OBJECTIVE

The aim of the study was to assess the incidence and clinical implications of increased plasma angiotensin II despite chronic ACE inhibitor therapy in patients with heart failure.

RESULTS

The studied population consisted of 70 patients (mean age 59+/-9 years). Plasma renin activity and plasma concentration of aldosterone, norepinephrine, atrial natriuretic peptide, angiotensin II, tumour necrosis factor, <em>interleukin</em>-6 and <em>interleukin</em>-1B were assessed at 6 months of ACE inhibitor therapy. Mean left ventricular ejection fraction was 24+/-5% and the end-systolic and end-diastolic diameters were 59+/-9 and 71+/-8 mm, respectively. Despite chronic enalapril or captopril therapy, 35 patients (50%) had increased plasma angiotensin II (median 33 pg. ml(-1), range 17-84), while it was in the normal range in the remaining 35 patients (median 10 pg. ml(-1), range 5-<em>15</em>). Plasma renin activity (P=0.005), <em>interleukin</em>-6 (P=0.004), New York Heart Association functional class III-IV (P=0. 006), furosemide dose (P=0.01), lack of beta-blocker therapy (P=0. 04) and norepinephrine (P=0.04) were univariately associated with increased angiotensin II. Multivariate regression analysis identified the plasma renin activity (0.0004), norepinephrine (0.02) and <em>interleukin</em>-6 (0.03) as independent predictors of plasma angiotensin II. During follow-up (35+/-29 months), nine (12.8%) patients died and 13 had new heart failure episodes. Increased plasma angiotensin II, despite ACE inhibitor therapy, was a significant predictor of death or heart failure according to the Kaplan-Meier survival method by log rank test (P=0.002).

CONCLUSIONS

Fifty per cent of patients with heart failure, ha increased plasma angiotension II despite chronic ACE inhibitor therapy. These patients had higher neurohormonal activation and poor prognosis.

1. Elevated levels of cytokines, especially <em>interleukin</em> (IL)-6 and IL-1ra, can be measured in the plasma of athletes after exhaustive long term exercise. 2. The present study investigates the kinetics of several cytokines and chemokines in ten male athletes before, during and after 2.5 h of treadmill running at 75 % of maximal oxygen consumption (VO2,max). Blood was sampled before, every half-hour during running and every hour in the following 6 h recovery period. 3. The plasma concentration of IL-6 increased after 30 min of running, and peaked at the end of running with a 25-fold increase compared with the pre-exercise value. IL-1ra increased only after running, and peaked after 2 h of rest with an 18-fold increase compared with the pre-exercise value. No changes were found in the concentrations of IL-1beta, tumour necrosis factor (TNF)alpha, IL-<em>15</em> and macrophage inflammatory protein (MIP)-1beta, and the concentrations of IL-8 and MIP-1alpha were below detection limits. 4. The results suggest that very early events in exercise trigger the release of IL-6, and that the cytokine response to exercise has similarities to that observed after trauma.

We estimated whether single collections of cord blood contained sufficient cells for hematopoietic engraftment of adults by evaluating numbers of cord blood and adult bone marrow myeloid progenitor cells (MPCs) as detected in vitro with steel factor (SLF) and hematopoietic colony-stimulating factors (CSFs). SLF plus granulocyte-macrophage (GM)-CSF detected 8- to 11-fold more cord blood GM progenitors [colony-forming units (CFU)-GM] than cells stimulated with GM-CSF or 5637 conditioned medium (CM), growth factors previously used to estimate cord blood CFU-GM numbers. SLF plus erythropoietin (Epo) plus <em>interleukin</em> 3 (IL-3) enhanced detection of cord blood multipotential (CFU-GEMM) progenitors <em>15</em>-fold compared to stimulation with Epo plus IL-3. Under the same conditions, bone marrow CFU-GM and CFU-GEMM were only enhanced in detection 2- to 4- and 6- to 8-fold. Increased detection of cord blood CFU-GEMM correlated directly with decreased detection of cord blood erythroid burst-forming units (BFU-E). In contrast, adult bone marrow CFU-GEMM and BFU-E numbers were both enhanced by SLF plus Epo plus IL-3. This suggests that most cord blood BFU-E may actually be CFU-GEMM. Cord blood collections (n = 17) contained numbers of MPCs (especially CFU-GM) similar to the number found in nine autologous bone marrow collections. To assess additional sources of MPCs, the peripheral blood of 1-day-old infants was assessed. However, average concentrations of MPCs circulating in these infants were only 30-46% that in their cord blood. Expansion of cord blood MPCs was also evaluated. Incubation of cord blood cells for 7 days with SLF resulted in 7.9-, 2.2-, and 2.7-fold increases in numbers of CFU-GM, BFU-E, and CFU-GEMM compared to starting numbers; addition of a CSF with SLF resulted in even greater expansion of MPCs. The results suggest that cord blood contains a larger number of early profile MPCs than previously recognized and that there are probably sufficient numbers of cells in a single cord blood collection to engraft an adult. Although the expansion data must be considered with caution, as human marrow repopulating cells cannot be assessed directly, in vitro expansion of cord blood stem and progenitor cells may be feasible for clinical transplantation.

