Preclinical evaluation of radiation and systemic, RGD-targeted, adeno-associated virus phage-TNF gene therapy in a mouse model of spontaneously metastatic melanoma.

The incidence of melanoma in the United States continues to rise, with metastatic lesions notoriously recalcitrant to therapy. There are limited effective treatment options available and a great need for more effective therapies that can be rapidly integrated in the clinic. In this study, we demonstrate that the combination of RGD-targeted adeno-associated virus phage (RGD-AAVP-TNF) with hypofractionated radiation therapy results in synergistic inhibition of primary syngeneic B16 melanoma in a C57 mouse model. Furthermore, this combination appeared to modify the tumor microenvironment, resulting in decreased Tregs in the draining LN and increased tumor-associated macrophages within the primary tumor. Finally, there appeared to be a reduction in metastatic potential and a prolongation of overall survival in the combined treatment group. These results indicate the use of targeted TNF gene therapy vector with radiation treatment could be a valuable treatment option for patients with metastatic melanoma.

A study on ovine pneumonic pasteurellosis: isolation and identification of Pasteurellae and their antibiogram susceptibility pattern in Haramaya District, Eastern Hararghe, Ethiopia.

BACKGROUND: Sheep constitute the second major component of livestock in Ethiopia. However, efficient utilization of this potential resource is hampered by combination of health problems, poor management and feed shortage. Haramaya district is one of the remote settings in Ethiopia where information about the livestock disease is not well documented. Hence this study was conducted to determine the causative agents and their antimicrobial susceptibility pattern of bacterial Pasteurella isolates among pneumonic ovine in Haramaya district, Eastern Hararghe, Ethiopia. RESULTS: Out of 256 samples examined, Pasteurella was isolated in 64 (25%), of which 38 (59.4%) were from lungs and 26 (40.6%) were from nasal cavities. 87.5% of the isolates were Mannheimia haemolytica and 12.5% were Pasteurella multocida. All of the isolates from the lungs were Mannheimia haemolytica whereas 69% of the isolates from nasals cavities were Mannheimia haemolytica. Age and body temperature were significantly associated with Pasteurella isolates from clinic (P < 0.05). Despite diverse in the site of origins, the isolates exhibited uniformity in sensitivity to a majority of the antibacterial agents. The most effective drug was Cholramphenicol (100%) followed by Sulfamethoxazole (89.1%) and Tetracycline (84.4%). Both species were completely resistant to Gentamycin and Vancomycin. CONCLUSION: Mannheimia haemolytica is the most common cause of ovine pneumonic pasteurellosis in the study area. The isolates were susceptible to limited antimicrobial agents. Therefore, the antimicrobial susceptibility test should be conducted before treatment, except for critical cases.

Estrogen and progesterone treatment: effects on muscarinic M(4) receptor subtype in the rat brain.

We investigated the effect of ovariectomy (OVX) and hormonal treatment for 10 weeks by estradiol and progesterone on muscarinic M(4) receptor subtype in different brain areas of female rats. Moreover, motor activity of OVX and hormone-treated rats was measured by automated open field exploration boxes. Receptor quantification in the hippocampus, frontal cortex, parietal cortex, amygdala and hypothalamus was done by receptor autoradiography using a selective ligand for muscarinic M(4) receptors. Ovariectomy up-regulated M(4) receptors in the dentate gyrus, CA1, CA3, frontal cortex and hypothalamus whereas the estrogen treatment restored M(4) binding to that of the sham group. Progesterone treatment had no effect on the ovariectomy-induced up-regulation of M(4) receptors. Ovariectomy significantly decreased the exploratory activity of the rats compared to the sham group. Estrogen treatment restored the exploratory behavior of the ovariectomized rats to that of the sham group whereas the progesterone-treated rats were less alert to the surrounding when compared to the sham and estrogen supplemented rats. The effect of estrogen on the hippocampal muscarinic M(4) receptor subtype is a novel finding and may have functional significance for cholinergic receptors especially in relation to postmenopausal memory problems and neurodegenerative disease like Alzheimer's disease.

Localization of M1 muscarinic receptors in rat brain using selective muscarinic toxin-1.

