By using a single monoclonal antibody, a novel glycoprotein complex composed of at least three distinct bands was defined on the surface of mitogen- or alloantigen-stimulated T cells. These bands (210,000, 165,000, and 130,000 Mr) were not disulfide linked and could be radio-labeled with 125I or 35S-methionine and readily detected by immunoprecipitation with the monoclonal antibody A-1A5. Only approximately <em>20</em>% of normal T cells (E rosette positive) and approximately 47% non-T cells (E rosette negative) were reactive with A-1A5. However, upon activation of T cells, the amount of A-1A5 binding per cell and the percentage of positive cells significantly increased. This increase was most pronounced in the activated cell subpopulations currently undergoing cell division (S, G2, and M phases), which became 79% A-1A5 positive (after PHA stimulation) and 99% A-1A5 positive (in long-term culture with alloantigen and <em>IL</em>2). Resting lymphocytes contained only the 130,000 Mr band reactive with A-1A5. The two other bands (210,000 and 165,000 Mr) were markers for T cell activation and only appeared several days after T cell stimulation and became especially prominent after the addition of exogenous <em>IL</em> 2. All T lymphoblastoid cell lines tested expressed at least the lower bands (130,000 Mr), and the T cell line HSB also expressed one of the activation-related larger proteins (165,000 Mr). B lymphoblastoid cell lines expressed only a very weak lower band (130,000 Mr), and the cell line U-937 (in the monocyte-macrophage lineage) expressed only a single band (145,000 Mr) not aligned with any of the bands found on lymphoid cells. The estimated number of A-1A5 binding sites per cell was much higher on U-937 (11 X 10(5)), and generally higher on other cell lines of myeloid lineage (1 to 4 X 10(5)) than on lymphoid cell lines (0.2 to 1.3 X 10(5)).

When purified populations of human natural killer (NK) cells were tested for cytotoxic activity in the presence of partially purified preparations of human interleukin 2 (<em>IL</em> 2), a definite, dose-dependent linear increase in reactivity was observed. To determine whether such augmentation by <em>IL</em> 2 might reflect an important aspect of the physiologic regulation of NK activity, we examined the effects of monoclonal antibodies against human <em>IL</em> 2 on spontaneous NK activity. The presence of such antibodies during the 4-hr cytotoxicity assay resulted in significant inhibition of NK activity, and when the NK cells were pretreated for 16 to <em>20</em> hr with anti-<em>IL</em> 2, little or no activity remained. These data suggest that the spontaneous cytotoxic activity of NK cells is dependent on their continued exposure to <em>IL</em> 2. The reduction in NK activity resulting from treatment with anti-<em>IL</em> 2 could be at least partially restored by exposure to only low amounts of partially purified <em>IL</em> 2. These data have provided the basis for formulating a novel model of NK cell activation.

OBJECTIVE

To test the hypothesis of a diurnal variation in circulating levels of interleukin-6 (IL-6) and/or tumour necrosis factor-alpha (TNF-alpha) in rheumatoid arthritis and other inflammatory connective tissue diseases.

METHODS

Serum levels of IL-6 and TNF-alpha were measured at three hour intervals from 7:30 to 22:30 in 48 patients with different rheumatic diseases as well as ten healthy controls. In four of the patients with rheumatoid arthritis, serum IL-6 levels were measured before and after one week of treatment with prednisolone 15-20 mg daily.

RESULTS

IL-6 and TNF-alpha could not be detected in serum from healthy controls. However, serum IL-6 levels were substantially increased in patients with rheumatoid arthritis. Furthermore, patients with rheumatoid arthritis showed a statistically significant circadian variation in levels of IL-6. Peak values appeared in the morning and low values in the afternoon and evening. In contrast, levels were low and stable in other connective tissue diseases. Levels of TNF-alpha were low in patients with rheumatoid arthritis and high in patients with other connective tissue diseases, but without circadian rhythm. After treatment with prednisolone, levels of serum IL-6 decreased significantly, but the circadian rhythm remained.

CONCLUSIONS

The circadian rhythm of circulating IL-6 might correspond to the circadian rhythm of symptoms in rheumatoid arthritis. The diurnal variation of IL-6, and possibly other cytokines, might explain the conflicting results previously reported on the inter-relationship between circulating IL-6 levels and disease activity in rheumatoid arthritis.

In sepsis, several cell types (e.g., lymphocytes) undergo apoptosis and have the potential to harm the host if not cleared by professional phagocytes. Apoptotic cells display "eat me" signals such as phosphatidylserine that can be readily recognized by phagocytes. For full engulfment of these cells, binding to integrin alpha(v)beta(3), mediated by the bridging protein, milk fat globule epidermal growth factor-factor VIII (MFG-E8), is necessary. We hypothesized that, in sepsis, phagocytosis of apoptotic cells is impaired due to decreased MFG-E8 expression and that adoptive transfer of exosomes containing MFG-E8 is beneficial. Sepsis was induced in rats by cecal ligation and puncture (CLP) and MFG-E8 expression assessed by Western blot <em>20</em> h later. Dendritic cells were generated from bone marrow cells, and secreted exosomes were collected and injected into CLP animals. Plasma cytokines (enzyme-linked immunosorbent assay) and thymocyte apoptosis (TC-Ao, annexin V) were assessed. The ability of peritoneal macrophages from septic animals to engulf apoptotic cells was determined in an ex vivo phagocytosis assay. A 10-day survival study was conducted. Cecal ligation and puncture reduced MFG-E8 protein levels in the spleen and liver by 48% and 70%, respectively, and increased TC-Ao by 1.6-fold. Injection of MFG-E8-containing exosomes, however, led to a 33% reduced detection of TC-Ao, without directly inhibiting apoptosis. In fact, peritoneal macrophages from exosome-treated rats displayed a 2.8-fold increased ability to phagocytose apoptotic thymocytes. Inhibition of MFG-E8 before injection of exosomes completely abrogated the enhanced phagocytosis. Treatment with bone marrow dendritic cell-derived exosomes also reduced plasma tumor necrosis factor alpha and interleukin (<em>IL</em>)-6 levels and improved survival from 44% to 81%. We conclude that, by providing the indispensable factor MFG-E8 for complete engulfment of apoptotic cells, these exosomes lead to an attenuation of the systemic inflammatory response and overall beneficial effect in sepsis.

