM-CSF) in stages IIIc and IV disease.

METHODS

Treatment involved intratumoral injection of up to 4 mL of 10(6) pfu/mL of JS1/34.5-/47-/GM-CSF followed 3 weeks later by up to 4 mL of 10(8) pfu/mL every 2 weeks for up to 24 treatments. Clinical activity (by RECIST [Response Evaluation Criteria in Solid Tumors]), survival, and safety parameters were monitored.

RESULTS

Fifty patients (stages IIIc, n = 10; IVM1a, n = 16; IVM1b, n = 4; IVM1c, n = <em>20</em>) received a median of six injection sets; 74% of patients had received one or more nonsurgical prior therapies for active disease, including dacarbazine/temozolomide or interleukin-2 (<em>IL</em>-2). Adverse effects were limited primarily to transient flu-like symptoms. The overall response rate by RECIST was 26% (complete response [CR], n = 8; partial response [PR], n = 5), and regression of both injected and distant (including visceral) lesions occurred. Ninety-two percent of the responses had been maintained for 7 to 31 months. Ten additional patients had stable disease (SD) for greater than 3 months, and two additional patients had surgical CR. On an extension protocol, two patients subsequently achieved CR by 24 months (one previously PR, one previously SD), and one achieved surgical CR (previously PR). Overall survival was 58% at 1 year and 52% at 24 months.

CONCLUSIONS

The 26% response rate, with durability in both injected and uninjected lesions including visceral sites, together with the survival rates, are evidence of systemic effectiveness. This effectiveness, combined with a limited toxicity profile, warrants additional evaluation of JS1/34.5-/47-/GM-CSF in metastatic melanoma. A US Food and Drug Administration-approved phase III investigation is underway.

OBJECTIVE

To establish the safety and efficacy of repeat infusions of tocilizumab (previously known as MRA), a humanized anti-interleukin-6 (IL-6) receptor antibody, alone and in combination with methotrexate (MTX), for the treatment of rheumatoid arthritis (RA).

METHODS

The study group comprised 359 patients with active RA in whom the response to MTX was inadequate. During a stabilization period, these patients received their current dose of MTX for at least 4 weeks. Following stabilization, they were randomized to 1 of 7 treatment arms, as follows: tocilizumab at doses of 2 mg/kg, 4 mg/kg, or 8 mg/kg either as monotherapy or in combination with MTX, or MTX plus placebo.

RESULTS

A 20% response (improvement) according to the American College of Rheumatology criteria (ACR20 response) was achieved by 61% and 63% of patients receiving 4 mg/kg and 8 mg/kg of tocilizumab as monotherapy, respectively, and by 63% and 74% of patients receiving those doses of tocilizumab plus MTX, respectively, compared with 41% of patients receiving placebo plus MTX. Statistically significant ACR50 and ACR70 responses were observed in patients receiving combination therapy with either 4 mg/kg or 8 mg/kg of tocilizumab plus MTX (P < 0.05). A dose-related reduction in the Disease Activity Score in 28 joints was observed from week 4 onward, in all patients except those receiving monotherapy with 2 mg/kg of tocilizumab. In the majority of patients who received 8 mg/kg of tocilizumab, the C-reactive protein level/erythrocyte sedimentation rate normalized, while placebo plus MTX had little effect on these laboratory parameters. Tocilizumab was mostly well tolerated, with a safety profile similar to that of other biologic and immunosuppressive therapies. Alanine transaminase and aspartate transaminase levels followed a sawtooth pattern (rising and falling between infusions). There were moderate but reversible increases in the nonfasting total cholesterol and triglyceride levels and reversible reductions in the high-density lipoprotein cholesterol and neutrophil levels. There were 2 cases of sepsis, both of which occurred in patients who were receiving combination therapy with 8 mg/kg of tocilizumab plus MTX.

CONCLUSIONS

These results indicate that targeted blockade of IL-6 signaling is a highly efficacious and promising means of decreasing disease activity in RA.

Embryonic stem (ES) cells have the potential to serve as an alternative source of hematopoietic precursors for transplantation and for the study of hematopoietic cell development. Using coculture of human ES (hES) cells with OP9 bone marrow stromal cells, we were able to obtain up to <em>20</em>% of CD34+ cells and isolate up to 10(7) CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8 to 9 days of culture. The hES cell-derived CD34+ cells were highly enriched in colony-forming cells, cells expressing hematopoiesis-associated genes GATA-1, GATA-2, SCL/TAL1, and Flk-1, and retained clonogenic potential after in vitro expansion. CD34+ cells displayed the phenotype of primitive hematopoietic progenitors as defined by co-expression of CD90, CD117, and CD164, along with a lack of CD38 expression and contained aldehyde dehydrogenase-positive cells as well as cells with verapamil-sensitive ability to efflux rhodamine 123. When cultured on MS-5 stromal cells in the presence of stem cell factor, Flt3-L, interleukin 7 (<em>IL</em>-7), and <em>IL</em>-3, isolated CD34+ cells differentiated into lymphoid (B and natural killer cells) as well as myeloid (macrophages and granulocytes) lineages. These data indicate that CD34+ cells generated through hES/OP9 coculture display several features of definitive hematopoietic stem cells.