Mambas, African snakes of the genus Dendroaspis, produce several types of toxins that are of pharmacological interest. The novel muscarinic toxin-1 (MT-1), from the green mamba Dendroaspis angusticeps, binds specifically to muscarinic M1 receptors in homogenates of rat cerebral cortex. Iodination of the toxin, 125I-muscarinic toxin-1 (125I-MT-1), renders the toxin selective for M1 muscarinic receptors. Quantitative measurement of 125I-MT-1 autoradiography in rat brain sections indicated highest labeling in the nucleus accumbens, striatum, and dentate gyrus. High densities of 125I-MT-1 binding sites were located in the CA1 region of the hippocampus, frontal, and parietal cortices. Moderate densities of binding sites were seen in temporal cortex, and hippocampal subregions CA2, CA3, and CA4, whereas low labeling was observed in the cerebellum and spinal cord.

Effects of nucleus basalis lesion on muscarinic receptor subtypes.

The cholinergic system in the central nervous system is an important component of the neural circuitry of learning, memory and cognition. A decline of cholinergic innervation in the human brain is a characteristic feature of dementia of Alzheimer's type. In this study, changes in cholinergic markers were studied after a unilateral lesion of the nucleus basalis magnocellularis (nbM). Acetylcholinesterase (AChE) histochemistry showed a loss of cortical AChE-containing neurons, and choline acetyltransferase (ChAT) immunohistochemistry demonstrated a loss of cholinergic cells in nbM. The localizations of muscarinic M1 and M2 receptors using [3H]pirenzepine ([3H]PZ) and [3H]AF-DX 384, respectively, were studied by quantitative autoradiography 1, 2, 4 and 6 weeks following unilateral ibotenic acid lesion of nbM. A significant decrease in [3H]PZ binding sites was observed at postlesion week 1 in the parietal and temporal cortices. The decrease in [3H]AF-DX 384 binding sites on the lesioned side was observed throughout frontal, parietal and temporal cortices after postlesion week 1, with a significant increase after 6 weeks, possibly as result of loss of presynaptic receptors and upregulation of postsynaptic ones. Moreover, laminar distribution after nbM lesion shows that M1 and M2 receptor binding sites are more affected in superficial layers (I,II,III) than in the deep layers (IV,V,VI), depending on ligand, postlesion period and cortical region. Furthermore, nbM lesion causes a higher deficit of M2 receptors than of M1 receptors. These data suggest the existence of a presynaptic population as well as a postsynaptic population of M1 and M2 receptors which are differently affected after unilateral nbM lesion.

Distribution of nicotinic receptors in human thalamus as visualized by 3H-nicotine and 3H-acetylcholine receptor autoradiography.

Nicotinic cholinergic receptors in human thalamus were measured using (-)3H-nicotine (20 nM) and 3H-acetylcholine (3H-ACh) (20 nM) as radioligands. The specific binding for 3H-nicotine to homogenates of thalamus was 51.6 +/- 8.3 pmol/g protein and for 3H-ACh 18.6 +/- 1.9 pmol/g protein. Receptor autoradiography indicated a high labelling of both 3H-Nicotine and 3H-ACh in the antero-ventral nucleus of thalamus and dorso-medial nucleus of thalamus, while the labelling was lower in the postero-lateral nucleus of thalamus and in the postero-lateral ventral nucleus of thalamus. Quantitative measurement of the 3H-nicotine autoradiograms showed highest labelling in the anteroventral nucleus of thalamus (17.34 +/- 0.76 pmol/g tissue). This study indicates a heterogeneous distribution of high-affinity nicotinic receptors in the human thalamus.

[3H]acetylcholine nicotinic recognition sites in human brain: characterization of agonist binding.

In the presence of a cholinesterase inhibitor to prevent hydrolysis and atropine to block muscarinic cholinergic receptors, [3H]acetylcholine ([3H]ACh) binding to human brain membranes showed highest levels of nicotinic binding sites in the thalamus. [3H]ACh, in the presence of atropine, binds to heterogeneous high-affinity binding sites in human thalamus. Scatchard analysis of the binding gave a Kd of 0.58 nM and a Bmax of 3.3 pmol/g protein for the 'super high-affinity' site and a Kd of 27 nM and a Bmax of 70 pmol/g protein for the 'high-affinity' site. Moreover, in competition studies nicotinic agonists such (-)-nicotine and carbachol displaceable [3H]ACh-specific binding sites consist of both a high- and a low-affinity population of sites. These results indicate that highest levels of [3H]ACh binding in human brain were found in the thalamus. Moreover, the human thalamus was found to have multiple high-affinity nicotinic agonist sites.