The noradrenergic system plays an integral role in the stress response and modulates expression of proinflammatory cytokines. Recent work from our laboratory and others has shown that certain stressors increase the expression of the proinflammatory cytokine interleukin-1beta (<em>IL</em>-1beta) in the hypothalamus and spleen. One goal of the following studies was to assess the role of norepinephrine in stress-elicited increases in <em>IL</em>-1beta. To do this, adult male Sprague-Dawley rats were injected with propranolol (<em>20</em> mg/kg i.p.) or desipramine (<em>20</em> mg/kg s.c.) and exposed to 80 inescapable footshocks (2.0 mA, 90 s variable ITI, 5 s each). We found that propranolol blocked the <em>IL</em>-1beta response to footshock in both the hypothalamus and the spleen, while the noradrenergic reuptake inhibitor desipramine significantly augmented the footshock-induced <em>IL</em>-1beta response in both of these sites. Our second goal was to determine whether these effects would also be blocked by administration of a putative microglial inhibitor (minocycline). Minocycline (40 mg/kg i.p.) completely reversed the footshock-induced increase in hypothalamic <em>IL</em>-1beta but had no effect on the <em>IL</em>-1beta response in the spleen. Moreover, lack of an effect of minocycline on conditioned fear responding suggests that the effect of this drug cannot be explained by nonspecific sedative properties produced by the drug. Together, these data suggest that NE powerfully modulates the hypothalamic and splenic <em>IL</em>-1beta response to stress, and that microglia may be a primary cellular source of central <em>IL</em>-1beta in response to footshock.

The hepatic drug-metabolizing cytochrome P-450 (CYP) enzymes are down-regulated during inflammation. In vitro studies with hepatocytes have shown that the cytokines released during inflammatory responses are largely responsible for this CYP repression. However, the signaling pathways and the cytokine-activated factors involved remain to be properly identified. Our research has focused on the negative regulation of CYP3A4 (the major drug-metabolizing human CYP) by interleukin 6 (<em>IL</em>-6) (the principal regulator of the hepatic acute-phase response). CYP3A4 down-regulation by <em>IL</em>-6 requires activation of the glycoprotein receptor gp130; however, it does not proceed through the JAK/STAT pathway, as demonstrated by the overexpression of a dominant-negative STAT3 factor by means of an adenoviral vector. The involvement of <em>IL</em>-6-activated kinases such as extracellular signal-regulated kinase ERK1/2 or p38 is also unlikely, as evidenced by the use of specific chemical inhibitors. It is noteworthy that <em>IL</em>-6 caused a moderated induction in the mRNA of the transcription factor C/EBPbeta (CCAAT-enhancer binding protein beta) and a marked increase in the translation of C/EBPbeta-LIP, a <em>20</em>-kDa C/EBPbeta isoform lacking a transactivation domain. Adenovirus-mediated expression of C/EBPbeta-LIP caused a dose-dependent repression of CYP3A4 mRNA, whereas overexpression C/EBPalpha and C/EBPb-LAP (35 kDa) caused a significant induction. Our results support the idea that <em>IL</em>-6 down-regulates CYP3A4 through translational induction of C/EBPbeta-LIP, which competes with and antagonizes constitutive C/EBP transactivators. From a clinical point of view, these findings could be relevant in the development of therapeutic cytokines with a less repressive effect on hepatic drug-metabolizing enzymes.

Hepatosteatosis is associated with increased expression of tumor necrosis factor alpha (TNF-alpha) and interleukin (<em>IL</em>)-12, major T helper (Th) 1 cytokines, and reduced hepatic natural killer T (NKT) cell numbers. The relationship between lipid accumulation, cytokine expression, and hepatic NKT cells is not known. This study was conducted to assess the role of <em>IL</em>-12 in the development of hepatic steatosis and its potential impact on liver NKT cells. Male C57Bl/6 wildtype (WT) and <em>IL</em>-12-deficient (<em>IL</em>-12(-/-)) mice were fed a choline-deficient diet (CDD) for 0, 10, or <em>20</em> weeks. CDD led to marked hepatosteatosis, reduced hepatic but not splenic NKT cell numbers and function, and increased hepatic expression of the T(h)1-type cytokines <em>IL</em>-12, interferon gamma (IFN-gamma), and TNF-alpha in WT mice. The absence of <em>IL</em>-12 resulted in similar CDD-induced hepatosteatosis, but preserved hepatic NKT cells and significantly reduced hepatic IFN-gamma and TNF-alpha expression. Treatment of CDD-fed mice with lipopolysaccharide led to a significant increase in hepatic <em>IL</em>-12 expression, and Kupffer cell (KC) depletion reduced liver <em>IL</em>-12 expression and restored NKT cells in CDD-induced fatty liver. Interestingly, KCs from CDD-fed mice failed to produce increased quantities of <em>IL</em>-12 upon activation in vitro when compared to similarly treated KCs from control fed mice, suggesting that secondary factors in vivo promote heightened <em>IL</em>-12 production. Finally, human livers with severe steatosis showed a substantial decrease in NKT cells.

