Binding of lipopeptide to CD14 induces physical proximity of CD14, TLR2 and TLR1

Lipoproteins or lipopeptides (LP) are bacterial cell wall components detected by the innate immune system. For LP, it has been shown that TLR2 is the essential receptor in cellular activation. However, molecular mechanisms of LP recognition are not yet clear. We used a FLAG-labeled derivative of the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine (Pam(3)CSK(4)) to study the roles of CD14, TLR2 and TLR1 in binding and signaling of LP and their molecular interactions in human cells. The activity of Pam(3)CSK(4)-FLAG was TLR2 dependent, whereas the binding was enabled by CD14, as evaluated by flow cytometry and confocal microscopy. Using FRET and FRAP imaging techniques to study molecular associations, we could show that after Pam(3)CSK(4)-FLAG binding, CD14 and Pam(3)CSK(4)-FLAG associate with TLR2 and TLR1, and TLR2 is targeted to a low-mobility complex. Thus, LP binding to CD14 is the first step in the LP recognition, inducing physical proximity of CD14 and LP with TLR2/TLR1 and formation of the TLR2 signaling complex.     

Functional and molecular characterization of a monoclonal antibody against human interleukin 2

Human recombinant interleukin 2 (r-IL2) was used as an immunizing antigen to yield a murine monoclonal antibody (mAb) termed BO-7. Although the antibody binds to r-IL2 more avidly, it also reacted strongly with IL2 from natural sources in an enzyme-linked immunosorbent assay (ELISA), allowing the detection of the purified lymphokine at sensitivity levels closely approaching those found with the IL2 biological assay. Binding to the antigen is specific, as deduced from the close correlation of ELISA immunoreactivity with IL2 biological activity and from immunoblot analysis of electrophoretically separated IL2 from various sources. Binding studies with synthetic IL2-derived peptides revealed the location of the epitope, which is recognized by mAb BO-7: A peptide representing amino acid residues 59-72 (peptide 84) is strongly reactive with the antibody, while an overlapping peptide (residues 48-69) is not. Peptide 84, moreover, can be applied for immunopurification of mAb BO-7 and competes for binding to the antibody with the intact IL2 molecule. In turn, another monoclonal anti-IL2 antibody (35H10), showing the same reactivity pattern with peptides, competes with mAb BO-7 for binding to IL2. The application of mAb BO-7 as a specific reagent for the quantitation of IL2 in a sandwich-type ELISA is demonstrated.     

Methods for in vivo haematoporphyrin derivative quantification: a review

Photodynamic therapy is a new treatment for early carcinomas. Although undergoing phase 1/2 clinical assays, clinical indications for this therapy remain rare mainly because of the approximate dosimetry of HPD uptake by tumour tissues in human beings.In this review we present the potential interest and limits of both direct fluorescence detection or dosimetry of HPD and in vivo measurements of singlet oxygen, produced during photodynamic therapy. Clinical applications of such measurements should represent one of the main conditions for the future development of photodynamic therapy.

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Résumé La photochimiothérapie est un nouveau traitement des cancers débutants. Alors que des essais cliniques de phase 1–2 sont entrepris, les indications pour ce type de traitement demeurent rares, principalement du fait d'une dosimétrie approximative de la captation de l'hématoporphyrine dérivée par les tissus cancéreux humains. La fluorescence émise par l'HPD peut Être utilisée in-vivo pour un diagnostique topographique de la répartition de l'HPD, mais aussi le dosage quantitatif des espèces fluorescentes présentes dans le mélange HPD. Le dosage de l'oxygène singulet, généré lors de la réaction photochimique, est nettement plus difficile à réaliser mais a été proposé pour le dosage in-vivo des formes porphyriniques actives présentes dans le milieu. Les applications cliniques de telles mesures représentent une condition essentielle pour le developpement de la photochimiothérapie car à côté des possibilités de diagnotiques offertes par l'analyse de la répartition intratumorale de l'HPD, un dosage précis permettrait d'optimiser le moment du traitement, arbitrairement fixé aujourd'hui à 72 heures.