Vascular endothelial growth factor (VEGF) has been shown to be a potent mediator of angiogenesis that functions as a survival factor for endothelial cells by up-regulating Bcl-2 expression. We have recently reported that human dermal microvascular endothelial cells (HDMECs) seeded in biodegradable sponges and implanted into severe combined immunodeficient (SCID) mice organize into functional human microvessels that transport mouse blood cells. In this study, we implanted sponges seeded with OSCC-3 (oral squamous cell carcinoma) or SLK (Kaposi's sarcoma) together with endothelial cells into SCID mice to generate human tumors vascularized with human microvessels. This model system was used to examine the role of both endothelial cell Bcl-2 and the proangiogenic chemokine <em>interleukin</em>-8 (IL-8) on tumor growth and intratumoral microvascular density. Coimplantation of HDMECs overexpressing Bcl-2 (HDMEC-Bcl-2) and tumor cells resulted in a 3-fold enhancement of tumor growth when compared with the coimplantation of control HDMECs and tumor cells. This was associated with increased intratumoral microvascular density and enhanced endothelial cell survival. To determine whether the enhanced neovascularization mediated by Bcl-2 overexpression in endothelial cells was influenced by the synthesis of endogenous mediators of angiogenesis, we screened these cells for expression of VEGF, basic fibroblast growth factor (bFGF), and IL-8 by ELISA. HDMEC-Bcl-2 cells and VEGF-treated HDMECs exhibited a <em>15</em>-fold and 4-fold increase, respectively, in the expression of the proangiogenic chemokine IL-8 in vitro, whereas the expression of VEGF and bFGF remained unchanged. Transfection of antisense Bcl-2 into HDMECs blocked VEGF-mediated induction of IL-8. Conditioned media from HDMEC-Bcl-2 induced proliferation and sprouting of endothelial cells in vitro and neovascularization in rat corneas. Anti-IL-8 antibody added to HDMEC-Bcl-2 conditioned media markedly reduced the potency of these responses. SCID mice bearing VEGF-producing tumor implants that were treated with anti-lL-8 antibody exhibited a 43% reduction in microvessel density and a 50% reduction in tumor weight compared with treatment with a nonspecific antibody. These results demonstrate that the up-regulation of Bcl-2 expression in endothelial cells that constitute tumor microvessels enhances intratumoral microvascular survival and density and accelerates tumor growth. Furthermore, endothelial cells that overexpress Bcl-2 have more angiogenic potential than control cells, and IL-8-neutralizing antibodies attenuate their angiogenic activity in vitro and in vivo.

Several methods have been developed to quantify cytokines and chemokines in biological fluids and tissue culture samples, including bioassays, enzyme-linked immunosorbent assay (ELISA), intracellular staining, ribonuclease protection assay (RPA) and polymerase chain reaction (PCR). However, each of these techniques possesses one or more significant limitations. Here, we describe a new multiplexed assay, using the FlowMetrix system, that can quantify multiple cytokines simultaneously in a small sample volume. This assay was found to be more accurate, sensitive and reproducible than the conventional microtitre ELISA procedure. Furthermore, the time and cost involved are comparable to, or less than, the ELISA. A key feature of the FlowMetrix assay is its ability to multiplex: here, we show that this assay can accurately quantitate <em>15</em> cytokines in a 100 microl sample volume while the same analysis by ELISA requires 1.5 ml (100 microl for each cytokine assay). By using this Flow Metrix assay, we could demonstrate that only T helper 1 (T(H)1)-deviated cells produce detectable levels of <em>interleukin</em> (IL)-2, while only T(H)2-deviated cells produce significant amounts of IL-4. Six other cytokines were produced by both T cell subsets, with the T(H)1 population producing more IL-3, granulocyte-monocyte colony stimulating factor (GM-CSF) and interferon (IFN)-gamma, and the T(H)2 population producing more IL-5, IL-10, and IL-13. Seven other cytokines were not produced in detectable amounts. This assay should prove to be a powerful tool in the quantitation of cytokines, or any other soluble product for which antibody pairs are available. It will also provide a more complete picture of the plethora of cytokines secreted during an immune response.

