Defective lymphotoxin-beta receptor-induced NF-kappaB transcriptional activity in NIK-deficient mice.

The role of NF-kappaB-inducing kinase (NIK) in cytokine signaling remains controversial. To identify the physiologic functions of NIK, we disrupted the NIK locus by gene targeting. Although NIK-/- mice displayed abnormalities in both lymphoid tissue development and antibody responses, NIK-/- cells manifested normal NF-kappaB DNA binding activity when treated with a variety of cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1), and lymphotoxin-beta (LTbeta). However, NIK was selectively required for gene transcription induced through ligation of LTbeta receptor but not TNF receptors. These results reveal that NIK regulates the transcriptional activity of NF-kappaB in a receptor-restricted manner.

Cutting edge: inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by toll-like receptor 2.

The agent of Lyme disease, Borrelia burgdorferi, produces membrane lipoproteins possessing potent inflammatory properties linked to disease pathology. The recent association of toll-like receptors (TLR) 2 and 4 with LPS responses prompted the examination of TLR involvement in lipoprotein signaling. The ability of human cell lines to respond to lipoproteins was correlated with the expression of TLR2. Transfection of TLR2 into cell lines conferred responsiveness to lipoproteins, lipopeptides, and sonicated B. burgdorferi, as measured by nuclear translocation of NF-kappaB and cytokine production. The physiological importance of this interaction was demonstrated by the 10-fold greater sensitivity of TLR2-transfected cells to lipoproteins than LPS. Futhermore, TLR2-dependent signaling by lipoproteins was facilitated by CD14. These data indicate that TLR2 facilitates the inflammatory events associated with Lyme arthritis. In addition, the widespread expression of lipoproteins by other bacterial species suggests that this interaction may have broad implications in microbial inflammation and pathogenesis.

IRAK-M is a novel member of the Pelle/interleukin-1 receptor-associated kinase (IRAK) family.

The interleukin-1 receptor-associated kinase (IRAK) was first described as a signal transducer for interleukin-1 (IL-1) and has later been implicated in signal transduction of other members of the Toll/IL-1 receptor family. We now report the identification and characterization of a novel IRAK-like molecule. In contrast to the ubiquitously expressed IRAK and IRAK-2, this new IRAK-like molecule is found mainly in cells of monomyeloic origin and is, therefore, designated IRAK-M. Although IRAK-M and IRAK-2 exhibit only a negligible autophosphorylation activity, they can reconstitute the IL-1 response in a 293 mutant cell line lacking IRAK. In addition, we show for the first time that members of the IRAK family are indispensable elements of lipopolysaccharide signal transduction. The discovery of IRAK-M adds another level of complexity to our understanding of signaling by members of the Toll/IL-1 receptor family.

Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2.

The life-threatening complications of sepsis in humans are elicited by infection with Gram-negative as well as Gram-positive bacteria. Recently, lipopolysaccharide (LPS), a major biologically active agent of Gram-negative bacteria, was shown to mediate cellular activation by a member of the human Toll-like receptor family, Toll-like receptor (TLR) 2. Here we investigate the mechanism of cellular activation by soluble peptidoglycan (sPGN) and lipoteichoic acid (LTA), main stimulatory components of Gram-positive bacteria. Like LPS, sPGN and LTA bind to the glycosylphosphatidylinositol-anchored membrane protein CD14 and induce activation of the transcription factor NF-kappaB in host cells like macrophages. We show that whole Gram-positive bacteria, sPGN and LTA induce the activation of NF-kappaB in HEK293 cells expressing TLR2 but not in cells expressing TLR1 or TLR4. The sPGN- and LTA-induced NF-kappaB activation was not inhibited by polymyxin B, an antibiotic that binds and neutralizes LPS. Coexpression together with membrane CD14 enhances sPGN signal transmission through TLR2. In contrast to LPS signaling, activation of TLR2 by sPGN and LTA does not require serum. These findings identify TLR2 as a signal transducer for sPGN and LTA in addition to LPS.

The type II IL-1 receptor interacts with the IL-1 receptor accessory protein: a novel mechanism of regulation of IL-1 responsiveness.