Interleukin 1 (<em>IL</em>-1) is a major soluble mediator of inflammation. Two human <em>IL</em>-1 genes, alpha and beta, have been isolated, which encode polypeptides with only <em>20</em>-30% amino acid sequence homology. Unlike most secreted proteins, the two cytokines do not have a signal sequence, an unexpected finding in view of their biological role. Here we show that <em>IL</em>-1 beta is actively secreted by activated human monocytes via a pathway of secretion different from the classical endoplasmic reticulum--Golgi route. Drugs which block the intracellular transport of <em>IL</em>-6, of tumour necrosis factor alpha and of other secretory proteins do not inhibit secretion of <em>IL</em>-1 beta. Secretion of <em>IL</em>-1 beta is blocked by methylamine, low temperature or serum free medium, and is increased by raising the culture temperature to 42 degrees C or by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol or carbonyl cyanide chlorophenylhydrazone. <em>IL</em>-1 beta is contained in part within intracellular vesicles which protect it from protease digestion. In U937 cells large amounts of <em>IL</em>-1 beta are made but none is secreted. In these cells <em>IL</em>-1 beta is not found in the vesicular fraction, and all the protein is accessible to protease digestion. This suggests that intracellular vesicles that contain <em>IL</em>-1 beta are part of the protein secretory pathway. We conclude that <em>IL</em>-1 beta is released by activated monocytes via a novel mechanism of secretion which may involve translocation of intracellular membranes and is increased by stress conditions.

We examined the hypothesis that insulin resistance in skeletal muscle promotes the development of atherogenic dyslipidemia, associated with the metabolic syndrome, by altering the distribution pattern of postprandial energy storage. Following ingestion of two high carbohydrate mixed meals, net muscle glycogen synthesis was reduced by approximately 60% in young, lean, insulin-resistant subjects compared with a similar cohort of age-weight-body mass index-activity-matched, insulin-sensitive, control subjects. In contrast, hepatic de novo lipogenesis and hepatic triglyceride synthesis were both increased by >2-fold in the insulin-resistant subjects. These changes were associated with a 60% increase in plasma triglyceride concentrations and an approximately <em>20</em>% reduction in plasma high-density lipoprotein concentrations but no differences in plasma concentrations of TNF-alpha, <em>IL</em>-6, adiponectin, resistin, retinol binding protein-4, or intraabdominal fat volume. These data demonstrate that insulin resistance in skeletal muscle, due to decreased muscle glycogen synthesis, can promote atherogenic dyslipidemia by changing the pattern of ingested carbohydrate away from skeletal muscle glycogen synthesis into hepatic de novo lipogenesis, resulting in an increase in plasma triglyceride concentrations and a reduction in plasma high-density lipoprotein concentrations. Furthermore, insulin resistance in these subjects was independent of changes in the plasma concentrations of TNF-alpha, <em>IL</em>-6, high-molecular-weight adiponectin, resistin, retinol binding protein-4, or intraabdominal obesity, suggesting that these factors do not play a primary role in causing insulin resistance in the early stages of the metabolic syndrome.

BACKGROUND

The anti-interleukin (<em>IL</em>) 6 receptor antibody tocilizumab inhibits signalling of <em>IL</em>6, a key cytokine in rheumatoid arthritis (RA) pathogenesis.

OBJECTIVE

To evaluate through the AMBITION study the efficacy and safety of tocilizumab monotherapy versus methotrexate in patients with active RA for whom previous treatment with methotrexate/biological agents had not failed.

METHODS

This 24-week, double-blind, double-dummy, parallel-group study, randomised 673 patients to either tocilizumab 8 mg/kg every 4 weeks, or methotrexate, starting at 7.5 mg/week and titrated to 20 mg/week within 8 weeks, or placebo for 8 weeks followed by tocilizumab 8 mg/kg. The primary end point was the proportion of patients achieving American College of Rheumatology (ACR) 20 response at week 24.

RESULTS

The intention-to-treat analysis demonstrated that tocilizumab was better than methotrexate treatment with a higher ACR20 response (69.9 vs 52.5%; p<0.001), and 28-joint Disease Activity Score (DAS28) <2.6 rate (33.6 vs 12.1%) at week 24. Mean high-sensitivity C-reactive protein was within the normal range from week 12 with tocilizumab, whereas levels remained elevated with methotrexate. The incidence of serious adverse events with tocilizumab was 3.8% versus 2.8% with methotrexate (p = 0.50), and of serious infections, 1.4% versus 0.7%, respectively. There was a higher incidence of reversible grade 3 neutropenia (3.1% vs 0.4%) and increased total cholesterol>> or =240 mg/dl (13.2% vs 0.4%), and a lower incidence of alanine aminotransferase elevations >3x-<5x upper limit of normal (1.0% vs 2.5%), respectively.

