In order to analyze the effects of interleukin (IL) 6 on human in vitro Ig production B lymphocytes were activated by Staphylococcus aureus Cowan strain I (SAC) in the presence of low concentrations of IL2 (1 U/ml) and dexamethasone (10(-7) M). Previously we showed that this model of B cell response is completely monocyte dependent. We here demonstrate that, under these experimental conditions, IL6 is able to replace monocytes and stimulate Ig production provided IL1 is also present. Dose-effect curves show that these two monokines act synergistically. This synergy is demonstrable only in the presence of dexamethasone, when B lymphocytes are activated (by SAC) and when T cell help (provided by IL2) is present. It results in the production of both IgM and IgG. Both IL1 and IL6 have to be present during the first 48 h of culture to exert an optimal effect. These results show that IL6 may act on early (as well as on late) stages of normal B lymphocyte differentiation. Moreover, glucocorticosteroids potentiate the synergistic effect of IL1 and IL6 on their B lymphocyte target, an effect comparable to that exerted on hepatocytes.

1. Hypothermia was investigated as a parameter indicating the severity of the acute effects of lipopolysaccharides (LPS) in BALB/c mice, and was compared with the induction of serum levels of IL1 beta, TNF alpha and IL6. 2. Hypothermia induced by low doses of LPS (10-50 micrograms/mouse IP LPS E. coli 0111:B4) peaked at 2 hr after LPS and then either plateaued (50 micrograms) or declined. LPS, 100 and 300 mu, induced greater degrees of hypothermia that plateaued or continued to increase with time for 8 hr. Higher doses of LPS induced similar levels of hypothermia until 4 hr but then continued to increase markedly until 8 hr. 3. TNF alpha levels peaked early (1-2 hr) and declined rapidly, IL6 levels peaked at 3 hr and then declined slowly, and IL1 beta levels peaked at 4 hr, declined at lower doses of LPS, plateaued at higher doses and continued to slowly increase at highest doses. 4. The peak levels of the cytokines (IL1 beta up to 4 hr) and hypothermia (4 hr) increased in relation to the dose of LPS and maximum responses were apparently achieved in all cases at 300-1000 micrograms LPS. 5. A similar parallel between hypothermia and induction of cytokines was observed in C57BL6 and OF1 mice, which were good and poor responders to LPS, respectively, and with the more potent Shigella dysenteria LPS in BALB/c mice. 6. In conclusion, hypothermia is a useful parameter for indicating the strength of the acute effects of LPS. Further studies are necessary to determine whether or not the cytokines studied here play a causative role in hypothermia.

We generated a novel mouse model of spinal cord injury (SCI) by hemisection of the right L1 lumbar spinal cord, measured the permeability of the blood-spinal cord barrier (BSCB), and tested the hypothesis that tumor necrosis factor alpha (TNF alpha) penetrates the injured BSCB by an enhanced transport system. SCI produced stereotypical sensorimotor deficits resembling the classically described Brown-Seqúard syndrome. Disruption of the BSCB was reflected by increased spinal cord uptake of radiolabeled albumin from blood; this was transient (immediately after SCI) and confined to the lumbar spinal cord. By contrast, specific increase in the entry of TNF alpha was detected in brain, cervical, thoracic, and lumbar spinal cord at 1 week after SCI, in addition to its immediate and transient increase consistent with barrier disruption. Lack of a second peak of increase in the entry of IL1 beta further supported the specificity of the TNF alpha response. Moreover, enhanced uptake of radiolabeled TNF alpha was suppressed by excess non-radiolabeled TNF alpha, indicating competition of entry via the known transport system for TNF alpha. Therefore, upregulation of the transport system after SCI probably mediates the increased permeation of TNF alpha across the BSCB. Enhanced entry of TNF alpha at 1 week after SCI was concurrent with sensorimotor and gait improvement of the mouse. We conclude that SCI by lumbar hemisection activates the transport system for TNF alpha at the BBB and suggest that selective permeation of TNF alpha may facilitate functional recovery.