Langerhans cells (LCs) represent a subset of immature dendritic cells (DCs) specifically localized in the epidermis and other mucosal epithelia. As surrounding keratinocytes can produce <em>interleukin</em> (IL)-<em>15</em>, a cytokine that utilizes IL-2Rgamma chain, we analyzed whether IL-<em>15</em> could skew monocyte differentiation into LCs. Monocytes cultured for 6 d with granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-<em>15</em> differentiate into CD1a(+)HLA-DR(+)CD14(-)DCs (IL<em>15</em>-DCs). Agents such as lipopolysaccharide (LPS), tumor necrosis factor (TNF)alpha, and CD40L induce maturation of IL<em>15</em>-DCs to CD83(+), DC-LAMP(+) cells. IL<em>15</em>-DCs are potent antigen-presenting cells able to induce the primary (mixed lymphocyte reaction [MLR]) and secondary (recall responses to flu-matrix peptide) immune responses. As opposed to cultures made with GM-CSF/IL-4 (IL4-DCs), a proportion of IL<em>15</em>-DCs expresses LC markers: E-Cadherin, Langerin, and CC chemokine receptor (CCR)6. Accordingly, IL<em>15</em>-DCs, but not IL4-DCs, migrate in response to macrophage inflammatory protein (MIP)-3alpha/CCL20. However, IL<em>15</em>-DCs cannot be qualified as "genuine" Langerhans cells because, despite the presence of the 43-kD Langerin, they do not express bona fide Birbeck granules. Thus, our results demonstrate a novel pathway in monocyte differentiation into dendritic cells.

The purpose of the study was to identify a comprehensive prognostic system of pretreatment clinical parameters in 425 patients (pts) with metastatic renal-cell carcinoma treated with different subcutaneous (s.c.) recombinant cytokine-based home therapies in consecutive trials. Treatment consisted of (A) s.c. interferon-alpha 2a (INF-alpha), s.c. <em>interleukin</em>-2 (IL-2) (n=102 pts), (B) s.c. IFN-alpha 2a, s.c. IL-2, and i.v. 5-fluorouracil (5-FU) (n=235 pts) or (C) s.c. IFN-alpha 2a, s.c. IL-2, and i.v. 5-FU combined with p.o. 13-cis-retinoic acid (13cRA) (n=88 pts). Kaplan-Meier survival analysis, log-rank statistics, and Cox regression analysis were employed to identify risk factors and to create a multiple risk factor model. The following pretreatment risk factors were identified by univariate analysis: (1) three and more metastatic sites, (2) presence of liver, lymph node or bone metastases, (3) neutrophil count>> or = 6500 cells microl(-1), (4) serum lactate dehydrogenase level (LDH)>> or = 220 U l(-1), and (5) serum C-reactive protein level (CRP)>> or = 11 mg l(-1). Cox regression analysis with forward stepwise variable selection identified neutrophil count as the major prognostic factor (hazard ratio=1.9, P<0.001), while serum levels of LDH and CRP, time between diagnosis of tumour and onset of metastatic disease, number of metastatic sites, and bone metastases were significant but somewhat less important prognostic variables within the multiple risk factor model (hazard ratio < or = 1.5). Patients were assigned to one of the three risk groups according to cumulative risk defined as the sum of simplified risk s.c.ores for six pretreatment variables. Low-, intermediate-, and high-risk patients achieved a median overall survival of 32+ months (95% CI 24, 43; 5-year survival of 27%), 18+ months (95% CI <em>15</em>, 20; 5-year survival of 11%), and 8+ months (95% CI 6, 10; 5-year survival of 5%), respectively. These prognostic categories are helpful both in individual patient care and in the assessment of patients entering prospective clinical trials.

In a previous study, it was shown that the Kaposi sarcoma-associated herpesvirus (KSHV) was specifically associated with monotypic (IgMlambda) plasmablasts in multicentric Castleman disease (MCD). The plasmablasts occur as isolated cells in the mantle zone of B-cell follicles but may form microlymphoma or frank plasmablastic lymphoma. To determine the clonality and cellular origin of the monotypic plasmablasts, the rearranged Ig genes in 13 patients with KSHV-related MCD, including 8 cases with microlymphomas and 2 with frank lymphomas, were studied. To investigate the role of the <em>interleukin</em> 6 (IL-6) receptor signaling in the pathogenesis of MCD and associated lymphoproliferative disorders, viral IL-6 and human IL-6 receptor expression was examined. KSHV-positive plasmablasts were polyclonal in MCD-involved lymphoid tissues in all cases and microlymphomas in 6 of 8 cases. Monoclonal KSHV-positive plasmablasts were seen in microlymphomas of 2 cases and in both frank lymphomas. Despite their mature phenotype, KSHV-positive plasmablasts did not harbor somatic mutations in the rearranged Ig genes, indicating origination from naive B cells. Viral IL-6 was expressed in 10% to <em>15</em>% of KSHV-positive plasmablasts, whereas the human IL-6 receptor was expressed in most KSHV-positive cells. Thus, KSHV infects monotypic but polyclonal naive B cells and is associated with a range of lymphoproliferative disorders from polyclonal isolated plasmablasts and microlymphomas to monoclonal microlymphoma and frank plasmablastic lymphomas in MCD patients. Activation of the IL-6 receptor signaling pathway may play a role in differentiation of KSHV-infected naive B cells into plasmablasts and development of lymphoproliferative lesions. (Blood. 2001;97:2130-2136)