CONCLUSIONS

Hepatosteatosis reduces the numbers of hepatic NKT cells in a KC-and IL-12-dependent manner. Our results suggest a pivotal and multifunctional role of KC-derived IL-12 in the altered immune response in steatotic liver, a process that is likely active within human nonalcoholic fatty liver disease.

<em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22, <em>IL</em>-24, <em>IL</em>-26, <em>IL</em>-28, and <em>IL</em>-29 are new members of the <em>IL</em>-10 interferon family. Monocytes are well-known sources of <em>IL</em>-19, <em>IL</em>-<em>20</em>, and <em>IL</em>-24. We demonstrated here that monocytes also expressed <em>IL</em>-29, and monocyte differentiation into macrophages (Mphi) or dendritic cells (DCs) strongly changed their production capacity of these cytokines. Maturation of DCs with bacterial stimuli induced high expression of <em>IL</em>-28/<em>IL</em>-29 and <em>IL</em>-<em>20</em>. Simulated T cell interaction and inflammatory cytokines induced <em>IL</em>-29 and <em>IL</em>-<em>20</em> in maturing DCs, respectively. Compared with monocytes, DCs expressed only minimal <em>IL</em>-19 levels and no <em>IL</em>-24. The differentiation of monocytes into Mphi reduced their <em>IL</em>-19 and terminated their <em>IL</em>-<em>20</em>, <em>IL</em>-24, and <em>IL</em>-29 production capacity. Like monocytes, neither Mphi nor DCs expressed <em>IL</em>-22 or <em>IL</em>-26. The importance of maturing DCs as a source of <em>IL</em>-28/<em>IL</em>-29 was supported by the much higher mRNA levels of these mediators in maturing DCs compared with those in CMV-infected fibroblasts, and the presence of <em>IL</em>-28 in lymph nodes but not in liver of lipopolysaccharide-injected mice. <em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22, <em>IL</em>-24, and <em>IL</em>-26 do not seem to affect Mphi or DCs as deduced from the lack of corresponding receptor chains. The significance of <em>IL</em>-<em>20</em> and <em>IL</em>-28/<em>IL</em>-29 coexpression in maturing DCs may lie in the broadly amplified innate immunity in neighboring tissue cells like keratinocytes. In fact, <em>IL</em>-<em>20</em> induced the expression of antimicrobial proteins, whereas <em>IL</em>-28/<em>IL</em>-29 enhanced the expression of toll-like receptors (TLRs) and the response to TLR ligands. However, the strongest response to TLR2 and TLR3 activation showed keratinocytes in the simultaneous presence of <em>IL</em>-<em>20</em> and <em>IL</em>-29.

The effects of vesicle size, lipid composition, and drug-to-lipid ratio on the biological activity of liposomal doxorubicin in mice have been investigated using a versatile procedure for encapsulating doxorubicin inside liposomes. In this procedure, vesicles exhibiting transmembrane pH gradients (acidic inside) were employed to achieve drug trapping efficiencies in excess of 98%. Drug-to-lipid ratios as high as 0.3:1 (wt:wt) could be obtained in a manner that is relatively independent of lipid composition and vesicle size. Egg phosphatidylcholine (EPC)/cholesterol (55:45; mol/mol) vesicles sized through filters with a <em>20</em>0-nm pore size and loaded employing transmembrane pH gradients to achieve a doxorubicin-to-lipid ratio of 0.3:1 (wt/wt) increased the LD50 of free drug by approximately twofold. Removing cholesterol or decreasing the drug-to-lipid ratio in EPC/cholesterol preparations led to significant decreases in the LD50 of liposomal doxorubicin whereas, the LD50 increased 4- to 6-fold when distearoylphosphatidylcholine was substituted for EPC. The results suggest that the stability of liposomally entrapped doxorubicin in the circulation is an important factor in the toxicity of this drug in liposomal form. In contrast, the antitumor activity of liposomal doxorubicin is not influenced dramatically by alterations in lipid composition. Liposomal doxorubicin preparations of EPC, EPC/cholesterol (55:45; mol:mol), EPC/egg phosphatidylglycerol (EPG)/cholesterol (27.5:27.5:45; mol:mol), and distearoylphosphatidylcholine/cholesterol (55:45; mol:mol) all demonstrated similar efficacy to that of free drug when given at doses of <em>20</em> mg/kg and below. Higher dose levels of the less toxic formulations could be administered, leading to enhanced increases in life span (<em>ILS</em>) values. Variations in vesicle size, however, strongly influenced the antitumor activity of liposomal doxorubicin. At a dose of <em>20</em> mg/kg, large EPC/cholesterol systems are significantly less effective than free drug (with <em>ILS</em> values of 65% and 145%, respectively). In contrast, small systems sized through filters with a 100-nm pore size are more effective than free drug, resulting in an <em>ILS</em> of 375% and a 30% long term (greater than 60 days) survival rate when administered at a dose of <em>20</em> mg/kg. Similar size-dependent effects are observed for distearoylphosphatidylcholine/cholesterol systems.

