Retinoic acids up-regulate functional eosinophil-driving receptor CCR3.

Eotaxins and their receptor CCR3 have a definitive role for tissue accumulation of eosinophils both under homeostatic and pathologic conditions. However, physiological stimuli that can up-regulate CCR3 in blood-derived human eosinophils have not been recognized. As a prior gene microarray study revealed up-regulation of CCR3 in eosinophils stimulated with retinoic acids (RAs), the expression of functional CCR3 was examined. We found that 9-cis RA and all-trans RA (ATRA) significantly induced surface CCR3 expression regardless of the presence of IL-3 or IL-5. Pharmacological manipulations with receptor-specific agonists and antagonists indicated that retinoic acid receptor-α activation is critical for CCR3 up-regulation. RA-induced CCR3 was associated with its functional capacity, in terms of the calcium mobilization and chemotactic response to eotaxin-1 (CCL11). Our study suggests an important role of vitamin A derivatives in the tissue accumulation of eosinophils.

Ex vivo application of carbon monoxide in UW solution prevents transplant-induced renal ischemia/reperfusion injury in pigs.

I/R injury is a major deleterious factor of successful kidney transplantation (KTx). Carbon monoxide (CO) is an endogenous gaseous regulatory molecule, and exogenously delivered CO in low concentrations provides potent cytoprotection. This study evaluated efficacies of CO exposure to excised kidney grafts to inhibit I/R injury in the pig KTx model. Porcine kidneys were stored for 48 h in control UW or UW supplemented with CO (CO-UW) and autotransplanted in a 14-day follow-up study. In the control UW group, animal survival was 80% (4/5) with peak serum creatinine levels of 12.0 +/- 5.1 mg/dL. CO-UW showed potent protection, and peak creatinine levels were reduced to 6.9 +/- 1.4 mg/dL with 100% (5/5) survival without any noticeable adverse event or abnormal COHb value. Control grafts at 14 days showed significant tubular damages, focal fibrotic changes and numerous infiltrates. The CO-UW group showed significantly less severe histopathological changes with less TGF-beta and p-Smad3 expression. Grafts in CO-UW also showed significantly lower early mRNA levels for proinflammatory cytokines and less lipid peroxidation. CO in UW provides significant protection against renal I/R injury in the porcine KTx model. Ex vivo exposure of kidney grafts to CO during cold storage may therefore be a safe strategy to reduce I/R injury.

Thioredoxin reduces C-C chemokine-induced chemotaxis of human eosinophils.

BACKGROUND: Human thioredoxin (TRX) is one of redox-active proteins that regulate reactive oxidative metabolisms. In recent study, we found that serum levels of TRX were elevated in asthmatic patients with exacerbation; however, few details are known about the physiological role of TRX in allergic inflammation, involving eosinophil infiltration. OBJECTIVE: In the present study, we examined whether TRX modulated C-C chemokine-induced chemotaxis of human eosinophils. METHODS: Eosinophils were isolated from subjects with mild eosinophilia by modified CD16 negative selection. After incubation with or without recombinant TRX, chemotaxis of human eosinophils was measured using Boyden chamber. RESULTS: Preincubation with TRX suppressed eotaxin- and regulated on activation, normal T-cell expressed and secreted (RANTES)-induced chemotaxis of eosinophils. Although, TRX had no effect on the expression of C-C chemokine receptor 3, which is a receptor of eotaxin and RANTES, we demonstrated that the activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases, which play an important role in eosinophil migration, was attenuated by the treatment with TRX. CONCLUSION: Our results suggest that the elicited TRX is beneficial to reduce allergic inflammation through negative regulation of eosinophil functions and has potential in the treatment of allergic diseases, such as asthma.

Soluble vascular cell adhesion molecule-1 induces human eosinophil migration.

