Chorioallantoic membrane angiogenesis model for tissue engineering: a new twist on a classic model

Tissue-engineering (TE) applications include the isolation, culture, and seeding of cells into a suitable matrix or scaffold before in vivo transplantation. After transplantation, vascularization of the scaffold is a principal limiting factor for cell viability for the first 6-8 days posttransplantation. A model for systematic analysis of this process has been developed. Fertilized White Leghorn eggs were incubated (at 37.8 degrees C in 60% relative humidity) and opened on day 3 of incubation. Preadipocyte-seeded fibrin constructs were implanted in a specially designed plastic cylinder and placed through the opening on the surface of the chorioallantoic membrane (CAM) on day 8 of incubation. Vascularization of the constructs by chorioallantoic blood vessels was assessed for up to 8 days posttransplantation. The survival rate for embryos receiving transplanted constructs was about 90%. Histology confirmed transplant cell viability at day 4 posttransplantation and vascularization of the constructs by avian endothelial cells began at this time. A new in vivo model to study the effect of angiogenesis in TE constructs, including assessments of viability, proliferation, and differentiation of transplanted cells and biomaterial properties, is presented. Advantages include easy access to the vascular network of the CAM, lack of immunocompetence, low costs, and avoidance of animal experiments.     

A European multicenter study of immunoblotting in serodiagnosis of lyme borreliosis

A European multicenter study of immunoblotting for the serodiagnosis of Lyme borreliosis showed considerable variation in results obtained from tests with a panel of 227 serum samples. Six laboratories used different immunoblot methods, and a wide range of bands was detected in all the assays. Multivariable logistic regression analysis of data from individual laboratories was used to determine the most discriminatory bands for reliable detection of antibodies to Borrelia burgdorferi sensu lato. These bands were used to construct individual interpretation rules for the immunoblots used in the six laboratories. Further analysis identified a subset of eight bands, which were important in all the laboratories, although with variations in significance. Possible European rules, all closely related, were formulated from these bands, although there was no single rule that gave high levels of sensitivity and specificity for all the laboratories. This is a reflection of the wide range of methodologies used, especially the use of different species and strains of B. burgdorferi sensu lato. The panel of European rules provides a framework for immunoblot interpretation which may be adapted in relation to the characteristics of Lyme borreliosis in local areas.     

Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines

Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4(+) helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-gamma enzyme-linked immunospot assay (ELISPOT) assays and HLA-peptide tetramer staining. Single-cell cloning resulted in both CD4(+) and CD8(+) T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8(+) and CD4(+) T cell epitopes.     

Fas expression by tumor stroma is required for cancer eradication

The contribution of molecules such as perforin, IFN-γ (IFNγ), and particularly Fas ligand (FasL) by transferred CD8⁺ effector T (T_E) cells to rejection of large, established tumors is incompletely understood. Efficient attack against large tumors carrying a surrogate tumor antigen (mimicking a “passenger” mutation) by T_E cells requires action of IFNγ on tumor stroma cells to avoid selection of antigen-loss variants. Because “cancer-driving” antigens (CDAs) are rarely counterselected, IFNγ may be expected to be dispensable in elimination of cancers by targeting a CDA. Here, initial regression of large, established tumors required neither IFNγ, FasL, nor perforin by transferred CD8⁺ T_E cells targeting Simian Virus (SV) 40 large T as CDA. However, cytotoxic T_E cells lacking IFNγ or FasL could not prevent relapse despite retention of the rejection antigen by the cancer cells. Complete tumor rejection required IFNγ-regulated Fas by the tumor stroma. Therefore, T_E cells lacking IFNγ or FasL cannot prevent progression of antigenic cancer because the tumor stroma escapes destruction if its Fas expression is down-regulated.     

Assessing the spatiotemporal evolution of neuronal activation with single-trial event-related potentials and functional MRI

The brain acts as an integrated information processing system, which methods in cognitive neuroscience have so far depicted in a fragmented fashion. Here, we propose a simple and robust way to integrate functional MRI (fMRI) with single trial event-related potentials (ERP) to provide a more complete spatiotemporal characterization of evoked responses in the human brain. The idea behind the approach is to find brain regions whose fMRI responses can be predicted by paradigm-induced amplitude modulations of simultaneously acquired single trial ERPs. The method was used to study a variant of a two-stimulus auditory target detection (odd-ball) paradigm that manipulated predictability through alternations of stimulus sequences with random or regular target-to-target intervals. In addition to electrophysiologic and hemodynamic evoked responses to auditory targets per se, single-trial modulations were expressed during the latencies of the P2 (170-ms), N2 (200-ms), and P3 (320-ms) components and predicted spatially separated fMRI activation patterns. These spatiotemporal matches, i.e., the prediction of hemodynamic activation by time-variant information from single trial ERPs, permit inferences about regional responses using fMRI with the temporal resolution provided by electrophysiology.     

Transmission of donor illness by stem cell transplantation: should screening be different in older donors?

With increasing donor age, the potential of transmitting diseases from donor to recipient reaches new dimensions. Potentially transmittable diseases from donors include infections, congenital disorders, and acquired illnesses like autoimmune diseases or malignancies of hematological or nonhematological origin. While established nonmalignant or malignant diseases might be easy to discover, early-stage hematological diseases like CML, light-chain multiple myelomas, aleukemic leukemias, occult myelodysplastic syndromes and other malignant and nonmalignant diseases might not be detectable by routine screening but only by invasive, new and/or expensive diagnostic tests. In the following article, we propose recommendations for donor work-up, taking into consideration the age of the donors. In contrast to blood transfusions, stem cells from donors with abnormal findings might still be acceptable for HCT, when no other options are available and life expectancy is limited. This issue is discussed in detail in relation to the available donor and stem cell source. Finally, the recommendations presented here aim at harmonized worldwide work-up for donors to insure high standard quality.