Enterotoxigenic Bacteroides fragilis (ETBF) secretes a <em>20</em>-kDa metalloprotease toxin termed B. fragilis toxin (BFT). ETBF disease in animals is associated with an acute inflammatory response in the intestinal mucosa, and lethal hemorrhagic colitis may occur in rabbits. In this study, we confirmed recent reports (J. M. Kim, Y. K. Oh, Y. J. Kim, H. B. Oh, and Y. J. Cho, Clin. Exp. Immunol. 123:421-427, <em>20</em>01; L. Sanfilippo, C. K. Li, R. Seth, T. J. Balwin, M. J. Menozzi, and Y. R. Mahida, Clin. Exp. Immunol. 119:456-463, <em>20</em>00) that purified BFT stimulates interleukin-8 (<em>IL</em>-8) secretion by human intestinal epithelial cells (HT29/C1 cells) and demonstrate that stimulation of <em>IL</em>-8 production is dependent on biologically active BFT and independent of serum. Induction of <em>IL</em>-8 mRNA expression occurs rapidly and ceases by 6 h after BFT treatment, whereas <em>IL</em>-8 secretion continues to increase for at least 18 h. Our data suggest that BFT-stimulated <em>IL</em>-8 secretion involves tyrosine kinase-dependent activation of nuclear factor-kappaB (NF-kappaB) as well as activation of the mitogen-activated protein kinases (MAPKs), p38 and extracellular signal-related kinase. Simultaneous activation of NF-kappaB and MAPKs appears necessary for secretion of <em>IL</em>-8 by HT29/C1 cells treated with BFT.

Apoptosis allows the clearance of unwanted cells from living tissues without causing inflammation. Processing of phagocytosed apoptotic cells yields Ags that access the cytosol and the MHC class I pathway of engulfing cells and are recognized by Ag-specific CTL. We show here that injection of apoptotic RMA cells, a syngeneic T cell lymphoma, into C57BL/6 mice results in priming of a functional and long-lasting tumor-specific immune response. Cross-priming of CTLs by apoptotic cells requires CD4+ T cell help. Apoptotic cells, however, are at least <em>20</em>-fold less immunogenic than nonreplicating live cells. Immunogenicity of apoptotic cells is proportional to the number of cells injected, correlates with the serum concentration of <em>IL</em>-10 and <em>IL</em>-1beta cytokines, and is enhanced in <em>IL</em>-10 knockout mice. Moreover, immunization with dendritic cells (DCs), but not macrophages (Mphi), pulsed with apoptotic cells primes tumor-specific CTLs and confers protection against a tumor challenge. Our findings demonstrate that tumor cells undergoing apoptosis are, though scarcely, immunogenic in vivo, outline the different roles of Mphi and DCs in the physiologic clearance of unwanted cells, and have implications in designing immunomodulating vaccines.

Inflammation-mediated dysregulation of the kynurenine pathway has been implicated as a contributor to a number of major brain disorders. Consequently, we examined the impact of a systemic inflammatory challenge on kynurenine pathway enzyme expression in rat brain. Indoleamine 2,3-dioxygenase (IDO) expression was induced in cortex and hippocampus following systemic lipopolysaccharide (LPS) administration. Whilst IDO expression was paralleled by increased circulating interferon (IFN)-gamma concentrations, IFN-gamma expression in the brain was only modestly altered following LPS administration. In contrast, induction of IDO was associated with increased central tumour necrosis factor (TNF)-alpha and interleukin (<em>IL</em>)-6 expression. Similarly, in cultured glial cells LPS-induced IDO expression was accompanied by increased TNF-alpha and <em>IL</em>-6 expression, whereas IFN-gamma was not detectable. These findings indicate that IFN-gamma is not required for LPS-induced IDO expression in brain. A robust increase in kynurenine-3-monooxygenase (KMO) expression was observed in rat brain 24h post LPS, without any change in kynurenine aminotransferase II (KAT II) expression. In addition, we report that constitutive expression of KAT II is approximately 8-fold higher than KMO in cortex and <em>20</em>-fold higher in hippocampus. Similarly, in glial cells constitutive expression of KAT II was approximately 16-fold higher than KMO, and expression of KMO but not KAT II was induced by LPS. These data are the first to demonstrate that a systemic inflammatory challenge stimulates KMO expression in brain; a situation that is likely to favour kynurenine metabolism in a neurotoxic direction. However, our observation that expression of KAT II is much higher than KMO in rat brain is likely to counteract potential neurotoxicity that could arise from KMO induction following an acute inflammation.

A chronically HIV-1-infected T cell clone (J1.1) derived from Jurkat cells was developed that possesses defects in CD3 signaling. This clone was phenotypically determined to be CD4- and express a reduced surface density of CD3 as compared with a pool of uninfected Jurkat clones. Although J1.1 could be induced with TNF-alpha to produce HIV-1 particles, stimulation via the CD3 (T3-Ti) complex, using mAb cross-linking, had no effect on viral production. Further investigation revealed that J1.1 secreted approximately <em>20</em>-fold less <em>IL</em>-2 than did uninfected Jurkat cells after anti-CD3 treatment. In addition, a separate defect in Ca2+ mobilization was noted in the HIV-1-infected J1.1 line when compared with uninfected Jurkat cells after anti-CD3 cross-linking. The cell line described offers a new model in which to study the mechanisms of several defects directly imposed by HIV-1 on CD3+ cells.