IL-1 binds to two types of receptors on the cell membrane, of which only type I (IL-1RI) transduces signals in concert with the coreceptor IL-1 receptor accessory protein (IL-1RAcP) while type II (IL-1RII) allegedly functions solely as ligand sink and decoy receptor without participating in IL-1 signaling. To investigate the regulatory role of IL-1RII on IL-1 responsiveness, a chimeric receptor encompassing the extracellular and transmembrane portions of IL-1RII and the cytoplasmic signal-transducing domain of IL-1RI was transfected into two murine EL-4-derived sublines that do or do not express IL-1RAcP, respectively. The chimeric receptor was able to transduce the IL-1 signal and induce IL-2 production only in the cell line which expressed IL-1RAcP, suggesting effective interaction between the extracellular domains of IL-1RII and IL-1RAcP in the presence of IL-1. The physical association of ligated IL-1RII with IL-1RAcP was proven by crosslinking experiments with radio-iodinated IL-1 and subsequent immunoprecipitations in normal human B cells and in EL-4 D6/76 cells transiently cotransfected with IL-1RII and IL-1RAcP, respectively. Based on these findings, it is proposed that upon IL-1 binding IL-1RII can recruit IL-1RAcP into a nonfunctional trimeric complex and thus modulate IL-1 signaling by subtracting the coreceptor molecule from the signaling IL-1RI. In this novel mechanism of coreceptor competition, the ratio between IL-1RII and IL-1RI becomes the central factor in determining the IL-1 responsiveness of a cell and the availability of IL-1RAcP becomes limiting for effective IL-1 signaling.

Human toll-like receptor 2 confers responsiveness to bacterial lipopolysaccharide.

Bacterial lipopolysaccharide (LPS) induces activation of the transcription factor nuclear factor kappaB (NF-kappaB) in host cells upon infection. LPS binds to the glycosylphosphatidylinositol (GPI)- anchored membrane protein CD14, which lacks an intracellular signaling domain. Here we investigated the role of mammalian Toll-like receptors (TLRs) as signal transducers for LPS. Overexpression of TLR2, but not TLR1, TLR4, or CD14 conferred LPS inducibility of NF-kappaB activation in mammalian 293 cells. Mutational analysis demonstrated that this LPS response requires the intracellular domain of TLR2. LPS signaling through TLR2 was dependent on serum which contains soluble CD14 (sCD14). Coexpression of CD14 synergistically enhanced LPS signal transmission through TLR2. In addition, purified recombinant sCD14 could substitute for serum to support LPS-induced TLR2 activation. LPS stimulation of TLR2 initiated an interleukin 1 receptor-like NF-kappaB signaling cascade. These findings suggest that TLR2 may be a signaling component of a cellular receptor for LPS.

Effects of overexpression of IL-1 receptor-associated kinase on NFkappaB activation, IL-2 production and stress-activated protein kinases in the murine T cell line EL4.

The association and activation of the IL-1 receptor-associated protein kinase (IRAK) to the IL-1 receptor complex is one of the earliest events detectable in IL-1 signal transduction. We generated permanent clones of the murine T cell line EL4 6.1 overexpressing human (h)IRAK to evaluate the role of this kinase in IL-1 signaling. Overexpression of hIRAK enhanced IL-1-stimulated activation of the transcription factor NFkappaB, whereas a truncated form (N-IRAK) specifically inhibited IL-1-dependent NFkappaB activity. In clones stably overexpressing hIRAK a weak constitutive activation of NFkappaB correlated with a low basal IL-2 production which was enhanced in an IL-1-dependent manner. Compared to the parental cell line the dose-response curve of IL-1-induced IL-2 production was shifted in both potency and efficacy. These results demonstrate that IRAK directly triggers NFkappaB-mediated gene expression in EL4 cells. Qualitatively different effects were observed for the IL-1-induced activation of stress-activated protein (SAP) kinases: permanent overexpression of IRAK did not affect the dose dependence but prolonged the kinetics of IL-1-induced activation of SAP kinases, suggesting that this signaling branch may be regulated by distinct mechanisms.

Effects of IL-1 receptor accessory protein on IL-1 binding.

Interleukin-1 (IL-1) is an important pro-inflammatory cytokine that exerts its diverse biological functions by binding to a receptor complex consisting of two transmembrane proteins, type I IL-1 receptor (IL-1RI) and IL-1 receptor accessory protein (IL-1RAcP). Both receptor chains are indispensable for IL-1 signaling, but only IL-1RI is able to bind the cytokine. The effects of IL-1RAcP on IL-1 binding are unclear. This study shows that although expression of IL-1RAcP does not alter the equilibrium dissociation constant of IL-1, it has an effect on the non-equilibrium binding modus, most likely due to changes in on/off rates. This defines an additional function of IL-1RAcP: it stabilizes the active IL-1 receptor complex.

The interleukin-1 receptor accessory protein (IL-1RAcP) is essential for IL-1-induced activation of interleukin-1 receptor-associated kinase (IRAK) and stress-activated protein kinases (SAP kinases).

