Intestinal lesions induced experimentally by methotrexate.

Mice were given up to 9 doses of methotrexate intermittently over a 3-week period. Inhibition of mitosis occurred in the duodenum, jejunum and ileum after the first injection, and after 2 doses the crypt epithelium showed megalocytosis and occasional abnormal mitotic figures. Further treatment produced degeneration of the epithelium of the villi, which became irregular and atrophic, and the amount of crypt tissue was greatly reduced. Focal ulceration and haemorrhage occurred in some animals. Changes in the caecum and colon developed later and were much milder. After withdrawal of methotrexate the intestinal mucosa rapidly recovered and was normal 1 week later.     

The effect of bromhexine on experimentally induced diabetic nephropathy.

Diabetes was induced in male Wistar rats by a single i.v. injection of streptozotocin (40 mg/kg). Animals were treated with bromhexine at 2 dose levels (2.5 mg/kg/day and 25 mg/kg/day) for 13 months thereafter and compared to non-diabetic controls and untreated diabetic animals. Renal pathology showed a significant increase in glomerular volume and basement membrane thickening in untreated diabetic animals. The higher dose bromhexine treated diabetic animals showed a significant decrease in glomerular volume as compared with diabetic animals not given bromhexine.     

Influence of taurocholate, taurochenodeoxycholate, and taurodehydrocholate on sulfobromophthalein transport into bile.

To test the hypothesis that incorporation of sulfobromophthalein (BSP) into mixed micelles could account for the increase in its biliary transport maximum (Tmax) by bile salts, we have compared in hamsters the influence on BSP Tmax of taurocholate and taurochenodeoxycholate (two micelle-forming physiological bile salts) to that of taurodehydrocholate, a bile salt which, in vitro, does not form micelles. In a first series of experiments, it was observed that taurocholate and taurochenodeoxycholate increased the secretion of phospholipid (40 and 53%, respectively), and cholesterol (50 and 110%, respectively), whereas taurodehydrocholate decreased the secretion of phospholipid (-31%) and cholesterol (-43%). This result suggests that, in vivo, taurodehydrocholate or its metabolites do not form mixed micelles. In a second series of experiments, it was seen that the three bile salts induced a similar increase in BSP Tmax (63% with taurocholate, 52% with taurochenodeoxycholate, and 51% with taurodehydrocholate). These results provide circumstantial evidence for the hypothesis that mixed micelle formation is not an important determinant of maximal BSP secretion into bile.     

Naturally occurring and experimentally induced mycoplasmal arthritis of cattle.

Mycoplasma agalactiae subsp. bovis strain Iowa 1136 was isolated from synovial fluids of a clinical case of arthritis in cattle on pasture in Iowa. When given to calves and cows by intra-articular or intravenous injection, it caused severe and persistent joint infections with fever, lameness, and swelling of the affected joints, plus synovitis, tendonitis, and fibrinous-purulent synovial fluids of high protein content. Intramammary administration of the organism caused severe mastitis. Calves nursing the cows developed severe mycoplasmal arthritis.     

Histopathological studies on experimentally induced pulmonary adenomatosis in guinea-pig lungs.

The main purpose of our experimental series was to induce, in experimental animals, diffuse pulmonary fibrosis resembling that in human lungs. In the lungs of guinea-pigs injected with a soluble immune complex and continuously exposed to a 40-60 per cent oxygen-rich atmosphere, diffuse pulmonary fibrosis occurred in many cases in the course of 2 to 3 months after the injection. After the 100th experimental day, multiple foci of pulmonary adenomatosis occurred. The morphology was similar to that of Jaagsiekte. Electron microscopic observations revealed that these hyperplastic cells originated from type II pneumoncytes.     

Migration inhibitory factor expression in experimentally induced endotoxemia.

Macrophage migration inhibitory factor (MIF) is an important constituent of the host response to stress and infection and is the first mediator that has been identified to be released from immune cells upon stimulation with glucocorticoids. MIF also has been shown to be secreted from the anterior pituitary gland, monocytes/macrophages, and T cells activated by various proinflammatory stimuli. Once released, MIF acts to counter-regulate the inhibitory effect of glucocorticoids on inflammatory cytokine production. To characterize more precisely the role of MIF in the host response to infection, we undertook a systematic analysis of MIF expression in various organs of the rat after endotoxin (lipopolysaccharide) administration. MIF protein and mRNA were analyzed by immunohistochemistry and in situ hybridization, respectively. MIF was found to be expressed constitutively in organs such as the lung, liver, kidney, spleen, adrenal gland, and skin. Significant quantities of MIF protein were detected preformed in various cell types and appeared to be released as a consequence of endotoxemia. In virtually all tissues examined, the loss of MIF protein 6 hours after lipopolysaccharide administration was accompanied by the induction of MIF mRNA and, at 24 hours, by the restoration of immunoreactive, intracellular MIF. The constitutive production of MIF by several cell and tissue types together with its rapid release from intracellular pools distinguishes MIF from other cytokines or hormonal mediators and significantly expands the physiological role of this unique counter-regulator of glucocorticoid action.     