Biliary tract malignancies represent challenges because of the lack of effective therapy and poor prognosis, in part because of the paucity of information regarding the mechanisms regulating their growth. We have recently identified a critical role for the p44/p42 mitogen-activated protein kinase (MAPK) pathway in <em>interleukin</em> 6 (IL-6)-stimulated growth of human cholangiocytes. Although IL-6 is a potential mitogen for cholangiocarcinoma, the role of this cytokine and its intracellular signaling pathways in cholangiocarcinoma growth is unknown. Thus, our aims were to determine the role of IL-6-mediated signaling mechanisms, and in particular the MAPK pathways, in the growth regulation of human cholangiocarcinoma. KMCH-1 cells (malignant cholangiocyte cells) secreted IL-6 constitutively, and increased IL-6 secretion in response to inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and IL-1beta. Stimulation with IL-6 resulted in proliferation of malignant cholangiocytes. These cells also possessed the IL-6 receptor complex subunits as directly assessed by immunoblot analysis. Furthermore, proliferation was completely inhibited by preincubation with anti-IL-6 neutralizing antibodies, indicating that the proliferative response to IL-6 involved receptor-mediated signaling. Both p38 and p44/p42 MAPKs were constitutively present and active in malignant cholangiocytes, and increased activity of both was observed within <em>15</em> minutes of stimulation with IL-6. Selective inhibition of either the p44/p42 MAPK pathway, by PD098059, or of the p38 MAPK pathway, by SB203580, blocked proliferation in response to IL-6. Thus, IL-6 can contribute to the autocrine and/or paracrine growth stimulation of malignant cholangiocytes via activation of either p38 or p44/p42 MAPK signaling pathways.

Multicentric Castleman disease (MCD) is a distinct type of lymphoproliferative disorder associated with inflammatory symptoms and <em>interleukin</em> 6 (IL-6) dysregulation. In the context of human immunodeficiency virus (HIV) infection, MCD is associated with Kaposi sarcoma-associated herpesvirus, also called human herpesvirus type 8 (KSHV/HHV8). Within a prospective cohort study on 60 HIV-infected patients with MCD, and a median follow-up period of 20 months, 14 patients developed KSHV/HHV8-associated non-Hodgkin lymphoma (NHL): 3 "classic" KSHV/HHV8(+) Epstein-Barr virus-positive (EBV(+)) primary effusion lymphoma (PEL), 5 KSHV/HHV8(+) EBV(-) visceral large cell NHL with a PEL-like phenotype, and 6 plasmablastic lymphoma/leukemia (3/3 KSHV/HHV8(+) EBV(-)). The NHL incidence observed in this cohort study (101/1000 patient-years) is about <em>15</em>-fold what is expected in the general HIV(+) population. MCD-associated KSHV/HHV8(+) NHL fell into 2 groups, suggesting different pathogenesis. The plasmablastic NHL likely represents the expansion of plasmablastic microlymphoma from the MCD lesion and progression toward aggressive NHL. In contrast, the PEL and PEL-like NHL may implicate a different original infected cell whose growth is promoted by the cytokine-rich environment of the MCD lesions.

OBJECTIVE

Apoptosis is a form of programmed cell death seen in a variety of developmental and disease states, including traumatic injuries. The main objective of this study was to determine whether apoptosis is observed after human spinal cord injury (SCI). The spatial and temporal expression of apoptotic cells as well as the nature of the cells involved in programmed cell death were also investigated.

METHODS

The authors examined the spinal cords of <em>15</em> patients who died between 3 hours and 2 months after a traumatic SCI. Apoptotic cells were found at the edges of the lesion epicenter and in the adjacent white matter, particularly in the ascending tracts, by using histological (cresyl violet, hematoxylin and eosin) and nuclear staining (Hoechst 33342). The presence of apoptotic cells was supported by staining with the terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling technique and confirmed by immunostaining for the processed form of caspase-3 (CPP-32), a member of the <em>interleukin</em>-1beta-converting enzyme/Caenorhabditis elegans D 3 (ICE/CED-3) family of proteases that plays an essential role in programmed cell death. Apoptosis in this series of human SCIs was a prominent pathological finding in 14 of the <em>15</em> spinal cords examined when compared with five uninjured control spinal cords. To determine the type of cells undergoing apoptosis, the authors immunostained specimens with a variety of antibodies, including glial fibrillary acidic protein, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), and CD45/68. Oligodendrocytes stained with CNPase and a number of apoptotic nuclei colocalized with positive staining for this antibody.

CONCLUSIONS

These results support the hypothesis that apoptosis occurs in human SCIs and is accompanied by the activation of caspase-3 of the cysteine protease family. This mechanism of cell death contributes to the secondary injury processes seen after human SCI and may have important clinical implications for the further development of protease inhibitors to prevent programmed cell death.

