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In the last few years, there has been an increasing tendency to use natural polymers for the fabrication of dressings for wound and burn management. Among them, alginate, a polysaccharide extracted primarily from marine algae, exhibits attractive properties being non-toxic, hydrophilic and biodegradable. The aim of this study was to characterize the in vitro biocompatibility and the efficacy of a composite polymeric material based on sodium alginate (NaAlg) and povidone iodine (PVPI) complex in a mouse model of wound healing. The developed material combines the excellent wound healing properties of alginates with the bactericidal and fungicidal properties of PVPI, providing a controlled antiseptic release. We demonstrated that the NaAlg/PVPI films are able to reduce the inflammatory response both in human foreskin fibroblasts after lipopolysaccharide (LPS) stimulus and in rodents after wound induction. Furthermore, the NaAlg/PVPI film-treated animals showed a significantly higher wound closure compared to untreated animals at each time point considered. Interestingly, the complete wound closure was achieved within 12 days only in the film-treated group, indicating that the full-thickness wounds healed more rapidly in these animals. The results demonstrate that the NaAlg/PVPI films are biocompatible and possess healing properties that accelerate the wound closure., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Phylogenetic studies of bacteria have been based so far either on a single gene (usually the 16S rRNA) or on concatenated housekeeping genes. For what concerns the genus Mycobacterium these approaches support the separation of rapidly and slowly growing species and the clustering of most species in well-defined phylogenetic groups. The advent of high-throughput shotgun sequencing leads us to revise conventional taxonomy of mycobacteria on the light of genomic data. For this purpose we investigated 88 newly sequenced species in addition to 60 retrieved from GenBank and used the Average Nucleotide Identity pairwise scores to reconstruct phylogenetic relationships within this genus., Results: Our analysis confirmed the separation of slow and rapid growers and the intermediate position occupied by the M. terrae complex. Among the rapid growers, the species of the M. chelonae-abscessus complex belonged to the most ancestral cluster. Other major clades of rapid growers included the species related to M. fortuitum and M. smegmatis and a large grouping containing mostly environmental species rarely isolated from humans. The members of the M. terrae complex appeared as the most ancestral slow growers. Among slow growers two deep branches led to the clusters of species related to M. celatum and M. xenopi and to a large group harboring most of the species more frequently responsible of disease in humans, including the major pathogenic mycobacteria (M. tuberculosis, M. leprae, M. ulcerans). The species previously grouped in the M. simiae complex were allocated in a number of sub-clades; of them, only the one including the species M. simiae identified the real members of this complex. The other clades included also species previously not considered related to M. simiae. The ANI analysis, in most cases supported by Genome to Genome Distance and by Genomic Signature-Delta Difference, showed that a number of species with standing in literature were indeed synonymous., Conclusions: Genomic data revealed to be much more informative in comparison with phenotype. We believe that the genomic revolution enabled by high-throughput shotgun sequencing should now be considered in order to revise the conservative approaches still informing taxonomic sciences., (Copyright © 2017 Elsevier B.V. All rights reserved.)

mGluR1 receptors are believed to play major roles in the pathophysiology of diseases such as anxiety and chronic pain and are being actively investigated as targets for drug development. Sequence polymorphisms can potentially influence the efficacy of drugs in patient populations and are therefore an important consideration in the drug development process. To identify DNA sequence variants of the mGluR1 receptor, comparative DNA sequencing was performed on DNA samples (n=186) from apparently healthy subjects representing two ethnic groups. In total, eight non-synonymous single nucleotide polymorphisms (SNPs) were identified and one SNP (c2977>T) was found to be particularly common, this SNP results in a proline to serine substitution at residue 993 (P993S). The WT (P993) and S993 variants were expressed in an inducible system which allowed us to titrate gene expression to equivalent levels and were pharmacologically characterized. We determined the potency and affinity of standard antagonist compounds as well as the potency and efficacy of the endogenous ligand glutamate and other agonist compounds at both receptor variants. Agonist evoked increases in intracellular Ca(2+) were measured by fluorometric imaging plate reader (FLIPR). The potency of mGluR1 antagonists was evaluated by their ability to inhibit quisqualate induced increases in intracellular Ca(2+), while their affinities were determined by radio-ligand binding studies. This study demonstrates that the Pro993Ser amino acid exchange is highly frequent in the human mGluR1 gene. This polymorphism however, does not appear to affect the potency of agonist compounds or the potencies or affinities of small molecule antagonist compounds.