BACKGROUND: Tissue eosinophilia is one of the hallmarks of allergic diseases and Th2-type immune responses including asthma. Adhesion molecules are known to play an important role in the accumulation of eosinophils in allergic inflammatory foci, and they contribute to eosinophil activation. Elevated levels of the soluble forms of adhesion molecules in the body fluid of asthmatic patients have been observed, although their pathophysiological significance remains to be fully elucidated. METHODS: Peripheral blood eosinophils were purified, and the effect of soluble vascular cell adhesion molecule-1 (sVCAM-1) on eosinophil migration was investigated using in vitro systems. RESULTS: We found that sVCAM-1 (1 to 10 mug/ml) induced eosinophil chemotaxis, rather than chemokinesis, in a concentration-dependent fashion. In addition, sVCAM-1 induced cell shape change and actin polymerization, which are necessary for cell movement. Manipulations with very late antigen (VLA)-4-neutralizing antibody and signal inhibitors indicated that the sVCAM-1-induced chemotaxis was mediated through ligand-dependent activation of tyrosine kinase Src, p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase (ERK) MAPK. Rapid phosphorylation of these signaling molecules was observed using a bead-based multiplex assay. CONCLUSION: Our results raise the possibility of sVCAM-1 in the fluid phase as a significant contributor to the heightened eosinophilic inflammatory response.

A population-based study of drug resistance and transmission of tuberculosis in an urban community.

SETTING: A low-income neighborhood of Sao Paulo, Brazil. OBJECTIVE: To determine the incidence, risk factors and transmission patterns of multidrug-resistant tuberculosis (MDR-TB). DESIGN: Prospective longitudinal study of patients with pulmonary TB (PTB). METHODS: Sputum culture-confirmed patients with PTB were recruited between March 2000 and May 2002. Bivariate and multivariate logistic regression analyses were performed to identify factors associated with MDR-TB. Mycobacterium tuberculosis isolates were tested for drug susceptibility and typed by IS6110-RFLP analysis. RESULTS: Of 420 patients, respectively 71% and 27% were new and previously treated; 15.5% of the patients' M. tuberculosis isolates were resistant to at least one drug; of these, 11% and 27% were found among new and previously treated cases, respectively. Respectively 1% and 16.7% of the new and previously treated cases were MDR-TB. RFLP analysis showed that new transmission of MDR strains was uncommon. By multivariate logistic regression analysis, previous TB and hospitalization in the 24 months before TB diagnosis were identified as independent predictors of MDR-TB. CONCLUSIONS: The results showed an intermediate level of MDR-TB incidence in a neighborhood of Sao Paulo and identified predictors that can be targeted for intervention by national and local TB control programs.

Activity-independent cell adhesion to tissue-type transglutaminase is mediated by alpha4beta1 integrin.

Transglutaminases (TGases) are enzymes which catalyze cross-link formation between glutamine residues and lysine residues in substrate proteins. We have previously reported that one of the TGases, blood coagulation factor XIIIa (FXIIIa), is capable of mediating adhesion of various cells. In this paper, we report for the first time that tissue-type transglutaminase (TGc) also has cell adhesion activity. TGc-coated plastic surface promoted adhesion and spreading of cells in a TGc concentration-dependent manner. However, there are some obvious differences between cell adhesion mediated by TGc and FXIIIa. As was reported previously, the adhesion to FXIIIa is dependent on its TGase activity. In contrast, the TGc-mediated cell adhesion is independent of its TGase activity: 1) The modification of the active center cysteine with iodoacetamide blocked the enzyme activity without any effect on cell adhesion; 2) the addition of Mg2+ did not induce the enzyme activity, but it was as effective as Ca2+ for cell adhesion; 3) the addition of NH4+ inhibited the enzyme activity but did not affect the cell adhesion significantly. The integrins involved in these cell adhesions are quite different. In the case of FXIIIa, alpha vbeta3 and alpha5beta1 integrins are involved and consequently the RGD peptide substantially inhibited the adhesion. On the other hand, the cell adhesion to TGc is mediated by alpha4beta1 integrin but not alpha5beta1; a CS-1 peptide, which represents the binding site of fibronectin to alpha4beta1 integrin, completely inhibited the cell adhesion to TGc. It is possible that TGc and FXIIIa may mediate cell adhesion under different physiological and pathological situations.