In sIBM, an inflammatory process mediated by cytotoxic T cells and cytokines in conjunction with a degenerative process, deposits of beta amyloid and misfolded proteins appear to be the main culprits in disease pathogenesis. IL-1β may play a key role because it is upregulated in sIBM myofibers, co-localizes with Amyloid Precursor Protein (APP) and promotes the production of APP and amyloid deposits. We performed a small, pilot study to examine whether anakinra, an IL1 receptor antagonist could benefit sIBM patients. Four patients with biopsy-proven sIBM received anakinra for a mean period of 7.7 months. No improvement in muscle strength or stabilization was noted in any of the patients based on grip strength and MRC measurements. The treatment failure may be due to insufficiency of anakinra to suppress the intramuscular IL1, the short study period, or the irrelevance of IL1 in the disease process.

A human astrocytoma cell line, U373, and its subclones showed a high proliferative response to IL1 alpha or IL1 beta with concomitant IL6 production and IL6 mRNA accumulation. IL1 itself raised neither the cAMP level nor the intracellular Ca2+ level nor did it induce phosphatidylinositol (PI) breakdown. Chorela toxin (CT) and pertussis toxin (PT)-pretreatment markedly inhibited IL1-induced proliferation, while CT enhanced IL1-induced IL6 mRNA expression significantly. Drugs raising cellular cAMP level or cAMP analogues augmented IL1-mediated IL6 mRNA expression but much less. Although cAMP is not directly involved in the IL1 action, cAMP thus has a modulatory effect on IL1-mediated IL6 gene activation.

BACKGROUND

Rheumatoid arthritis (RA), an autoimmune disease, is characterized by pronounced inflammation and cell accumulation within affected joints. Beneficial effects of active ingredients of the astragalus root (Radix astrogali) in treatment of immunological diseases have been previously observed, but the mechanisms are not well understood. This study aims to evaluate therapeutic effects and the mechanisms of astragalus polysaccharides (APS) on adjuvant-induced arthritis (AA) in rats.

METHODS

Effects of treatment of AA rats with increasing doses of APS, Tripterygium glycosides (positive control) and saline (negative control) on swelling, arthritic index, synovial cell accumulation, serum concentrations of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β), synovial apoptosis and immunostaining for Bcl-2 and Bax were determined.

RESULTS

APS treatment reduced cell accumulation, swelling and arthritic index of the joints and serum concentrations of TNF-α and IL1-β in a dose-dependent manner in AA rats. Synovial cell apoptosis was elevated in response to APS treatment and accompanied by increased staining for pro-apoptotic Bax protein and decreased staining for anti-apoptotic Bcl-2 protein.

CONCLUSIONS

APS treatment reduced multiple indices of arthritis in rats with AA. Results support further investigation of therapeutic effects of APS in treatment of RA and other autoimmune diseases.

The oxidative stress responsive transcription factor nuclear factor-kappa B (NF-kappa B) consists of a p50 (50 kDa) and p65/RelA (65 kDa) component and can be activated in vitro by TNF alpha, IL1 beta, hydrogen peroxide and oxygen radicals. All of the above factors are also known to be elevated at certain times after transient global ischemia. The present study was performed to determine if NF-kappa B was activated in vivo by transient global forebrain ischemia. Adult male rats were subjected to 30 min of 4-vessel occlusion (4-VO) and sacrificed at selected post-ischemic time points. Levels of NF-kappa B p50 and p65 subunits were determined by immunocytochemistry, Western blot and electrophoretic mobility-shift analysis. The enhancer complex was also confirmed by immuno-gel-shift analysis. Specific labeling of DNA strand breaks and DNA fragmentation was examined in situ by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Western blot analysis of hippocampus showed induction of p50 and p65. A time course of NF-kappa B induction in hippocampus showed a p50-specific band at 6 h that increased in intensity over 12, 48 h and then decreased by 96 h post-ischemia. Immunocytochemistry revealed at 24 h post-ischemia that p65 and p50 immunoreactivity was present in neuronal nuclei of hippocampal CA1 neurons as well as all other hippocampal regions and several other forebrain regions which were not vulnerable to transient forebrain ischemia. At 72 h post-ischemia, nuclear NF-kappa B immunoreactivity had disappeared in all brain areas except in hippocampal CA1 neurons which were degenerating. No evidence for DNA fragmentation as revealed by TUNEL staining could be observed at 24 h. However, at 72 h, hippocampal CA1 neurons were heavily labeled. The results of this study demonstrate that global forebrain ischemia causes a transient activation of NF-kappa B in many forebrain regions. NF-kappa B remains persistently activated in the vulnerable hippocampal CA1 sector. Because of the persistent activation of NF-kappa B in these neurons, the possibility exists that NF-kappa B has a role in programmed cell death in hippocampal CA1 neurons.

