Transfection of human macrophages by lipoplexes via the combined use of transferrin and pH-sensitive peptides

The crucial function of macrophages in a variety of biological processes and pathologies render these cells important targets for gene therapeutic interventions. Commonly used synthetic gene delivery vectors have not been successful in transfecting these non-dividing cells. A combination strategy involving cationic liposomes to condense and carry DNA, transferrin to facilitate cellular uptake, and the pH-sensitive peptide GALA to promote endosome destabilization, resulted in significant expression of a luciferase gene. Transfection of macrophages was dependent on the degree of differentiation of the cells. The quaternary complexes of cationic liposomes, DNA, transferrin, and GALA exhibited a net negative charge, which may obviate a limitation of cationic synthetic vectors in vivo. The lack of cytotoxicity and the expected lack of immunogenicity of these complexes may render them useful for gene delivery to macrophages in vivo.     

The ultrastructure of the pre-meiotic and meiotic stages of spermatogenesis in Plagioscion squamosissimus (Teleostei,Perciformes, Sciaenidae)

Spermatogenesis of 'corvina' P. squamosissimus starts from a stem cell that gives rise to germ cells. These cells are enveloped by Sertoli cells, forming cysts. The germ cells in the cysts are all at the same stage of development and are interconnected by cytoplasmic bridges. Spermatogonia are the largest germ cells. In the cysts, these cells differentiate into primary spermatogonia and secondary spermatogonia. The primary spermatogonia are isolated in the cyst and give rise to the secondary spermatogonia. After several mitotic divisions, they produce spermatocytes I, which can be identified by synaptonemal complexes in the nucleus. The spermatocytes I enter the first phase of meiosis to produce the spermatocytes II. These are not very frequently seen because they rapidly undergo a second phase of meiosis to produce spermatids.     

Atypical lipid-dependent Malassezia species isolated from dogs with otitis externa

During a survey of the occurrence of Malassezia species in the external ear canals of dogs with chronic otitis externa, lipid-dependent Malassezia species were isolated in three dogs. These species were identified as Malassezia furfur and M. obtusa but showed atypical assimilation patterns. To our knowledge, this is the first report of the isolation of lipid-dependent species of the genus Malassezia in association with canine otitis.     

Response of jejunal Na+, K+-ATPase to 5-hydroxytryptamine in young and adult rats: effect of fasting and refeeding

The present study is aimed to evaluate the effects of 5-hydroxytryptamine (5-HT) upon jejunal Na+,K+-ATPase in young (20-day-old) and adult (60-day-old) rats, and determine the effect of food intake on the response of the sodium pump to the amine. Basal Na+,K+-ATPase activity in jejunal epithelial cells from young rats was twice that in adult animals and responded to 5-HT with stimulation. In adult rats, fasting reduced by 25% basal jejunal Na+, K+-ATPase activity, whereas in young rats, no such change was observed. The sensitivity of jejunal Na+,K+-ATPase to 5-HT in young fasted rats was similar to that observed in fed animals. The effect of refeeding in young rats was a 2-fold increase in jejunal Na+, K+-ATPase activity, this being accompanied by insensitivity to 5-HT. In adult rats, refeeding was accompanied by an increase in jejunal Na+,K+-ATPase activity. It is concluded that the stimulatory effect of 5-HT upon jejunal Na+,K+-ATPase activity is a phenomenon dependent on both age and type of diet. In young rats, it is the food intake that plays an important role in development of insensitivity of Na+,K+-ATPase to stimulation by 5-HT, while in adult animals fasting or fasting followed by refeeding does not play a major role in regulating its sensitivity to the amine.     

5-HT mechanisms of median raphe nucleus in the conditioned freezing caused by light/foot-shock association

We have shown that 5-HT mechanisms of the median raphe nucleus (MRN) are involved in contextual fear-conditioning processes as electrolytic or neurotoxic lesions with N-methyl-D-aspartate (NMDA) or injections of 8-hydroxy-2-(di-n-propilamino)-tetralin (8-OH-DPAT) into this structure inhibit freezing behavior in a contextual fear paradigm. In this work, we extend these studies by analyzing the behavioral responses in a classical fear-conditioning paradigm (light or tone/foot-shock association) in rats with either neurochemical lesion with NMDA or injected with 8-OH-DPAT into the MRN. The animals received NMDA or 8-OH-DPAT or saline microinjections into the MRN and were submitted to conditioning trials in an experimental chamber, where they received 10 foot-shocks (0.6 mA, 1 s, variable interval between 10 and 50 s) paired with tone or light (CS). On the next day, they were tested in a different experimental chamber, with or without CS presentation, where the duration of freezing and the number of rearing episodes were recorded. Light or tone alone caused a significant amount of freezing. NMDA lesions or 8-OH-DPAT injections into the MRN clearly inhibited freezing behavior in rats conditioned to light/foot-shock association, but not in the conditioning sessions with tones. Besides the proposed role in contextual fear conditioning, these results clearly show that MRN is involved in the fear conditioning with light as conditioned stimuli. Distinct neural substrates seem to subserve conditioning fear with acoustic stimuli.     

