Adenosine A2A Receptor Agonists Promote More Rapid Wound Healing Than Recombinant Human Platelet–Derived Growth Factor (Becaplermin Gel)

Animal studies of the topical application of adenosine A _2A receptor agonists show that it promotes wound closure. To further confirm the efficacy of adenosine A _2A receptor agonists as promoters of wound healing, we compared the effect of MRE0094, a novel selective adenosine A _2A receptor agonist, to CGS-21680, a reference selective adenosine A _2A receptor agonist, as well as to recombinant human platelet–derived growth factor (0.01% Becaplermin gel), an agent currently used to promote healing of diabetic ulcers, on wound closure in healthy BALB/C mice. Wounds (12 mm diameter) were created on the dorsum of mice (two per mouse) and then treated daily with vehicle, 0.01% Becaplermin gel, or different doses of the adenosine A _2A receptor agonists. The wound margins were traced onto plastic sheets, and the wound areas were digitized, quantitated, and compared. We found that application of MRE0094 (1 g/wound and 10 g/wound) and CGS-21680 (1 g/wound and 5 g/wound) achieved 50% wound closure significantly more rapidly than control application (day 1.9, 1.9, 3.5, 3.2, respectively, versus control day 4, p < 0.05 ANOVA). Surprisingly, neither higher nor lower concentrations of CGS-21680 affected the rate of wound closure, as compared to control. In contrast, Becaplermin gel did not increase the rate at which wounds closed (50% closure by day 7.2, p = NS versus control). These data confirm our prior observations that adenosine A _2A receptor agonists promote wound closure, and they suggest that these agents may be as effective if not more effective than Becaplermin gel for the treatment of poorly healing wounds.     

Slow inward current in single cells isolated from adult human ventricles

Characteristics of the slow inward current (I _si) in human ventricular myocytes isolated from septal specimens obtained in patients undergoing corrective cardiac surgery were studied using the whole-cell clamp method. A first series of experiments was performed under normal standard superfusion. Clamping from –60 mV evoked an inward current with a threshold at about –35 mV, a maximum around +10 mV and an apparent reversal potential at about +55 mV. No overlapping transient or background outward currents were detected in the –60 to +30 mV potential range, but time-dependent and steady-state outward currents were elicited at potentials above +30 mV. An overlap of steady-state activation and inactivation curves was present between –30 and +10 mV and a slight relief from inactivation was observed for voltages positive to +10mV. The time course of inactivation consisted of fast and slow phases with time constants differing by a factor of eight. Slow time constants of inactivation were shorter at potentials that elicited larger I _si, and longer at potentials inducing smaller I _si. Recovery from inactivation evolved slowly with 100% reactivation occurring in about 4000 ms. Switching the holding potential from –60 to –40 mV led to a reversible decline of I _si without any change of the decay time constants. I _si was significantly increased by 0.1 M isoproterenol. Total or partial inhibition by inorganic (2 mM Mn²⁺, 3 mM Co²⁺, 1 mM Cd²⁺) and organic (1 M methoxyverapamil, 5 M diltiazem) calcium antagonists did not unmask any transient outward current. However, a consistent increase of I _si was reversibly observed with 3 mM 4-aminopyridine while using standard solutions. A second series of experiments carried out with K⁺- and Na⁺-free solutions did not demonstrate any significant change from data observed with standard solutions except a reduction of outward currents at steps above +30 mV and alteration of inactivation kinetics. In this experimental setting, 4-aminopyridine also increased I _si but to a lesser degree. We conclude that I _si, as compared to the outward currents, is dominant in the diseased human ventricular cells we have studied.     

Effects of Cannabinoids on the Immune System and Central Nervous System

This review aims to improve understanding of the modulatory effects that cannabinoids exert on the immune system and CNS. Cannabinoids possess immunomodulatory activity, are neuroprotective in vivo and in vitro and can modify the production of inflammatory mediators, such as nitric oxide, prostanoids and cytokines, that are expressed by, and act on, the immune system and the brain. The mechanisms of cannabinoid actions are not fully understood, but appear to involve complex interactions between cannabinoid receptors and a number of signal transduction pathways. Endogenous cannabinoid ligands appear to act as local modulators of immune/inflammatory reactions. Cannabinoid-induced immunosuppression may have implications for the treatment of neurological disorders that are associated with excess immunological activity, such as multiple sclerosis and Alzheimer's disease. There is anecdotal evidence that cannabis use improves the symptoms of multiple sclerosis, and studies with animal models are beginning to provide evidence for the mechanism of such effects. The development of nonpsychotropic cannabinoid analogues and modulators of the metabolism of endogenous cannabinoid ligands may lead to novel approaches to the treatment of neurodegenerative disorders.     

Nonmyeloablative stem cell transplantation is an effective therapy for refractory or relapsed hodgkin lymphoma: results of a spanish prospective cooperative protocol.

