Modeling immunotherapy for allergy

Type I hypersensitivity, which functions to protect the organism from parasites, is caused by binding of antigen to IgE antibodies pre-attached to the cell surface of tissue mast cells and their circulating counterparts, the basophils. In “allergy,” type I hypersensitivity is inappropriately induced by protein-based foreign substances (such as pollen) or protein components of insect stings, which in the normal course of events would be cleared from the organism without causing any damage. Paradoxically, a successful clinical treatment of allergy involves repeated immunization of allergic persons with low doses of the allergen—immunotherapy. Investigation of the available experimental evidence leads to the conclusion that the phenomena of immunotherapy are best addressed in terms of the interplay among the mechanism(s) of immune memory—Th1/Th2 cross-regulation—and the physical compart-mentalization of the immune system. These conclusions are illustrated with a numerical simulation.     

Large-scale purification of xylanase T-6

Bacillus stearothermophilus T-6 produces an extracellular thermostable xylanase that can bleach paper pulp optimally at 60°C and pH 9.0. We developed an efficient method for purifying the enzyme from the cell broth by using successive steps of batch adsorption on the cellulosic cation exchanger SE-52. The optimal pH values for adsorption and elution are 5.5 and 9 respectively. The conductivity of the cell broth should remain below 7 mS/cm² and the suitable temperature range for adsorption is 15–60°C. The adsorption parameters are: maximum capacity, 118 mg enzyme/g adsorbent; dissociation constant, 0.6 mg/ml; partition coefficient 0.988. On the basis of these results, a large-scale (12 000 l) purification process was carried out resulting in a 55% final yield and over 95% pure enzyme.     

The role of arachidonic acid in vasogenic brain edema

Arachidonic acid is released rapidly from cellular membrane phospholipids after pathological insults associated with the delayed development of brain edema. Intracerebral injection of arachidonic acid caused significant increases in brain water and sodium content with decreases in potassium content and Na+,K+-ATPase activity. The 125I-labeled bovine serum albumin spaces in brain (a measure of blood-brain barrier permeability) rose threefold 24 h after arachidonic acid injection. There was gross and microscopic evidence of edema. Saturated fatty acids and monounsaturated fatty acids were not effective. These data indicate that the endothelial cells of the blood-brain barrier are target sites for the action of arachidonic acid. It is hypothesized that the increased permeability of endothelial cells to macromolecules and water results from alterations of membrane phospholipids and increased vesicular transport, changes that are responsible for the delayed development of vasogenic edema.     

Influence of gender and castration on liver and CNS N-demethylation of morphine in rats1

Rats were injected with combinations of morphine-N-¹⁴CH₃ and morphine-6³H and the isotope content of the brain and liver was measured by combustion in a tissue oxidizer. The liver of intact male rats showed a significant increase in the ³H to ¹⁴C isotope ratio relative to the blood reflecting the existence of N-demethylation in this organ. This increase was not observed in the liver of either intact females or castrated males or females. Centrally, the hypothalamus, medial thalamus, and corpus striatum of both intact and castrated male and female rats exhibited increases in ³H to ¹⁴C isotope ratios indicating the presence of N-demethylation in these tissues. These results indicate that testicular hormones serve to increase the hepatic N-demethylation of morphine, but apparently reduce the comparable reaction in the CNS.     

Intracellular alkaline phosphatase activity in cultured human cancer cells

Summary The effect of saponin treatment in demonstrating intracellular portion of alkaline phosphatase activity in human cancer cell lines was evaluated. Previous reports using standard lead-salt techniques visualized enzyme almost exclusively on the plasma membrane and sometimes in the lysosomes. However, by treating cells with saponin before or during the cytochemical incubation, intracellular alkaline phosphatase became demonstrable at the endoplasmic reticulum, Golgi apparatus, Golgi-derived vesicles and mitochondria as well as lysosomes and plasma membrane. These intracellular catalytic activities were significantly inhibited by the specific amino acid inhibitors characteristic for each cell line, and this suggested that intracellular alkaline phosphatase is the same isoenzyme as that present in the plasma membrane. The results of our current and previous studies therefore indicate that saponin reveals latent intracellular alkaline phosphatase activity by changing the membrane's physical state; thereby increasing the availability of both catalytic and antigenic sites of the enzyme to substrate and to antibody respectively.This work was supported by National Institutes of Health Grant No. CA 21967     

Similar content of phospholipids and gangliosides in normal and homozygous familial hypercholesterolemia fibroblasts.

The cellular content of total and individual phospholipids and gangliosides was measured in fibroblasts cultured from four normal subjects, three patients with lysosomal lipid storage diseases, and two subjects with homozygous familial hypercholesterolemia. Measurements were made on cells grown in medium containing fetal calf serum under conditions in which normal cells derive cholesterol for cell growth from low density lipoprotein present in the fetal calf serum, whereas familial hypercholesterolemia homozygote cells, which lack cell surface low density lipoprotein receptors, derive cholesterol from endogenous synthesis. No difference was observed in the cellular content of total or individual phospholipids and gangliosides in the normal and familial hypercholesterolemia homozygote cells. In contrast, cells from a patient with Niemann-Pick disease and a patient with Sandhoff disease showed elevations in the content of sphingomyelin and complex gangliosides, respectively.     

Determination of sialidase activities in HeLa cells using gangliosides specifically labeled in N-acetylneuraminic acid.

Radioactive gangliosides, N-[¹⁴C]-acetylneuraminylgalactosylglucosylceramide ([¹⁴C]G_M3) and N- [¹⁴C]-acetylneuraminylgalactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl]-galactosylglucosylceramide ([¹⁴C]G_D1a), were synthesized from CMP-[¹⁴C]sialic acid and the appropriate precursor glycolipid using specific sialyltransferase activities. These compounds were isolated and used as substrates to assay sialidase activity in HeLa cells. Although sodium butyrate added to the culture medium increased G_M3 biosynthesis in HeLa cells, sialidase activity, as well as that of other glycohydrolases, was the same in control and butyrate-treated HeLa cells. The same sialidase activity appeared to hydrolyze both [¹⁴C]G_M3 and [¹⁴C]G_D1a, but not fetuin; the enzyme had a pH optimum of 5.0 and a K_m of 75 μm for the ganglioside substrates. Although the cells contained a high sialidase activity (4–7 nmol/mg of protein/h) and could bind exogenously added [¹⁴C]G_M3, no “ecto”-sialidase activity would be detected in intact cells under conditions where a close to physiological pH is maintained. The results indicate that ganglioside sialidase is not involved directly in the morphological and biochemical differentiation induced in HeLa cells by exposure to sodium butyrate.