Mechanical Characterization of an In Vitro Device Designed to Quantitatively Injure Living Brain Tissue

Due to the nonlinear, viscoelastic material properties of brain, its mechanical response is dependent upon its total strain history. Therefore, a low strain rate, large strain will likely produce a tissue injury unique from that due to a high strain rate, moderate strain. Due to a lack of current understanding of specific in vivo physiological injury mechanisms, a priori assumptions cannot be made that a low strain rate injury induced by currently employed in vitro injury devices is representative of clinical, nonimpact, inertial head injuries. In the present study, an in vitro system capable of mechanically injuring cultured tissue at high strain rates was designed and characterized. The design of the device was based upon existing systems in which a clamped membrane, on which cells have been cultured, is deformed. However, the present system incorporates three substantial improvements: (1) noncontact measurement of the membrane deflection during injury; (2) precise and independent control over several characteristics of the deflection; and (3) generation of mechanical insults over a wide range of strains (up to 0.65) and strain rates (up to 15s^–1). Such a system will be valuable in the elucidation of the mechanisms of mechanical trauma and determination of injury tolerance criteria on a cellular level utilizing appropriate mechanical injury parameters.     

Declining density of intimal smooth muscle cells and age as preconditions for atheronecrosis in the basilar artery

The aging basilar artery has some differences and some similarities when compared with the aorta and coronary arteries. As the non-necrotic intimal thickness increases over time, the number of smooth muscle cells reaches a steady state around age 25–30 years in the coronaries and aorta, but continues to increase in the basilar artery, even to 90 years of age. The numbers of cells per unit of tissue (the cell density) declines with age, and the patterns of decline are quantitatively similar in all three arterial segments. All arteries so far examined behave alike in showing that atheronecrosis emerges in those specimens that have sufficiently low density of intimal smooth muscle cells. These results identify low intimal cell density as a criterion for recognizing arteries that are prone to atheronecrosis. One possible explanation is that depopulation of the fibrotically thickened and aged intima, by spreading apart the smooth muscle cells with expanding matrix materials, could be the conditioning factor that brings about the intrusion of atheronecrosis.     

Direct and indirect effects of soluble extracts of Schistosoma mansoni eggs on fibroblast proliferation in vitro.

The possibility that soluble products of Schistosoma mansoni eggs might participate in the pathogenesis of hepatic fibrosis in schistosomiasis was investigated. Both crude saline extracts of eggs (soluble egg antigen [SEA]) and a partially purified SEA fraction contained activity which stimulated guinea pig and human dermal fibroblasts to proliferate in vitro, as measured by uptake of [3H]thymidine. Maximum activity was present in fractions which eluted from Sephacryl S-200 with an apparent molecular weight of less than or equal to 12,500 and in fractions which had an estimated pI 8, as determined by preparative isoelectric focusing of partially purified SEA. Activity in crude SEA was not removed by chromatography on concanavalin A-Sepharose 4B. When concanavalin A-binding glycoproteins lacking intrinsic fibroblast-stimulating activity were incubated with spleen cells from infected or uninfected mice, fibroblasts-stimulating activity was detected in the culture supernatants. Thus, SEA contains two functionally distinct molecular species. One of these directly stimulates fibroblasts, whereas the other induces the release of a fibroblast-stimulating activity from lymphocytes or macrophages or both. Since these fibroblast-stimulating factors might be elaborated in the livers of infected individuals, these observations suggest a potential role of soluble schistome products in the pathogenesis of hepatic fibrosis in schistosomiasis.     

Poly(lactide-co-glycolide) microspheres as a moldable scaffold for cartilage tissue engineering

This study demonstrates the use of biodegradable poly(lactide-co-glycolide) (PLG) microspheres as a moldable scaffold for cartilage tissue engineering. Chondrocytes were delivered to a cylindrical mold with or without PLG microspheres and cultured in vitro for up to 8 weeks. Cartilagenous tissue formed using chondrocytes and microspheres maintained thickness, shape, and chondrocyte collagen type II phenotype, as indicated by type II collagen staining. The presence of microspheres further enhanced total tissue mass and the amount of glycosaminoglycan that accumulated. Evaluation of microsphere composition demonstrated effects of polymer molecular weight, end group chemistry, and buffer inclusion on tissue-engineered cartilage growth. Higher molecular weight PLG resulted in a larger mass of cartilage-like tissue formed and a higher content of proteoglycans. Cartilage-like tissue formed using microspheres made from low molecular weight and free carboxylic acid end groups did not display increases in tissue mass, yet a modest increased proteoglycan accumulation was detected. Microspheres comprised of PLG with methyl ester end groups yielded a steady increase in tissue mass, with no real increase in matrix accumulation. The microencapsulation of Mg(OH)(2) had negative effects on tissue mass and matrix accumulation. The data herein reflect the potential utility of a moldable PLG-chondrocyte system for tissue-engineering applications.     

A modified human ELISPOT assay to detect specific responses to primary tumor cell targets

Background  

The desired outcome of cancer vaccination is to induce a potent T cell response which can specifically recognize and eliminate autologous tumor cells in vivo. Accordingly, immunological assays that demonstrate recognition of native tumor cells (tumor-specific) may be more clinically relevant than assays that demonstrate recognition of tumor protein or peptide (antigen-specific).     

Cell adhesion-related proteins as specific markers of sponge cell types involved in allogeneic recognition

Sponge immunocyte identification is of interest to comparative immunologists since characterizing these cells will allow investigations into the mechanisms of non-self recognition in the oldest animal phylum. Here, we report that polyclonal antibodies raised against the core protein of a proteoglycan involved in cell adhesion in the marine sponge Microciona prolifera are specific markers for archaeocytes, the totipotent sponge cells. Archaeocytes are mobilized upon allogeneic contact and they accumulate in the contact zone. A second type of cell, the gray cells, are specifically recognized by monoclonal antibodies raised against CD44, a hyaluronan receptor. Gray cells do also accumulate in the contact area. Specific staining of a third sponge cell type, the rhabdiferous cells, shows that these do not accumulate upon allografting. These specific cell markers allow tracking of archaeocytes and gray cells, and show that they play an active role in sponge allogeneic reactions.     

Genetic diagnoses and associated anomalies in fetuses prenatally diagnosed with esophageal atresia

Esophageal atresia (EA) is a congenital anomaly occurring in 2.3 per 10,000 live births. Due to advances in prenatal imaging, EA is more readily diagnosed, but data on the associated genetic diagnoses, other anomalies, and postnatal outcome for fetuses diagnosed prenatally with EA are scarce. We collected data from two academic medical centers (n = 61). Our data included fetuses with suspected EA on prenatal imaging that was confirmed postnatally and had at least one genetic test. In our cohort of 61 cases, 29 (49%) were born prematurely and 19% of those born alive died in the first 9 years of life. The most commonly associated birth defects were cardiac anomalies (67%) and spine anomalies (50%). A diagnosis was made in 61% of the cases; the most common diagnoses were vertebral defects, anal atresia, cardiac anomalies, tracheoesophageal fistula with esophageal atresia, radial or renal dysplasia, and limb anomalies association (43%, although 12% met only 2 of the criteria), trisomy 21 (5%), and CHARGE syndrome (5%). Our findings suggest that most fetuses with prenatally diagnosed EA have one or more additional major anomaly that warrants a more comprehensive clinical genetics evaluation. Fetuses diagnosed prenatally appear to represent a cohort with a worse outcome.     

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.