OBJECTIVE

Radiotherapy is used for the treatment of lung cancer, but at the same time induces acute pneumonitis and subsequent pulmonary fibrosis, where TGF-β signaling is considered to play an important role.

METHODS

We irradiated thoraces of C57BL/6 mice (single dose, <em>20</em> Gy) and administered them a novel small-molecule TGF-β receptor I serine/threonine kinase inhibitor (LY2109761) orally for 4 weeks before, during, or after radiation. Noninvasive lung imaging including volume computed tomography (VCT) and MRI was conducted 6, 16, and <em>20</em> weeks after irradiation and was correlated to histologic findings. Expression profiling analysis and protein analysis was conducted in human primary fibroblasts.

RESULTS

Radiation alone induced acute pulmonary inflammation and lung fibrosis after 16 weeks associated with reduced life span. VCT, MRI, and histology showed that LY2109761 markedly reduced inflammation and pulmonary fibrosis resulting in prolonged survival. Mechanistically, we found that LY2109761 reduced p-SMAD2 and p-SMAD1 expression, and transcriptomics revealed that LY2109761 suppressed expression of genes involved in canonical and noncanonical TGF-β signaling and downstream signaling of bone morphogenetic proteins (BMP). LY2109761 also suppressed radiation-induced inflammatory [e.g., interleukin (IL)-6, IL-7R, IL-8] and proangiogenic genes (e.g., ID1) indicating that LY2109761 achieves its antifibrotic effect by suppressing radiation-induced proinflammatory, proangiogenic, and profibrotic signals.

CONCLUSIONS

Small-molecule inhibitors of the TGF-β receptor I kinase may offer a promising approach to treat or attenuate radiation-induced lung toxicity or other diseases associated with fibrosis.

Hemophagocytic syndrome (HPS) is characterized by an uncontrolled and poorly understood activation of T-helper 1 (Th-1) lymphocytes and macrophages. We studied <em>20</em> patients with HPS secondary to infections, autoimmune disease, lymphoma, or cancer and observed that the concentrations of serum interleukin 18 (<em>IL</em>-18), a strong inducer of Th-1 responses, interferon gamma (IFN-gamma) production, and stimulation of macrophages and natural killer (NK) cells were highly increased in HPS but not in control patients. In contrast, concentrations of its natural inhibitor, the <em>IL</em>-18 binding protein (<em>IL</em>-18BP), were only moderately elevated, resulting in a high level of biologically active free <em>IL</em>-18 in HPS (4.6-fold increase compared with controls; P < .001). Free <em>IL</em>-18 but not <em>IL</em>-12 concentrations significantly correlated with clinical status and the biologic markers of HPS such as anemia (P < .001), hypertriglyceridemia, and hyperferritinemia (P < .01) and also with markers of Th-1 lymphocyte or macrophage activation, such as elevated concentrations of IFN-gamma and soluble <em>IL</em>-2 and tumor necrosis factor alpha (TNF-alpha) receptor concentrations. Despite high <em>IL</em>-18 elevation, in vitro NK-cell cytotoxicity was severely impaired in HPS patients, in part due to NK-cell lymphopenia that was observed in a majority of patients but also secondary to an intrinsic NK-cell functional deficiency. We concluded that a severe <em>IL</em>-18/<em>IL</em>-18BP imbalance results in Th-1 lymphocyte and macrophage activation, which escapes control by NK-cell cytotoxicity and may allow for secondary HPS in patients with underlying diseases.

BACKGROUND

Damage control orthopedic surgery has recently been advocated for the management of femoral shaft fractures in severely injured patients because surgical procedures were found to represent a second-hit phenomenon regarding the operative burden. It has been attempted to determine the operative burden by means of proinflammatory cytokines. In this study in clinically stable patients with multiple injuries, the effects induced by different types of primary fracture stabilization on the systemic release of proinflammatory cytokines were evaluated.

METHODS

This was a prospective, randomized, multicenter intervention study. Inclusion criteria were long bone shaft fracture of the lower extremity; age 18 to 65 years; Injury Severity Score>> 16 or more than three extremity injuries (Abbreviated Injury Scale [AIS] score of 2 or more) in association with another injury (AIS score of 2 or more); and thoracic AIS score < 4. After informed consent, randomization for the treatment of the femoral shaft fracture was performed at admission. Groups were as follows: group I degrees FN (primary, < 24 hours) intramedullary nailing, and group DCO (DCO, I degrees ex.fix.) damage control orthopedic surgery and external fixation. In DCO patients, measurements were also performed at the time of conversion to the intramedullary procedure (DCO II degrees FN). Parameters included clinical parameters and complications (acute respiratory distress syndrome, multiple organ failure, sepsis). From serially sampled central venous blood, the perioperative concentrations of interleukin <em>IL</em>-1, <em>IL</em>-6, and <em>IL</em>-8 were determined. RESULTS Thirty-five patients were included (I degrees FN, n = 17; DCO, n = 18). In I degrees FN-patients, a perioperative increase of <em>IL</em>-6 levels was measured (preoperatively, 55 +/- 33 pg/dL; 24 hours postoperatively, +254 +/- 55 pg/dL; p = 0.03), which was not found in subgroup DCO I degrees Ex.fix.: preoperatively, 71 +/- 42 pg/dL; 24 hours postoperatively, 68 +/- 34 pg/dL; not significant [NS] or in group DCO II degrees FN: preoperatively, 36 +/- 21 pg/dL; 24 hours postoperatively, +39 +/- 25 pg/dL; NS. Likewise, in I degrees FN patients, a perioperative increase of <em>IL</em>-8 levels was measured only at the 7-hour time point (preoperatively, 35 +/- 29 pg/dL; 7 hours postoperatively, 95 +/- 23 pg/dL; p < 0.05), which was not found in group DCO I degrees Ex.fix.: preoperatively, 43 +/- 38 pg/dL; 24 hours postoperatively, 69 +/- 39 pg/dL; NS or in group DCO II degrees FN: preoperatively, 25 +/- <em>20</em> pg/dL; 24 hours postoperatively, 36 +/- 29 pg/dL; NS. There were no differences in the complication rate in terms of acute respiratory distress syndrome, sepsis, or multiple organ failure.