We report the results of reduced-intensity conditioning allogeneic stem cell transplantation (allo-RIC) in patients with advanced Hodgkin lymphoma (HL). Forty patients with relapsed or refractory HL were homogeneously treated with an RIC protocol (fludarabine 150 mg/m(2) intravenously plus melphalan 140 mg/m(2) intravenously) and cyclosporin A and methotrexate as graft-versus-host disease (GVHD) prophylaxis. Twenty-one patients (53%) had received >2 lines of chemotherapy, 23 patients (58%) had received radiotherapy, and 29 patients (73%) had experienced treatment failure with a previous autologous stem cell transplantation. Twenty patients (50%) were allografted in resistant relapse, and 38 patients received hematopoietic cells from an HLA-identical sibling. Five patients (12%) died from early transplant-related mortality (before day +100 after allo-RIC). One-year transplant-related mortality was 25%. Acute GVHD developed in 18 patients (45%). Chronic GVHD developed in 17 (45%) of the 31 evaluable patients. The response rate 3 months after the allo-RIC was 67% (21 [52%] complete remissions and 6 [15%] partial remissions). Eleven patients received donor lymphocyte infusions (DLIs) for disease relapse. The response rate after DLI was 54% (3 complete remissions and 3 partial remissions). Overall survival (OS) and progression-free survival (PFS) were 48% +/- 10% and 32% +/- 10% at 2 years, respectively. Refractoriness to chemotherapy was the only adverse prognostic factor for both OS (63% +/- 12% versus 35% +/- 13%; P = .05) and PFS (55% +/- 16% versus 10% +/- 9%; P = .006). For patients with failure of a prior autologous hematopoietic stem cell transplantation, results were especially good for those who experienced late relapses (>/=12 months: 2-year OS and PFS were 75% +/- 16% and 70% +/- 18%, respectively). These data suggest that allo-RIC is feasible in heavily pretreated HL patients and has an acceptable early transplant-related mortality. Results are better in patients allografted in sensitive disease. Both responses observed after the development of GVHD and DLI may suggest a graft-versus-HL effect. Allo-RIC has to be considered an effective therapeutic approach for patients who have had treatment failure with a previous autologous hematopoietic stem cell transplantation.     

In vitro models for the assessment of inflammatory and immuno-modulatory effects of the volatile organic compound chlorobenzene

An in vitro cell culture system based on an air/liquid culture technique was developed which allows a direct exposure of cells to volatile chemicals without medium coverage. For the establishment of the experimental system, chlorobenzene was used as a model compound. Chlorobenzene is a volatile organic compound which is mainly used as a solvent. Beside other adverse health effects, chlorobenzene exposure has been shown to be associated with respiratory tract irritations, Th2 differentiation, and allergic sensitizations. Human peripheral blood mononuclear cells (PBMC) and lung epithelial cells (A549) were exposed to chlorobenzene via gas phase for 20 h. Additionally, PBMC were incubated with culture supernatants from exposed lung epithelial cells. High chlorobenzene concentrations (100 g/m(3)) induced IL-8 production in A549 cells, whereby lower concentrations (10 microg/m(3)-1 g/m(3)) stimulated the secretion of the monocyte chemoattractant protein-1 (MCP-1). A direct effect of chlorobenzene on the cytokine secretion of PBMC was not found. However, if PBMC were incubated with culture supernatants of exposed lung cells, an enhanced production of the Th2 cytokine IL-13 was observed. This induction was prevented in the presence of an anti-MCP-1 antibody. Our data suggest that chlorobenzene induces the production of inflammatory mediators in lung cells. The primary chlorobenzene caused release of MCP-1 in lung epithelial cells may secondarily result in a Th2 differentiation in T lymphocytes. These findings may contribute to the understanding of how chlorobenzene mediates the development of inflammatory reactions in the airways and contributes to the development of an allergic reactivity.     

Formation of 8-oxoguanine in cellular DNA of Escherichia coli strains defective in different antioxidant defences

This paper examines the relationship in Escherichia coli betweenthe in vivo content of 8-oxoguanine (8-oxoG) in chromosomalDNA and deficiencies of various key antioxidant defences. Thestructural genes for catalases (katG and katE), cytosolic superoxidedismutases (sodA and sodB) or formamidopyrimidine-DNA glycosylase(fpg) were inactivated to obtain bacterial strains lacking thescavenger enzymes for H₂O₂ or O₂·^– or the DNA repairprotein for 8-oxoG. Wild-type bacteria showed 5-fold increasedsensitivity to both lethality and mutagenesis by H₂O₂ in K medium(1 % casamino acids and 1 % glucose), as compared with nutrientbroth. This higher sensitivity was associated with increasedchromosomal oxidative damage, estimated as the 8-oxodG content,and with a marked decrease in both catalase and SOD activities.Bacteria lacking both cytosolic SODs (sodA sodB mutant) displayedincreased 8-oxodG content in chromosomal DNA (2.8-fold thatof the wild-type) when grown under standard aerated conditions.Comparatively, no significant difference in 8-oxodG contentwas observed in cells grown without aeration. Bacteria totallydevoid of catalase activity (katG katE mutant) showed wild-typecontents of 8-oxodG in chromosomal DNA when grown under aeratedconditions. Nevertheless, the protective role of catalase inpreventing formation of 8-oxodG in chromosomal DNA became evidentunder oxidative stress conditions: growth under hyperoxygenationand, particularly, following H₂O₂ exposure. Catalase deficiencyresulted in a dramatic decrease in viability after H₂O₂ exposure.A deficiency of Fpg protein also sensitized E.coli to H₂O₂ lethality,though to lesser extent than a deficiency of catalase activity.However, the scavenger enzyme and the DNA repair protein protectedequally against 8-oxoG formed in vivo upon H₂O₂ treatment. ¹To whom correspondence should be addressed. Tel: 57 218695; Fax: 57 218688; Email: bblpucuc{at}uco.es     

