Engineering porcine arteries: effects of scaffold modification

Techniques have been developed to culture bovine or porcine vascular cells on polyglycolic acid (PGA) scaffolds to form engineered vessels. Previously, it was shown that smooth muscle cells (SMCs) that were in close proximity to PGA remnants after 8 weeks of culture had lower expression of SMC markers of differentiation and were more mitotic compared with SMCs that were distant from polymer residuals. Modifications of PGA were explored as a means to minimize residual polymer fragments after culture. To hasten degradation, polymer was treated with heat, NaOH, or gamma-irradiation. Differential scanning calorimetry, mass and tensile strength degradation, and inherent viscosity were used to assess polymer characteristics. When polymer was maintained in aqueous conditions, tensile strength of treated PGA degraded to zero within 3 weeks for each treatment. Engineered vessel constructs cultured on NaOH and gamma-treated polymer displayed smooth muscle alpha-actin throughout the vessel wall. Scaffold treatment impacted graft morphology, cellular differentiation, and mechanical integrity.     

Cytogenetic and molecular studies of a familial renal cell carcinoma.

In a previously studied family with inherited renal cell carcinoma (RCC), RCC was shown to segregate with a constitutional balanced t(3;8)(p14.2;q24.1). In addition, we recently showed that in a RCC tumor from this family the constitutional translocation became unbalanced, suggesting a genetic mechanism that may be associated with the primary genetic events of tumorigenesis. We now report that the RCC tumor cells from this case showed additional cytogenetic alterations, possibly related to tumor progression, which include an additional tumor-specific translocation involving band 14 of chromosome 13. Because this band contains the retinoblastoma (RB) gene, we examined the tumor for aberrations in the RB gene using DNA sequence polymorphism analysis and pulsed-field gel electrophoresis (PFGE), but did not detect alterations in the RB gene.     

A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer

BACKGROUND: There is a continued need to develop more effective cancer immunotherapy strategies. Exosomes, cell-derived lipid vesicles that express high levels of a narrow spectrum of cell proteins represent a novel platform for delivering high levels of antigen in conjunction with costimulatory molecules. We performed this study to test the safety, feasibility and efficacy of autologous dendritic cell (DC)-derived exosomes (DEX) loaded with the MAGE tumor antigens in patients with non-small cell lung cancer (NSCLC). METHODS: This Phase I study enrolled HLA A2+ patients with pre-treated Stage IIIb (N = 4) and IV (N = 9) NSCLC with tumor expression of MAGE-A3 or A4. Patients underwent leukapheresis to generate DC from which DEX were produced and loaded with MAGE-A3, -A4, -A10, and MAGE-3DPO4 peptides. Patients received 4 doses of DEX at weekly intervals. RESULTS: Thirteen patients were enrolled and 9 completed therapy. Three formulations of DEX were evaluated; all were well tolerated with only grade 1-2 adverse events related to the use of DEX (injection site reactions (N = 8), flu like illness (N = 1), and peripheral arm pain (N = 1)). The time from the first dose of DEX until disease progression was 30 to 429+ days. Three patients had disease progression before the first DEX dose. Survival of patients after the first DEX dose was 52-665+ days. DTH reactivity against MAGE peptides was detected in 3/9 patients. Immune responses were detected in patients as follows: MAGE-specific T cell responses in 1/3, increased NK lytic activity in 2/4. CONCLUSION: Production of the DEX vaccine was feasible and DEX therapy was well tolerated in patients with advanced NSCLC. Some patients experienced long term stability of disease and activation of immune effectors.     

Use of isolated immature-stage B cells to understand negative selection and tolerance induction at the molecular level

Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative selection. This process positions the B cell antigen receptor (BCR) in a central role for initiating the process of negative selection and suggests developmental regulation of its signaling. The observation that immature B cells are more susceptible to negative selection than are mature B cells has been demonstrated in a number of in vitro and in vivo model systems and support the idea of developmental regulation of BCR-initiated responses. Since identical antigen receptors are expressed on immature and mature B cells, the critical fate-determining distinction between these developmental stages must lie downstream of the receptor-ligand interaction itself, in the form of different BCR-linked signaling processes or with different secondary events occurring subsequent to BCR cross-linking. To address the first possibility, our laboratory and others have sought to define the differences in BCR-mediated signal transduction in immature and mature B lymphocytes. In this review article we will discuss current in vitro systems to study this question in primary, nontransformed murine B lymphocytes. In addition, we will discuss our previously published work in order to illustrate how these model systems have been useful in beginning to unravel the molecular basis for immune B cell negative selection and tolerance.     

