Identification of B- and T-Cell Epitopes of BB, a Carrier Protein Derived from the G Protein of Streptococcus Strain G148

Most conventional vaccines consist of killed organisms or purified antigenic proteins. Such molecules are generally poorly immunogenic and need to be coupled to carrier proteins. We have identified a new carrier molecule, BB, derived from the G protein of Streptococcus strain G148. We show that BB is able to induce strong antibody responses when conjugated to peptides or polysaccharides. In order to localize T and B cell epitopes in BB and match them with the albumin-binding region of the molecule, we immunized mice with BB, performed B and T pepscan analyses, and compared the results with pepscan done with sera and cells from humans. Our results indicate that BB has two distinct T helper epitopes, seven linear B-cell epitopes, and one conformational B-cell epitope in BALB/c mice. Four linear B-cell epitopes were identified from human sera, three of which overlapped mouse B-cell epitopes. Finally, three human T-cell epitopes were detected on the BB protein. One of these T-cell epitopes is common to BALB/c mice and humans and was localized in the region that contains the albumin-binding site. These data are of interest for the optimization of new carrier molecules derived from BB.     

Biomimetic apatite sintered at very low temperature by spark plasma sintering: Physico-chemistry and microstructure aspects

Nanocrystalline apatites analogous to bone mineral are very promising materials for the preparation of highly bioactive ceramics due to their unique intrinsic physico-chemical characteristics. Their surface reactivity is indeed linked to the presence of a metastable hydrated layer on the surface of the nanocrystals. Yet the sintering of such apatites by conventional techniques, at high temperature, strongly alters their physico-chemical characteristics and biological properties, which points out the need for “softer” sintering processes limiting such alterations. In the present work a non-conventional technique, spark plasma sintering, was used to consolidate such nanocrystalline apatites at non-conventional, very low temperatures (T < 300 °C) so as to preserve the surface hydrated layer present on the nanocrystals. The bioceramics obtained were then thoroughly characterized by way of complementary techniques. In particular, microstructural, nanostructural and other major physico-chemical features were investigated and commented on. This work adds to the current international concern aiming at improving the capacities of present bioceramics, in view of elaborating a new generation of resorbable and highly bioactive ceramics for bone tissue engineering.     

Nerve regeneration and elastin formation within poly(glycerol sebacate)-based synthetic arterial grafts one-year post-implantation in a rat model

The objective of this study was to evaluate the long-term performance of cell-free vascular grafts made from a fast-degrading elastic polymer. We fabricated small arterial grafts from microporous tubes of poly(glycerol sebacate) (PGS) reinforced with polycaprolactone (PCL) nanofibers on the outer surface. Grafts were interpositioned in rat abdominal aortas and characterized at 1 year post-implant. Grafts remodeled into “neoarteries” (regenerated arteries) with similar gross appearance to native rat aortas. Neoarteries mimic arterial tissue architecture with a confluent endothelium and media and adventita-like layers. Patent vessels (80%) showed no significant stenosis, dilation, or calcification. Neoarteries contain nerves and have the same amount of mature elastin as native arteries. Despite some differences in matrix organization, regenerated arteries had similar dynamic mechanical compliance to native arteries in vivo. Neoarteries responded to vasomotor agents, albeit with different magnitude than native aortas. These data suggest that an elastic vascular graft that resorbs quickly has potential to improve the performance of vascular grafts used in small arteries. This design may also promote constructive remodeling in other soft tissues.     

Comparison of pulsed-field gel electrophoresis and amplified fragment length polymorphism techniques for investigating outbreaks of enteritis due to campylobacters

Campylobacters are the most commonly reported cause of acute bacterial enteritis in the United Kingdom and United States, with poultry, milk, and water implicated as sources or vehicles of infection. The majority of campylobacter infections are sporadic, although outbreaks may occur, and these provide an opportunity to evaluate genotypic fingerprinting techniques. In this study, pulsed-field gel electrophoresis (PFGE) was compared with single-enzyme-amplified fragment length polymorphism (SAFLP). The results for the three separate episodes indicated that SAFLP and PFGE both clustered the strains from the first incident as 100% homologous. The strains from the second and third incidents clustered as distinct from both the first incident and from each other. PFGE is well recognized as a discriminatory fingerprinting technique for campylobacters; however, SAFLP has proven to be equally discriminatory, but far less labor intensive and with the added advantages of less "hands-on" time and inexpensive equipment, it is an excellent alternative to PFGE for investigation of outbreaks.     

Effects of interleukin-6 on extravillous trophoblast invasion in early human pregnancy

Invasion of uterine tissues by extravillous trophoblast cells (EVT) is essential for successful human pregnancy. EVT invasion is tightly regulated by a number of factors, including growth factors and cytokines, but the mechanisms that underlie their regulatory effect remain poorly understood. Interleukin (IL)-6 has been suggested to play a role in controlling EVT invasion. We hypothesized that IL-6 produced by cells in uterine decidua would regulate EVT invasiveness via IL-6Rα and gp130 receptors expressed by trophoblast cells. The effect of IL-6 on EVT signalling and cytokine production was also studied. Supernatants from disaggregated 'total' decidual cells, CD8(+) T cells, CD10(+) decidual stromal cells, CD14 macrophages, CD56(+) uterine natural killer cells, cytotrophoblast and EVT cells contained large quantities of IL-6 protein at both 8-10 and 12-14 weeks gestational age. IL-6Rα and gp130 were immunolocalized to EVT in placental bed biopsies from 8 to 20 weeks gestation and IL-6Rα expression was confirmed by western blotting. IL-6 had no effect on the invasive potential of EVT from chorionic villi or the immortalized EVT cell line HTR-8/SVneo in a Matrigel(?) invasion assay. IL-6 stimulated phosphorylation of several cell signalling proteins in EVT (8-14 weeks' gestation), although significance was lost after correction for multiple comparisons. Incubation with IL-6 decreased secretion of regulated upon activation, normal T-cell expressed and secreted (RANTES) by EVT cells. In conclusion, although IL-6 did not affect trophoblast cell invasion, it stimulated EVT cellular cascades and inhibited secretion of RANTES involved in a number of cellular processes.     

