TGF-Beta inhibits the in vitro induction of lymphokine-activated killing activity

Summary Employing serum-free media, human peripheral blood mononuclear cells, and purified recombinant interleukin-2 (IL-2), conditions were observed in which the development of IL-2-driven cytotoxic activity was suppressed. The cytotoxic activity of such IL-2-generated lymphokine activated killing (LAK) was tested against natural killer-resistant cultured tumor cells (Daudi, Raji, and a glioma). LAK generation was inhibited by addition of some normal sera, normal platelets, or some tumor cells. Because recent reports have indicated that transforming growth factor-beta (TGF-beta)-like factors are often secreted by tumors and the acidic alpha granules of platelets and can be present in sera, we tested the effect of purified human TGF-beta on the activation of LAK. Our results indicated that TGF-beta is very suppressive for LAK induction, and can completely prevent both the IL-2-driven proliferation and cytotoxicity at concentrations as low as 5 ng/ml. Titrations of IL-2 and of TGF-beta indicated that the suppression is dose-dependent and can be avoided by employing higher levels of IL-2. It was also found that the suppressive effect of TGF-beta can be overcome by washing suppressed cell populations and further culture in low levels of IL-2. Collectively, these data indicate that TGF-beta can be a potent inhibitor of LAK generation under standard activation conditions, but that this effect is regulated by the relative level of IL-2 and may be overcome and/or reversed in vitro.     

Donnan potentials from striated muscle liquid crystals. Sarcomere length dependence.

Donnan potentials from A-bands and I-bands were measured as a function of sarcomere length in skinned long-tonic muscle fibers of the crayfish. These measurements were made using standard electrophysiological technique. Simultaneously, the relative cross-sectional area of the fibers was determined. Lattice plane spacings and hence unit-cell volumes were determined by low-angle x-ray diffraction. At a sarcomere length at which the myosin filaments and actin filaments nominally do not overlap, measurements of potential, relative cross-sectional area, and unit-cell volume were used in conjunction with Donnan equilibrium theory to calculate the effective linear charge densities along the myosin filament (6.6 X 10(4) e-/mu) and actin filament (6.8 X 10(3) e-/mu). Using these linear charge densities, unit-cell volumes and Donnan equilibrium theory, an algorithm was developed to predict A-band and I-band potentials at any sarcomere length. Over the range of sarcomere lengths investigated, the predicted values coincide with the experimental data. The ability of the model to predict the data demonstrates the applicability of Donnan equilibrium theory to measurements of electrochemical potential from liquid-crystalline systems.     

Preliminary characterization of a Chinese hamster ovary cell glycosylation mutant isolated by screening for low intracellular lysosomal enzyme activity

A novel screening procedure was developed for isolating Chinese hamster ovary cell mutants altered in the early steps of the biosynthesis of asparagine-linked glycoproteins. This procedure identifies cells with low intracellular levels of two lysosomal hydrolases, beta-glucuronidase and alpha-iduronidase. One mutant cell line isolated in this way, CHB 11-1-3, has low intracellular levels of seven lysosomal enzymes as compared to wild-type cells. Although CHB 11-1-3 synthesizes mannosylphosphoryldolichol and [Man]₅[NAcG1cNH₂]₂-P-P-lipid, it fails to utilize these lipid intermediates to make normal amounts of [Glc]₃[Man]₉[NAcG1cNH₂]₂P-P-lipid. As a consequence of this glycosylation defect, this mutant transfers oligosaccharides of a different structure than wild type to the lysosomal enzyme beta-hexosaminidase. In addition, it underglycosylates its proteins.     

Preparation and structure of heparin lyase-derived heparan sulfate oligosaccharides

Porcine intestinal mucosal heparan sulfate was exhaustivelydepolymerized on a large scale using beparin lyase II (heparinaseII) or heparin lyase III (heparitinase, EC 4.2.2.8 [EC] ). The oligosaccharidemixtures formed with each enzyme were fractionated by low pressuregel permeation chromatography. Size-uniform mixtures of disaccharides,tetrasaccharides, and hexasaccharides were obtained. Each size-fractionatedmixture was then purified on the basis of charge by repetitivesemipreparative strong-anion-exchange high-performance liquidchromatography. This approach has led to the isolation of 13homogenous oligosaccharides. The purity of each oligosaccharidewas demonstrated by the presence of a single peak on analyticalstrong-anion-exchange high-performance liquid chromatographyand reversed polarity capillary electrophoresis. The structuresof these oligosaccharides were established using 500 MHz one-and two-dimensional nuclear magnetic resonance spectroscopy.Three of the thirteen structures that were solved were novelwhile the remaining 10 have been previously described. All ofthe structures obtained using heparin lyase III contained a     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl⁻ and Na⁺ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.     