     

In situ dormancy of B lymphocytes programmed for an IgE antibody response and their sudden release from unresponsiveness under in vitro conditions

Priming of CBA/J mice with different doses of antigen has aprofound effect on the ratio of IgE versus IgG antibodies appearingupon Immunization. Repeated injections of minute doses induceIgG and high titers of IgE antibodies. Large doses elicit ahigh IgG but a very low IgE antibody titer. In order to studythe modalities for activation and inactivation of IgE-producingB cells, an in vitro culture system was established in whichspleen cells from animals primed with keyhole limpet hemocyaninwere re-stliulated with antigen. In contrast to the expectationfrom the in vivo situation, spleen cells from animals Immunizedwith large doses of antigen and virtually lacking IgE antibodiesproduce high amounts of IgE antibodies upon re-stimulation invitro. The titers in spleen cell cultures from mice primed withminute doses remain proportional to the response measured asserum antibodies. In accordance with the induction of high amountsof IgE antibodies in spleen cell cultures from mice primed withlarge doses, the frequency of IgE antibody-secreting cells wasraised drastically, 1000-fold. The in vitro response is a trueanamnestic response. The sudden appearance in high frequencyof IgE antibody-forming cells among spleen cells isolated fromprimed mice which have high IgG but virtually no IgE antibodytiters is as yet unexplained and the origin of the B memorycells has not yet been traced. The answer might be crucial forour understanding of the down-regulation of the IgE Immune responses.     

Cost comparison of sevoflurane with isoflurane anesthesia in arthroscopic menisectomy surgery

PURPOSE: To determine the "real world" cost of sevoflurane compared with isoflurane in balanced general anesthesia for daycare arthroscopic menisectomy, we prospectively investigated perioperative drug requirement and expense as well as recovery time. METHODS: Following intravenous induction, 40 consenting adult patients randomly received either sevoflurane- or isoflurane-based anesthesia with a standardized gas inflow rate of 3 l x min. Recovery was assessed in the postanesthetic recovery room (PARR) in a double-blind manner at 15 min intervals using the Aldrete scoring system until patients met discharge criteria. RESULTS: Patient demographics, anesthetic duration, volatile potency and adjunct drug requirements were similar in the two groups. Total perioperative drug cost per patient was CAN$38.10+/-10.13 (mean +/- SD) for the sevoflurane group and $23.87+/-6.59 for the isoflurane group (P<0.01). Although the nonvolatile drug cost was comparable between the two groups, the volatile drug cost per patient was $19.40+/-8.80 for sevoflurane and $4.50+/-1.90 for isoflurane (P<0.01). This four-fold sevoflurane-to-isoflurane cost difference was the product of two ratios, both based on the volume of liquid anesthetic: the ratio of consumption, 2.1; and the ratio of institutional price, 2.1. Intraoperative hemodynamic response, time until discharge from the PARR and incidences of postoperative nausea and vomiting did not significantly differ between the two groups. CONCLUSIONS: When used to maintain equipotent balanced general anesthesia for daycare arthroscopic menisectomy, volatile consumption and cost were greater for sevoflurane compared with isoflurane. Nonvolatile perioperative drug cost and recovery times were similar, however, in the two groups.     

MRI findings in Hirayama’s disease: flexion-induced cervical myelopathy or intrinsic motor neuron disease?

Hirayama’s disease is a benign juvenile form of focal amyotrophy affecting the upper limbs. Previous studies have suggested that the disorder is a neck flexion induced cervical myelopathy. We report clinical and magnetic resonance imaging findings in nine patients with Hirayama’s disease. Cervical imaging of seven patients revealed spinal cord changes consisting of focal atrophy and foci of signal alterations. On neck flexion a forward movement and mild reduction in the anteroposterior diameter of the lower cervical cord against the vertebral bodies was noted in affected individuals as well as in five normal controls. In contrast to earlier reports, none of our patients showed complete obliteration of the posterior subarachnoid space. Measurement of the anteroposterior spinal cord diameter in each vertebral segment (C4–C7) revealed no significant differences in the degree of spinal cord flattening between the two groups. Furthermore, two of our patients had significant degenerative changes in the cervical spine (disc herniation, retrospondylosis) contralateral to the clinically affected side. These degenerative changes resulted in a marked cord compression on neck flexion but were not associated with ipsilateral clinical abnormalities or spinal cord alterations. Our results argue against a flexion-induced cervical myelopathy and support the view that Hirayama’s disease is an intrinsic motor neuron disease. Received: 15 March 1999 Received in revised form: 25 May 1999 Accepted: 1 June 1999     