CONCLUSIONS

In this prospective, randomized, multicenter study, a sustained inflammatory response was measured after primary (<24 hours) intramedullary femoral instrumentation, but not after initial external fixation or after secondary conversion to an intramedullary implant. These findings may become clinically relevant in patients at high risk of developing complications. It confirms previous studies in that damage control orthopedic surgery appears to minimize the additional surgical impact induced by acute stabilization of the femur.

Simian adenoviral vectors (SAd) offer an attractive alternative to standard human adenovirus serotype 5 (AdH5) subunit vaccination, due to pre-existing immunity affecting vaccine performance. We have used a mouse model of liver-stage malaria to test the efficiency of three chimpanzee-origin adenoviral vectors, AdC6, AdC7 and AdC9 containing ME.TRAP as an insert. AdC7 and AdC9 elicited strong immunogenicity ( approximately <em>20</em>% of CD8(+) T cells in spleen), equivalent to or outperforming AdH5 and inducing sterile protection in 92% (C9), 83% (H5 and C7) and 67% (C6) of the mice, providing the first evidence of single-dose protection to Plasmodium berghei. Protection was afforded by the SAd despite high levels of pre-existing immunity to AdH5. Phenotypic analysis showed that all adenoviral vectors (Ad) elicited CD8(+) T cell responses with an effector memory T cell (T(EM)) phenotype. By contrast, vaccination with poxviral vectors did not confer protection to P. berghei and induced a predominantly CD8(+) central memory T cell (T(CM)) response. Multifunctional CD8(+) T cell responses (co-expressing IFN-gamma, TNF-alpha and <em>IL</em>-2) were also induced by the Ad in higher percentages than the poxviral vectors. Our data suggest that T(EM) cells are important as a first line of defense against fast-replicating pathogens such as murine Plasmodium and demonstrate the potential of replication-defective SAd as future malaria vaccines for humans.

Sera from 61 Indian patients with visceral leishmaniasis caused by infection with Leishmania donovani were tested for the presence of T helper 1 (Th1) cell-(interferon-gamma [IFN-gamma]) and Th2 cell-associated cytokines (interleukin-4 [<em>IL</em>-4] and <em>IL</em>-10). The IFN-gamma activity was detected in 53%. <em>IL</em>-4 in 84%, and <em>IL</em>-10 in 56% of patient samples. Sera from 10 healthy Indian controls showed detectable IFN-gamma in 90%. <em>IL</em>-4 in 10%, and <em>IL</em>-10 in <em>20</em>%; corresponding percentages for sera from eight healthy American controls were 100%, 12%, and 0%, respectively. Quantitative data for the 61 patients compared with the 10 Indian controls indicated comparable mean levels of IFN-gamma, but three- and 13-fold increases in <em>IL</em>-10 and <em>IL</em>-4, respectively. Undetectable IFN-gamma activity, observed in 47% of patients, was associated with the presence <em>IL</em>-4 alone or in combination with <em>IL</em>-10 but not with <em>IL</em>-10 alone. In patients who had failed prior therapy (n = 29) compared with previously untreated patients (n = 32). IFN-gamma levels were 67% lower and <em>IL</em>-4 levels were two-fold higher, <em>IL</em>-10 activity was comparable. These results using peripheral blood support the presence of a suppressive Th2 cell-associated immune response in symptomatic Indian kala-azar and point to a possible role for <em>IL</em>-4.

A number of epidemiological studies have associated increased cardiopulmonary mortality and hospital admissions with episodes of high particulate air pollution. Inhaled particles, with a mass median aerodynamic diameter <10 microm (PM10) reach the lower respiratory tract where they are phagocytized by alveolar macrophages (AM). Depending on particle composition, exposed AM may produce reactive oxygen species and inflammatory mediators resulting in vascular permeability changes, airway constriction, tissue injury, and inflammation. In the present study human and rat AM were reacted with a range of environmental particles, including oil fly ash (OFA), diesel dust (DD), and ambient air particles (UAP) collected in four urban centers. AM were tested for a chemiluminescence response induced by the particles as well as <em>IL</em>-6 and TNF production. While OFA in a dose range of 1000-10 microg/2-3 x 10(5) AM caused acute cytotoxicity above 100 microg in both human and rat AM (LDH release at 2 hr), DD and UAP were found to be nontoxic in the same dose range. However, after <em>20</em> hr of coincubation, UAP concentrations >167 microg/ml were also cytotoxic. Subcytotoxic concentrations of OFA induced a strong immediate chemiluminescence response by AM. A small but significant chemiluminescence response was induced by two out of three UAP tested, while no chemiluminescence was generated in response to DD. The magnitude of particle-induced chemiluminescence was not predictive of a cytokine response by either human or rat AM. TNF and <em>IL</em>-6 production was strongly induced by UAP over a range of noncytotoxic concentrations of particles. OFA induced only small amounts of TNF in a subset of human AM preparations, but not in rat AM. The AM cytokine response to UAP was partly inhibitable by polymyxin B, but not by the iron chelator deferoxamine, indicating that endotoxins but not transitional iron were cytokine-inducing moieties in the tested UAP preparations.

