Molecular analysis of T-cell receptor repertoire in bone marrow transplant recipients: evidence for oligoclonal T-cell expansion in graft-versus-host disease lesions

We have analyzed the T-cell receptor (TCR) V beta repertoire using polymerase chain reaction (PCR) in a cohort of eight patients receiving allogeneic bone marrow transplantation (BMT) from related and unrelated donors at the City of Hope. Results of PCR studies from graft-versus-host disease (GVHD) skin lesions show a bias in the usage of TCR V beta families, whereas examination of peripheral blood (PB) withdrawn at the same time did not reveal a similar phenomenon. In one such family, TCR V beta 2 is predominantly expressed in 7 of 7 biopsy specimens examined. V beta 2 TCR expression from these patients was analyzed more extensively using a combination of individual TCR gene cloning, followed by sequence analysis. We found evidence of oligoclonal expansion of single V beta 2-bearing TCRs in GVHD lesions, and in the PB of some patients after diagnosis of GVHD. In contrast, GVHD-negative biopsy samples showed no evidence for clonotypic TCR amplification. Sequence-specific TCR CDR3 region probes were derived from analysis of the predominant expressed TCR in GVHD lesions, and used to probe Southern blots of amplified V beta 2 TCR mRNA from PB and tissue from BMT recipients and their respective donors. In most cases the probes are highly specific in detecting TCR expression from GVHD lesions alone, although in several instances expression could be detected in PB after GVHD diagnosis. These data provide supporting evidence for the hypothesis that acute GVHD is associated with expansion of T-cell clones expressing antigen-specific TCRs that may contribute to the disease pathology.     

Influenza virus hemagglutinin stimulates the protein kinase C activity of human polymorphonuclear leucocytes

Human polymorphonuclear leukocytes (PMNL) incubated with influenza virus, A/USSR/90/77 (H1N1) or its hemagglutinin produced a significant increase in their PKC activity when compared with untreated PMNL. The activated kinase translocated from cytosol to cellular membrane. The calcium-dependent enzyme activity was inhibited by a specific inhibitor suggesting that alpha and/or beta isoforms of PKC were involved.     

Mash1 and neurogenin1 expression patterns define complementary domains of neuroepithelium in the developing CNS and are correlated with regions expressing notch ligands

Genetic studies in Drosophila and in vertebrates have implicated basic helix-loop-helix (bHLH) genes in neuronal fate determination and cell type specification. We have compared directly the expression of Mash1 and neurogenin1 (ngn1), two bHLH genes that are expressed specifically at early stages of neurogenesis. In the PNS these genes are expressed in complementary autonomic and sensory lineages. In the CNS in situ hybridization to serial sections and double-labeling experiments indicate that Mash1 and ngn1 are expressed in adjacent and nonoverlapping regions of the neuroepithelium that correspond to future functionally distinct areas of the brain. We also showed that in the PNS several other bHLH genes exhibit similar lineal restriction, as do ngn1 and Mash1, suggesting that complementary cascades of bHLH factors are involved in PNS development. Finally, we found that there is a close association between expression of ngn1 and Mash1 and that of two Notch ligands. These observations suggest a basic plan for vertebrate neurogenesis whereby regionalization of the neuroepithelium is followed by activation of a relatively small number of bHLH genes, which are used repeatedly in complementary domains to promote neural determination and differentiation.     

Nutritional knowledge and consumption frequency of foods--a case study

The relationship between nutritional knowledge and the consumption frequency of preferred food-types was studied among one sample of Uruguayan consumers. A locally-adapted version of Parmenter & Wardle's General Nutrition Knowledge Questionnaire and a food consumption survey based on 39 food groups were completed by a total of 270 participants. Cluster Analysis enabled the identification of two clusters showing different levels of nutritional knowledge--cluster 1 (n = 177) and cluster 2 (n = 93), providing an average of 73.6% and 52.9% of correct answers, respectively. These clusters differed significantly (p < or = 0.05) in age and educational distribution--cluster 1 was composed mainly by older adults and persons with a higher educational level. A number of areas were identified where nutritional knowledge was extremely poor, as was the case with the recommended daily fruit and vegetable intake and the caloric content of the nutrients. Overall, nutritional knowledge was found to have a positive influence on food preferences and consumption frequency, those participants with a higher nutritional knowledge reporting a higher consumption of fruits, vegetables and low-fat products, in addition to a lower consumption of high-fat and high-sugar foods.     

Calcium Silicate Hydrates Studied by Small-Angle Neutron Scattering (SANS)

Small-angle neutron scattering (SANS) was used to study calcium silicate hydrate (C-S-H) formed via pozzolanic reaction between calcium oxide and ultrafine silica in water or polymer solutions at a temperature of 20°C. The SANS profile of this product was consistent with a structure that consisted of platelets with maximum diameters of ∼20 nm (± 5 nm) in the x – y plane, which was similar to a structure that had been deduced in previous work via X-ray diffractometry. The presence of two different types of superplasticizer in solution (at a concentration of 10 g/L) had no significant effect on its formation kinetics or its SANS profile.     