Classification of 'activation' antibodies against integrin beta1 chain.

We compared the effects of two anti-beta1 integrin activating antibodies, TS2/16 and AG89, on K562 cell adhesion to fibronectin. Though both antibodies effectively induced cell adhesion, the EC50 for AG89 was more than 200-fold higher than that for TS2/16. Scatchard analysis of the data from [125I]Fab fragment binding to the cells revealed that the TS2/16 epitope is exposed constitutively on all the beta1 integrin molecules, while only 3% of the beta1 integrins on resting K562 cells bear the AG89 epitope. Calculation of the actual number of each antibody bound to the cell during the cell adhesion assay revealed that induction of cell adhesion can be accomplished by binding much fewer AG89 molecules compared to TS2/16. Thus, AG89 and TS2/16 represent distinct classes of anti-integrin activating antibodies that show completely different binding characteristics as well as different activation effects on the integrin molecule upon binding.

Anti-serotonin action in combination with noradrenaline-stimulating action is important for inhibiting muricide in midbrain raphe-lesioned rats.

The present study was designed to examine the possible involvement of both an anti-serotonin action and a catecholamine-stimulating action in the mechanism of the inhibition of the muricide in rats with lesions of the midbrain raphe. Serotonin antagonists, such as cyproheptadine (10 mg/kg), cinanserin (10 mg/kg) and pirenperone (1 mg/kg), given alone showed little suppression of muricide in rats with raphe lesions, although the first two drugs were inhibitory at very large doses. Methamphetamine showed no inhibition of muricide at 0.32 mg/kg (i.p.), but exerted a marked inhibition of muricide when combined with the above serotonin antagonists. In addition, the dose-response curve for cyproheptadine and cinanserin was shifted markedly to the left when combined with L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) (100 mg/kg i.p.), but not with lisuride (0.32 mg/kg i.p.). Similarly, pirenperone produced a marked inhibition of muricide at doses of 0.32-1.8 mg/kg (i.p.) when combined with L-threo-DOPS, but not when combined with lisuride. These results suggest that the combination of an anti-serotonin action with noradrenergic activation is important for inhibiting muricide, at least in rats with raphe lesions. A similar mechanism also seems to be valid for the anti-muricidal effect of antidepressant drugs.

Influence of excitatory amino acid receptor antagonists and of baclofen on synaptic transmission in the optic nerve to the suprachiasmatic nucleus in slices of rat hypothalamus.

Electrical stimulation of the optic nerve evoked two positive waves with short latency, followed by a large negative wave in the suprachiasmatic nucleus of slices of hypothalamus of the rat. The latency to peak of the two positive waves and the large negative wave were 2.7 +/- 0.1, 6.1 +/- 0.1 and 10.3 +/- 0.5 msec, respectively. Only the large negative wave disappeared in low calcium Ca2+-high magnesium (Mg2+) Krebs solution and with the addition of tetrodotoxin (1 microM) all the waves disappeared. Baclofen inhibited the large negative wave in a dose-dependent manner but not the two positive waves. Excitatory amino acid antagonists also inhibited only the large negative wave, i.e. it was reduced to about 70% by 1 mM glutamic acid diethyl ester and to about 50% by both 1 mM 2-amino-4-phosphonobutyric acid and 1 mM DL-2-amino adipic acid. All waves were unaffected by 0.1 mM atropine, hexamethonium and curare. These results indicate that two positive waves, induced by stimulation of the optic nerve are attributed to nerve conduction and the large negative wave to the neurons of the suprachiasmatic nucleus, and that the neuronal pathway from the optic nerve to the suprachiasmatic nucleus may include aspartate and/or glutamate as an excitatory neurotransmitter.

Effect of monoamines on field potentials in the suprachiasmatic nucleus of slices of hypothalamus of the rat evoked by stimulation of the optic nerve.