The heparin-containing mast cells that reside in the connective tissue of the mouse, but not the chondroitin sulfate-containing mast cells in the gastrointestinal mucosa, stain with safranin when exposed to alcian blue/safranin. Mouse bone marrow-derived mast cells (BMMC), the probable in vitro counterparts of in vivo mucosal mast cells, were cultured for 14 days with mouse skin-derived 3T3 fibroblasts in RPMI 1640 medium containing 10% fetal calf serum and 50% WEHI-3 conditioned medium. Although the BMMC adhered to the fibroblast monolayer, they continued to divide, probably due to the presence of <em>interleukin</em> 3 in the conditioned medium. The mast cells remained viable throughout the period of coculture, since they failed to release lactate dehydrogenase and because they increased their histamine content approximately <em>15</em>-fold. After 12-14 days of coculture, greater than 50% of the BMMC changed histochemically to become safranin+; 30-40% of the 35S-labeled glycosaminoglycans on the proteoglycans synthesized by these cocultured mast cells were heparin, whereas heparin was not detected in the initial BMMC. In the absence of WEHI-3 conditioned medium, BMMC adhered to the fibroblast monolayer, and after 8 days of coculture, the number of mast cells did not change and their histamine content remained the same. However, these mast cells also became safranin+ and synthesized 40% heparin glycosaminoglycans. Thus, coculture of BMMC with fibroblasts induces a phenotypic change so that the resulting mast cells stain safranin+ and synthesize heparin proteoglycans, whereas the presence of WEHI-3 conditioned medium stimulates proliferation and an increase in histamine content.

OBJECTIVE

To evaluate the safety of anakinra (a recombinant human interleukin-1 receptor antagonist) in a large population of patients with rheumatoid arthritis (RA), typical of those seen in clinical practice.

METHODS

A total of 1,414 patients were randomly assigned to treatment with 100 mg of anakinra or placebo, administered daily by subcutaneous injection. Background medications included disease-modifying antirheumatic drugs, corticosteroids, and nonsteroidal antiinflammatory drugs, alone or in combination. The primary end point was safety, which was evaluated by adverse events (including infections), discontinuation from study due to adverse events, and death.

RESULTS

Safety was evaluated in 1,399 patients (1,116 in the anakinra group and 283 in the placebo group; 15 patients were randomized but did not receive any study drug) during the initial 6-month, double-blind, placebo-controlled phase of this long-term safety study. Baseline demographics, disease characteristics, and concomitant medications were similar between the 2 groups. The study group included patients with numerous comorbid conditions and a wide range of RA disease activity. Serious adverse events occurred at a similar rate in the anakinra group and the placebo group (7.7% and 7.8%, respectively). Serious infectious episodes were observed more frequently in the anakinra group (2.1% versus 0.4% in the placebo group). The rate of withdrawal due to adverse events was 13.4% in the anakinra group and 9.2% in the placebo group.

CONCLUSIONS

Results from this large, placebo-controlled safety study demonstrate that anakinra is safe and well tolerated in a diverse population of patients with RA, including those with comorbid conditions and those using multiple combinations of concomitant therapies. Although the frequency of serious infection was slightly higher in the anakinra group, no infection was attributed to opportunistic microorganisms or resulted in death.

Tumors often escape immune-mediated destruction by suppressing lymphocyte infiltration or effector function. New approaches are needed that overcome this suppression and thereby augment the tumoricidal capacity of tumor-reactive lymphocytes. The cytokine <em>interleukin</em>-<em>15</em> (IL-<em>15</em>) promotes proliferation and effector capacity of CD8(+) T cells, natural killer (NK) cells, and NKT cells; however, it has a short half-life and high doses are needed to achieve functional responses in vivo. The biological activity of IL-<em>15</em> can be dramatically increased by complexing this cytokine to its soluble receptor, IL-<em>15</em>R alpha. Here, we report that in vivo delivery of IL-<em>15</em>/IL-<em>15</em>R alpha complexes triggers rapid and significant regression of established solid tumors in two murine models. Despite a marked expansion of IL-2/IL-<em>15</em>R beta(+) cells in lymphoid organs and peripheral blood following treatment with IL-<em>15</em>/IL-<em>15</em>R alpha complexes, the destruction of solid tumors was orchestrated by tumor-resident rather than newly infiltrating CD8(+) T cells. Our data provide novel insights into the use of IL-<em>15</em>/IL-<em>15</em>R alpha complexes to relieve tumor-resident T cells from functional suppression by the tumor microenvironment and have significant implications for cancer immunotherapy and treatment of chronic infections.