The etiology of Parkinson's disease is not known. Nevertheless a significant body of biochemical data from human brain autopsy studies and those from animal models point to an on going process of oxidative stress in the substantia nigra which could initiate dopaminergic neurodegeneration. It is not known whether oxidative stress is a primary or secondary event. Nevertheless, oxidative stress as induced by neurotoxins 6-hydroxydopamine and MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) has been used in animal models to investigate the process of neurodegeneration with intend to develop antioxidant neuroprotective drugs. It is apparent that in these animal models radical scavengers, iron chelators, dopamine agonists, nitric oxide synthase inhibitors and certain calcium channel antagonists do induce neuroprotection against such toxins if given prior to the insult. Furthermore, recent work from human and animal studies has provided also evidence for an inflammatory process. This expresses itself by proliferation of activated microglia in the substantia nigra, activation and translocation of transcription factors, NF kappa-beta and elevation of cytotoxic cytokines TNF alpha, IL1-beta, and IL6. Both radical scavengers and iron chelators prevent LPS (lipopolysaccharide) and iron induced activation of NF kappa-B. If an inflammatory response is involved in Parkinson's disease it would be logical to consider antioxidants and the newly developed non-steroid anti-inflammatory drugs such as COX2 (cyclo-oxygenase) inhibitors as a form of treatment. However to date there has been little or no success in the clinical treatment of neurodegenerative diseases per se (Parkinson's disease, ischemia etc.), where neurons die, while in animal models the same drugs produce neuroprotection. This may indicate that either the animal models employed are not reflective of the events in neurodegenerative diseases or that because neuronal death involves a cascade of events, a single neuroprotective drug would not be effective. Thus, consideration should be given to multi-neuroprotective drug therapy in Parkinson's disease, similar to the approach taken in AIDS and cancer therapy.

The form of (1-3)-beta-D glucan found in the cell walls of the anamorphic Trichocomaceae that grow on damp building materials is considered to have potent toxic and inflammatory effects on cells of the respiratory system. It is also considered to have a potential role in the development of non-allergenic respiratory health effects. While human studies involving experimental exposures all point to the inflammatory potential of pure curdlan, a linear (1-3)-beta-D glucan in a triple helix configuration, animal experiments result in conflicting conclusions concerning the inflammatory potency of this glucan. However, because mice appear to be a better model than guinea pigs for exploring the respiratory effects of curdlan and because molecular mechanisms associated with this glucan remain largely unknown, we conducted further work to clarify the role of curdlan on the inflammatory response using our mouse model of lung disease. This study used in situ hybridization (ISH) to probe dectin-1 mRNA transcription with a digoxigenin-labeled cDNA probe, with reverse transcription (RT)-PCR based arrays used to measure inflammation gene and receptor transcriptional responses. Also, immunohistochemistry (IHC) was used to probe dectin-1 as well as anti-mouse Ccl3, Il1-alpha, and TNF-alpha expression to evaluate dose and time-course (4 and 12 h) postexposure (PE) response patterns in the lungs of intratracheally instilled mice exposed to a single 50 mul dose of curdlan at 10(-7), 10(-8), 10(-9), and 10(-10) M/animal (=4 mug to 4 ng curdlan/kg lung wt). Dectin-1 mRNA transcription and expression was observed in bronchiolar epithelium, alveolar macrophages (AMs), and alveolar type II cells (ATIIs) of lungs exposed to 4 mug to 40 ng curdlan/kg lung wt, at both time points. Compared to controls, array analysis revealed that 54 of 83 genes assayed were significantly modulated by curdlan. mRNA transcription patterns showed both dose and time dependency, with highest transcription levels in 10(-7) and 10(-8) M treatment animals, especially at 4-h PE. Nine gene mRNA transcripts (Ccl3, Ccl11, Ccl17, Ifng, Il1alpha, Il-20, TNF-alpha, Tnfrsf1b, and CD40lg) were significantly expressed at all doses suggesting they may have a central role in immunomodulating curdlan exposures. IHC revealed Ccl3, Il1-alpha, and TNF-alpha expression in bronchiolar epithelium, AMs and ATIIs illustrate the important immunomodulatory role that these cells have in the recognition of, and response to glucan. Collectively, these results confirm the inflammatory nature of curdlan and demonstrate the complex of inflammation-associated gene responses induced by (1-3)-beta-D glucan in triple helical forms. These observations also provide a biological basis for the irritant and inflammatory response to curdlan observed in humans and animals in experimental studies.

