Surface chemistry modulates focal adhesion composition and signaling through changes in integrin binding

Biomaterial surface properties influence protein adsorption and elicit diverse cellular responses in biomedical and biotechnological applications. However, the molecular mechanisms directing cellular activities remain poorly understood. Using a model system with well-defined chemistries (CH₃, OH, COOH, NH₂) and a fixed density of the single adhesive ligand fibronectin, we investigated the effects of surface chemistry on focal adhesion assembly and signaling. Surface chemistry strongly modulated integrin binding and specificity—₅β₁ integrin binding affinity followed the pattern OH>NH₂=COOH>CH₃, while integrin _Vβ₃ displayed the relationship COOH>NH₂OH=CH₃. Immunostaining and biochemical analyses revealed that surface chemistry modulates the structure and molecular composition of cell-matrix adhesions as well as focal adhesion kinase (FAK) signaling. The neutral hydrophilic OH functionality supported the highest levels of recruitment of talin, -actinin, paxillin, and tyrosine-phosphorylated proteins to adhesive structures. The positively charged NH₂ and negatively charged COOH surfaces exhibited intermediate levels of recruitment of focal adhesion components, while the hydrophobic CH₃ substrate displayed the lowest levels. These patterns in focal adhesion assembly correlated well with integrin ₅β₁ binding. Phosphorylation of specific tyrosine residues in FAK also showed differential sensitivity to surface chemistry. Finally, surface chemistry-dependent differences in adhesive interactions modulated osteoblastic differentiation. These differences in focal adhesion assembly and signaling provide a potential mechanism for the diverse cellular responses elicited by different material properties.     

Safety of various dosage regimens during induction of sublingual immunotherapy. A preliminary study

BACKGROUND: Sublingual immunotherapy (SLIT) has been demonstrated to be a viable alternative to injection immunotherapy. Administration of high doses of allergens to ensure efficacy has been shown to be well tolerated. The aim of the present study was the first step to address the issue of fast-induction regimens using various induction SLIT regimens in paediatric and adult patients. METHODS: Sixty-four patients (age range 5-46 years) with grass pollen rhinoconjunctivitis were enrolled in an 8-month double-blind, placebo-controlled trial of SLIT. Sixty-three patients were randomized to four groups and evaluated at the end of the study. One group received placebo (n = 16) and the other three groups (n = 47) received five grass pollen extracts according to three different induction regimens: regimen 1 starting with 3 IR tablets (n = 15), regimen 2 starting with 10 IR (n = 16) and regimen 3 starting with 30 IR (n = 16). The maintenance phase was made with sublingual-swallow drops at the same concentration of 300 IR/ml for all the patients. Adverse events were recorded on diary cards. RESULTS: During induction phase, 25/47 patients in the SLIT groups had adverse reactions in comparison to 2/16 patients in the placebo group (p < 0.05). The rate of adverse reactions was 33.3% (11.8-61.6) (95% CI) for regimen 1, 31.3% (11.0-58.7) for regimen 2, 43.8% (19.8-70.1) for regimen 3 and 12.5% (1.6-38.3) for placebo. Fifty-seven reactions were local reactions involving the oral region (54 SLIT, 3 placebo) and 13 were systemic reactions (all in the SLIT groups). 11/13 reactions were mild (gastrointestinal disorders, rhinoconjunctivitis), 1/13 consisted of moderate asthma and 1/13 consisted of severe abdominal pain. No urticaria, angioedema or life-threatening events were observed. CONCLUSIONS: These preliminary data showed that various induction regimens for SLIT are generally well tolerated and could allow a fast build-up phase of SLIT.     

Identification and typing of Streptomyces strains: evaluation of interspecific, intraspecific and intraclonal differences by RAPD fingerprinting

The suitability of random amplified polymorphic DNA PCR for the detection of differences between Streptomyces species and strains was evaluated. For this purpose, a protocol of RAPD specific for Streptomyces DNA, i.e. suitable for DNA presenting a high G+C content, was developed using S. ambofaciens ATCC23877. Among the 30 primers tested, all containing 80% G+C, 17 gave a pattern with this strain. Six oligonucleotides were chosen to compare 12 strains belonging to six species of Streptomyces. These oligonucleotides were then used to determine whether these strains could be differentiated at the DNA level with this method. All fingerprints obtained with six primers differed from one species to another. We showed that the RAPD method could be used to reveal intraspecific and intraclonal polymorphisms. Thus, RAPD allows for the rapid, sensitive and specific detection of genetic diversity among species and strains of Streptomyces.     