Genetic basis for conversion of rough-to-smooth colony morphology in Actinobacillus actinomycetemcomitans

The basis of the rough-to-smooth conversion of Actinobacillus actinomycetemcomitans was examined. Smooth variants often contained mutations at the flp promoter region. Replacing the mutated flp promoter with the wild-type promoter restored the rough phenotype. The expression level of the flp promoter was approximately 100-fold lower in smooth than in rough strains. Mutations of the flp promoter are a cause of the rough-to-smooth conversion.     

Practical Evaluation of Methods for Detection and Specificity of Autoantibodies to Extractable Nuclear Antigens

Detection and specificity of autoantibodies against extractable nuclear antigens (ENA) play a critical role in the diagnosis and management of autoimmune disease. Historically, the detection of these antibodies has employed double immunodiffusion (DID). Autoantibody specificity was correlated with diagnoses by this technique. Enzyme immunoassays have been developed by multiple manufacturers to detect and identify the specificity ENA autoantibodies. To address the relationship of ENA detection by DID and enzyme immunoassay, the performances of five immunoassays were compared. These included two DID and three enzyme-linked immunoassays (ELISA) (both screening and individual antigen profile kits). The sample set included 83 ENA-positive, antinuclear-antibody (ANA)-positive specimens, 77 ENA-negative, ANA-positive specimens, and 20 ENA- and ANA-negative specimens. Sensitivity and specificity were calculated by two methods: first, by using the in-house DID result as the reference standard, and second, by using latent class analysis, which evaluates each kit result independently. Overall, the results showed that the ELISA methods were more sensitive for detection of ENA autoantibodies than DID techniques, but presence and/or specific type of ENA autoantibody did not always correlate with the patient''s clinical presentation. Regardless of the testing strategy an individual laboratory uses, clear communication with the clinical staff regarding the significance of a positive result is imperative. The laboratory and the clinician must both be aware of the sensitivity and specificity of each testing method in use in the clinical laboratory.A diagnosis of autoimmune disease in patients is based upon clinical history, physical examination, and laboratory detection of antinuclear antibodies (ANAs). A particular class of ANAs specific for extractable nuclear antigens (ENA) was initially described in 1959 (3). Since that time, many different anti-ENA antibodies have been described. The detection of these autoantibodies and identification of their specificity have become well-established tools for the laboratory diagnosis of several autoimmune diseases. Studies of patients with ENA antibodies have shown that detection of these autoantibodies may have both diagnostic and prognostic significance, and the detection of anti-ENA antibodies has assumed an important role in the management of these patients (5, 16, 22). In most cases, ENA testing is ordered after an initial ANA screen. The indications for use are to establish a diagnosis in patients with suggestive clinical symptoms, to exclude a diagnosis of autoimmune disease in patients with few or uncertain clinical signs, to subclassify patients with a known diagnosis, and to monitor disease activity.Testing for anti-ENA antibodies has historically relied on gel-based immunoprecipitation techniques such as double immunodiffusion (DID) and counterimmunoelectrophoresis (2, 14). The associations of specific types of ENA autoantibodies with rheumatological diseases were established by using these gel-based immunoassay techniques (15). In the last decade, enzyme-linked immunoassay (ELISA) systems have been developed to detect and determine the specificity of anti-ENA antibodies. ELISA systems permit more rapid processing of more specimens with a faster turnaround time than gel-based assays. ELISA-based methods may also have increased sensitivity for detection of ENA antibodies. However, the increased sensitivity of these ELISAs may influence the clinical relevance of their detection because diagnostic specificity may be reduced (10, 12, 17, 24). As yet, a set of reference standards with known antibody specificities against defined antigen preparations is not available for evaluation of various methods or kits. Serum reference panels are available from the Association of Medical Laboratory Immunologists (4), but the specificities of these sera were determined by consensus results from multiple laboratories. The purpose of this study was to address the relationship between DID and ELISA methods for the detection and identification of anti-ENA antibodies by evaluating and comparing two DID kits and three ELISA kits. We evaluated both screening ELISAs and monospecific antigen ELISAs to determine anti-ENA specificity.     

Estimating the relative frequency of leukodystrophies and recommendations for carrier screening in the era of next‐generation sequencing

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A‐related leukodystrophy, Peroxisomal biogenesis disorders, POLR3‐related Leukodystrophy, Vanishing White Matter, and Pelizaeus‐Merzbacher Disease. Despite the relative frequency of these conditions, carrier‐screening laboratories regularly test only 20 of the 55 leukodystrophy‐related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening.