An ozone-generating system and chamber for testing injury to cultured cells

A system was constructed which alternately exposed cultured cells to specific concentrations of ozone in the gas phase and then to cell culture medium. It was designed to produce a constant mass transfer between the gas phase and the exposed cell phase. Monolayers of neonatal rat lung fibroblasts cultured in miniature glass dishes were used to test for ozone toxicity. The cells were alternately exposed to ozone (0.05, 0.15, 0.45, 1.35, and 4.05 ppm) for 30 sec and then to culture medium for 30 sec over a 1-hr period. Toxicity was measured as inhibition of cell growth 4 days after exposure to ozone. Growth was significantly inhibited at all concentrations with a 50% inhibitory concentration of 0.8 ppm. The system described was effective for quantifying ozone toxicity for cultured lung cells.     

Histotopic of glycosidases in the oviduct of the quail (Coturnix coturnix japonica) (author's transl)]

The activities of 6 glycosidases (n-acetyl-beta-glucosaminidase, beta-galactosidase, beta-glucuronidase, alpha-galactosidase, alpha-fucosidase and alpha-mannosidase) in the oviduct of the quail (Coturnix coturnix japonica) were studied with histochemical methods. Alpha-galactosidase and alpha-fucosidase showed a weak to moderate activity in the surface epithelium and in most of the glands of the oviduct. A Distinct reactivity of beta-glucuronidase was observed in the surface epithelium of the whole oviduct and in the glands of the uterovaginal-region. A moderate to distinct reactivity of n-acetyl-beta-glucosaminidase cound be demonstrated in the epithelium and in the glands of all regions of the oviduct. The comparatively highest activity of this enzyme was found in the glands of the magnum and in the surface epithelium of the uterus. The possible functions of the glycosidases in the oviduct are discussed briefly.     

Myelopathy due to intracranial dural arteriovenous fistulas draining intrathecally into spinal medullary veins. Report of three cases

Arteriovenous malformations (AVM's) of the spine commonly cause progressive myelopathy. Occasionally, myelography reveals serpentine filling defects characteristic of a spinal AVM, but an AVM or arteriovenous (AV) fistula cannot be demonstrated arteriographically, despite selective catheterization of all vessels known to have the potential of supplying the spinal cord and spinal dura. Often, and particularly in the setting of subacute or acute deterioration, this has been attributed to spontaneous thrombosis of the veins (the Foix-Alajouanine syndrome). Three patients are reported in whom intracranial dural AV fistulas, supplied by branches of the internal and external carotid arteries, drained into spinal veins and produced myelopathy. In one patient, motor and sensory deficits were limited to the lower extremities. In all three patients, disconnection of the fistula from its spinal venous drainage permitted arrest of a rapidly progressive myelopathy and partial recovery. These findings indicate that some patients who appear to have spinal cord AVM's but exhibit negative spinal arteriography are suffering from cranial dural AV fistulas and therefore need carotid as well as spinal arteriography. The considerable distance of these fistulas from the level of neurological expression supports venous hypertension as a pathophysiological mechanism of spinal cord injury. Interruption of a cranial dural fistula draining into spinal veins permits recovery of the myelopathy.     

Diphtheria toxin effects on brain-tumor xenografts. Implications for protein-based brain-tumor chemotherapy

A model was developed to determine whether protein-based chemotherapeutic agents can cross the blood-brain barrier and successfully treat brain tumors. The human small-cell lung carcinoma N417D was grown as a solid tumor in the nude rat brain, and diphtheria toxin (DT) was administered intravenously as therapy. Because rat cells lack functional DT receptors and are 1000 to 10,000 times less sensitive to DT than human cells, a therapeutic window exists between the implanted human tumor and the nude rat host. The pharmacokinetic and pharmacodynamic characteristics of DT were defined. Within 6 hours, more than 90% of the initial DT concentration was removed from the blood. The blood-to-tumor transfer constant Ki for DT in small N417D tumors was 0.49 microliters/gm-min, one-fourth to one-fifth the reported values for permeability to proteins in other experimental tumor models. Despite the toxin's short plasma half-life and the relatively intact blood-tumor barrier, DT administered intravenously as a single dose significantly extended animal survival. Untreated nude rats developed solid parenchymal tumors and died in 11 to 16 days (median 15 days). When administered at 0.1 micrograms/animal, DT increased the median survival time to 19 days (p less than 0.0016) while 1.0-microgram doses extended median survival times to 26.5 days (p less than 0.0002). A higher dose of DT (3.0 micrograms) had no further beneficial effect on survival (26.1 days). Blood-brain barrier constraints to successful monoclonal antibody-based therapies of brain tumors may have been overestimated since antibody conjugates have plasma half-lives longer than DT, and the permeability of N417D tumors to DT is equal to or less than the permeability of other experimental tumors to large proteins. Recently developed immunotoxins that have the higher potency of DT and a therapeutic window as wide as DT has in this nude rat/human tumor paradigm may be effective in treating brain tumors despite limited blood-tumor permeability.     

Inhibition of heparin activity in plasma by soluble fibrin: evidence for ternary thrombin-fibrin-heparin complex formation

The ability of heparin to dramatically enhance the inactivation of thrombin (IIa) by antithrombin III (ATIII) in buffer is negated through formation of a IIa-fibrin-heparin ternary complex (Hogg and Jackson, Proc Natl Acad Sci USA 86:3619, 1989; Hogg and Jackson, J Biol Chem 265:241, 1990). IIa, in this ternary complex, is protected from inactivation by ATIII. Our aim was to determine whether fibrin also compromises heparin efficacy in plasma. We found that soluble fibrin ablated the heparin-mediated prolongation of the thrombin time with half-maximal effect at 60 nmol/L fibrin. The heparin-mediated prolongation of the activated partial thromboplastin time (APTT) was also reduced by fibrin with half-maximal effects at 140 nmol/L fibrin using 0.12 U/mL heparin and 500 nmol/L fibrin using 0.25 U/mL heparin. The mechanism of inhibition of heparin activity by fibrin in plasma was determined by measuring IIa-ATIII complexes by enzyme-linked immunosorbent assay (ELISA). Fibrin was found to inhibit the heparin- catalyzed inactivation of IIa by ATIII with half-maximal effect at 97 +/- 19 nmol/L fibrin. Fibrin had no effect on the heparin-catalyzed inactivation of factor Xa by ATIII in plasma, using either standard heparin, a heparinoid preparation (Orgaran; Organon, Lane Cove, Sydney, Australia), or low-molecular weight heparin. These findings imply that fibrin is a potent modulator of heparin activity in vivo by inhibiting heparin-catalyzed IIa-ATIII complex formation through formation of ternary IIa-fibrin-heparin complexes.