Interleukin-1 (IL-1) is a central mediator of the immune system involved in acute and chronic inflammatory responses. Although the sequences of two types of IL-1 receptors are known, the exact molecular events resulting in signal transduction and coupling to downstream signaling elements remain unclear. The recently cloned IL-1 receptor accessory protein (IL-1RAcP) has been suggested as a co-receptor molecule for IL-1RI, supported by the observation that its expression correlates to IL-1 responsiveness. We transfected the EL-4 subline D6/76 with IL-1RAcP cDNA. This cell line is an IL-1 non-responder expressing IL-1RI but lacking constitutive IL-1RAcP expression. The expression of IL-1RAcP in EL-4 D6/76 was sufficient to restore IL-1-induced activation of interleukin-1 receptor-associated kinase and of stress-activated protein kinases, translocation of the transcription factors NFkappaB and IL-1 NF to the nucleus, and induction of IL-2 mRNA synthesis. These results proved that IL-1RAcP is an indispensible molecule in the IL-1 receptor signal transduction complex, necessary to link events on the plasma membrane level to downstream signaling pathways, allowing IL-1-dependent activation of transcription factors and gene expression.

A critical role for interleukin-1 receptor accessory protein in interleukin-1 signaling.

Interleukin-1 (IL-1) is a central molecule in inflammation and immune responses whose pleiotropic activities are mediated by the type I IL-1 receptor (IL-1RI). The IL-1RI alone on the cell surface is silent after binding of the ligand. We show that the recently identified IL-1RI accessory protein (IL-1RAcP) converts the silent into a fully functional IL-1RI complex. Although transfection of IL-1RAcP into IL-1RAcP-deficient EL4D6/76 cells did not alter the binding kinetics or dissociation constants of the 125I-labeled IL-1alpha/IL-1RI complex, a very early event, internalization of the activated receptor complex, and a late event, IL-1-stimulated IL-2 production, were successfully restored. Therefore, recruitment of IL-1RAcP is a critical early step in the signaling cascade mediated by the IL-1RI activation complex.

Overexpression of the bcl-2 oncogene in the mouse pre-B cell line SPGM-1 protects from apoptosis, but does not affect blocked B-lineage differentiation and lineage switch towards macrophage like cells.

The CD5(+) mouse pre-B cell line SPGM-1 is able to undergo lineage conversion towards a macrophage like cell type. Although one of the kappa-light chain alleles is rearranged in V-Jkappa4 configuration, no protein is expressed nor could the expression be induced. Infection with and expression of the human bcl-2 gene protected SPGM-1 cells from apoptosis, allowing the rearrangement of their second kappa-allele in V-Jkappa5 configuration. mRNA transcripts of the V-Jkappa regions were detected only in SPGM-1 x bcl-2 cells, but not in SPGM-1, while kappa-light chain protein was not found in any of the cell lines. The myeloid differentiation potential of SPGM-1 cells was not affected by overexpression of the human bcl-2 gene. Upon appropriate stimulation, SPGM-1xbcl-2 cells became enlarged, adhered to the plastic surfaces and lost their immunoglobulin mu heavy chain expression.

MyD88: an adapter that recruits IRAK to the IL-1 receptor complex.

IL-1 is a proinflammatory cytokine that signals through a receptor complex of two different transmembrane chains to generate multiple cellular responses, including activation of the transcription factor NF-kappaB. Here we show that MyD88, a previously described protein of unknown function, is recruited to the IL-1 receptor complex following IL-1 stimulation. MyD88 binds to both IRAK (IL-1 receptor-associated kinase) and the heterocomplex (the signaling complex) of the two receptor chains and thereby mediates the association of IRAK with the receptor. Ectopic expression of MyD88 or its death domain-containing N-terminus activates NF-kappaB. The C-terminus of MyD88 interacts with the IL-1 receptor and blocks NF-kappaB activation induced by IL-1, but not by TNF. Thus, MyD88 plays the same role in IL-1 signaling as TRADD and Tube do in TNF and Toll pathways, respectively: it couples a serine/threonine protein kinase to the receptor complex.

Co-expression of mRNA for type I and type II interleukin-1 receptors and the IL-1 receptor accessory protein correlates to IL-1 responsiveness.

Three cell surface molecules participate in Interleukin-1 (IL-1) binding and signal generation, the two distinct types of receptors (type I IL-1R and type II IL-1R) and the IL-1 receptor accessory protein (IL-1RAcP). Low surface expression hampers the detection of all three components on a protein level in most cell types, thus the highly sensitive RT-PCR was used to analyse the mRNA expression in a panel of 18 murine cell types of different hemopoietic lineages and fibroblasts. The transcription of both types of IL-1 receptors was detected in all cell lines tested. In most cell lines the IL-1RAcP was co-expressed with the IL-1 receptors, and only these lines responded to IL-1. However, in three cell lines no mRNA for the IL-1RAcP could be detected, and these cells did not respond to IL-1. These results suggest that the expression of the IL-1RAcP correlates with IL-1 responsiveness and they point to a pivotal role for the IL-1RAcP in IL-1 signal generation.