Interactions between Polymeric Dispersants and Calcium Silicate Hydrates

To better understand the mechanism of interaction between hydrating silicate-based cements and polymeric dispersants of the type used as superplasticizers in modern construction concretes, two different types of polymeric dispersant were added (at concentrations of 1 and 10 g/L) during the synthesis of calcium silicate hydrate (C-S-H) via the pozzolanic reaction in dilute slurries of lime and reactive silica, at Ca/Si ratios in the range of 0.66–1.50. Although both polymers gave degrees of adsorption of >79% in all cases studied, no significant structural modifications of the resulting C-S-H products were observed via X-ray diffraction or ²⁹Si magic angle spinning–nuclear magnetic resonance. These results differ from recent work in which it was shown that similar types of polymer could intercalate into the interlayers of C-S-H that was made using an alternative process. It is suggested that the process by which the C-S-H is formed may have a strong influence on whether C-S-H can intercalate polymers. This observation is relevant to understanding the fate of such polymers in concrete.     

Constructal flow distributor as a bipolar plate for proton exchange membrane fuel cells

A plate-type constructal flow distributor is implemented as a gas distributor for a proton exchange membrane fuel cell. A 3D complete model is simulated using CFD techniques. The fuel cell model includes the gas flow channels, the gas diffusion layers and the membrane-electrode assembly (MEA). The governing equations for the mass and momentum transfer are solved including the pertinent source terms due to the electrochemical reactions in the different zones of the fuel cell. Three constructal flow configurations were studied; each pattern is a fractal expansion of the original design, therefore, the only difference between them is the number of branches in the geometry. It was found that the number of branches is the key parameter in the performance of a fuel cell when using the constructal distributors as flow channels. The performance of the fuel cell is reported in I-V curves, power curves, and overpotential curves in order to determine which irreversibility is the main cause of energy losses. In terms of flow analysis, it was found that the constructal flow distributor presents a low pressure drop for a wide range of Reynolds number conditions at the inlet, as well as an excellent uniformity of flow distribution. Regardless of the outstanding hydrodynamic performance of the constructal distributors and the large current density values obtained, the implementation of these designs as flow patterns for PEMFCs need further optimization; first, the manufacturing of the plates have to be addressed in an efficient way; and secondly, the application in stacks will require an elaborate design to accomplish this task.     

Growth and morphogenesis of epithelial cell organoids from peripheral and medial mammary parenchyma of prepubertal heifers

Collagen gel cell cultures were used to test the hypothesis that cells from peripheral parenchymal zones of the developing bovine mammary gland have greater proliferative and morphogenic potential than cells from the medial parenchymal mass. Tritiated thymidine was incorporated to assess cell proliferation. Dose responses for insulin-like growth factor one (IGF-I, 0 to 50 ng/ml) and transforming growth factor beta one (0 to 5 ng/ml) were determined for each cell population. Cells from the peripheral regions were two to three times more sensitive to the mitogenic action of IGF-I or serum. Transforming growth factor beta one concentrations of 12.5 to 500 pg/ml stimulated cell proliferation, but concentrations above 1 ng/ml were inhibitory. Peripheral cells showed a distinct biphasic response to addition of transforming growth factor beta one. Morphology of organoids was evaluated daily during culture and by histologic examination at the end of culture. Peripheral zone cells formed multi-layered cell structures that resembled native mammary parenchyma with serum or mammary gland extract. Our results indicate that epithelial cells of the peripheral and medial parenchymal zones in the ruminant mammary gland can differ in their proliferative and morphogenetic response to growth factor treatment in vitro. These differences likely reflect local tissue regulation necessary for sequential ductular and lobulo-alveolar development in vivo.     

Air classification of pea flour–analytical studies

The pea-milling and subsequent air classification of whole peas was investigated on laboratory scale equipment. Two approximately linear but divergent calibration graphs were constructed for the air classifier based on two different definitions of particle size cut-point. At high classifying speed (11 000 rev min^?1) a fine fraction or protein concentrate was produced containing 55.1% protein (cf. 22% in original flour) although this represented only 29% of the total protein. The yield of protein in the fine fraction could be increased by using a three-stage milling and classifying procedure, although the actual protein content of the concentrate remained more or less the same. This procedure also had the effect of reducing protein contamination of the starch (coarse) fraction. Other effects of air classifying observed included the concentration of the lipid component into the fine fraction and the rough fractionation of the fibre constituent between the coarse (testa) and fine (cell wall material) fractions. Various aspects and problems associated with the use of air classification for the preparation of pea protein concentrates are discussed.