Histamine enhances TGF-beta1-mediated suppression of Th2 responses.

Susceptibility of T cells to TGF-beta1 produced by regulatory T cells has an important impact on the induction and maintenance of peripheral tolerance and therefore on the development of autoimmunity, cancer, and allergy. Histamine not only mediates the deleterious effects of allergic reactions, it can also modulate the Th1/Th2 cell balance. We demonstrate that histamine dose-dependently enhanced TGF-beta1-mediated suppression and TGF-beta1 responsiveness of CD4+ T cells. This effect was mediated by the histamine 2 receptor (H2R), as demonstrated by receptor-specific agonists and antagonists. Furthermore, the histamine effect on TGF-beta1 responsiveness was cAMP/PKA dependent. This pathway is activated by the H2R, which is preferentially expressed on Th2 cells. Thus a higher additive effect of histamine on TGF-beta1 responsiveness was found in Th2 cells compared with Th1 cells. In fact, findings are confirmed by analysis of cytokine regulation, since activation of the H2R/cAMP pathway promoted TGF-beta1-mediated IL-4 inhibition but was ineffective in suppressing IFN-gamma. These results demonstrate that histamine supports TGF-beta1 susceptibility of T cells. Moreover, Th2 cells are more affected by histamine-enhanced TGF-beta1 suppression, which is particularly important for the regulation of allergen-specific T cells in allergic immune responses.

LacSwitch II regulation of connexin43 cDNA expression enables gap-junction single-channel analysis.

Metabolic and electrical coupling through gap junction channels is implicated in cell differentiation, tissue homeostasis, and electrotonic propagation of signals in excitable tissues. The characterization of gating properties of these channels requires electrophysiological recordings at both single- and multiple-channel levels. Hence, a system that is able to control connexin expression by external means would provide a useful tool. To regulate the expression of connexins in cells, plasmids encoding a transactivator and/or a lac-operon IPTG response-dependent Cx43 target gene were transfected into communication-deficient N2a neuroblastoma cells. Immunoblotting, dye coupling, and electrophysiological methods revealed that expression of Cx43 in selected clones could be tightly regulated. After 15-20 h of acute induction with IPTG, cell-to-cell communication reached its peak with junctional conductances of 15-30 nS. Chronic induction at specific doses of IPTG produced constant, controlled levels of Cx43 expression, which were reflected by predictable junctional coupling levels. These conditions allowed prolonged recordings from either lowly or highly coupled cells, making lac operon an ideal regulatory system for channel gating studies at a single-channel level.

A simple automated method for the fluorometric titration of calcium in biological fluids,.

A new automatic device has been developed for the determination of the calcium content in biological fluids. The method is based on a fluorometric titration technique, in which calcium is titrated with ethyleneglycolbis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). The end-point of the titration is indicated by quenching of the fluorescence of a calcium-calcein complex, in which calcein is the fluorescein-complexone indicator. To obtain proper results we found it necessary to use a 1 molar potassium hydroxide solution as titration medium. The results are printed out directly in concentration units (mmol/l). In order to carry out these determinations, several electronic and mechanical devices, including a sample-changer, titration cabinet with built-in fluorometer, control unit and electron titrator, were developed. Together they form the new calcium automatic device of which several qualitites such as precision, recovery and correlation have been studied. The disturbing influence of bilirubin has been minimized by using an excitation wavelength of 492 nm, at which there is nearly no absorption by bilirubin. At a concentration of bilirubin of as much as 500 micronmol/l, the recovery of calcium is still about 96%.

Two kinins isolated from an extract of the venom reservoirs of the solitary wasp Megascolia flavifrons.

From an extract of the venom reservoirs of the wasp Megascolia flavifrons two kinins have been isolated. The sequences of amino acids are: Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg (Thr6-bradykinin) and Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-Lys-Ala (Thr6-bradykinin-Lys-Ala). The bradykinin-like effects of the venom on a number of vertebrate smooth muscle preparations can be explained by the actions of these kinins.

B2-kininergic action of linear and cyclic tryptophan6- and tyrosine6-kallidin.

This study was undertaken to determine the importance of the central aromatic moiety in the kallidin and cyclokallidin molecules, using the relaxation of the isolated duodenum of the rat. Replacement in kallidin of the central phenylalanine by tryptophan increased the potency from an EC50 of 3 x 10(-10)M to 2 x 10(-12)M. Replacement by tyrosine decreased the potency to an EC50 of 8 x 10(-8)M. In cyclo-kallidin (EC50: 10(-8)M) the potencies were decreased: cyclo-Trp6-kallidin showed an EC50 of 10(-6)M and cyclo-Tyr6-kallidin of 3 x 10(-7)M. The relaxation of the rat duodenum by linear and cyclic kinins was potentiated by the bradykinin potentiating peptide BPP5a and antagonized by the B2 antagonist HOE-140. At a concentration of 10(-9)M, HOE-140 significantly decreased the potencies of bradykinin and cyclo-kallidin, but not of the B1 agonist desArg9-bradykinin.