Evaluation of experimentally induced Fusobacterium necrophorum infections in mice.

Two strains of mice, Swiss Webster and DBA/2Cr, were injected intraperitoneally or intravenously with varying dosages of Fusobacterium necrophorum. The ability to eliminate the infection was assessed by quantitative enumeration of the organisms present in the blood, liver, and spleen, Three- to 4-week-old DBA/2Cr mice were highly resistant to both routes of injection. The intraperitoneal injection of older mice failed to demonstrate a dose-effect relationship whereas an intravenous injection of as few as 10(4) cells of F. necrophorum produced progressively necrotic leg abscesses, apparently involving the lymphonodus ischiadicus which filters the site of injection. Mortality was increased with sensitization by a previous sublethal injection. Also, an ethanol-killed cell vaccine delayed the onset of lethal infection, whereas repeated sublethal live cell injections provided nonspecific protection since mice vaccinated with the growth medium were equally protected. The development of leg abscesses after intravenous injection visibly demonstrated the pathogenicity of F. necrophorum and may provide a suitable model for the evaluation of vaccines and the effectiveness of antibiotics.     

Lymphocyte subtypes in experimentally induced early-stage bovine tuberculous lesions.

The identity of the lymphocyte subtypes constituting the lymphocytic mantle within developing early-stage lesions of bovine tuberculosis was investigated immunohistochemically in calves inoculated intranasally with 2 x 10(7) colony-forming units of a field isolate of Mycobacterium bovis. Pulmonary lesions were examined 7, 14, 21, 28 and 42 days after inoculation, and bronchial lymph node lesions at 35 days. The immunolabelling results reported were obtained with monoclonal antibodies against two T-cell epitopes (WC1+ gamma delta and CD2+) and against B-cell epitopes. Large numbers of CD2+ T-lymphocytes were observed around developing areas of necrosis throughout the study; WC1+ gamma delta cells, however, were more numerous at these sites up to and including day 21. On the other hand, aggregates of B lymphocytes did not become prominent in areas adjacent to lesions until day 42. The results suggest that these lymphocyte phenotypes play a role in the pathogenesis of early-stage lesions.     

Binding of ubiquitin to experimentally induced murine AA amyloid.

Amyloid enhancing factor (AEF) activity has recently been demonstrated in ubiquitin purified from amyloidotic murine tissues and Alzheimer brain extract. Since AEF is known to bind to amyloid fibrils and 'fibril-AEF' on passive transfer induces accelerated amyloidogenesis in the recipient animals, it was of interest to investigate whether ubiquitin binds to amyloid. Immunohistological studies were carried out on liver sections from amyloidotic mice. Biotin-strepavidin-peroxidase methods using monospecific rabbit anti-mouse AA amyloid IgG (RAAG) and rabbit anti-bovine ubiquitin IgG (RABU) antibodies were employed to immunostain the amyloid and ubiquitin deposits, respectively. RABU-treated liver sections were counterstained with thioflavine S. RAAG reacted strongly with the amyloid, indicating that it is AA type, and RABU-positive immunodeposits were found bound to the thioflavine-S-positive AA deposits. Treatment of the liver sections with 0.1 M sodium acetate containing 0.5 M NaCl, pH 4, for 2-3 h at 37 degrees C nearly completely desorbed the AA amyloid-bound ubiquitin. Since ubiquitin demonstrates AEF activity in vivo and binds non-covalently to AA amyloid, we suggest that ubiquitin may indeed be 'fibril-AEF' and may play a crucial role in the pathogenesis of amyloidosis. To our knowledge, this is the first time that ubiquitin bound to extracellularly deposited amyloid has been demonstrated.     

Combined action of phosphomycin with streptomycin and gentamicin.

