Rat bone marrow stromal cell osteogenic differentiation and fibronectin adsorption on chitosan membranes: the effect of the degree of acetylation

Cell adhesion, migration, and proliferation of a few anchorage-dependent cells cultured on chitosan (Ch) matrices are influenced by the degree of N-acetylation (DA) of Ch. In the present work, we examined the influence of the DA on the attachment, spreading, proliferation, and osteogenic differentiation of rat bone marrow stromal cells (rBMSCs). Ch membranes were characterized in terms of surface morphology, roughness, and wettability, and in terms of adsorption of an adhesive protein, fibronectin (Fn). Chs with DAs in the range of 4 to 49% were used. Among the Ch samples, the DA of 4% led to the highest Fn surface concentration, both from single protein solution and from diluted serum. Furthermore, the levels of Fn adsorbed from serum found for this DA were threefold higher than for the tissue culture polystyrene control, indicating that in the presence of competitive proteins Ch is more specific toward Fn adsorption than tissue culture polystyrene. rBMSCs cultured on Ch carrying a DA of 4% were able to spread, proliferate, and differentiate, reaching a higher level of osteogenic differentiation than on the control, despite the lower cell attachment observed for all Ch samples. Because the Ch sample with a DA of 4% showed the highest Fn adsorption from serum, we suggest that cell adhesion, spreading, and osteogenic differentiation of rBMSCs on Ch may be mediated by the adsorbed layer of Fn.     

Copolymers of trimethylene carbonate and epsilon-caprolactone for porous nerve guides: synthesis and properties

Copolymers of trimethylene carbonate and epsilon-caprolactone were synthesized and characterized with the aim of assessing their potential in the development of a flexible and slowly degrading artificial nerve guide for the bridging of large nerve defects. The effect of the monomer ratio on the physical properties of the polymers and its influence on the processability of the materials was investigated. Under the applied polymerization conditions (130 degrees C, 3 days using stannous octoate as a catalyst) high molecular weight polymers (Mn above 93 000) were obtained. All copolymers had glass transition temperatures below room temperature. At trimethylene carbonate contents higher than 25 mol% no crystallinity was detected. A decrease in crystallinity resulted in the loss of strength and decrease in toughness, as well as in an increased polymer wettability. Amorphous poly(trimethylene carbonate), however, showed excellent ultimate mechanical properties due to strain-induced crystallization (Tm = 36 degrees C). Low crystallinity copolymers could be processed into dimensionally stable porous structures by means of immersion precipitation and by combination of this technique with the use of porosifying agents. Porous membranes of poly(trimethylene carbonate) could be prepared when blended with small amounts of high molecular weight poly(ethylene oxide). Poly(trimethylene carbonate) and poly(trimethylene carbonate-co-epsilon-caprolactone) copolymers with high epsilon-caprolactone content possess good physical properties and are processable into porous structures. These materials are most suitable for the preparation of porous artificial nerve guides.     

Alternative methods to analyse the impact of HIV mutations on virological response to antiviral therapy

Background  

Principal component analysis (PCA) and partial least square (PLS) regression may be useful to summarize the HIV genotypic information. Without pre-selection each mutation presented in at least one patient is considered with a different weight. We compared these two strategies with the construction of a usual genotypic score.     

New microsatellite multiplex PCR for Candida albicans strain typing reveals microevolutionary changes

Five new microsatellite loci were described and characterized for use as molecular markers for the identification and genetic differentiation of Candida albicans strains. Following the typing of 72 unrelated clinical isolates, the analysis revealed that they were all polymorphic, presenting from 5 to 30 alleles and 8 to 46 different genotypes. The discriminatory power obtained by combining the information generated by three microsatellites used in a multiplex PCR amplification strategy was 0.99, the highest ever reported. The multiplex PCR was later used to test a total of 114 C. albicans strains, including multiple isolates from the same patient collected from different body locations and along episodes of vulvovaginal infections. Three different scenarios for strain relatedness were identified: (i) different isolates that were revealed to be the same strain, (ii) isolates that were the same strain but that apparently underwent a process of microevolution, and (iii) isolates that corresponded to different strains. Analysis of the microevolutionary changes between isolates from recurrent infections indicated that the genotype alterations observed could be the result of events that lead to the loss of heterozygosity (LOH). In one case of recurrent infection, LOH was observed at the CAI locus, and this could have been related to exposure to fluconazole, since such strains were exposed to this antifungal during treatment. The analysis of microsatellites by a multiplex PCR strategy was found to be a highly efficient tool for the rapid and accurate differentiation of C. albicans strains and adequate for the identification of fine microevolutionary events that could be related to strain microevolution in response to environmental stress conditions.     

Recombinant dissection of myosin heavy chain of Toxocara canis shows strong clustering of antigenic regions

Myosins from nematode parasites elicit strong humoral and cellular immune responses and have been investigated as vaccine candidates. In this study we cloned and sequenced a cDNA coding for myosin heavy chain from Toxocara canis, a nematode parasite of canids which may also infect humans and cause various unspecific symptoms. To determine the major antigenic regions the myosin heavy chain was systematically dissected into ten overlapping recombinant fusion polypeptides which were purified by metal chelate chromatography. Single fragments were then tested for their IgG reactivity in sera from toxocarosis patients and healthy probands. Two regions, one region at the mid to carboxy-terminal end of the head domain and one region in the rod domain, were identified as major antigens, which in combination were positive with 86% of the sera. The other domains were less reactive. This shows that the patients' IgG reactivity was not directed evenly against all parts of the molecule, but was rather clustered in few regions.     

