Differentiation of myoblasts and CNS cells grown either separately or as co-cultures on microcarriers

Dispersed neuronal and muscular elements from fetal or neonatal origin, can organize and mature in culture when grown on positively charged cylindrical microcarriers (MCS), to a stage which simulatein vivo maturation. Cells arrange themselves on the MCS to form aggregates which remain floating in the nutrient medium. In such a tridimensional organization, the neuronal tissue is capable of regenerating a network of nerve fibers which establish synapse interconnections and undergo myelination. Oligodendrocytes organize on MCS in a tridimensional pattern and produce extensive myelin-like membranes. Myoblasts in MC-cultures fuse into polynucleated myotubes which become striated and contract spontaneously. Creatine kinase and acetylcholine receptor (AChR) are formed during myogenesis in similar quantities in MC-cultures and in monolayers. When both neuronal and muscle tissues are prepared from the same fetus (autologous nerve-muscle co-cultures) and are cultured on MCS, they interconnect to form neuro-muscular junctions. Cells from both tissues, exhibit better differentiation, for longer periods in MC-cultures than they do in monolayers. The floating functional entities are easy to sample and can be harvested for ultrastructural, immunocytochemical and biochemical analysis. In addition, MC-cultures can be used as a good tool for the study of acute and chronic exposures to toxicological agents, as well as for implantation into demyelinated, injured or dystrophic tissues. In this case the MCS in the implanted entities will serve as identifiable markers.     

Temperature-Mediated Interaction of Tetanus Toxin with Cerebral Neuron Cultures: Characterization of a Neuraminidase-Insensitive Toxin-Receptor Complex

Abstract: Energy-dependent internalization of ¹²⁵I-labeled tetanus toxin into cultured neural cells is shown to follow an energy-independent binding process. A three-step model, involving receptor-mediated binding followed by sequestration and internalization is proposed. In the first step, binding of toxin is enhanced in appearance under low ionic strength medium, at 0–4°C; it is suppressed, however, with increasing incubation temperature under physiological salt concentrations. Cell-bound toxin is displaced by approximately 35.5% when high-salt medium (physiological concentrations) is added to cells at 0–4°C; the effect is further amplified at 37°C. Addition of disialoganglioside G_D1b (1–5 μg/ml) also lowers the amount of cell-associated toxin. The fraction of ¹²⁵I-labeled toxin retained by the cells after exposure to high-salt medium at 0–4°C or after addition of G_D1b is operationally defined as sequestered toxin. This second step, characterized by a stable association of the toxin with the neural cells, is affected by both physiological salt and by 37°C conditions. Lastly, an energy-dependent phenomenon of firm association of tetanus toxin with neural cells, compatible with internalization, is described. The toxin residing in this fraction is bioactive and cannot be removed by salts, gangliosides, or by treatment with protease or neuraminidase. Binding, sequestration, and internalization are mutually dependent, as they are all blocked by pretreatment of cells with neuraminidase and by an enhanced energy-independent sequestration event, which results in enhanced tetanus toxin internalization by an energy-dependent process.     

DNA ploidy as a prognostic factor in rhabdomyosarcoma. Analysis of 35 cases with image cytometry

OBJECTIVE: To correlate DNA ploidy in rhabdomyosarcoma (RMS) with other prognostic factors and patient survival and to search for possible reasons for inconsistent conclusions in similar, published studies. STUDY DESIGN: DNA content was measured in archival specimens obtained from 35 patients (23 children and 12 adults) with RMS. Cell suspensions were prepared by the modified Hedley technique, stained by the modified Feulgen-thionin method and analyzed by automated high-resolution image cytometry. DNA ploidy was assessed on the basis of DNA index values. We used the chi 2 test to correlate DNA ploidy with other prognostic factors, Kaplan-Meier procedure to estimate overall survival in terms of individual prognostic factors, log-rank test to calculate differences in survival between groups and Cox multivariate regression analysis to determine the independence of variables in relation to survival. RESULTS: A statistically significant correlation was found only between DNA ploidy and histologic subtype of RMS, patient sex and patient age. A hyperdiploid DNA pattern predominated among patients with embryonal RMS, and a tetraploid pattern dominated among patients with alveolar RMS. The highest 5-year survival rate was seen among patients with hyperdiploid RMS, followed by those with diploid, tetraploid and hypertetraploid RMS. Although DNA ploidy was a significant prognostic factor in univariate analysis, it did not retain its independent prognostic value in multivariate analysis, in which patient age, tumor size and histologic subtype were the only significant factors. We found 12 articles reporting on the association between DNA ploidy and survival of patients with RMS: 6 found a correlation, and 6 did not. The main reasons for the discrepancies seem to be the inclusion of chemotherapy-treated and nontreated patients, low number of patients and differences in grouping DNA histograms. CONCLUSION: The precise prognostic value of DNA ploidy in RMS remains equivocal. Larger, cooperative studies could give statistically more reliable results.     

Chromosome aberration and environmental physical activity: Down syndrome and solar and cosmic ray activity, Israel, 1990–2000

The possibility that environmental effects are associated with chromosome aberrations and various congenital pathologies has been discussed previously. Recent advances in the collection and computerization of data make studying these potential associations more feasible. The aim of this study was to investigate a possible link between the number of Down syndrome (DS) cases detected prenatally or at birth yearly in Israel over a 10-year period compared with the levels of solar and cosmic ray activity 1 year before the detection or birth of each affected child. Information about 1,108,449 births was collected for the years 1990–2000, excluding 1991, when data were unavailable. A total of 1,310 cases of DS were detected prenatally or at birth—138 in the non-Jewish community and 1,172 in the Jewish population. Solar activity indices—sunspot number and solar radio flux 2,800 MHz at 10.7 cm wavelength for 1989–1999—were compared with the number of DS cases detected. Pearson correlation coefficients (r) and their probabilities (P) were established for the percentage of DS cases in the whole population. There was a significant inverse correlation between the indices of solar activity and the number of cases of DS detected—r=–0.78, P=0.008 for sunspot number and r=–0.76, P=0.01 for solar flux. The possibility that cosmophysical factors inversely related to solar activity play a role in the pathogenesis of chromosome aberrations should be considered. We have confirmed a strong trend towards an association between the cosmic ray activity level and the incidence of DS.     

GIRK channel activation involves a local rearrangement of a preformed G protein channel complex

G protein-coupled signaling is one of the major mechanisms for controlling cellular excitability. One of the main targets for this control at postsynaptic membranes is the G protein-coupled potassium channels (GIRK/Kir3), which generate slow inhibitory postsynaptic potentials following the activation of Pertussis toxin-sensitive G protein-coupled receptors. Using total internal reflection fluorescence (TIRF) microscopy combined with fluorescence resonance energy transfer (FRET), in intact cells, we provide evidence for the existence of a trimeric G protein-channel complex at rest. We show that activation of the channel via the receptor induces a local conformational switch of the G protein to induce channel opening. The presence of such a complex thus provides the means for a precise temporal and highly selective activation of the channel, which is required for fine tuning of neuronal excitability.     

Isolation and Chimerization of a Highly Neutralizing Antibody Conferring Passive Protection against Lethal Bacillus anthracis Infection

Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD₅₀ B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD₅₀ of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.