OBJECTIVE

To evaluate the safety and pharmacokinetics of multiple infusions of a humanized anti-interleukin-6 (IL-6) receptor antibody, MRA, in patients with rheumatoid arthritis (RA).

METHODS

In an open label trial, 15 patients with active RA were intravenously administered 3 doses (2, 4, or 8 mg/kg) of MRA biweekly for 6 weeks, and pharmacokinetics were assessed. Patients continued on MRA treatment for 24 weeks, and were then assessed for safety and efficacy.

RESULTS

The treatment was well tolerated at all doses with no severe adverse event. Increased total serum cholesterol was detected as an MRA related reaction in 10/15 (66%) patients. There was no statistically significant difference in the frequency of adverse events among the 3 dose groups. There were no new observations of antinuclear antibody or anti-DNA antibody, and no anti-MRA antibody was detected. The T1/2 increased with repeated doses and as the dose increased. T1/2 after the 3rd dose of 8 mg/kg reached 241.8 +/- 71.4 h. In 12/15 (80%) patients whose serum MRA was detectable during the treatment period, objective inflammatory indicators such as C-reactive protein, erythrocyte sedimentation rate, and serum amyloid A were completely normalized at 6 weeks, although there was no statistically significant difference in efficacy among the 3 dose groups. Nine of 15 patients achieved ACR 20 at 6 weeks. At 24 weeks, 13 patients achieved ACR 20 and 5 achieved ACR 50.

CONCLUSIONS

Repetitive treatment with MRA was safe and normalized acute phase response in patients with RA. Optimal dosing schedule was not defined in this small study, but maintenance of serum MRA concentration seemed important to achieve efficacy.

BACKGROUND

How microbial infections exacerbate immune complex glomerulonephritis remains speculative. Toll-like receptors (TLRs) may be involved in this phenomenon, because TLRs have potent immunostimulatory functions when exposed to selected pathogen-associated molecules.

METHODS

We addressed this issue by characterizing the expression of TLR1-9 in MRLlpr/lpr mice that spontaneously develop immune complex glomerulonephritis as part of a systemic lupus-like autoimmune syndrome.

RESULTS

Five-week-old healthy MRLlpr/lpr mice expressed TLR3 mRNA in kidneys at comparable levels as in the spleen, while all other TLRs were expressed at low levels in the kidney. In <em>20</em>-week-old nephritic MRLlpr/lpr mice, renal mRNA levels had increased for TLR1-9. Renal TLR mRNA originated at least in part from glomeruli as evidenced by real-time RT-PCR from laser capture microdissected glomeruli. Immunostaining for TLR3, TLR7 and TLR9 revealed their expression by F4/80-positive infiltrating macrophages in <em>20</em>-week-old nephritic MRLlpr/lpr mice. In addition, TLR3 localized to glomerular mesangial cells. Cultured mesangial cells expressed TLR1-4 and TLR6, while murine macrophages expressed TLR1-9. TNF-alpha and IFN-gamma induced TLR2, TLR3 and TLR6 mRNA in mesangial cells, while they down-regulated TLR1-9 mRNA in macrophages. Stimulation of both cell types with ligands for TLR1-4, TLR5, TLR7 and TLR9 induced <em>IL</em>-6 production consistent with their respective TLR expression patterns. TNF-alpha and IFN-gamma enhanced ligand-induced <em>IL</em>-6 production in both cell types irrespective of their modulatory effect on respective TLR mRNA levels.

CONCLUSIONS

Thus, cell-type-specific expression and regulation of TLRs may be involved in infection-associated exacerbation of immune complex glomerulonephritis of MRLlpr/lpr mice.

OBJECTIVE

Behçet's disease (BD) is asystemic immunoinflammatory disorder and the aetiopathogenesis is to be specified. Cytokines play a role in immune response and in many inflammatory diseases. The aim of this case-control study is to investigate serum pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha, interleukin-1beta (IL-1beta), soluble IL-2 receptor (sIL-2R), IL-6, and chemokine IL-8 levels in patients with BD. We also determined the end product of lipid peroxidation (malondialdehyde (MDA)) in BD patients as an index for oxidative stress.

METHODS

A total of 37 patients (19 men, 18 women) with BD (active, n = 17; inactive, n = 20) and 20 age-matched and sex-matched healthy control subjects (11 men, nine women) included in this cross-sectional, blinded study. Serum TNF-alpha, IL-1beta, sIL-2R, IL-6 and IL-8 levels were determined by a spectrophotometer technique using the immulite chemiluminescent immunometric assay. Lipid peroxidation was evaluated by Wasowicz et aL The levels of cytokines and lipid peroxidation in the active period were compared with the inactive period of the disease. Results are expressed as mean +/- standard error.