A number of studies suggest the involvement of melanocortins in nociception, and although the mechanism through which this occurs is still unknown, experimental evidence would suggest an involvement of melanocortin MC(4) receptors. We investigated the effect of melanocortin receptor agonist and antagonists on nociceptive behaviour induced by formalin in the mouse. The intrathecal injection of the melanocortin receptor agonist MTII ([Ac-Nle(4),Asp(5),D-Phe(7),Lys(10)]cyclo-alpha-MSH-(4-10) amide) (5 nmol; P<0.05) significantly increased nociception in both phases of the formalin test, whereas the synthetic melanocortin receptor antagonists, SHU9119 ([Ac-Nle(4),Asp(5),D-2-Nal(7),Lys(10)]cyclo-alpha-MSH-(4-10) amide) (5 nmol), HS014 ([Ac-Cys(11),D-2-Nal(14),Cys(18)]beta-MSH-(11-22)amide) (5 nmol), and JKC-363 (cyclic [Mpr(11),D-Nal(14),Cys(18),Asp(22)-NH(2)]beta-MSH-11-22)) (5 nmol), and the endogenous receptor antagonist Agouti-related protein (AgRP) (1.5 nmol) were effective in reducing nociception in the late phase of the formalin test (50-60% of reduction in licking/flinching response; P<0.05). The present findings further support the involvement of the melanocortin system in the control of nociception. Moreover, considering that melanocortin MC(4) receptors are the only melanocortin subtype receptors present in the spinal cord, we can assume that the activity of the peptides in the formalin model is mediated through melanocortin MC(4) receptors.

Nociceptin, acting through the opioid receptor-like 1 (ORL1) receptor, produces anti-nociception in several models of neuropathy. We examined the involvement of the ORL1 receptor system in the allodynia developed after sciatic nerve ligation. Allodynic rats were selected by the von Frey hair and treated intrathecally with nociceptin or morphine. The peptide induced dose-dependent anti-allodynic activities, while morphine was effective at the higher dose only. By the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay, the two described ORL1 receptor isoforms were up-regulated in the allodynic animals, but unmodified in non-allodynic rats. Both short and long ORL1 receptor mRNA isoforms increased in the ipsilateral lumbar enlargement (by 50% and 100%, respectively), while 50% and 60% increases were found in the ipsilateral L5-L6 dorsal root ganglia, respectively. No significant changes were observed for either the nociceptin precursor or mu-opioid receptor expression. Thus, the ORL1 receptor system seems to regulate the mechano-allodynia that developed after nerve damage, suggesting its potential role in the treatment of neuropathic pain.

We have studied the effects of selective and non-selective adenosine receptor agonists and antagonists in audiogenic-seizure-sensitive DBA/2 mice, an animal model of generalized reflex epilepsy. With the exception of the adenosine A3 receptor agonist, N6-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine (IB-MECA), all the agonists studied prevented the development of audiogenic seizures in a dose-dependent manner. The ED50 values against the clonic phase of the audiogenic seizures were low, that is: 0.06 mg/kg, i.p., for the adenosine A1 receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), 0.02 and 0.03 mg/kg, i.p., for the adenosine A2A receptor agonists, 2-(4-(2-carboxyethyl)-phenylamino)-5'-N-ethylcarboxamidoadenosine (CGS 21680) and 2-hexynyl-5'-N-ethyl-carboxamidoadenosine (2-HE-NECA), and 0.7 mg/kg, i.p., for the adenosine A1/A3 receptor agonist, N6-2-(4-aminophenyl)ethyladenosine (APNEA). Conversely, the non-selective agonist, N-ethyl-carboxamidoadenosine (NECA), was highly potent, the ED50 being 0.0005 mg/kg, i.p. In the absence of auditory stimulation, the adenosine receptor antagonists increased the incidence of both clonic and tonic seizures in DBA/2 mice. The ED50 values were: for caffeine, 207.5 mg/kg, i.p., for the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 327.8 mg/kg i.p., for the adenosine A2A receptor antagonists, 3,7-dimethyl-1-propylxanthine (DPMX), 86.7 mg/kg i.p., for the (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3-dipropylxanthine (KF 17837), 69.1 mg/kg i.p., and 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-(4,3-c)1,2,4-triazolo(1,5 -c)-pyrimidine (SCH 58261), 321.8 mg/kg i.p. The rank order of convulsant potency in our epileptic model, following intracerebroventricular administration, was DPCPX > DMPX > 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC) > KF 17837 > Caffeine > SCH 58261 > 5-amino-9-chloro-2-(2-furyl)-1,2,4-triazolo(1,5-c)quinazoline (CGS 15943). Following a subconvulsant audiogenic stimulus of 83 dB, all adenosine receptor antagonists induced both tonic and clonic seizures. The ED50 values for such proconvulsant effects were: for caffeine 0.04 mg/kg, i.p., for the adenosine A receptor antagonist, DPCPX, 5.84 mg/kg, i.p., for the adenosine A2A receptor antagonists, DMPX, 0.02 mg/kg, i.p., CGS 15943, 0.29 mg/kg i.p., KF 17837, 0.57 mg/kg, i.p., CSC 0.12 mg/kg, i.p. and SCH 58261 0.07 mg/kg, i.p., respectively. These data suggest that stimulation of adenosine A1 and A2A receptors is involved in the suppression of seizures.