CONCLUSIONS

Tocilizumab monotherapy is better than methotrexate monotherapy, with rapid improvement in RA signs and symptoms, and a favourable benefit-risk, in patients for whom treatment with methotrexate or biological agents has not previously failed.

BACKGROUND

Several lines of evidence suggest that inflammatory mechanisms contribute to AD.

OBJECTIVE

To examine whether several markers of inflammation are associated with cognitive decline in African-American and white well-functioning elders.

METHODS

The authors studied 3,031 African-American and white men and women (mean age 74 years) enrolled in the Health, Aging, and Body Composition Study. Serum levels of interleukin-6 (IL-6) and C-reactive protein (CRP) and plasma levels of tumor necrosis factor-alpha (TNFalpha) were measured at baseline; cognition was assessed with the Modified Mini-Mental State Examination (3MS) at baseline and at follow-up. Cognitive decline was defined as a decline of >5 points.

RESULTS

In age-adjusted analyses, participants in the highest tertile of IL-6 or CRP performed nearly 2 points lower (worse) on baseline and follow-up 3MS (p < 0.001 for all) and declined by almost 1 point over the >2 years (p = 0.01 for IL-6 and p = 0.04 for CRP) compared with those in the lowest tertile. After multivariate adjustment, 3MS scores among participants in the highest tertile of IL-6 and CRP were similar at baseline but remained significantly lower at follow-up (p < or = 0.05 for both). Those in the highest inflammatory marker tertile were also more likely to have cognitive decline compared with the lowest tertile for IL-6 (26 vs 20%; age-adjusted odds ratio [OR] = 1.34; 95% CI 1.06 to 1.69) and for CRP (24 vs 19%; OR = 1.41; 95% CI 1.10 to 1.79) but not for TNFalpha (23 vs 21%; OR = 1.12; 95% CI 0.88 to 1.43). There was no significant interaction between race and inflammatory marker or between nonsteroidal anti-inflammatory drug use and inflammatory marker on cognition.

CONCLUSIONS

Serum markers of inflammation, especially IL-6 and CRP, are prospectively associated with cognitive decline in well-functioning elders. These findings support the hypothesis that inflammation contributes to cognitive decline in the elderly.

A murine monoclonal antibody (H4/18) raised against cultured human endothelial cells (HEC) prestimulated by the monokine interleukin 1 (<em>IL</em> 1) recognizes a cell surface molecule inducible by <em>IL</em> 1 or by the distinct monokine tumor necrosis factor (TNF) in primary or serially passaged HEC. H4/18 binding is not basally expressed or inducible by <em>IL</em> 1 in an SV-40 transformed HEC line, in human dermal fibroblasts, or in blood leukocytes. Expression of this molecule by HEC in response to <em>IL</em> 1 can be blocked by protein and RNA synthesis inhibitors but not by cyclooxygenase inhibitors. In addition, H4/18 can immunoprecipitate two biosynthetically labeled polypeptides (Mr 100,000 and 1<em>20</em>,000) from HEC stimulated with <em>IL</em> 1 but not from control HEC. Thus, the H4/18 binding site appears to be an inducible surface protein specific for HEC. The majority of HEC in a culture can be induced to express the H4/18 binding protein, but expression is transient (peak 4 to 6 hr) and over the next 24 hr declines to near basal levels either in the continued presence of or upon removal of <em>IL</em> 1. The magnitude of the peak response depends upon <em>IL</em> 1 concentration (peak 5 to 10 U/ml), and the response is optimized by the continued presence of <em>IL</em> 1 during the initial 4- to 6-hr induction period. The time of peak H4/18 binding does not appear to be a function of <em>IL</em> 1 concentration. The decline of H4/18 binding from peak levels is prevented by cycloheximide, a protein synthesis inhibitor. HEC maintained in the presence of <em>IL</em> 1 for 24 hr become refractory to restimulation by <em>IL</em> 1; however, <em>IL</em> 1-stimulated cells rested in the absence of <em>IL</em> 1 for <em>20</em> hr can be stimulated by fresh <em>IL</em> 1. HEC expression of the H4/18 binding protein is not induced by interleukin 2 or by interferon-alpha, -beta, or -gamma. Induction of H4/18 binding by TNF is also concentration dependent, transient, and dependent upon protein and RNA synthesis. Several observations suggest that <em>IL</em>1 and TNF act independently on HEC. Our TNF is a recombinant protein, expressed from a cloned cDNA and thus free of <em>IL</em> 1 contamination; it also has no activity in a highly sensitive <em>IL</em> 1 assay. Our standard <em>IL</em> 1 preparation is affinity purified and lacks TNF activity on L929 cells. Thus, our monokine preparations are not cross-contaminated. Most interestingly, HEC incubated with <em>IL</em> 1 and refractory to <em>IL</em>1 restimulation can be restimulated by TNF to express H4/18 binding and vice versa.(ABSTRACT TRUNCATED AT 400 WORDS)