Red wine inhibits the cell-mediated oxidation of LDL and HDL

OBJECTIVE: We compared the in vitro effects of red wine, white wine and ethanol on the cell mediated oxidation of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) by three frequently-used assays. METHODS: LDL and HDL isolated from normolipidemic human serum were incubated with J774.A1 macrophages in DMEM with copper, with or without red wine, white wine or ethanol (equivalent to 0.2 mg ethanol/ml). Lipoprotein oxidation was assessed by conjugated diene formation as measured by changes in absorbance at 234 nm (deltaA234), thiobarbituric-acid-reactive-substance (TBARS) production and trinitrobenzene-sulfonic-acid (TNBS) reactivity. RESULTS: Red wine (0.2 mg ethanol/mL) inhibited LDL oxidation as indicated by an 85.7% decrease in absorbance at 234 nm, a 96.5% decrease in TBARS production and complete prevention of the decrease in TNBS reactivity. White wine and ethanol did not have any significant effect at 0.2 mg/mL. White wine at 1.0 mg ethanol/mL inhibited TBARS production from LDL by 84.1%. Red wine (0.2 mg ethanol/mL) inhibited HDL oxidation as indicated by a 78.9% decrease in deltaA234, an 81.7% decrease in TBARS production and by no change in TNBS reactivity. White wine and ethanol had no effect at 0.2 mg/mL. White wine at 1.0 mg ethanol/mL inhibited TBARS production from HDL by 66.4%. CONCLUSIONS: These results indicate that red wine inhibits the cell mediated oxidation of lipoproteins, that white wine is not as effective as red wine and that the effect of the red wine is not due to its ethanol content.     

Influence of selective phosphodiesterase inhibitors on human neutrophil functions and levels of cAMP and Cai

Summary Chromatographic analysis of 3,5-cyclic nucleotide phosphodiesterase (PDE) isoenzymes in the cytosol of human neutrophils shows the predominant presence of PDE IV (cAMP specific) and PDE V (cGMP specific). PDE IV is characterized by (1) cAMP selectivity, (2) a K_M for cAMP of 1.2 M and (3) a typical rank order of IC ₅₀-values for PDE inhibitors: 0.13, 0.17, 47 and 9.5 M for PDE IV selective rolipram, PDE III/IV selective zardaverine, PDE III selective motapizone and unselective 3-isobutyl-l-methylxanthine (IBMX), respectively. Functions of polymorphonuclear leukocytes (PMN) such as N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide release and fMLP/thimerosal elicited leukotriene (LT) biosynthesis are inhibited by these PDE inhibitors with the same rank order and even lower IC₅₀-values. Measurements of changes in cytosolic Ca_i in Fura-2 loaded PMN demonstrate a transient Ca_i increase after stimulation with 0.1 M fMLP and an additional sustained elevation of Ca_i levels in the presence of thimerosal. PDE inhibitors suppress this sustained phase of Ca_i release with the same rank order of IC₅₀-values as LT biosynthesis. The correlation between fMLP/thimerosal-induced LT biosynthesis and Ca_i levels reveal a Ca_i threshold of 150 nM for arachidonic acid metabolism. cAMP levels in PMN were elevated by PDE inhibitors alone by less than 2-fold. In the presence of fMLP however, cAMP was increased up to 10-fold and the efficacy of PDE inhibitors to increase cAMP paralleled their potency to inhibit PDE IV. It is concluded that (1) suppression of PMN functions is achieved by PDE IV inhibition, (2) necessary cAMP elevations are within 50% increase, (3) superoxide release was affected by cAMP/protein kinase A (PKA) directly whereas (4) for inhibition of LT biosynthesis a cAMP related reduction of Ca-influx is involved. Send offprint requests to Ch. Schudt at the above address     

Oestradiol enhances the vulnerability threshold for schizophrenia in women by an early effect on dopaminergic neurotransmission

In a representative sample of 392 first hospital admissions for schizophrenia from a population of 1.5 million we assessed the "true" age of onset by a semi-standardized interview "IRAOS". We demonstrated that the mean age at onset of the disease is 3-4 years higher in females than in males, with the lifetime risk being exactly equal. In males, the rates of onset show a steep increase - starting from school age and reaching their maximum value in the age group 15-24 years - followed by a steady decrease. Females reach the first peak with a clear delay between 20 and 29 years. After the decrease, a second smaller peak is observed consistently in females within the age group 45-49 years and over. After having excluded competing explanations, we hypothesized that the effect of oestradiol on the dopaminergic system enhances the vulnerability threshold, which is lowered again during the menopause. Alternatively, we assumed that testosterone reduces the vulnerability threshold and thus furthers the earlier onset of the disease in males. We tested the hypotheses in three animal models by examining the effect of gonadal hormones on haloperidol-induced catalepsy and on apomorphine-induced stereotypies in both neonatal and adult rats. No clear influence by testosterone was shown. Oestradiol caused a significant reduction of both dopamine-agonist and dopamine-antagonist induced behaviour. The effects were stronger in neonatal rats. Since oestradiol caused the dopamine (DA) receptor affinity for sulpiride to be reduced by a factor of 2.8, we assumed that the behavioural changes due to oestradiol were accounted for by a down-regulation of DA receptor sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)