Highlights in cellular and molecular mechanisms of allergic diseases. XXVth Congress of the European Academy of Allergology and Clinical Immunology in Vienna.

This year, the annual congress of the European Academy of Allergology and Clinical Immunology was held on 10-14 June in Vienna. More than 6,000 delegates, practicing bench or bedside work or both, gathered from over 50 countries throughout the world. Health professionals, basic scientists and fellows in training could choose between a variety of activities in plenary, main, educational and workshop sessions, highlights of the past year, pros and cons, and oral abstract and poster sessions, and met with experts. A total of 1,713 abstracts, 31 symposia, and 54 workshops were presented, ranging from basic science to clinical trials and modern treatment of allergic diseases. Here, we summarize the highlights of cellular and molecular mechanisms of allergic disease.

High-altitude climate therapy reduces local airway inflammation and modulates lymphocyte activation.

High-altitude climate therapy is a well-established therapeutic option, which improves clinical symptoms in asthma. However, little is known about the underlying immunological mechanisms. The study investigates the influence of high-altitude climate therapy on airway inflammation and cellular components of specific and unspecific immune response. Exhaled NO significantly decreased within 3 weeks of therapy in patients with allergic and intrinsic, moderate and severe asthma. Interleukin-10 (IL-10)-secreting peripheral blood mononuclear cells (PBMC) increased within 3 weeks of therapy in six of 11 patients, whereas transforming growth factor-beta(1)-secreting PBMC remained stable. Furthermore, monocyte activation, assessed by CD80 expression significantly decreased during therapy. The frequency of CRTH2-expressing T cells decreased, while regulatory T cells (T(reg)) remained stable. FOXP3 and GATA-3 mRNA expression in CD4(+) T cells did not change, while interferon-gamma and IL-13 mRNA expression decreased in eight of 10 patients. The current data demonstrate that high-altitude climate therapy reduces local airway inflammation. Furthermore, monocytes switch towards a tolerogenic phenotype under high-altitude climate therapy. The T(reg)/Th2 ratio increases; however, because of the absence of antigens/allergens, no de novo differentiation of Th2 nor T(reg) cells is observed. The high-altitude climate therapy therefore may form the immunological basis for the endogenous control of allergen-driven diseases.

Cholinergic antagonists in a solitary wasp venom.

The venom of the solitary wasp Philanthus triangulum contains a cholinergic antagonist of the nicotinic receptor of the rectus abdominis muscle of the frog, Xenopus laevis. The venom of African P. triangulum contains two different cholinergic factors, a competitive and a non-competitive antagonist. The venom of the European P. triangulum may not contain a competitive antagonist of the nicotinic receptor of X. laevis, but only a very strong non-competitive antagonist. The possible non-synonymity of both groups of P. triangulum is discussed.

Megascoliakinin, a bradykinin-like compound in the venom of Megascolia flavifrons Fab. (Hymenoptera: Scoliidae).

The pharmacological and immunological properties of the venom of Megascolia flavifrons are compared with those of bradykinin and a number of bradykinin analogues. It is postulated that this venom contains a peptide, megascoliakinin, with a bradykinin-like sequence of amino acids, elongated at the C-terminal end with an unknown structure.

Ion channel block in rat diaphragm by the venom of the digger wasp Philanthus triangulum.

In the rat phrenic nerve-diaphragm preparation the twitch tension and tetanic contractions, already reduced by d-tubocurarine, succinylchloride or neostigmine, are further reduced by the venom of the digger wasp Philanthus triangulum F. The venom reduces the temperature- and voltage-sensitivity of the acetylcholine-receptor-activated ion channels, at the motor end-plate, and shortens the decay time of the miniature end-plate currents, analogous to a block described for the purified toxin, delta-PTX, on insect glutamate-activated channels. Preliminary results show that delta-PTX has an effect similar to that of the total venom on the decay phase of miniature end-plate currents.

The pharmacology of Microbracon venom.

1. Wasps of the genus Microbracon generally paralyse larvae of Lepidoptera. The wasp larva is exophagous on the paralysed host. The venom acts slowly and causes a flaccid paralysis. The venom of M. hebetor is extremely active in Lepidoptera, less active in honeybee workers, much less active in locusts and mealworms and probably inactive in non-insects. 2. In Lepidoptera and in locusts the venom presynaptically blocks the excitatory, but not the inhibitory, neuromuscular transmission. M. hebetor venom contains two high mol. wt toxins. A-MTX and B-MTX, with essentially the same action on neuromuscular transmission as the crude venom. Differences in dose-response curves of the two toxins, as well as differences in sensitivity to the two toxins between insect species are described. In wax moth larvae both A-MTX and B-MTX cause a slow and transient paralysis with a maximal effect after about 20 hr. The rate of recovery is dose-dependently decreased by the toxins.