Distribution of glycoconjugates during cochlea development in mice: light microscopic lectin study

During development, different epithelial cells in the mouse cochlea express different cell surface glycoconjugates, which may reflect membrane specialization. Some of the lectins tested in this study (SBA, succ-WGA, and PSA) labeled the sensory cells of the cochlea around birth. Other lectins (WGA, Con A, RCA-II, and PHA-E) labeled surfaces of the sensory cells, particularly the stereocilia, from early stages of development (gestation day (GD) 16) through 21 days after birth. These may be adhesion molecules needed to attach the newly forming tectorial membrane (TM) to the stereocilia. Lectin staining of the developing TM revealed that the substructures of the TM are biochemically distinct. Lectin staining also showed the temporal sequence of the expression of cytoplasmic glycoconjugates of the cochlear epithelium during development. Biochemical changes during development are probably the result of different cells being involved in the production of glycoconjugates, and may have functional significance, specifically with regard to the expression of adhesion and/or signaling molecules.     

Correlations of Soluble Interleukin-2 and Tumor Necrosis Factor Type II Receptors with Immunologic and Virologic Responses Under HAART

We assessed the correlations between some plasma markers of immune activation (soluble receptors of interleukin 2 (sIL2-R) and TNFp75 (sTNFII-R) and usual markers of HIV infection in patients treated with protease-inhibitors (PI). Forty-six PI-naive HIV-1-infected adults were included in a 1-year prospective cohort from the initiation of a PI-containing regimen (M0). Measurements of CD4+cell count, plasma HIV-RNA, sIL2-R and sTNFII-R were performed at M0, M6, and M12. The evolution of sIL2-R from baseline to M12 was significantly different between immunological responders (IR) (CD4+count above 200/mm³ for subject having less than 200 CD4 +/mm³ at inclusion, or increase of at least 50 CD4+/mm³ for others) (58 UI/ml) and non-IR (+28 UI/ml) (P =0.01). The evolution of sTNFII-R between M0 and M12 was significantly different between virological responders (VR) (plasma HIV-1 RNA less than 500 copies/ml at M12) (–2.5 ng/ml) and non-VR (+0.2 ng/ml) (P =0.02). Our study shows significative correlations between the evolutions of soluble interleukin-2 and TNFR-II receptors and those of CD4+T-lymphocytes or HIV-RNA responses in patients under HAART.     

Microhardness of superficial and deep sound human dentin

Our purpose in this study was to determine the microhardness of superficial and deep dentin by means of two indentation methods (Knoop and Vickers) under two different applied loads. Twelve dentin discs approximately 2-mm thick were obtained from both superficial and deep dentin by transversally sectioning the crowns of sound, extracted human third molars with a diamond blade under water irrigation. Dentin surfaces were sequentially polished, and indentations (n = 20 per surface) were performed with either Vickers indentor at loads of 300 and 500 g, respectively, or Knoop indentor at loads of 50 and 100 g, respectively. Average Vickers hardness number (VHN) and Knoop hardness number (KHN) were calculated and treated with two-way analysis of variance (ANOVA) and Student's t test. Microhardness of dentin was not influenced by the different loads applied for both indentation methods. Knoop hardness was significantly higher for superficial than for deep dentin (p < 0.05). Conversely, Vickers hardness was not significantly different for both substrates (p > 0.05). Differences in dentin hardness as a function of depth exist, but they might not be relevant, and no alteration of the distribution of stresses along the adhesive interface is expected.     

Sublingual immunotherapy for hazelnut food allergy: a randomized, double-blind, placebo-controlled study with a standardized hazelnut extract

BACKGROUND: Food allergy may be life-threatening, and patients affected need to receive accurate diagnoses and treatment. Hazelnut has often been implicated as responsible for allergic reactions, and trace quantities can induce systemic reactions. OBJECTIVE: The aim of this study was to evaluate the efficacy and tolerance of sublingual immunotherapy with a standardized hazelnut extract in patients allergic to hazelnut. METHODS: This was a randomized, double-blind, placebo-controlled study. Inclusion criteria were a history of hazelnut allergy and positive skin prick test and double-blind placebo-controlled food challenge results. Patients were then randomly assigned into 2 treatment groups (hazelnut immunotherapy or placebo). Efficacy was assessed by double-blind, placebo-controlled food challenge after 8 to 12 weeks of treatment. Blood samples were drawn for measurement of specific IgE, IgG(4), and serum cytokines before and after treatment. RESULTS: Twenty-three patients were enrolled and divided into 2 treatment groups. Twenty-two patients reached the planned maximum dose at 4 days. Systemic reactions were observed in only 0.2% of the total doses administered. Mean hazelnut quantity provoking objective symptoms increased from 2.29 g to 11.56 g (P = .02; active group) versus 3.49 g to 4.14 g (placebo; NS). Moreover, almost 50% of patients who underwent active treatment reached the highest dose (20 g), but only 9% in the placebo. Laboratory data showed an increase in IgG(4) and IL-10 levels after immunotherapy in only the active group. CONCLUSION: Our data confirm significant increases in tolerance to hazelnut after sublingual immunotherapy as assessed by double-blind, placebo-controlled food challenge, and good tolerance to this treatment.     

Prognostic factors in adult community-acquired bacterial meningitis: a 4-year retrospective study

The aim of this 4-year, observational, single-center study was to identify prognostic factors and evaluate the need for intensive care in cases of bacterial meningitis. During the study period, 60 cases of adult bacterial meningitis were identified. Fifty-one patients were transferred to the intensive care unit at various times during their hospital stay. In the multivariate analysis, factors significantly associated with the need for mechanical ventilation and/or vasopressive drugs included comorbidity and a Glasgow coma score of less than 12 at hour 6 following presentation. The results indicate patients with a decreased level of consciousness, neurological deficit or comorbidity should be admitted to the intensive care unit at an early stage of illness. When patients lack these criteria 6 h following presentation, admission to the medical ward is reasonable.