The impaired infection control related to the functional immaturity of the neonatal immune system is an important cause of infection in preterm newborns. We previously reported that constitutive Toll-like receptor (TLR) 4 expression and cytokine secretion on lipopolysaccharide (LPS) stimulation increases with gestational age. Here, we analyzed constitutive monocyte TLR2 expression and evaluated the expression profiles of the proximal downstream adapter molecule myeloid differentiation factor 88 (MyD88). We further investigated activation of protein kinases p38 and extracellular regulated kinsase (ERK) 1/2 in CD14 monocytes after ex vivo stimulation with bacterial TLR ligands (LPS and lipoteichoic acid [LTA]). The functional outcome of the stimulation was determined by cytokine secretion. Monocytes from 31 preterm newborns (<30 weeks of gestation, n=16; 30-37 weeks of gestation, n=<em>15</em>), 10 term newborns, and 12 adults were investigated. In contrast to TLR4 expression, TLR2 levels did not differ between age groups. However, MyD88 levels were significantly lower in preterm newborns. Activation of p38 and ERK1/2 was impaired in all newborn age groups after stimulation with TLR-specific ligands. Accordingly, after LTA stimulation, the levels of <em>interleukin</em> (IL)-1 beta , IL-6, and IL-8 cytokine production were substantially lower (P<.001) in preterm newborns than in adults. The reduced functional response to bacterial cell wall components appears to be part of the functional immaturity of the neonatal immune system and might predispose premature newborns to bacterial infection.

HLA-mismatched natural killer (NK) cells have shown efficacy in acute myeloid leukemia, and their adoptive transfer in patients with other malignancies has been proven safe. This phase I clinical trial was designed to evaluate safety (primary endpoint) and possible clinical efficacy (secondary endpoint) of repetitive administrations of allogeneic, in vitro activated and expanded NK cells along with chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). Patients with unresectable, locally advanced/metastatic NSCLC receiving 1st/2nd line chemotherapy were eligible to receive 2-4 doses of activated NK cells from two relative donors. Donor's CD56(+) cells were cultured for 20-23 days with <em>interleukin</em>-<em>15</em> (IL-<em>15</em>) and hydrocortisone (HC) and administered intravenously between chemotherapy cycles. Premedication with corticosteroids and/or H1 inhibitors was allowed. Sixteen patients (performance status 0-1) with adenocarcinoma (n = 13) or squamous cell carcinoma (n = 3) at stage IIIb (n = 5) or IV (n = 11) receiving 1st (n = 13) or 2nd (n = 3) line treatment were enrolled. Fifteen patients received 2-4 doses of allogeneic activated NK cells (0.2-29 × 10(6)/kg/dose, median 4.<em>15</em> × 10(6)/kg/dose). No side effects (local or systemic) were observed. At a median 22-month follow-up (range, 16.5-26 months) 2 patients with partial response and 6 patients with disease stabilization were recorded. Median progression free survival and overall survival were 5.5 and <em>15</em> months, respectively. A 56% 1-year survival and a 19% 2-year survival were recorded. In conclusion, repetitive infusions of allogeneic, in vitro activated and expanded with IL-<em>15</em>/HC NK cells, in combination with chemotherapy are safe and potentially clinically effective.

<em>Interleukin</em>-<em>15</em> (IL-<em>15</em>) is crucial for the generation of multiple lymphocyte subsets (natural killer (NK), NK-T cells, and memory CD8 T cells), and transpresentation of IL-<em>15</em> by monocytes and dendritic cells has been suggested to be the dominant activating process of these lymphocytes. We have previously shown that a natural soluble form of IL-<em>15</em>R alpha chain corresponding to the entire extracellular domain of IL-<em>15</em>R alpha behaves as a high affinity IL-<em>15</em> antagonist. In sharp contrast with this finding, we demonstrate in this report that a recombinant, soluble sushi domain of IL-<em>15</em>R alpha, which bears most of the binding affinity for IL-<em>15</em>, behaves as a potent IL-<em>15</em> agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-<em>15</em>R beta/gamma heterodimer, whereas it does not affect IL-<em>15</em> binding and function of the tripartite IL-<em>15</em>R alpha/beta/gamma membrane receptor. Our results suggest that, if naturally produced, such soluble sushi domains might be involved in the IL-<em>15</em> transpresentation mechanism. Fusion proteins (RLI and ILR), in which IL-<em>15</em> and IL-<em>15</em>R alpha-sushi are attached by a flexible linker, are even more potent than the combination of IL-<em>15</em> plus sIL-<em>15</em>R alpha-sushi. After binding to IL-<em>15</em>R beta/gamma, RLI is internalized and induces a biological response very similar to the IL-<em>15</em> high affinity response. Such hyper-IL-<em>15</em> fusion proteins appear to constitute potent adjuvants for the expansion of lymphocyte subsets.