Symbiodinium diversity among host clionaid sponges from Caribbean and Pacific reefs: Evidence of heteroplasmy and putative host-specific symbiont lineages

Among the Porifera, symbiosis with Symbiodinium spp. (i.e., zooxanthellae) is largely restricted to members of the family Clionaidae. We surveyed the diversity of zooxanthellae associated with sponges from the Caribbean and greater Indo-Pacific regions using chloroplast large subunit (cp23S) domain V sequences. We provide the first report of Clade C Symbiodinium harbored by a sponge (Cliona caesia), and the first report of Clade A Symbiodinium from an Indo-Pacific sponge (C. jullieni). Clade A zooxanthellae were also identified in sponges from the Caribbean, which has been reported previously. Sponges that we examined from the Florida Keys all harbored Clade G Symbiodinium as did C. orientalis from the Indo-Pacific, which also supports earlier work with sponges. Two distinct Clade G lineages were identified in our phylogenetic analysis; Symbiodinium extracted from clionaid sponges formed a monophyletic group sister to Symbiodinium found in foraminiferans. Truncated and 'normal' length variants of 23S rDNA sequences were detected simultaneously in all three morphotypes of C. varians providing the first evidence of chloroplast-based heteroplasmy in a sponge. None of the other sponge species examined showed evidence of heteroplasmy. As in previous work, length variation in cp23S domain V sequences was found to correspond in a highly precise manner to finer resolution of phylogenetic topology among Symbiodinium clades. On a global scale, existing data indicate that members of the family Clionaidae that host zooxanthellae can form symbiotic associations with at least four Symbiodinium clades. The majority of sponge hosts appear to harbor only one cladal type of symbiont, but some species can harbor more than one clade of zooxanthellae concurrently. The observed differences in the number of partners harbored by sponges raise important questions about the degree of coevolutionary integration and specificity of these symbioses. Although our sample sizes are small, we propose that one of the Clade G lineages identified in this study is comprised of sponge-specialist zooxanthellae. These zooxanthellae are common in Caribbean sponges, but additional work in other geographic regions is necessary to test this idea. Sponges from the Indo-Pacific region harbor zooxanthellae from Clades A, C, and G, but more sponges from this region should be examined.     

Eastern equine encephalitis virus rapidly infects and disseminates in the brain and spinal cord of cynomolgus macaques following aerosol challenge

Eastern equine encephalitis virus (EEEV) is mosquito-borne virus that produces fatal encephalitis in humans. We recently conducted a first of its kind study to investigate EEEV clinical disease course following aerosol challenge in a cynomolgus macaque model utilizing the state-of-the-art telemetry to measure critical physiological parameters. Here, we report the results of a comprehensive pathology study of NHP tissues collected at euthanasia to gain insights into EEEV pathogenesis. Viral RNA and proteins as well as microscopic lesions were absent in the visceral organs. In contrast, viral RNA and proteins were readily detected throughout the brain including autonomic nervous system (ANS) control centers and spinal cord. However, despite presence of viral RNA and proteins, majority of the brain and spinal cord tissues exhibited minimal or no microscopic lesions. The virus tropism was restricted primarily to neurons, and virus particles (~61–68 nm) were present within axons of neurons and throughout the extracellular spaces. However, active virus replication was absent or minimal in majority of the brain and was limited to regions proximal to the olfactory tract. These data suggest that EEEV initially replicates in/near the olfactory bulb following aerosol challenge and is rapidly transported to distal regions of the brain by exploiting the neuronal axonal transport system to facilitate neuron-to-neuron spread. Once within the brain, the virus gains access to the ANS control centers likely leading to disruption and/or dysregulation of critical physiological parameters to produce severe disease. Moreover, the absence of microscopic lesions strongly suggests that the underlying mechanism of EEEV pathogenesis is due to neuronal dysfunction rather than neuronal death. This study is the first comprehensive investigation into EEEV pathology in a NHP model and will provide significant insights into the evaluation of countermeasure.