The effects of the application of serotonin, histamine, noradrenaline and dopamine to the bath on field potentials in the suprachiasmatic nucleus evoked by stimulation of the optic nerve were studied using a hypothalamic slice. Stimulation of the contralateral optic nerve evoked fast positive and late large negative waves in the suprachiasmatic nucleus. The monoamines produced a dose dependent suppression of the amplitude of the negative wave but did not affect that of the positive waves, and the order of potency was serotonin greater than noradrenaline greater than dopamine greater than or equal to histamine. The negative wave was suppressed by phenylephrine (0.1-10 microM) in a dose-dependent manner, whereas it was unaffected by isoproterenol (0.1-10 microM). The suppression of the negative wave produced by the application of histamine and noradrenaline was antagonized by the H1-receptor antagonist, diphenhydramine and the alpha 1-receptor antagonists, phenoxybenzamine and phentolamine. Therefore, the suppression of the negative wave by histamine and noradrenaline was mediated by the H1-receptor and alpha 1-receptor, respectively. The present study indicates that monoamines may play an inhibitory role in the regulation of neurotransmission in the retinohypothalamic pathway to the suprachiasmatic nucleus.

Effect of L-glutamate, injected into the posterior hypothalamus, on blood pressure and heart rate in unanesthetized and unrestrained rats.

In unanesthetized and unrestrained rats, microinjection of L-glutamate into the posterior hypothalamus produced hypertension and tachycardia. These cardiovascular responses were mostly accompanied with behavioral excitation such as searching the cage, rearing and sniffing. The cardiovascular responses elicited by L-glutamate were attenuated by prior injection with propranolol and hexamethonium but not with glutamate diethylester, phentolamine and atropine. The behavioral responses to L-glutamate were suppressed by propranolol, hexamethonium and phentolamine. These results suggest that catecholaminergic and/or cholinergic systems in the posterior hypothalamus may be involved in the mediation of the cardiovascular and behavioral responses induced by L-glutamate.

Noradrenergic involvement in catalepsy induced by delta 9-tetrahydrocannabinol.

In order to elucidate the role of the catecholaminergic system in the cataleptogenic effect of delta 9-tetrahydrocannabinol (THC), the effect of pretreatment with 6-hydroxydopamine (6-OHDA) or with desipramine and 6-OHDA and lesions of the locus coeruleus were investigated in rats. The cataleptogenic effect of THC was significantly reduced in rats treated with 6-OHDA and in rats with lesions of the locus coeruleus but not in rats treated with desipramine and 6-OHDA, as compared with control rats. On the contrary, the cataleptogenic effect of haloperidol was significantly reduced in rats treated with desipramine and 6-OHDA but not in rats treated with 6-OHDA or in rats with lesions of the locus coeruleus. These results indicate that noradrenergic neurons have an important role in the manifestation of catalepsy induced by THC, whereas dopaminergic neurons are important in catalepsy induced by haloperidol.

Involvement of the dorsal hippocampus in mediation of the antianxiety action of tandospirone, a 5-hydroxytryptamine1A agonistic anxiolytic.

The effect of tandospirone, a 5-hydroxytryptamine (5-HT)1A agonist/anxiolytic, injected directly into dorsal hippocampus, on Vogel-type conflict behavior in rats was investigated and the findings were compared with the effects of diazepam and zopiclone. Tandospirone (30 micrograms/2 microliters and 60 micrograms/2 microliters) and diazepam (40 micrograms/2 microliters) but not zopiclone (20 micrograms/2 microliters), produced a potent anticonflict action in rats. The anticonflict action of tandospirone (30 micrograms/2 microliters), injected into the dorsal hippocampus, was significantly blocked by (-)-propranolol (5 mg/kg i.p.). The present findings provide evidence that suggests that tandospirone has an antianxiety action, presumably by stimulating 5-HT1A receptors in the dorsal hippocampus.

Conflict behavior and dynamics of monoamines of various brain nuclei in rats.