The combined action of phosphomycin with streptomycin and gentamicin against 41 strains of Gram-positive micrococci (S. aureus and Str. faecalis) and 87 strains of Gram-negative bacilli (including 38 strains of Pseudomonas) was evaluated. The combination of phosphomycin with streptomycin acted synergistically against 12 strains of Gram-positive micrococci, and the combination of phosphomycin with gentmicin against 10 strains. The combination of phosphomycin with streptomycin acted synergistically against 31% of strains (except Pseudomonas), and the combination of phosphomycin with gentamicin against 65% of strains. Only six of 38 Pseudomonas strains were acted synergistically by phosphamycin with streptomycin, and four by phosphomycin with gentamicin. The results indicate that the combination of phosphomycin with aminoglycoside antibiotics shows greater synergistic activity against Gram-negative strains than against Gram-positive micrococci.     

The effect of bromhexine on experimentally induced diabetic nephropathy

Diabetes was induced in male Wistar rats by a single i.v. injection of streptozotocin (40 mg/kg). Animals were treated with bromhexine at 2 dose levels (2.5 mg/kg/day and 25 mg/kg/day) for 13 months thereafter and compared to non-diabetic controls and untreated diabetic animals. Renal pathology showed a significant increase in glomerular volume and basement membrane thickening in untreated diabetic animals. The higher dose bromhexine treated diabetic animals showed a significant decrease in glomerular volume as compared with diabetic animals not given bromhexine.     

Heterotransplantation of experimentally induced dog stomach adenocarcinoma to nude mice.

Heterotransplantation of gastric adenocarcinoma, induced in dog by N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) has been attempted to nude mouse. Biopsied materials from Borrmann 3 type carcinoma, which showed signet-ring cell carcinoma at the subcardiac region of a beagle dog, were inoculated into the muscles of the hind legs of BALB/c-nu/nu nude mice. Serial transmission was obtained in 2 lives, so far 4-5 passages. The histological findings of the grafts were mainly poorly differentiated adenocarcinomas.     

Reversibility of hepatic fibrosis in experimentally induced cholestasis in rat.

The reversibility of hepatic fibrosis was investigated in an experimental model of extrahepatic cholestasis in the rat after common bile duct ligation for 2 weeks, followed by bilioduodenal anastomosis for 3 weeks. Bile duct ligation resulted in a transitory marked elevation in the serum concentration of 5'-nucleotidase, alkaline phosphatase, and bilirubin during the first 3 days. Then these levels decreased to threefold, twofold, and 100-fold the normal values, respectively, during the following 4 weeks. Histologic examination of the liver disclosed extensive bile duct proliferation and the formation of periportal fibrosis, with only slight inflammation and necrosis. The distribution of the major components of the hepatic extracellular matrix was analyzed 2 weeks after bile duct ligation, using the indirect immunoperoxidase method. Fibrous septa were found to be strongly stained for collagens I, pro-III, III and IV, fibronectin, and laminin. The most intense staining was found in enlarged periportal areas, collagen IV and laminin being particularly abundant around newly formed bile ducts. These changes paralleled high steady-state levels of alpha 1(I) and alpha 1(IV) collagen and B2 chain laminin mRNAs. Relief of the obstruction for 2 weeks resulted in a shift in the serum concentration of 5'-nucleotidase, alkaline phosphatase, and bilirubin toward normal values. A dramatic resorption of bile duct proliferations and periportal fibrosis were observed. Three weeks after bile duct repermeabilization, immunohistochemical study showed that the pattern of distribution of extracellular matrix components was almost normal, except for collagen IV, which remained abundant in the sinusoids when compared with the normal liver. In parallel, the steady-state B2-chain laminin mRNA level became lower than in cholestatic livers, whereas alpha 1(I) and alpha 1(IV) mRNAs were almost undetectable. These results show that hepatic fibrosis induced by experimental extrahepatic cholestasis in rat disappears in less than 3 weeks after relief of bile duct obstruction, suggesting that an active degradation of matrix protein occurs, except for collagen IV in the sinusoid.     

Depressed immunological defence mechanisms in mice with experimentally induced diabetes.

Persistent diabetes mellitus with marked hyperglycemia was induced in mice by the administration of streptozotocin. In these streptozotocin-induced diabetic mice, resistance to tubercle bacillus challenge and primary as well as secondardy humoral immune responses against foreign erythrocytes were markedly depressed. The T-cell function in delayed hypersensitivity to 2,4-dinitro-1-fluorobenzene and bacterial phagocytic activity or peritoneal macrophages were markedly depressed. In contrast, the B-cell function in antibody production against T-independent antigen and the intracellular killing of bacteria in peritoneal macrophages were intact. We concluded that depression of the T-cell function or the phagocytic activity of macrophages or both may be the main immunological defect in these mice.