We reported previously that the core oligosaccharide region of the lipopolysaccharide (LPS) is essential for optimal adhesion of Actinobacillus pleuropneumoniae, an important swine pathogen, to respiratory tract cells. Rough LPS and core LPS mutants of A. pleuropneumoniae serotype 1 were generated by using a mini-Tn10 transposon mutagenesis system. Here we performed a structural analysis of the oligosaccharide region of three core LPS mutants that still produce the same O-antigen by using methylation analyses and mass spectrometry. We also performed a kinetic study of proinflammatory cytokines production such as interleukin (IL)-6, tumor necrosis factor-alpha, IL1-beta, MCP-1, and IL8 by LPS-stimulated porcine alveolar macrophages, which showed that purified LPS of the parent strain, the rough LPS and core LPS mutants, had the same ability to stimulate the production of cytokines. Most interestingly, an in vitro susceptibility test of these LPS mutants to antimicrobial peptides showed that the three core LPS mutants were more susceptible to cationic peptides than both the rough LPS mutant and the wild type parent strain. Furthermore, experimental pig infections with these mutants revealed that the galactose (Gal I) and d,d-heptose (Hep IV) residues present in the outer core of A. pleuropneumoniae serotype 1 LPS are important for adhesion and overall virulence in the natural host, whereas deletion of the terminal GalNAc-Gal II disaccharide had no effect. Our data suggest that an intact core-lipid A region is required for optimal protection of A. pleuropneumoniae against cationic peptides and that deletion of specific residues in the outer LPS core results in the attenuation of the virulence of A. pleuropneumoniae serotype 1.

The mechanisms for the initiation of immune reactions in the central nervous system are poorly understood. In this report, we describe the presence of intercellular adhesion molecule-1 (ICAM-1) and Lgp 55 (suggested mouse homologue of human intercellular adhesion molecule-2, ICAM-2) on the surface of brain microvessel endothelium (EN) cells and show in vitro induction of ICAM-1 molecules on EN cells with pro-inflammatory cytokines. ICAM-1 expression was detected using flow cytometry analysis with biotinylated anti-ICAM-1 antibody (YN1/1.7.4). Lgp 55 expression was characterized using PA3 monoclonal antibody. According to our results, 30-40% of the non-activated brain EN cells expressed ICAM-1 and 15-20% expressed Lgp 55 molecules. The ICAM-1 molecule expression was increased after the activation of the cells with recombinant murine gamma interferon (IFN-gamma), tumor necrosis factor (TNF-alpha), and interleukin-1 alpha (IL1-alpha) in a dose-dependent manner. The increased ICAM-1 expression was detected as early as 2 h following the cytokine treatment and reached its maximum after 24 h. Transforming growth factor-beta (TGF-beta) did not influence the expression of ICAM-1 molecule. Lgp 55 molecule does not seem to be regulated by pro-inflammatory cytokines. ICAM-1 and Lgp 55 expression was found to be polarized on the luminal surface of EN by confocal laser microscopy suggesting accessibility for leukocytes. Inducible ICAM-1 expression may play a critical role in formation of inflammatory reactions inside the central nervous system.

Bacterial magnetic particles (BacMPs) are efficient platforms of proteins for surface display systems. In this study, mononuclear cells from peripheral blood were separated using BacMPs expressing protein A on the BacMP membrane surface (protein A-BacMPs), which were complexed with the Fc fragment of anti-mouse IgG antibody. The procedure of positive selection involves incubation of mononuclear cells and mouse monoclonal antibodies against different cell surface antigens (CD8, CD14, CD19, CD20) prior to treatment with protein A-BacMP binding with rabbit anti-mouse IgG secondary antibodies. Flow cytometric analysis showed that approximately 97.5 +/- 1.7% of CD19(+) and CD20(+) cells were involved in the positive fraction after magnetic separation. The ratio of the negative cells in the negative fraction was approximately 97.6 +/-1.4%. This indicates that CD19(+) and CD20(+) cells can be efficiently separated from mononuclear cells. Stem cell marker (CD34) positive cells were also separated using protein A-BacMP binding with antibody. May-Grunwald Giemsa stain showed a high nuclear/cytoplasm ratio, which indicates a typical staining pattern of stem cells. The separated cells had the capability of colony formation as hematopoietic stem cells. Furthermore, the inhibitory effect of magnetic cell separation on CD14(+) cells was evaluated by measurement of cytokine in the culture supernatant by ELISA when the cells were cultured with or without lipopolysaccharide (LPS). The induction of IL1-beta, TNFalpha, and IL6 was observed in the presence of 1 ng/mL LPS in all fractions. On the other hand, in the absence of LPS, BacMPs had little immunopotentiation to CD14(+) cells as well as that of artificial magnetic particles, although TNFalpha and IL6 were slightly induced in the absence of LPS in the positive fraction.