The present study was an attempt to clarify the role of noradrenaline (NA) and of the 5-hydroxytryptamine (5-HT) system in various nuclei in brain, as a component of a proposed neural circuit in the mediation of conflict behavior and the anticonflict action of anxiolytics. The authors investigated changes in the concentrations of NA, 3-methoxy-4-hydroxyphenylethyleneglycol, 5-HT and 5-hydroxyindoleacetic acid in discrete regions of the brain in rats, in correlation with conflict behavior and also the effects of diazepam and suriclone. Noradrenergic neural activity diminished with a conflict situation, in the frontal cortex, central amygdala, mammillary body and dorsal hippocampus. 5-Hydroxytryptaminergic neural activity increased with a conflict situation in the frontal cortex, central amygdala, basolateral amygdala and medial septum. These changes in the frontal cortex, central amygdala, mammillary body and dorsal hippocampus were not observed when diazepam 20 mg/kg (p.o.) and suriclone 40 mg/kg (p.o.) produced anticonflict action. Suriclone normalized the increased 5-HT-ergic activity in the medial septum. The suppression of NA-ergic and the activation of 5-HT-ergic (except for the mammillary body) neural activity in the frontal cortex, central amygdala, mammillary body and dorsal hippocampus seemed to be linked to the mediation of conflict behavior. The facilitatory and inhibitory action on NA and 5-HT (except for the mammillary body) neurons, respectively, in these regions of the brain, may be involved in mechanisms underlying the anticonflict action of anxiolytics (diazepam or suriclone).

Synthesis and pharmacological activity of a phosphate ester of delta8-tetrahydrocannabinol.

A water-soluble phosphate ester of delta8-tetrahydrocannabinol (delta8-THC) was synthesized and its pharmacological activities were examined. The cataleptogenic and thiopental sleep-potentiating effects of delta8-THC phosphate in the mouse was approximately 10 and 7% of those of delta8-THC, respectively. However this phosphate showed almost the same potency and a longer duration of hypothermic effect, as compared with delta8-THC in the mouse. The acute toxicity of this phosphate was far lower than that of delta8-THC. delta 8-THC phosphate was difficultly hydrolyzed by alkaline phosphatase or mouse liver homogenate in vitro. The mode of action of the phosphate derivative is discussed in connection with this enzymatically difficult hydrolysis.

Behavioral pharmacology of zopiclone.

The pharmacological properties of zopiclone, a cyclopyrrolone derivative, have been studied in comparison with diazepam, nitrazepam, and flurazepam, on behavioral tests in mice and rats, including open-field activity, Skinner box conflict test, hyperemotionality and muricidal behavior of olfactory bulbectomized or raphectomized rats, pentetrazol or electroshock convulsions, inclined screen, rotarod, and thiopental-, ether-, or ethanol-induced anesthesia. Zopiclone exhibited pharmacological properties qualitatively similar to those of benzodiazepines especially in conflict, aggressivity, and pentetrazol-induced convulsion tests. On the other hand, its myorelaxant activity was somewhat weaker than that of the reference drugs. The pharmacological effects of zopiclone were of short duration.

Inhibitory effect of a selective kappa receptor agonist, U-50, 488H, on methamphetamine-elicited ipsilateral circling behavior in rats with unilateral nigral lesions.

The present study was designed to investigate the effect of U-50, 488H, a highly selective kappa opioid agonist, on ipsilateral and contralateral circling behavior induced by methamphetamine and by apomorphine, respectively. U-50, 488H (3.2-10 mg/kg IP) by itself failed to induce either ipsilateral or contralateral circling in rats with unilateral nigral lesions produced by an injection of 6-hydroxydopamine. U-50, 488H produced a significant dose-dependent inhibition of methamphetamine (1.0 mg/kg SC)-elicited ipsilateral circling. However, it had no effect on contralateral circling induced by apomorphine (0.5 mg/kg SC). The present results indicate that U-50, 488H presynaptically inhibits the release of dopamine in the rat striatum.