Murine epidermal Langerhans cells do not express inducible nitric oxide synthase

In Leishmania-infected macrophages (MΦ), the formation of reactive nitrogen intermediates by the inducible isoform of nitric oxide synthase (iNOS) is critical for the killing of the intracellular parasites. We have recently shown that, in addition to MΦ, epidermal Langerhans cells (LC) can phagocytose Leishmania major, but they do not allow parasite replication. Therefore, we analyzed whether LC and MΦ display the same leishmanicidal effector mechanism. Unlike MΦ, stimulation of unselected epidermal cells with interferon-γ/lipopoly-saccharide did not lead to the release of nitric oxide (NO), and inhibition of NO production had no effect on the rate of infection of LC. iNOS mRNA was clearly detectable in MΦ as well as unselected epidermal cells (the majority of which consists of keratinocytes) after stimulation with different cytokines. In contrast, pure LC obtained by single-cell picking from cytokine-activated or L. major-infected epidermal cells did not express iNOS mRNA. Addition of the NO donor S-nitroso-N-acetylpenicillamine to already-infected LC did not alter their rate of infection, indicating that LC do not utilize exogenous NO for the control of intracellular Leishmania. These results suggest that in the L. major-infected skin, activated MΦ and keratinocytes, but not LC have the ability to express iNOS activity. Therefore, an as yet unidentified, NO-independent mechanism appears to be responsible for the control of parasite replication in LC.     

Polyene sequence distribution in modified poly(vinyl chloride) after thermal degradation

The products resulting from the reaction of PVC with sodium benzenethiolate were degraded to 0,3% at 180°C in the solid state and at 160°C in solution in trichlorobenzene. The polyene distribution of the polymers after degradation was studied by both UV-visible and resonance Raman spectroscopies, as a function of the degree of substitution. The results show that there are two types of behaviour: that of the PVC sample prior to the substitution reaction together with the samples modified up to a definite degree of substitution which depends on the starting isotactic content, and that of samples with higher degrees of substitution. The former group exhibits not only a steady improvement in thermal stability but also a preferential formation of polyenes of 7 – 9 double bonds whose concentration decreases with increasing degree of substitution. Conversely, for the second group of samples the thermal stability decreases with the degree of substitution and no specific absorption bands are observed. On the basis of earlier work on the selective substitution of the isotactic GTTG and heterotactic TTTG triads during the first stage of the reaction, the present results show that the bands at 393, 416, and 437 nm are related to specific polyenes which result from initiation by the above quoted conformations in PVC, a conclusion for which confirmatory evidence was obtained by resonance Raman spectroscopic examination of the samples. There is, therefore, clear evidence for the occurrence of two distinct degradation mechanisms, one involving initiation by the unstable triad conformations and the other via random initiation at stable and normal structures. To this may be added the initiation by defect structures, which have been extensively documented in the literature.     

Fourteen novel OPA1 mutations in autosomal dominant optic atrophy including two de novo mutations in sporadic optic atrophy

The OPA1 gene, encoding a dynamin-related GTPase that plays a role in mitochondrial biogenesis, is implicated in most cases of autosomal dominant optic atrophy (ADOA). Sixty-nine pathogenic OPA1 mutations have been reported so far. Most of these are truncating mutations located in the GTPase domain coding region (exons 8-16) and at the 3'-end (exons 27-28). We screened 44 patients with typical ADOA using PCR-sequencing. We also tested 20 sporadic cases of bilateral optic atrophy compatible with ADOA. Of the 18 OPA1 mutations found, 14 have never been previously reported. The novel mutations include one nonsense mutation, 3 missense mutations, 6 deletions, one insertion and 3 exon-skipping mutations. Two of these are de novo mutations, which were found in 2 patients with sporadic optic atrophy. The recurrent c.2708_2711delTTAG mutation was found in 2 patients with a severe congenital presentation of the disease. These results suggest that screening for OPA1 gene mutations may be useful for patients with optic atrophy who have no affected relatives, or when the presentation of the disease is atypical as in the case of early onset optic atrophy.