Oxidized low-density lipoprotein (oxLDL)-regulated secretion of inflammatory cytokines in smooth muscle cells (SMCs) is regarded as an important step in the progression of atherosclerosis; however, its underlying mechanism remains unclear. This study investigated the role of toll-like receptor 4 (TLR4) in oxLDL-induced expression of inflammatory cytokines in SMCs both in vivo and in vitro. We found that the levels of TLR4, interleukin 1-β (IL1-β), tumor necrosis factor-α (TNFα), monocyte chemoattractant protein 1 (MCP-1) and matrix metalloproteinase-2 (MMP-2) expression were increased in the SMCs of atherosclerotic plaques in patients with femoral artery stenosis. In cultured primary arterial SMCs from wild type mice, oxLDL caused dose- and time-dependent increase in the expression levels of TLR4 and cytokines. These effects were significantly weakened in arterial SMCs derived from TLR4 knockout mice (TLR4-/-). Moreover, the secretion of inflammatory cytokines was blocked by TLR4-specific antibodies in primary SMCs. Ox-LDL induced activation of p38 and NFκB was also inhibited in TLR4-/- primary SMCs or when treated with TLR4-specific antibodies. These results demonstrated that TLR4 is a crucial mediator in oxLDL-induced inflammatory cytokine expression and secretion, and p38 and NFκB activation.

BACKGROUND

The immunoreceptor tyrosine-based activation motif (ITAM) pathway provides osteoclast co-stimulatory signals and regulates proliferation, survival and differentiation of effector immune cells. In the osteoclast, the receptors Triggering Receptor Expressed on Myeloid cells 2 (TREM2) and Osteoclast Associated Receptor (OSCAR) and their respective adaptor proteins, DAP12 and FcRγ mediate ITAM signals and induce calcium signaling and the crucial transcription factor, NFATc1. In rheumatoid arthritis (RA), OSCAR expression by monocytes is inversely correlated with disease activity. Additionally, serum levels of OSCAR are reduced in RA patients versus healthy controls suggesting that expression and secretion or cleavage of soluble (s) OSCAR is immune modulated. Recent data suggest that endothelial cells may also be a source of OSCAR.

METHODS

ITAM receptors, their adaptor proteins, and NFATc1 and cathepsin K were detected in human synovial tissues by immunohistochemistry. Synovial tissues from patients with active RA were compared with tissue from patients in remission, osteoarthritis (OA) patients and healthy individuals. OSCAR was measured by immunoassay in synovial fluids recovered from active RA and OA patients. Endothelial cells were cultured with or without 5 ng/mL TNF-α or IL-1β over 72 hours. Temporal expression of OSCAR mRNA was assessed by qRT PCR and OSCAR protein in the supernatant was measured by ELISA.

RESULTS

Significantly higher (P < 0.05) NFATc1-positive inflammatory cell aggregates were found in active RA tissues than in healthy synovial tissue. Similarly, the percentage of OSCAR, FcRγ, DAP12 and TREM2 positive cells was significantly higher in active RA tissues compared to the healthy synovial tissue. Notably, OSCAR was strongly expressed in the microvasculature of the active RA tissues (9/9), inactive RA (8/9) weakly in OA (4/9) but only in the lumen of healthy synovial tissue (0/8). OSCAR levels were detected in synovial fluids from both RA (47 to 152 ng/mL) and OA (112 to 145 ng/mL) patients. Moreover, OSCAR mRNA expression and soluble OSCAR release was stimulated by TNF-α and IL1-β in cultured endothelial cells.