Interleukin 10 is an antiinflammatory cytokine and inhibits the production of tumor necrosis factor. We have previously found that intracerebroventricular (i.c.v.) administration of recombinant human interleukin 10 inhibits brain tumor necrosis factor production induced by an i.c.v. injection of lipopolysaccharide in mice. In view of its possible pharmacological use, we have now studied whether interleukin 10 administered peripherally could inhibit brain tumor necrosis factor production. Mice were injected with recombinant human interleukin 10 (20 microg/mouse, i.v.) 10 min-24 h before lipopolysaccharide (2.5 microg, i.c.v.). Tumor necrosis factor was measured, using a bioassay, in brain homogenates 90 min after lipopolysaccharide. Recombinant human interleukin 10 administered i.v. between 10 min and 6 h before lipopolysaccharide markedly inhibited brain tumor necrosis factor production. We also measured the production of tumor necrosis factor by whole blood of these mice, and it was also markedly inhibited by recombinant human interleukin 10 treatment. In conclusion, systemic recombinant human interleukin 10 administration inhibits brain tumor necrosis factor production. suggesting its usefulness in tumor necrosis factor-mediated pathologies of the central nervous system.

The protective effects of the selective adenosine A1 receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA), the selective adenosine A2A receptor agonist, 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA), and the non-selective agonist, 5'-N-ethylcarboxamidoadenosine (NECA) were studied against lethal seizures induced by intraperitoneal (i.p.) injection of pentylenetetrazole (80 mg/kg). In acute studies there was a dose-dependent reduction of lethal seizures, as shown by the low dose's protecting 50% of animals (PD50): 0.11, 0.05 and 0.05 mg/kg i.p. for CCPA, 2HE-NECA and NECA, respectively. In the repeated administration studies the animals received either vehicle or drug i.p. twice daily for 12 days. The drug doses were twice the PD50 value: 0.3 mg/kg for CCPA or 0.1 mg/kg for both 2HE-NECA and NECA. 2HE-NECA and NECA maintained their protective activity against pentylenetetrazole-induced seizures (63% or 60% vs. 60% or 58% in acute studies, respectively). Conversely, repeated treatment with CCPA resulted in a marked decrease of its effects (67% vs. 30% in acute studies; P < 0.05). The data indicate that in addition to adenosine A1 the A2A receptors also appear to be involved in the protection from seizures. The anticonvulsant effects induced by repeated stimulation of adenosine A1 receptors are subject to tolerance, whereas effects depending on adenosine A2A receptor activation are maintained.

The anticonvulsant activity of felbamate against sound-induced seizures was studied in the DBA/2 mouse model. Felbamate (10-300 mg/kg, i.p.) produced dose-dependent effects with ED50 values for the suppression of tonic, clonic and wild running phases of 23.1, 48.8 and 114.6 mg/kg, respectively. Felbamate also protected DBA/2 mice from N-methyl-D-aspartate (NMDA)-induced seizures with ED50 values of 12.1 and 29 mg/kg for tonus and clonus, respectively. Pretreatment with glycine, an agonist to the glycine/NMDA receptors, shifted the dose-response effect of felbamate to the right (ED50 = 56.8 against tonus and 94.8 mg/kg versus clonus). Similarly, D-serine, an agonist at the glycine site, shifted the ED50 of felbamate against the tonic component of audiogenic seizures from 23.1 to 78.1, and that against clonus from 48.8 to 90.3 mg/kg. Felbamate was also potent to prevent seizures induced by administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), an AMPA/kainate receptor agonist (ED50 = 11.8 and 20.9 mg/kg, against tonus and clonus, respectively). The data indicate that felbamate is an effective anticonvulsant drug in the genetic model of seizure-prone DBA/2 mice. Our findings suggest that the anticonvulsant properties of felbamate depend upon its interaction with neurotransmission mediated by both the glycine/NMDA and the AMPA/kainate receptor complex.