We have previously demonstrated that short-term exposure to diesel exhaust (DE) for 1 h induced a marked leukocytic infiltration in the airways of healthy human volunteers involving neutrophils, lymphocytes, and mast cells along with increases in several inflammatory mediators. We hypothesized that the leukocyte infiltration and the various inflammatory responses induced by DE were mediated by enhanced chemokine and cytokine production by resident cells of the airway tissue and lumen. To investigate this, <em>15</em> healthy human volunteers were exposed to diluted DE and air on two separate occasions for 1 h each in an exposure chamber. Fiberoptic bronchoscopy was performed 6 h after each exposure to obtain endobronchial biopsies and bronchial wash (BW) cells. Using reverse transcriptase/polymerase chain reaction enzyme-linked immunosorbent assay (RT-PCR ELISA), a novel and sensitive technique to quantify relative amounts of cytokine mRNA gene transcripts, and immunohistochemical staining with computer-assisted image analysis to quantify expression of cytokine protein in the bronchial tissue, we have demonstrated that DE enhanced gene transcription of <em>interleukin</em>-8 (IL-8) in the bronchial tissue and BW cells along with increases in IL-8 and growth-regulated oncogene-alpha (GRO-alpha) protein expression in the bronchial epithelium, and an accompanying trend toward an increase in IL-5 mRNA gene transcripts in the bronchial tissue. There were no significant changes in the gene transcript levels of <em>interleukin</em>-1B (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interferon gamma (IFN-gamma), and granulocyte macrophage colony-stimulating factor (GM-CSF) either in the bronchial tissue or BW cells after DE exposure at this time point. These observations suggest an underlying mechanism for DE-induced airway leukocyte infiltration and offer a possible explanation for the association observed between ambient levels of particulate matter and various respiratory health outcome indices noted in epidemiological studies.

OBJECTIVE

Interleukin-10 (IL-10) is a potent stimulator of B lymphocytes in vitro. In vivo dysregulation of IL-10 gene expression was therefore analyzed in patients with rheumatoid arthritis (RA), primary Sjögren's syndrome (SS), and systemic lupus erythematosus (SLE).

METHODS

Spontaneous production of IL-10 by peripheral blood mononuclear cells was measured using reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay in untreated patients with either RA (n = 10), SS (n = 10), or SLE (n = 10), and in 15 normal control subjects.

RESULTS

IL-10 production was dramatically higher in RA, SS, and SLE patients than in controls. In each group, both B lymphocytes and monocytes, but not T lymphocytes, produced IL-10.

CONCLUSIONS

IL-10 production is increased in RA, SS, and SLE. It may play a role in B lymphocyte hyperactivity and in the development of autoimmunity.

<em>Interleukins</em>-2 and -<em>15</em> (IL-2 and IL-<em>15</em>) are cytokines with overlapping but distinct biological effects. Their receptors share two subunits (the IL-2R beta and -gamma chains) that are essential for signal transduction. The IL-2 receptor requires an additional IL-2-specific alpha subunit for high affinity IL-2 binding. Recently, a murine IL-<em>15</em>-specific alpha subunit was identified, cloned, and shown to be structurally related to IL-2R alpha. However, the murine IL-<em>15</em>R alpha alone bound IL-<em>15</em> with a 1000-fold higher affinity than that seen with IL-2R alpha and IL-2. We now extend these studies into the human system with the isolation of three differentially spliced human IL-<em>15</em>R alpha variants that are all capable of high affinity binding of IL-<em>15</em>. The cytoplasmic domain of IL-<em>15</em>R alpha, like that of IL-2R alpha, is dispensable for mitogenic signaling, suggesting that the primary role of the alpha chains is to confer high affinity binding. At high concentrations, IL-<em>15</em>, like IL-2, is able to signal through a complex of IL-2R beta and -gamma in the absence of the alpha subunit. Furthermore, the IL<em>15</em>RA and IL2RA genes have a similar intron-exon organization and are closely linked in both human and murine genomes. However, the distribution of expression of the IL-<em>15</em>R alpha is much wider than that of the IL-2R alpha, suggesting a broader range of cellular targets for IL-<em>15</em>.

The amyloid precursor protein (APP) has been associated with Alzheimer's disease (AD) because APP is processed into the beta-peptide that accumulates in amyloid plaques, and APP gene mutations can cause early onset AD. Inflammation is also associated with AD as exemplified by increased expression of <em>interleukin</em>-1 (IL-1) in microglia in affected areas of the AD brain. Here we demonstrate that IL-1alpha and IL-1beta increase APP synthesis by up to 6-fold in primary human astrocytes and by <em>15</em>-fold in human astrocytoma cells without changing the steady-state levels of APP mRNA. A 90-nucleotide sequence in the APP gene 5'-untranslated region (5'-UTR) conferred translational regulation by IL-1alpha and IL-1beta to a chloramphenicol acetyltransferase (CAT) reporter gene. Steady-state levels of transfected APP(5'-UTR)/CAT mRNAs were unchanged, whereas both base-line and IL-1-dependent CAT protein synthesis were increased. This APP mRNA translational enhancer maps from +55 to +144 nucleotides from the 5'-cap site and is homologous to related translational control elements in the 5'-UTR of the light and and heavy ferritin genes. Enhanced translation of APP mRNA provides a mechanism by which IL-1 influences the pathogenesis of AD.

BACKGROUND

A comprehensive characterization of the effects of cigarette smoke on systemic soluble immune/inflammatory markers may provide insight into the mechanisms through which smoking causes disease.

METHODS

Levels of 78 inflammation, immune, and metabolic markers were measured using multiplex immune assays in 1819 Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO) participants aged 55 to 74 years from three existing nested case-control studies. These data were made representative of the entire PLCO screening arm through reweighting with weights estimated in logistic regression models. We assessed associations between smoking status, cigarettes smoked per day, and time since quitting with dichotomized marker levels using adjusted weighted logistic regression models.

RESULTS

Current smoking was associated with 10 inflammation markers after correcting for multiple testing, encompassing several components of the immune/inflammation response. Levels of seven of these markers (<em>interleukin</em> [IL]-<em>15</em>, IL-1RA, IL-1β, IL-16, stem cell factor, soluble <em>interleukin</em> 6 receptor, and soluble vascular endothelial growth factor receptor 3) were lower among current smokers (n = 414) when compared with never smokers (n = 548), with odds ratios (ORs) ranging from 0.44 to 0.27, while levels of CC motif ligand (CCL)/thymus and activation regulated chemokine (CCL17/TARC) (OR = 4.08, 95% confidence interval [CI] = 2.01 to 8.25), CCL11/EOTAXIN (OR = 2.57, 95% CI = 1.45 to 4.55), and C-reactive protein (CRP) (OR = 2.54, 95% CI = 1.29 to 4.98) were elevated. These markers were not associated with cigarettes per day among current smokers, but there were trends in IL-<em>15</em>, IL-1RA, IL-1β, CCL17/TARC, CCL11/EOTAXIN, and CRP levels across categories of years since quitting smoking.

CONCLUSIONS

Smoking is associated with a broad range of alterations in systemic immune and inflammation marker levels among older, long-term smokers. Smoking cessation may result in marker levels reverting back to those of never smokers over time.

BACKGROUND

Posttraumatic stress disorder (PTSD) is associated with dysregulation of the hypothalamus pituitary adrenal (HPA) axis. Alterations include various responses to HPA axis stimulation, different basal hormone levels, and changes in glucocorticoid receptor (GR) numbers on lymphocytes. The functional significance of these latter changes remains elusive.

METHODS

Twelve Bosnian war refugees with PTSD and 13 control subjects were studied. On 2 consecutive days, they collected saliva samples after awakening and at 11, <em>15</em>, and 20 hours. Glucocorticoid (GC) sensitivity was measured by dexamethasone (DEX) inhibition of lipopolysaccharide (LPS)-induced <em>interleukin</em>-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) production in whole blood.

RESULTS

The PTSD patients showed no cortisol response after awakening and had lower daytime cortisol levels (F = 14.57, p <.001). Less DEX was required for cytokine suppression in PTSD patients (IL-6: t = -2.82, p =.01; TNF-alpha: t = 5.03, p <.001), reflecting higher GC sensitivity of pro-inflammatory cytokine production. The LPS-stimulated production of IL-6, but not TNF-alpha, was markedly increased in patients (IL-6: F = 10.01, p <.004; TNF-alpha: F =.89, p =.34).

CONCLUSIONS

In refugees with PTSD, hypocortisolism is associated with increased GC sensitivity of immunologic tissues. Whether this pattern reflects an adaptive mechanism and whether this is sufficient to protect from detrimental effects of low cortisol remains to be investigated.

OBJECTIVE

The term chronic widespread pain refers to a group of painful diseases of poorly understood pathophysiology. One major subgroup is fibromyalgia (FM), as defined by the criteria of the American College of Rheumatology. Among other hypotheses, a potential pathophysiologic role of cytokines in chronic widespread pain has been proposed. We undertook this study to investigate whether cytokine profiles differ in patients with chronic widespread pain and controls.

METHODS

We analyzed cytokine expression patterns in 40 patients with chronic widespread pain (26 of whom had FM), 40 age- and sex-matched healthy controls, and an additional <em>15</em> patients with chronic widespread pain who were recruited from a different center. Expression of messenger RNA (mRNA) for <em>interleukin</em>-2 (IL-2), IL-4, IL-8, IL-10, tumor necrosis factor alpha (TNFalpha), and transforming growth factor beta1 (TGFbeta1) in peripheral blood was analyzed using quantitative real-time polymerase chain reaction (PCR). Serum protein levels were measured by enzyme-linked immunosorbent assay.

RESULTS

We found significantly lower relative gene expression (P < 0.0001 for IL-4; P = 0.03 for IL-10) and lower levels of serum protein concentrations (P < 0.0001 for IL-4; P = 0.04 for IL-10) of the Th2 cytokines IL-4 and IL-10 in patients with chronic widespread pain than in the control group. This finding was corroborated in an additional group of <em>15</em> patients with chronic widespread pain. There were no significant differences between the groups in levels of mRNA for IL-2, IL-8, TNFalpha, or TGFbeta1. Protein data paralleled the real-time PCR results.

CONCLUSIONS

Chronic widespread pain is associated with a lack of antiinflammatory and analgesic Th2 cytokine activity, which may contribute to its pathogenesis.