In vitro inhibitory effects of polymer-linked methotrexate derivatives on tetrahydrofolate dehydrogenase and murine L5178Y cells.

In vitro studies were made on four synthetic polymeric derivatives of the antitumor agent methotrexate (MTX): 1) divinylether-maleic anhydride-MTX (DIVEMA-MTX), 2) poly-L-lysine-MTX (PL-MTX), 3) polyethyleneimine-MTX (PEI-MTX), and 4) carboxymethyl cellulose-MTX (CMC-MTX). They were tested for their ability to inhibit tetrahydrofolate dehydrogenase (dihydrofolate reductase). Their growth inhibition of murine L5178Y leukemia cells was also studied. 1wo of these polymers, DIVEMA-MTX and PEI-MTX, had similar or only slightly reduced activity compared to equivalent concentrations of MTX, whereas PL-MTX and CMC-MTX had significantly higher (1--3 logs) minimal inhibitory concentrations.

Relative biochemical aspects of low and high doses of methotrexate in mice.

During low infusion rates of methotrexate (1.0 microng/hr/mouse; plateau plasma concentration, 2 X 10(-8) M), [3H]deoxyuridine incorporation into DNA was inhibited to a significant degree in small intestine and femur marrows. However, incorporation of [3H]thymidine into intestinal DNA was stimulated at this low infusion rate. During high infusion rates of methotrexate (10 microng/hr/mouse, plateau plasma concentration, 4 X 10(-7) M), inhibition of the incorporation of [3H]deoxyuridine at the steady state levels of plasma methotrexate in both the small intestine and femur marrow was significant. In contrast to stimulation at the low infusion rate, incorporation of [3H]thymidine into intestinal DNA at this high infusion rate was inhibited to a significant degree. Inhibition was not statistically significant in femur marrow DNA. The inhibition of [3H]thymidine into intestinal DNA could be reversed by the simultaneous infusion of inosine. Thus, in the in vivo system, an antipurine effect on DNA Synthesis at high methotrexate plasma concentration in the small intestine was observed. This antipurine effect was not apparent at the lower concentrations. The lower concentration, however, could still inhibit [3H]deoxyuridine incorporation into intestinal and femur marrow DNA to a significant enough degree that, if prolonged, it would resultin lethality to the mice. The thymineless state can be maintained for at most 60 hr in mice without lethal toxicity, whereas the antipurine state can be maintained for no longer than 18 hr in mice without some lethal toxicity. These data have important implications in rescue studies using thymidine or leucovorin.

In vitro transformation of Syrian hamster epidermal cells by N-methyl-N'-nitro-N-nitrosoguanidine.

The selection of Syrian hamster epidermal cells which do not terminally differentiate has provided a quantitative focus assay for in vitro chemical transformation. One-day-old Syrian hamster epidermal cells plated at 5 x 10(6)/100-mm dish were treated for 5 hr with various concentrations of N-methyl-N'-nitro-N-nitrosoguanidine. After 4 weeks, the normal epidermal cells began to terminally differentiate to keratinized squamous cells and died, but transformed epidermal colonies grew to higher cells densities and appeared as darker areas against a lightly stained normal cell background. Transformed epidermal foci were isolated and subcultured for at least 15 passages, whereas normal epidermal cells could not be subcultured under the same conditions. The transformed cells assumed the typical cobblestone-like morphology of epithelial cells, retained desmosomes and tonofilaments, and were able to use citrulline in place of arginine. Argininosuccinate synthetase (EC 6.3.4.5) activity was significantly higher in the epidermal cells than in fibroblasts. The injection of 5 x 10(6) cells of two transformed epidermal cell lines into athymic nude mice resulted in the formation of tumors which were identified as keratinizing squamous carcinomas.

The acute phase response in the rodent.