CONCLUSIONS

Increased levels of ITAM related factors were present in synovial tissue from active RA joints compared to OA and healthy joints. OSCAR was strongly expressed by the vasculature of active RA patients and membrane bound and soluble OSCAR was stimulated by inflammatory mediators in endothelial cells in vitro.

BACKGROUND

Biomarkers, such as C-reactive protein [CRP] and procalcitonin [PCT], are insufficiently sensitive or specific to stratify patients with sepsis. We investigate the prognostic value of pancreatic stone protein/regenerating protein (PSP/reg) concentration in patients with severe infections.

METHODS

PSP/reg, CRP, PCT, tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL1-β), IL-6 and IL-8 were prospectively measured in cohort of patients ≥ 18 years of age with severe sepsis or septic shock within 24 hours of admission in a medico-surgical intensive care unit (ICU) of a community and referral university hospital, and the ability to predict in-hospital mortality was determined.

RESULTS

We evaluated 107 patients, 33 with severe sepsis and 74 with septic shock, with in-hospital mortality rates of 6% (2/33) and 25% (17/74), respectively. Plasma concentrations of PSP/reg (343.5 vs. 73.5 ng/ml, P < 0.001), PCT (39.3 vs. 12.0 ng/ml, P < 0.001), IL-8 (682 vs. 184 ng/ml, P < 0.001) and IL-6 (1955 vs. 544 pg/ml, P < 0.01) were significantly higher in patients with septic shock than with severe sepsis. Of note, median PSP/reg was 13.0 ng/ml (IQR: 4.8) in 20 severely burned patients without infection. The area under the ROC curve for PSP/reg (0.65 [95% CI: 0.51 to 0.80]) was higher than for CRP (0.44 [0.29 to 0.60]), PCT 0.46 [0.29 to 0.61]), IL-8 (0.61 [0.43 to 0.77]) or IL-6 (0.59 [0.44 to 0.75]) in predicting in-hospital mortality. In patients with septic shock, PSP/reg was the only biomarker associated with in-hospital mortality (P = 0.049). Risk of mortality increased continuously for each ascending quartile of PSP/reg.

CONCLUSIONS

Measurement of PSP/reg concentration within 24 hours of ICU admission may predict in-hospital mortality in patients with septic shock, identifying patients who may benefit most from tailored ICU management.

Alternative splicing generates protein isoforms that are conditionally or differentially expressed in specific tissues. The discovery of factors that control alternative splicing might clarify the molecular basis of biological and pathological processes. We found that IL1-α-dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III-transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel-interacting protein (KCNIP4). The alternative KCNIP4 isoform cannot interact with the γ-secretase complex, resulting in modification of γ-secretase activity, amyloid precursor protein processing, and increased secretion of β-amyloid enriched in the more toxic Aβ x-42 species. Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels. This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer's disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain.

Gene expression profiles permit analysis of host immune response at the transcriptome level. We used the Pax gene Blood RNA (PAX) System and Affymetrix microarrays (HG-U133A&B) to survey profiles in basic military trainees and to classify them as healthy, febrile respiratory illness (FRI) without adenovirus, FRI with adenovirus, and convalescent from FRI with adenovirus. We assessed quality metrics of RNA processing for microarrays. Class prediction analysis discovered nested sets of transcripts that could categorize the phenotypes with optimized accuracy of 99% (nonfebrile vs febrile, P<0.0005), 87% (healthy vs convalescent, P=0.001), and 91% (febrile without vs with adenovirus, P<0.0005). The discovered set for classification of nonfebrile vs febrile patients consisted of 40 transcripts with functions related to interferon induced genes, complement cascades, and TNF and IL1 signaling. The set of seven transcripts for distinguishing healthy vs convalescent individuals included those associated with ribosomal structure, humoral immunity, and cell adhesion. The set of 10 transcripts for distinguishing FRI without vs with adenovirus had functions related to interferon induced genes, IL1 receptor accessory protein, and cell interactions. These results are the first in vivo demonstration of classification of infectious diseases via host signature transcripts and move us towards using the transcriptome in bio-surveillance.