Germline mutation in BRAF codon 600 is compatible with human development: de novo p.V600G mutation identified in a patient with CFC syndrome

Champion KJ, Bunag C, Estep AL, Jones JR, Bolt CH, Rogers RC, Rauen KA, Everman DB. Germline mutation in BRAF codon 600 is compatible with human development: de novo p.V600G mutation identified in a patient with CFC syndrome. BRAF, the protein product of BRAF, is a serine/threonine protein kinase and one of the direct downstream effectors of Ras. Somatic mutations in BRAF occur in numerous human cancers, whereas germline BRAF mutations cause cardio‐facio‐cutaneous (CFC) syndrome. One recurrent somatic mutation, p.V600E, is frequently found in several tumor types, such as melanoma, papillary thyroid carcinoma, colon cancer, and ovarian cancer. However, a germline mutation affecting codon 600 has never been described. Here, we present a patient with CFC syndrome and a de novo germline mutation involving codon 600 of BRAF, thus providing the first evidence that a pathogenic germline mutation involving this critical codon is not only compatible with development but can also cause the CFC phenotype. In vitro functional analysis shows that this mutation, which replaces a valine with a glycine at codon 600 (p.V600G), leads to increased ERK and ELK phosphorylation compared to wild‐type BRAF but is less strongly activating than the cancer‐associated p.V600E mutation.     

Emotion management skills in sexually maltreated and nonmaltreated girls: a developmental psychopathology perspective

Research has demonstrated that children who experience familial sexual maltreatment are at risk for developing psychological difficulties characterized by emotional and behavioral dysregulation. Surprisingly, however, little attention has been directed toward identifying processes in emotional development that differ in maltreated and nonmaltreated children. From a developmental psychopathology perspective, the present study examined emotion management skills (i.e., emotional understanding, emotion regulation) in 21 sexually maltreated girls and their nonmaltreated peers to determine how the experience of sexual maltreatment may interfere with normative emotional development. Findings indicated that sexually maltreated girls, in comparison to their nonmaltreated peers, demonstrate lower emotional understanding and decreased ability to regulate their emotions in accordance with cultural expectations. Further, maltreated girls expected less emotional support and more relational conflict from parents in response to sadness displays and from parents and peers in response to anger displays. These findings will be discussed from the functionalist approach to emotional development, emphasizing the importance of social context (e.g., maltreating, nonmaltreating) in the development of children's emotion management skills.     

The development of monoclonal human rabies virus-neutralizing antibodies as a substitute for pooled human immune globulin in the prophylactic treatment of rabies virus exposure

To provide a more defined and safer replacement for the human rabies immune globulin (HRIG) from pooled serum which is currently used for treatment of exposure to rabies virus we have developed a series of human rabies virus-specific monoclonal antibodies. Mouse-human heterohybrid myeloma cells producing rabies virus-specific human monoclonal antibodies were prepared using B cells obtained from volunteers recently-immunized with a commercial rabies virus vaccine (HDCV). Cell lines producing antibody which neutralized the Evelyn-Rokitnicki-Abelseth (ERA) rabies virus strain in vitro were cloned and the resulting monoclonal antibodies characterized for isotype, specificity against a variety of rabies virus isolates, and neutralization capacity. The ability of the monoclonal antibodies to neutralize a variety of rabies virus strains in vitro correlated with their binding specificity for these viruses in an enzyme-linked immunoadsorbant assay (ELISA). A number of these antibodies have proven suitable for the formulation of a prophylactic human monoclonal antibody-based reagent which would provide significant advantages to the HRIG in having defined, reproducible specificity, lessened possibility of contamination with viral pathogens, and consistent availability.     

Autoantibodies against the platelet glycoprotein IIb/IIIa complex in patients with chronic ITP

Chronic idiopathic thrombocytopenic purpura (ITP) is caused by an antibody reactive with platelet-associated antigens. The present studies provide direct evidence that some patients with chronic ITP have autoantibodies against the platelet glycoprotein (GP) IIb/IIIa complex. Microtiter wells, coated with a monoclonal antibody (2G12) specific for GPIIb/GPIIIa were reacted with GPIIb/GPIIIa contained in a platelet extract. Control wells containing the same antibody were reacted with a cell extract containing no GPIIb/GPIIIa. After washing, the wells were reacted with patient or control plasma, and IgG binding was detected using 125I-Fab2-anti-human IgG. Assay values were expressed as binding ratios (cpm GPIIb/GPIIIa wells/cpm control wells). Plasma from 5 of 56 patients with chronic ITP had ratios (1.36-3.14) greater than 3 standard deviations above the mean (+/- SD) of control plasmas--0.93 +/- 0.12. Elevated values were also noted in two patients with anti-P1A1 antibody (ratios greater than 30) and in one patient with Hodgkin's disease and an ITP-like syndrome (ratio 1.53). Normal values were noted in 34 patients with a variety of immune and nonimmune diseases. Plasma from two of the positive ITP patients was reacted with 125I-surface-labeled platelets and, after solubilization, the IgG and bound antigen were precipitated with Staph-A. Autoradiographs from SDS- PAGE electrophoresis of the Staph-A-bound proteins shows two radioactive bands consistent in size with GPIIb and GPIIIa.