The effect of general anaesthesia on extracellular levels of acetylcholine (ACh) in the caudate-putamen of freely moving rats was studied by microdialysis. ACh concentrations were determined in the same perfusate samples by radioenzymatic and HPLC/electrochemical procedures in order to compare the assays. The concentration of ACh in perfusate samples was estimated to be 0.30 microM in conscious unrestrained rats. However, when these rats were administered chloral hydrate (400 mg/kg i.p.), the level of ACh was decreased immediately by 50%, attaining a value of 0.06 microM within 20-40 min following the injection. Upon recovery of the righting reflex, ACh levels were once again re-elevated. The levels of choline (Ch), the precursor of ACh, were unaffected by anaesthesia. It was apparent that the level of consciousness (i.e. awake vs. anaesthetized) is an important factor determining ACh overflow. Radioenzymatic and HPLC assays proved to give identical results for the analysis of ACh and Ch.

Two major systems of cholinergic projection neurons are found within the centrum of the mammalian brain: the basal nuclear complex, projecting predominantly to the cerebral cortex, amygdala, and hippocampus, and the pontomesencephalotegmental network, innervating primarily the thalamus. Neurons comprising the latter network also project to the basal forebrain, but the functional properties of that fiber connection, if any, are unknown. In an attempt to address this issue, the extracellular concentration of acetylcholine was measured in the basal nuclear complex of freely moving rats, both singularly and in combination with lesions and pharmacologic manipulations. Acetylcholine release monitored in the presence of physostigmine sulfate in the basal forebrain was (a) calcium-dependent, (b) increased by systemic scopolamine injection, the rise persisting in the presence of quisqualate lesions of the basal nuclear complex, (c) blocked by tetrodotoxin, and (d) abolished by ablation of cell bodies in the pontomesencephalic tegmentum, which also produced a decrease of choline acetyltransferase activity in the nucleus basalis/substantia innominata region, but not by quisqualate lesions of the basal forebrain. It is concluded from these data that the calcium-dependent release of acetylcholine in the basal nuclear complex (a) is largely axonal in nature, (b) derives substantially from axons of the cholinergic pontomesencephalic tegmentum, and (c) appears to be controlled by presynaptic muscarinic receptors on axon terminals of the latter system. The pontomesencephalotegmental cholinergic complex might thus influence cortical acetylcholine release, in part at least, by means of serial-order cholinergic-cholinergic interactions in the basal nuclear complex.

Experiments were carried out to gain additional evidence that l-fenfluramine reduces the dopamine-mediated effects in intact animals. l-Fenfluramine 5 and 10 mg/kg i.p. dose dependently raised the levels of homovanillic acid in the striatum and nucleus accumbens of rats 1 h after injection. The effect of 5 mg/kg l-fenfluramine disappeared and was actually reversed 4 and 8 h after injection. The effect of 10 mg/kg l-fenfluramine, administered 48 h after the last haloperidol dose, was completely antagonized in both striatum and nucleus accumbens of animals made tolerant to the effect of haloperidol on homovanillic acid levels (through repeated treatment with 1 mg/kg haloperidol i.p. twice daily for 11 days). Unlike haloperidol (0.25 mg/kg), l-fenfluramine in various doses (2.5-20 mg/kg i.p.) did not modify the levels of striatal 3-methoxytyramine or change the decrease induced by a s.c. injection of 0.5 mg/kg apomorphine. The effect of apomorphine was not antagonized by 10 or 20 mg/kg l-norfenfluramine, an active metabolite of l-fenfluramine but 20 mg/kg l-norfenfluramine significantly raised striatal 3-methoxytyramine levels. l-Fenfluramine 20 mg/kg (but not 10 mg/kg) significantly enhanced the output of striatal acetylcholine assessed by trans-striatal microdialysis, for 60 min after injection. Apomorphine 1 mg/kg i.p. completely antagonized the increase of acetylcholine caused by 1 mg/kg haloperidol or 20 mg/kg l-fenfluramine. The results confirm that the l isomer of fenfluramine produces effects on the responses to dopamine and acetylcholine similar to those of neuroleptics by a mechanism not involving direct blockade of receptors.