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 behavior of novel hydrophilic composite bone cements in simulated body fluids

Composite bone cements were formulated with bioactive glass (MgO--SiO(2)--3CaO. P(2)O(5)) as the filler and hydrophilic matrix. The matrix was composed of a starch/cellulose acetate blend (SCA) as the solid component and a mixture of methylmethacrylate/acrylic acid (MMA/AA) as the liquid component. The curing parameters, mechanical properties, and bioactive behavior of these composite cements were determined. The addition of up to 30 wt % of glass improved both compressive modulus and yield strength and kept the maximum curing temperature at the same value presented by a typical acrylic-based commercial formulation. The lack of a strongly bonded interface (because no coupling agent was used) had important effects on the swelling and mechanical properties of the novel bone cements. However, bone cements containing AA did not show a bioactive behavior, because of the deleterious effect of this monomer on the calcium phosphate precipitation on the polymeric surfaces. Formulations without AA were prepared with MMA or 2-hydroxyethyl methacrylate (HEMA) as the liquid component. Only these formulations could form an apatite-like layer on their surface. These systems, therefore, are very promising: They are bioactive, hydrophilic, partially degradable, and present interesting mechanical properties. This combination of properties could facilitate the release of bioactive agents from the cement, allow bone ingrowth in the cement, and induce a press-fitting effect, improving the interfaces with both the prosthesis and the bone.     

Fluorescence in situ hybridization analysis of HER-2/neu in brushings of normal oral mucosa

Oncogene alterations have been clearly demonstrated to be related to the carcinogenesis and progression of oral squamous cell carcinoma (OSCC). However, the analysis of these alterations for screening and early diagnostic purposes generally requires invasive techniques for surgical removal of pathological epithelium. The aim of the present study was to assess the feasibility of fluorescence in situ hybridization (FISH) analysis of HER-2/neu amplification in oral mucosa brushings and to compare the HER-2/neu status with the history and smoking and drinking habits of healthy subjects. Cells obtained by centrifugation of oral brushings from 21 subjects (overall no. of cells: 5125) were suspended in physiological saline and fixed onto two slides for cytological evaluation and FISH analysis (dual-target, dual-color fluorescence assay) of the HER-2/neu gene and CEP17 centromere. A mean of 89.8% of the cells showed two HER-2/neu signals and a mean of 94% had two CEP17 signals at fluorescent microscopy. Finally, a mean of 96% of cells with HER-2/neu / CEP17 had a ratio equal to 1. No association between smoking and drinking habits, age and the HER-2/neu and CEP17 characteristics evaluated by FISH was found.     

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.     

Platelet-activating factor receptor deficiency delays elimination of adult worms but reduces fecundity in Strongyloides venezuelensis-infected mice

We describe the parasitological kinetics and histopathological and immunological alterations in platelet-activating factor receptor-deficient (PAFR(-/-)) and wild-type mice after a single Strongyloides venezuelensis infection (subcutaneous inoculation of 500 L3 larvae). There was no difference in the numbers of worms that reached and became established in the small intestines of PAFR(-/-) and wild-type mice. However, at 12 days after infection, significantly more worms were recovered from PAFR(-/-) mice. Although PAFR(-/-) infected mice showed a delay in elimination of adult worms, worms established in the small intestine of these mice produced a significantly lower number of eggs due to a reduction in worm fecundity. There were also significant reductions in the number of circulating and tissue eosinophils and tumor necrosis factor levels in the small intestines of PAFR(-/-) mice infected for 7 days compared to the number and level in wild-type mice. Histological analysis confirmed the reduced inflammatory process and revealed that the PAFR(-/-) mice had a smaller number of goblet cells. The concentrations of the type 2 cytokines interleukin-4 (IL-4), IL-5, and IL-10 were lower in small intestine homogenates and in supernatants of antigen-stimulated lymphocytes from spleens or mesenteric lymph nodes of PAFR(-/-) mice than in the corresponding preparations from wild-type mice. Thus, in S. venezuelensis-infected PAFR(-/-) mice, decreased intestinal inflammation is associated with enhanced worm survival but decreased fecundity. We suggest that although a Th2-predominant inflammatory response decreases worm survival, the worm may use factors produced during this response to facilitate egg output and reproduction. PAFR-mediated responses appear to modulate these host-derived signals that are important for worm fecundity.