Role of macrophages,interferon gamma and procoagulant activity in the resistance of genetic heterogeneous mouse populations to mouse hepatitis virus infection

Summary Genetic heterogeneous mouse populations selected for high (H_III) and low (L_III) antibody response were used to study some aspects of mouse hepatitis virus 3 (MHV3) infection, such as the resistance pattern, virus replication in the liver and peritoneal exudate or in cultured peritoneal macrophages, the interferon (IFN) synthesis in the serum and peritoneal exudate and the procoagulant activity (PCA) of the peritoneal exudate (PEC) and spleen cells (SC). The H_III mice, when compared to their L_III mice counterparts, were susceptible to MHV3 infection showing higher virus titres in the liver and peritoneal exudate, comparable IFN alpha/beta or IFN gamma titres in the peritoneal exudate or in the serum, and higher levels of PCA of PEC and SC. A higher virus titre was detected in the supernatants of H_III mouse macrophages infected with MHV3. The activation of H_III mouse macrophages with LPS, IFN alpha/beta or IFN gamma, in contrast to that of L_III mouse macrophages, did not induce an antiviral effect with partial restriction of the MHV3 replication. The LPS antiviral activity was shown to be partially exerted by IFN alpha/beta synthesis. The IFN gamma was shown to be more effective in inducing an antiviral state in L_III macrophages, when compared to IFN alpha/beta. The data obtained are consistent with the notion that the resistance mechanisms to the MHV3 infection involve the PCA and the sensitivity of macrophages to IFN.     

Inhibition of nitric oxide synthase increases synaptophysin mRNA expression in the hippocampal formation of rats

Synaptophysin is a protein involved in the biogenesis of synaptic vesicles and budding. It has been used as an important tool to investigate plastic effects on synaptic transmission. Nitric oxide (NO) can influence plastic changes in specific brain regions related to cognition and emotion. Experimental evidence suggests that NO and synaptophysin are co-localized in several brain regions and that NO may change synaptophysin expression. Therefore, the aim of the present work was to investigate if inhibition of NO formation would change synaptophysin mRNA expression in the hippocampal formation. Male Wistar rats received single or repeated (once a day for 4 days) i.p. injections of saline or l-nitro-arginine (l-NOARG, 40 mg/kg), a non-selective inhibitor of nitric oxide synthase (NOS). Twenty-four hours after the last injection the animals were sacrificed and their brains removed for ‘in situ’ hybridization study using ³⁵S-labeled oligonucleotide probe complementary to synaptophysin mRNA. The results were analyzed by computerized densitometry. Acute administration of l-NOARG induced a significant (p < 0.05, ANOVA) increase in synaptophysin mRNA expression in the dentate gyrus, CA1 and CA3. The effect disappeared after repeated drug administration. No change was found in the striatum, cingulated cortex, substantia nigra or nucleus accumbens. These results reinforce the proposal that nitric oxide is involved in plastic events in the hippocampus.     

Evaluation of dot enzyme-linked immunosorbent assay for mucocutaneous leishmaniasis and comparison with microplate enzyme immunoassay.

A dot enzyme-linked immunosorbent assay (dot ELISA) was evaluated and compared with a standard microplate ELISA (immunoglobulin G [IgG] ELISA) for the serological diagnosis of mucocutaneous leishmaniasis. The two assays were used to test 113 serum specimens from the following groups: normal individuals and patients with deep mycoses, toxoplasmosis, mucocutaneous leishmaniasis, visceral leishmaniasis, Chagas' disease, malaria, and schistosomiasis. Both tests exhibited cross-reactivity when testing specimens from cases of visceral leishmaniasis and Chagas' disease. The dot ELISA proved to be economical with respect to use of reagents and was easy to perform. Interpretation could easily be made by visual inspection of reaction endpoints in the nitrocellulose disks, obviating the need for spectrophotometric readings. There were no significant differences in sensitivity between the dot ELISA and the IgG ELISA at a cutoff level either of 20 or 40. However, its most remarkable feature was the high specificity compared with that of the IgG ELISA. Because of its ease of performance and high sensitivity and specificity, the dot ELISA should be an excellent test to be executed in the field during seroepidemiological surveys.