In the rodent, the general response to acute inflammation and tissue damage is characterized by a complex rearrangement in the pattern of concentrations of proteins in the plasma leading to an increase in the sedimentation rate of erythrocytes, an increase in leukocyte concentration in the bloodstream, and a decrease in the hematocrit. Body temperature changes only slightly or not at all. The reasons for the change in plasma concentrations of proteins are changes in their rates of synthesis in the liver. Degradation of plasma proteins is not affected. The details of the acute phase response evolved in the interaction of species with their environment. Therefore, it is not surprising to find differences in the details of the acute phase response among species. For example, alpha 2-macroglobulin is a strongly positive acute phase reactant in the rat, but not in the mouse; C-reactive protein is a strongly positive acute phase protein in the mouse, but is not found in the rat. An inducible acute phase cysteine proteinase inhibitor system, which has evolved from a primordial kininogen gene, has been observed so far only in the rat. The changes in the synthesis rates of acute phase proteins during inflammation are closely reflected by corresponding changes in intracellular mRNA levels. In the liver, the capacity to induce the acute phase pattern of synthesis and secretion of plasma proteins probably develops around birth. Changes in mRNA levels are brought about by changes in transcription rates or by changes in mRNA stability. Kinetics of mRNA changes during the acute phase response differ for individual proteins. The main signal compound for eliciting the acute phase response in liver seems to be interleukin-6/interferon-beta 2/hepatocyte stimulating factor, whereas interleukin-1 leads to typical acute phase changes in mRNA levels only for alpha 1-acid glycoprotein, albumin, and transthyretin. Plasma protein genes are expressed in various extrahepatic tissues, such as the choroid plexus, the yolk sac, the placenta, the seminal vesicles, and other sites. All these tissues are involved in maintaining protein homeostasis in associated extracellular compartments by synthesis and secretion of proteins. Synthesis and secretion of plasma proteins in paracompartmental organs other than the liver is not influenced by the acute phase stimuli.

mRNA for kininogens and thiostatins (major acute phase alpha 1-proteins) in the liver of kininogen-deficient rats.

mRNAs for low and high molecular weight kininogens (1.6 and 3.0 kb in size, respectively) and for two thiostatins (1.6 kb in size) were found in the liver of kininogen-deficient Brown-Norway (BN/Mai Pfd) rats. The levels of mRNAs for thiostatins, but not those for low and high molecular weight kininogens (arising from a single kininogen gene), increased strongly during acute inflammation. The pattern of DNA restriction sites for the kininogen gene and the thiostatin genes in the mutant rat strain was identical to that in at least four other rat strains.

In vivo methotrexate transport in murine Lewis lung tumor.

Methotrexate uptake by murine Lewis lung tumor was measured in vivo over a wide dose range. The data were analyzed according to a model previously developed for tissues in which methotrexate uptake is rate limited by transport across the cell membrane. Methotrexate transport in this tumor followed Michaelis-Menten kinetics with a rate constant for permeability (k/K) of 0.012 min-1. The methotrexate binding capacity of dihydrofolate reductase in the tumor was not exceeded at any dose studied. A low membrane permeability in conjunction with a high dihydrofolate reductase level explains the resistance of this tumor to methotrexate.

Basal and pentagastrin stimulated 6-keto prostaglandin F1 alpha secretion in dog stomach.

The effect of pentagastrin on gastric secretion of prostacyclin (PGI2) was studied by measuring the output of 6-keto-prostaglandin F1 alpha in the gastric juice of 8 dogs before and after an intramuscular injection of pentagastrin. A similar study was done in 7 control dogs not given pentagastrin. Pentagastrin was found to significantly increase the gastric secretory output of acid and 6-keto PGF1 alpha during the first 30 minutes after the pentagastrin injection. This was highly significant when compared to the control group. It is concluded that pentagastrin stimulates gastric prostacyclin secretion.

Gluten enteropathy occurring in chronic hepatitis.

Steatorrhoea and subtotal villus atrophy due to gluten enteropathy was found to occur in a 27 year old female, who had histologically proven chronic active hepatitis six years ago. She had abnormal elevation of immunoglobulins IgG, IgM, and IgA, decreased level of complement C4 and a positive test for HLA-B8. The SGOT was persistently mildly elevated, but her latest liver biopsy (6 years after onset) showed only changes of chronic persistent hepatitis. She had good symptomatic and histological (jejunal biopsy) improvement with gluten-free diet.