Blocking the binding of TNF-alpha to TNF receptor subtype-1 (TNFR1) is an important strategy for the treatment of rheumatoid arthritis (RA). We recently succeeded in developing a TNFR1-selective antagonistic TNF mutant, R1antTNF. Here, we report the anti-inflammatory effects of R1antTNF in a murine collagen-induced arthritis model. To improve the in vivo stability of R1antTNF, we first engineered PEG (polyethylene glycol)-modified R1antTNF (PEG-R1antTNF). In prophylactic protocols, PEG-R1antTNF clearly improved the incidence, and the clinical score of arthritis due to its long plasma half-life. Although, the effect of PEG-R1antTNF on the incidence and production of IL1-beta was less than that of the existing TNF-blocking drug Etanercept, its effect on severity was almost as marked as Etanercept. Interestingly, in therapeutic protocols, PEG-R1antTNF showed greater therapeutic effect than Etanercept. These data suggest that the anti-inflammatory effects of PEG-R1antTNF depend on the stage of arthritis. Recently, there has been much concern over the reactivation of viral infection caused by TNF blockade. Unlike Etanercept, PEG-R1antTNF did not reactivate viral infection. Together, these results indicate that selective inhibition of TNF/TNFR1 could be effective in treating RA and that PEG-R1antTNF could serve as a promising anti-inflammatory drug for this purpose.

Interleukin-1 beta (IL1β) is a proinflammatory cytokine that mediates arthritic pathologies. Our objectives were to evaluate pain and limb dysfunction resulting from IL1β over-expression in the rat knee and to investigate the ability of local IL1 receptor antagonist (IL1Ra) delivery to reverse-associated pathology. IL1β over-expression was induced in the right knees of 30 Wistar rats via intra-articular injection of rat fibroblasts retrovirally infected with human IL1β cDNA. A subset of animals received a 30 µl intra-articular injection of saline or human IL1Ra on day 1 after cell delivery (0.65 µg/µl hIL1Ra, n = 7 per group). Joint swelling, gait, and sensitivity were investigated over 1 week. On day 8, animals were sacrificed and joints were collected for histological evaluation. Joint inflammation and elevated levels of endogenous IL1β were observed in knees receiving IL1β-infected fibroblasts. Asymmetric gaits favoring the affected limb and heightened mechanical sensitivity (allodynia) reflected a unilateral pathology. Histopathology revealed cartilage loss on the femoral groove and condyle of affected joints. Intra-articular IL1Ra injection failed to restore gait and sensitivity to preoperative levels and did not reduce cartilage degeneration observed in histopathology. Joint swelling and degeneration subsequent to IL1β over-expression is associated limb hypersensitivity and gait compensation. Intra-articular IL1Ra delivery did not result in marked improvement for this model; this may be driven by rapid clearance of administered IL1Ra from the joint space. These results motivate work to further investigate the behavioral consequences of monoarticular arthritis and sustained release drug delivery strategies for the joint space.

Our recent studies indicate that lipocortin 1 (LC1), a putative second messenger protein for the anti-inflammatory steroids in peripheral tissues, may also contribute to the regulatory actions of the glucocorticoids on the hypothalamo-pituitary-adrenal axis. In the present study we have used in vitro and in vivo models to compare the effects of adrenalectomy, LC1 and dexamethasone on the cytokine-induced secretion of the 41-amino acid corticotrophin releasing factor (CRF-41) and arginine vasopressin (AVP) by the rat hypothalamus. In addition, western blot analysis was used to examine the influence of dexamethasone on the expression of LC1 in the hypothalamus. In vitro, interleukins- (IL-) 1 alpha (100 and 200 pg/ml), 1 beta (0.5 and 1.0 ng/ml) and 8 (0.25-1.0 ng/ml) readily initiated the release of CRF-41 and AVP from hypothalami removed from intact rats. IL-6 (10 and 20 ng/ml) was also an effective CRF-41 secretagogue but, unlike the other interleukins tested, it was ineffective with regard to AVP. Adrenalectomy 7-14 days prior to autopsy increased significantly (p < 0.01) the magnitude of the CRF-41 responses to IL-1 alpha, IL-1 beta and IL-6 but not to IL-8. In contrast however, while hypothalamic tissue from adrenalectomized rats, unlike that from intact animals, responded to IL-6 (5-20 ng/ml) with a pronounced hypersecretion of AVP, the AVP responses to IL-1 alpha and IL-1 beta were largely unaffected by adrenalectomy as too were those to IL-8. The marked increases in CRF-41 and AVP release from hypothalami from adrenalectomized rats initiated in vitro by IL1 alpha, IL-1 beta, IL-6 and IL-8 were readily overcome by preincubation of the tissue with dexamethasone (10(-7) M). In addition, the steroid caused 'externalization' of two species of immunoreactive (ir-) LC1 (37 and 58 kDa) by the hypothalamic cells but failed to influence the total LC1 content of the tissue. The inhibitory effects of dexamethasone on the cytokine-induced release of CRF-41 in vitro were mimicked by LC1 (10 ng/ml) which alone had no effect on the basal release of the peptide. However, unlike dexamethasone, LC1 failed to influence the concomitant release of AVP from the hypothalamic tissue elicited by IL1 alpha, IL-1 beta or IL-8 and potentiated that evoked by IL-6.(ABSTRACT TRUNCATED AT 400 WORDS)