RESULTS

IL-1beta levels were below the detection limits of the assay (< 5 pg/ml) in all samples. Mean levels of MDA (8.1+/-0.7 micromol/l), sIL-2R (800+/-38 U/ml), IL-6 (12.6+/-1.1 pg/ml), IL-8 (7.2+/-0.4 pg/ml), and TNF-alpha (7.9+/-0.5 pg/ml) in active BD patients were significantly higher than those in inactive patients (4.3+/-0.5 micromol/l, p < 0.01; 447+/-16 U/ml, p < 0.001; 8.3+/-0.6 pg/ml, p = 0.006; 5.3+/-0.1 pg/ml, p < 0.001; and 5.1 0.2 pg/ml, p < 0.001; respectively) or control subjects (2.1+/-0.2 micromol/l, p < 0.001; 446+/-20 U/ml, p < 0.001; 6.4+/-0.2 pg/ml, p < 0.001; 5.4+/-0.1 pg/ml, p < 0.001; and 4.7+/-0.1 pg/ml, p < 0.001, respectively). On the contrary, only the mean IL-6 level was significantly different between inactive BD and control subjects (p = 0.02). All acute phase reactants were significantly higher in active BD than in inactive period (for each, p < 0.01).

CONCLUSIONS

High levels of sIL-2R, IL-6, IL-8 and TNF-alpha indicate the activation of immune system in BD. Serum sIL-2R, IL-6, IL-8 and TNF-alpha seem to be related to disease activity. Increased lipid peroxidation suggests oxidative stress in BD and therefore tissue damage in such patients. Amelioration of clinical manifestations would be envisaged by targeting these cytokines, chemokines and lipid peroxidation with pharmacological agents.

Nanoparticles can be administered via nasal, oral, intraocular, intratracheal (pulmonary toxicity), tail vein and other routes. Here, we focus on the time-dependent translocation and potential damage of TiO(2) nanoparticles on central nervous system (CNS) through intranasal instillation. Size and structural properties are important to assess biological effects of TiO(2) nanoparticles. In present study, female mice were intranasally instilled with two types of well-characterized TiO(2) nanoparticles (i.e. 80 nm, rutile and 155 nm, anatase; purity>99%) every other day. Pure water instilled mice were served as controls. The brain tissues were collected and evaluated for accumulation and distribution of TiO(2), histopathology, oxidative stress, and inflammatory markers at post-instillation time points of 2, 10, <em>20</em> and 30 days. The titanium contents in the sub-brain regions including olfactory bulb, cerebral cortex, hippocampus, and cerebellum were determined by inductively coupled plasma mass spectrometry (ICP-MS). Results indicated that the instilled TiO(2) directly entered the brain through olfactory bulb in the whole exposure period, especially deposited in the hippocampus region. After exposure for 30 days, the pathological changes were observed in the hippocampus and olfactory bulb using Nissl staining and transmission electron microscope. The oxidative damage expressed as lipid peroxidation increased significantly, in particular in the exposed group of anatase TiO(2) particles at 30 days postexposure. Exposure to anatase TiO(2) particles also produced higher inflammation responses, in association with the significantly increased tumor necrosis factor alpha (TNF-alpha) and interleukin (<em>IL</em>-1 beta) levels. We conclude that subtle differences in responses to anatase TiO(2) particles versus the rutile ones could be related to crystal structure. Thus, based on these results, rutile ultrafine-TiO(2) particles are expected to have a little lower risk potential for producing adverse effects on central nervous system. Although understanding the mechanisms requires further investigation, the present results suggest that we should pay attention to potential risk of occupational exposure for large-scaled production of TiO(2) nanoparticles.

Recent studies revealed that two novel interleukin (<em>IL</em>)-12-related cytokines, <em>IL</em>-23 and <em>IL</em>-27, have potent antitumor activities. However, the antitumor effects were mainly evaluated in relatively highly immunogenic tumors and have not been fully evaluated against nonimmunogenic or poorly immunogenic tumors. In this study, we investigated the antitumor efficacies of <em>IL</em>-23 and <em>IL</em>-27 on poorly immunogenic B16F10 melanoma and found that the antitumor responses mediated by <em>IL</em>-23 and <em>IL</em>-27 were clearly different. In syngeneic mice, mouse single-chain (sc) <em>IL</em>-23-transfected B16F10 (B16/<em>IL</em>-23) tumors exhibited almost the same growth curve as B16F10 parental tumor about until day <em>20</em> after tumor injection and then showed growth inhibition or even regression. In contrast, sc<em>IL</em>-27-transfected B16F10 (B16/<em>IL</em>-27) tumors exhibited significant retardation of tumor growth from the early stage. In vivo depletion assay revealed that the antitumor effect of B16/<em>IL</em>-23 was mainly mediated by CD8+ T cells and IFN-gamma whereas that of B16/<em>IL</em>-27 mainly involved natural killer cells and was independent of IFN-gamma. We also found that antitumor effects of B16/<em>IL</em>-23 and B16/<em>IL</em>-27 were synergistically enhanced by treatment with <em>IL</em>-18 and <em>IL</em>-12, respectively. Furthermore, B16/<em>IL</em>-23-vaccinated mice developed protective immunity against parental B16F10 tumors but B16/<em>IL</em>-27-vaccinated mice did not. When combined with prior in vivo depletion of CD25+ T cells, 80% of B16/<em>IL</em>-23-vaccinated mice completely rejected subsequent tumor challenge. Finally, we showed that the systemic administration of neither <em>IL</em>-23 nor <em>IL</em>-27 induced such intense toxicity as <em>IL</em>-12. Our data support that <em>IL</em>-23 and <em>IL</em>-27 might play a role in future cytokine-based immunotherapy against poorly immunogenic tumors.