Some simplified methods for the photography of equipment and apparatus.

A simplified approach to the photography of equipment and apparatus is described. A number of examples are shown of the use of portable flash equipment, fluorescent light boxes and 'north-light' (daylight from North-facing windows), as light sources.

Tetanus immunity in older adults.

The frequency of tetanus has declined little in the past decade. The majority of cases occur in persons over 50 years of age. A sample of 225 blood donors, between the ages of 50 and 68, from a mixed rural-suburban county, were tested for tetanus antitoxin levels. Only 14% had protective levels. This low rate indicates the need for increased attention to tetanus immunizations in older adults.

Transdermal delivery of mepindolol and propranolol in normal man. 1st communication: study design, clinical and pharmacodynamic aspects.

In the present study the beta-adrenoreceptor blocking effects of acute and repeated transdermally delivered mepindolol (20 mg/9.8 cm2 patch) and propranolol (40 mg/9.8 cm2 patch) on supine and stimulated circulatory function (isoprenaline infusion, isometric hand grip, delayed auditory feed-back) in nine healthy male volunteers were assessed. The study was conducted in a placebo-controlled double-blind cross-over fashion, with three one-week treatment courses randomly allocated in a period-balanced fashion. Treatment phases were separated by a one-week wash-out phase. The placebo and mepindolol patch were well tolerated, whereas the propranolol patch caused skin irritation and itching in most subjects, and even vesicular lesions in 3/9 subjects. Acute and repeated 24 h application of the propranolol patch caused only small and clinically not relevant changes of heart rate and blood pressure. Acute application of the mepindolol patch induced only mild blunting of the diastolic blood pressure and heart rate responses to isoprenaline i.v. infusion at 8 h. However, the isoprenaline responses were nearly abolished after one week repeated 24 h application of the mepindolol patch, in a stable and protracted fashion. The circulatory responses to isometric handgrip and delayed auditory feedback tended to be reduced, but to a smaller extent than for the isoprenaline test. Repeated application of mepindolol via the investigational device for transdermal drug delivery (BIO TSD) thus resulted in stable and protracted beta-adrenoreceptor blockade.

Pharmacokinetics and pharmacodynamics of single oral doses of ibopamine, quinidine and their combination in normal man.

The pharmacokinetics and haemodynamics (phono- and impedance cardiography) of single oral doses of 200 mg ibopamine (SK&F 100168), 400 mg quinidine sulphate, and their combination, have been assessed in 6 healthy male volunteers. No significant differences in the mean pharmacokinetic parameters of either drug were seen between the single and combined doses. Ibopamine caused an increase in mean estimated stroke volume (SV +29% for the maximum change from baseline and +15% cumulatively over the first h) with no change in mean heart rate (HR) or QTc. Quinidine administered concomitantly blunted the response of SV. A tendency to a lower mean concentration of epinine early after administration of the combination may have contributed to the difference. Quinidine alone hardly affected SV (-3% from baseline over the first h), but it did increase mean HR (+6 beats.min-1) and mean QTc (+30 ms). These changes were sustained up to 8 h after dosing, and were not affected by concurrent ibopamine.

Temelastine, a novel histamine H1-receptor antagonist, does not induce oxidative hepatic enzyme activity in man.

Temelastine (SK&F 93944) is a novel histamine H1-receptor antagonist with a high degree of protein binding and predominant hepatic elimination. The pharmacokinetics of antipyrine were assessed in eight male normal subjects after single oral doses of 10 mg/kg antipyrine, administered prior to chronic dosing with temelastine 100 mg twice daily for 2 weeks, and 48 hours after the last dose of temelastine. After the first dose of antipyrine the mean maximum plasma concentration (Cmax) was 78.3 mumol/l (range: 60.3 to 95.8), the mean AUC (0-infinity) was 1330 mumol.h/l (range: 1030 to 2074), the mean apparent terminal disposition rate constant K was 0.0656 h-1 (range: 0.0428 to 0.0769) and the mean residence time (MRT) was 16.7 h on average (range: 14.4 to 24.1). After the second dose the mean Cmax, AUC (0-infinity) and MRT had slightly increased by on average 11, 5 and 7% respectively whereas the mean K had decreased by 9%. None of the differences between the means of the log-transformed parameters for second-first dose reached statistical significance. Temelastine therefore did not appear to induce hepatic oxidative enzyme activity in man, as judged by antipyrine as a model substance.