The venom of Ampulex compressa--effects on behaviour and synaptic transmission of cockroaches.

1. The solitary wasp Ampulex compressa stings a cockroach, Periplaneta americana, twice. 2. The first sting into the ventral thorax results in a transient paralysis. During this paralysis the wasp stings the suboesophageal ganglion, which gradually results in a permanent deactivation. 3. The venom gland is a paired and highly branched organ, with a common ductus venatus. The large lumen is lined with a folded cuticula. No venom reservoir is present. 4. Extract of the venom gland induces a slow contraction of the guinea pig ileum. 5. The agonist present in the venom cannot be identified with a known agonist. 6. Venom gland extract blocks synaptic transmission from the cercal nerve to giant neurons in the sixth abdominal ganglion of the cockroach. 7. The block develops gradually, like the gradual appearance of the effects of the sting into the suboesophageal ganglion on the behaviour of the cockroach.

Kinins in ant venoms--a comparison with venoms of related Hymenoptera.

1. Venom preparations have been made of six ant, one pompilid wasp, two mutillid wasp, and four social wasp species. 2. The venoms were analysed pharmacologically in order to detect kinin-like activity. 3. Due to the small amounts of venoms available only a cascade of smooth muscle preparation could be used. 4. Kinin activities have been found in five ant venoms and in four social wasp venoms. 5. No kinin activity has been found in the venoms of the pompilid and mutillid wasps. 6. All ant venoms also contain unidentified agonists for vertebrate smooth muscle preparations.

Synthesis and biological activities of head-to-tail cyclic bradykinin analogues of varying ring size.

Syntheses of cyclic kinin analogues with different backbone atom numbers are described. Cyclization, by either the O-benzotriazolyl-N,N,N',N'-tetramethyluronium tetrafluoroborate/1-hydroxybenzotriazole/diisopropylethyl amine (TBTU-HOBt-DIPEA) or the diphenylphosphoryl azide (DPPA) procedure of linear peptides prepared by the solid-phase method based on the g-fluorenyl methyloxycarbonyl chemistry, was used for preparing cyclo-Gly-Ile-Ile-Gly-bradykinin, cyclo-Lys-kallidin (cyclo-Lys-Lys-bradykinin) and cyclo-des Arg-bradykinin. Peptides were characterized by amino acid analysis, optical rotation, analytical high-performance liquid chromatography and matrix-assisted laser desorption ionization-time flight mass spectrometry. Pharmacological experiments showed that cyclo-Gly-Ile-Ile-Gly-bradykinin (39 backbone atoms) and cyclo-Lys-bradykinin (30 backbone atoms) are about equipotent, when tested on the relaxation of the isolated rat duodenum preparation. The potency of cyclo-des Arg-bradykinin is at least three orders of magnitude lower. The potency of cyclo-Lys-Lys-bradykinin (33 backbone atoms) is one tenth the activity of bradykinin but about 10 times higher than the potency of the above-mentioned cyclokinins and makes the latter analogue the most potent end-to-end cyclic analogue known currently. The present results, in agreement with data from earlier reports, seem to indicate that the enhancement of the number of backbone atoms in the cyclic kinins first increases and subsequently decreases the potency, whereas a reduction in the atom number from 27 to 24 causes a dramatic decrease in potency.

Smooth muscle contracting compounds in the venom of Megascolia flavifrons (Hym: Scoliidae) with notes on the stinging behaviour.

1. The wasp Megascolia flavifrons stings larvae of the stag beetle Oryctes nasicornis on the ventral side of all segments, except the last three, which do not contain nerve ganglia. 2. Experiments indicate a central rather than a peripheral action of the venom. 3. From pharmacological analysis it is concluded that the venom does not contain cholinergic or serotonergic activity, but contains histamine- and bradykinin-like substances. 4. The presence of histamine was confirmed by a radioenzymatic method.

Smooth muscle contracting compounds in venoms of sphecid wasps (Hym: Sphecidae).

1. The presence of smooth muscle contracting substances in venom preparations made from six species of sphecid wasps was investigated using a number of standard pharmacological techniques. 2. In addition, acetylcholine was identified by enzymatic degradation as well as by mass fragmentography, while histamine was determined using a radio-enzymatic method. 3. The venom reservoir of P. triangulum contains about 400 ng acetylcholine, but no histamine-, 5-HT- or bradykinin-like activity. 4. The venoms of the five other species contain histamine in amounts varying from 20 to 2000 ng per venom reservoir, but no acetylcholine-, 5-HT- or bradykinin-like activity.