The clinical course of cystic fibrosis (CF) varies considerably among patients carrying the same CF-causing gene mutation. Additional genetic modifiers may contribute to this variability. As airway inflammation is a key component of CF pathophysiology, we investigated whether major cytokine variants represent such modifiers in young CF patients. We tested 13 polymorphisms in 8 genes that play a key role in the inflammatory response: tumor necrosis factor, lymphotoxin alpha, interleukin (IL) 1B, IL1 receptor antagonist, IL6, IL8, IL1beta 1 (TGFB1), for an association with lung disease progression and nutritional status in 329 CF patients. Variants in the TGFB1 gene at position +869T/C demonstrated a significant association with lung function decline. A less pronounced rate of decline in forced expiratory volume in 1 sec (FEV(1)) and forced vital capacity (FVC) were observed in patients heterozygous for TGFB1 +869 (+869CT), when compared to patients carrying either TGFB1 +869TT or +869CC genotypes. These findings support the concept that TGFB1 gene variants appear to be important genetic modifiers of lung disease progression in CF.

Meningococcal lipopolysaccharide (LPS) is of crucial importance for the pathogenesis of invasive infection. We show that sialylation and elongation of the alpha-chain effectively shields viable unencapsulated Neisseria meningitidis from recognition by human dendritic cells (DC). In contrast, beta- and gamma- chain of the LPS carbohydrate moiety play only a minor role in the interaction with DC. The protective function of the LPS for the bacteria can be counteracted in vivo by phase variation of the lgtA gene encoding LPS glycosyltransferase A. Capsule expression protects N. meningitidis efficiently from recognition and phagocytosis by DC independent of the LPS structure. Despite the significant impact of LPS composition on the adhesion and phagocytosis of N. meningitidis no differences were found in terms of cytokine levels secreted by DC for IL1-beta, IL-6, IL-8, TNF-alpha, IFN-gamma and GM-CSF. However, significantly lower levels of the regulatory mediator IL-10 were induced by encapsulated strains in comparison to isogenic unencapsulated derivatives. IL-10 secretion was shown to depend on phagocytosis because poly alpha-2,8 sialic acid did not influence IL-10 secretion. The use of truncated LPS isoforms in vaccine preparations can therefore not only result in attenuation but also in more efficient targeting of DC.

Delta 9-Tetrahydrocannabinol (THC) injection modulates immune cell function, but the significance of this in altering host resistance to infection is not understood. In addition, exposure to THC and other drugs of abuse during infection is associated with an acute mortality syndrome. We examined the effect of THC injection on the survival of mice infected with Legionella pneumophila (Lp). Mice given two injections of THC (8 mg/kg)-one 24 hr before and the second 24 hr after a sublethal Lp infection-experienced acute collapse and death. The drug injection after infection caused death; deaths occurred within 30 min after the injection, and neither one nor two drug injections before infection resulted in death. The THC-induced mortality resembled cytokine-mediated shock in both kinetics and symptoms; therefore, sera from drug-treated animals were measured for the acute-phase cytokines tumor necrosis factor (TNF) and interleukin 6 (IL6). The level of each cytokine was significantly elevated by THC treatment, suggesting a role in the observed mortality. To directly test this role, mice were administered a single injection of either anti-TNF alpha, anti-IL6, or a mixture of anti-IL1 alpha and -IL1 beta antibodies 1 hr before the second THC injection. Results showed that each antibody treatment protected the mice, with anti-IL6 being the most effective. Fluctuations in blood granulocytes levels also supported a role of acute-phase cytokines in THC-induced mortality. These results show that THC injection increases the blood levels of acute-phase cytokines in infected animal and that these elevated levels, at least in part, account for the mortality induced by THC injection.