Effective of acetylsalicylic acid, indomethacin, phenylbutazone, paracetamol, ibuprofen and naproxen on canine gastric mucosa: a histological and ultrastructural study.

The effect of various non-steroidal analgesic drugs on gastric mucosal cells was studied in 48 dogs with 2 other dogs as control. Each drug was instilled into the stomach of 8 anaesthetised dogs through a gastrostomy. Gastric biopsies, obtained at 0, 1, 2 and 3 hours after administration of the drug, were subjected to histological and electron microscopical examination. Histological analysis did not show any significant changes in gastric mucosal cells in any of the drug-related groups as compared to controls. Electron microscopy, however, showed significant changes in the gastric mucous cells and parietal cells of all test groups, as compared to controls. Ultrastructural abnormalities in mucous cells included dilation of cisternae of endoplasmic reticulum, loss of granules and microvilli, and thickening of the mucous layer and were most pronounced with acetylsalicylic acid, moderate with phenylbutazone, indomethacin and naproxen, but mild with paracetamol and ibuprofen. Parietal cell changes consisting of reduction in the concentration of tubulovesicles, which canaliculi, were found to be severe with indomethacin and phenylbutazone, moderate with paracetamol and naproxen, but only minimal with acetylsalicylic acid and ibuprofen.

Endoscopic, histological and ultrastructural correlations in the diagnosis of colitis.

The accuracy of the endoscopic diagnosis, found at fiberoptic distal-colonoscopy, was assessed by correlation with the histological findings of multiple colo-rectal biopsies. Of 25 subjects with normal colon at endoscopy, 18 (72%) had normal histology, while 7 (28%) had histological evidence of mild non-specific colitis (6 cases) or Crohn's colitis (one case). Of 15 cases with endoscopic appearance of mild colitis, histological sections showed mild non-specific colitis in 12 (80%), idiopathic ulcerative colitis in one and Crohn's colitis in another. In 12 patients with an endoscopic diagnosis of moderate-severe colitis, histology confirmed moderate to severe idiopathic ulcerative colitis in 9 (75%) and severe Crohn's colitis in 2. Pseudomembranous colitis was confirmed by histology in 2 out of 3 cases detected by endoscopy. Electron-microscopy, although amplifying the histological findings, did not provide any specific diagnostic information. It is concluded that histological studies of multiple colo-rectal biopsies can significantly improve the diagnostic accuracy of distal-colonoscopy. Biopsies should therefore be obtained routinely in colonoscopy even if the appearances look normal.

P3 amylase isoenzyme in the diagnosis of pancreatitis.

P3 amylase isoenzyme was found to be present in the plasma of 43 out of 140 patients with suspected pancreatitis. Of 43 patients with the P3 isoenzyme, pancreatitis was definitely confirmed in 21 by laparotomy. Of the remainder, 19 had probable pancreatitis based on a combination of clinical, biochemical, endoscopic (ERCP), ultrasonic and computerised tomographic evidence, while 3 had possible pancreatitis based on clinical evidence alone. Of 97 patients without the P3 isoenzyme 1 patient had evidence of probable pancreatitis and 1 had evidence of possible pancreatitis. Statistical analysis (excluding the 4 cases of possible pancreatitis) showed that the P3 amylase isoenzyme had a sensitivity of 98%, a specificity of 100% and an efficiency of 99%. The predictive values of a positive and negative test were 100% and 99% respectively. Hyperamylasemia was not a pre-requisite to the presence of the P3 isoenzyme, since 11 out of 13 patients, with normal total amylase levels, were found to have the P3 band and confirmation of their pancreatitis. It is concluded that the P3 amylase isoenzyme is a specific and sensitive test for pancreatitis, and appears also to be superior to the total plasma amylase.