Chronic pneumonia despite adaptive immune response to Mycobacterium bovis BCG in MyD88-deficient mice

To assess the role of Toll-like receptor (TLR) signalling in host response to mycobacterial infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) were infected with the vaccine strain Mycobacterium bovis (BCG), and the immune response and bacterial burden were investigated. Macrophages and dendritic cells from MyD88-deficient mice stimulated in vitro with BCG mycobacterial antigens produced very low levels of proinflammatory cytokines, while the expression of costimulatory molecules such as CD40 and CD86 was preserved. Upon systemic infection with BCG (2 x 10(6) CFU i.v.) MyD88-deficient mice developed confluent chronic pneumonia with two log higher CFU than wild-type mice. Interestingly, the infection was controlled in liver and spleen and there was efficient systemic T-cell priming with high IFNgamma production by CD4+ splenic T cells in MyD88-deficient mice. Lung infiltrating cells showed IFNgamma production by pulmonary CD4+ T cells upon specific restimulation, and a reduced capacity to produce nitric oxide and IL-10. In summary, despite the dramatic reduction of the innate immune response, MyD88-deficient mice were able to mount an efficient T-cell response to mycobacterial antigens, which was however insufficient to control infection in the lung, resulting in chronic pneumonia in MyD88-deficient mice.     

'Fat mass and obesity associated' gene (<Emphasis Type="Italic">FTO</Emphasis>): No significant association of variant rs9939609 with weight loss in a lifestyle intervention and lipid metabolism markers in German obese children and adolescents

Background  

We have previously identified strong association of six single nucleotide polymorphisms (SNPs) in FTO (fat mass and obesity associated gene) to early onset extreme obesity within the first genome wide association study (GWA) for this phenotype. The aim of this study was to investigate whether the obesity risk allele of one of these SNPs (rs9939609) is associated with weight loss in a lifestyle intervention program. Additionally, we tested for association of rs9939609 alleles with fasting blood parameters indicative of glucose and lipid metabolism.     

Evidence for high-velocity smooth pursuit in the trained cat

It is generally accepted that in cats smooth pursuit velocity of the eye never exceeds a few degrees per second. This is in contrast with observations in primates, where smooth pursuit velocity can reach values as high as 100°/s. Cats were trained to fixate and pursue spots of light appearing on a translucent screen. Spots were moved in the horizontal and vertical planes at different constant velocities up to 80°/s. Eye position was recorded with the sclerai search coil technique. Naive cats did not pursue moving targets with high efficiency. Smooth eye movement velocity saturated at 5°/s. After a few days of training, smooth-pursuit eye velocity increased with target velocity and saturated at 25°/s on average. However, velocities twice as high have been observed frequently. When the target was unexpectedly extinguished, smooth eye movement velocity dropped to values close to 0°/s in approximately 350 ms. After a short training period (usually 5 times the same target presentation), the eye continued to move smoothly until the target reappeared. These data suggest that smooth pursuit eye movements of the cat are qualitatively similar to those of primates, but reach lower velocities and are more variable in their characteristics.     

Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial

BACKGROUND: DC derived-exosomes are nanomeric vesicles harboring functional MHC/peptide complexes capable of promoting T cell immune responses and tumor rejection. Here we report the feasability and safety of the first Phase I clinical trial using autologous exosomes pulsed with MAGE 3 peptides for the immunization of stage III/IV melanoma patients. Secondary endpoints were the monitoring of T cell responses and the clinical outcome. PATIENTS AND METHODS: Exosomes were purified from day 7 autologous monocyte derived-DC cultures. Fifteen patients fullfilling the inclusion criteria (stage IIIB and IV, HLA-A1+, or -B35+ and HLA-DPO4+ leukocyte phenotype, tumor expressing MAGE3 antigen) were enrolled from 2000 to 2002 and received four exosome vaccinations. Two dose levels of either MHC class II molecules (0.13 versus 0.40 x 1014 molecules) or peptides (10 versus 100 mug/ml) were tested. Evaluations were performed before and 2 weeks after immunization. A continuation treatment was performed in 4 cases of non progression. RESULTS: The GMP process allowed to harvest about 5 x 1014 exosomal MHC class II molecules allowing inclusion of all 15 patients. There was no grade II toxicity and the maximal tolerated dose was not achieved. One patient exhibited a partial response according to the RECIST criteria. This HLA-B35+/A2+ patient vaccinated with A1/B35 defined CTL epitopes developed halo of depigmentation around naevi, a MART1-specific HLA-A2 restricted T cell response in the tumor bed associated with progressive loss of HLA-A2 and HLA-BC molecules on tumor cells during therapy with exosomes. In addition, one minor, two stable and one mixed responses were observed in skin and lymph node sites. MAGE3 specific CD4+ and CD8+ T cell responses could not be detected in peripheral blood. CONCLUSION: The first exosome Phase I trial highlighted the feasibility of large scale exosome production and the safety of exosome administration.     

Genetic analysis of cercarial emergence rhythms ofSchistosoma mansoni

Using two chronobiological variants ofSchistosoma mansoni (a blood fluke infecting man) from Guadeloupe (French West Indies), we carried out experimental crossbreeding between schistosomes with an early and those with a late cercarial shedding pattern. The results obtained on the F₁ (intermediate shedding patterns) and F₂ generations (early, intermediate, and late patterns) demonstrate that the cercarial emergence rhythms of schistosomes are genetically determined. This genetic variability is interpreted as a consequence of the selective pressure exerted by the two different hosts (man and rat) implicated in the life cycle ofS. mansoni from the Guadeloupean focus of schistosomiasis.This work received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases and the CNRS.