Establishment of specific pathogen-free (SPF) rat colonies using gnotobiotic techniques.

Gnotobiotic Wistar rats were produced using gnotobiotic techniques, which were established in the production of a SPF mouse colony, in order to establish a barrier-sustained colony. One strain of Escherichia coli, 28 strains of Bacteriodaceae (B-strains), three strains of Lactobacillus (L-strains) and a chloroform-treated fecal suspension (CHF, Clostridium mixture) were prepared from conventional Wistar rats as the microflora source. Two groups of limited-flora rats, E. coli plus B-strains and E. coli plus CHF, were produced. After confirmation that Clostridium difficile was not detected in the CHF-inoculated rats, two groups of limited-flora rats were transferred to an isolator and housed together in a cage. These rats were then orally inoculated with L-strains. The gnotobiotic rats showed colonization resistance to Pseudomonas aeruginosa, and the number of E. coli in the feces was 10(5) to 10(6)/g. The gnotobiotic rats were transferred to a barrier room as a source of intestinal flora for SPF colonies. In the SPF rats, basic cecal flora was mainly composed of Bacteroidaceae, clostridia, fusiform-shaped bacteria and lactobacilli, and did not change over a long period. Their flora became similar to that of conventional rats.

Establishment of specific pathogen-free guinea-pig colonies using limited-flora guinea-pigs associated with conventional guinea-pig flora, and monitoring of their cecal flora.

Six groups of limited flora (LF) Hartley guinea-pigs were produced by inoculation of hysterectomy-derived GF guinea-pigs with various combinations of cecal bacteria of conventional (CV) guinea-pigs to determine the effective bacterial cocktails for the establishment of a specific pathogen free (SPF) colony. Bifidobacterium magnum (Bif) isolated from CV guinea-pigs was used for pretreatment. The mortality of LF guinea-pigs inoculated with only Bif was 75%, and that of those inoculated with Bif plus chloroform-treated cecal suspension (CHF) or Bif plus CHF plus 32 isolates from CV guinea-pigs was 40 to 66.7%. These three groups were in an unhealthy condition with mucoid enteritis-like diarrhea. However, the mortality of LF guinea-pigs inoculated with the anaerobic growth on EG plates injected with 10(-5) dilution of cecal contents (CF) or inoculated with Bif plus CF was 6.3 and 15%, respectively. These latter two groups of LF guinea-pigs were transferred to separate barrier rooms and some of the LF guinea-pigs were maintained in isolators as a source of intestinal flora for SPF guinea-pigs. The composition of cecal flora of LF guinea-pigs was stable for a long time, and bacteroidaceae and peptococcaceae were maintained as predominant components. The basic composition of the cecal flora of SPF guinea-pigs originated from LF guinea-pigs, which consists mainly of the anaerobic bacteria, was not changed over a long period, and the flora composition became similar to that in CV guinea-pigs. Guinea-pig-specific pathogens from the SPF colonies were not detected during experiments.

[Prognostic factors of hepatocellular carcinoma: analysis by the proportional hazard model].

Five hundred fifty patients hospitalized to our hospital during 1990 to 1999 were studied. Subjects consisted of 413 males and 102 females and mean age was 62.1 years. Association with HBV infection, HCV infection and both infection was 11.1%, 78.4% and 2.5% respectively. According to the criteria based in Liver Cancer Study of Japan, 5 year survival rate in clinical stage I, II, III was 42.3%, 38.8% and 17.5%. Tumor morphology was nodular type in 78.9%, massive type in 9.1% and diffuse type in 10.5% of cases. Portal tumor thrombus and distal metastasis were observed in 19.9% and 6.3% of all cases. The 1-, 3- and 5-year survival rate in patients received any therapy was 80.8%, 44.6% and 28.7%, respectively. Analysis by the proportional hazard model showed that HBV infection, advanced clinical stage, multiple tumors, a tumor diameter in excess of 5 cm and AFP positivity were shown as significant factors on poor prognosis.

Possible relationship between hyperinsulinemia and glomerular hypertrophy in nephrosclerosis.

Hyperinsulinemia is potentially associated with the development of vascular sclerosis. On the other hand, the relationship between hyperinsulinemia and nephrosclerosis has not been elucidated. In this investigation clinicopathological studies were performed in 40 patients with nephrosclerosis, with special attention to the relationship between hyperinsulinemia and glomerular hypertrophy. Forty patients with biopsy-proven nephrosclerosis were divided into two groups by the 75-g oral glucose tolerance test (OGTT): group A, 2-hr plasma glucose concentration > 140 mg/dL (n = 25); group B, 140 < or = 2-hr plasma glucose < 200 mg/dL (n = 15). Patients with diabetes mellitus or diabetic nephropathy were not included. Morphometric analysis of the glomeruli revealed a significantly larger mean glomerular volume in subjects with nephrosclerosis in both subgroups. In addition, the mean glomerular volume was significantly correlated with the fasting insulin level, while no significant correlation was observed between the mean glomerular volume and creatinine clearance or degree of global sclerosis. These results indicate that hyperinsulinemia may be intimately related to glomerular hypertrophy in patients with nephrosclerosis.

[Successful treatment of interstitial pneumonitis with cyclosporin A in a patient with rheumatoid arthritis accompanied by acute interstitial nephritis].

A 49-year-old male was admitted to our hospital because of acute renal failure. He had been treated by a local doctor for rheumatoid arthritis (RA) during the past eight years. We treated him with steroid pulse therapy, because of suspected acute interstitial nephritis. We confirmed this diagnosis by renal biopsy and steroid pulse therapy markedly improved his renal dysfunction. Immunohistochemical studies revealed that interstitial infiltrating leukocytes consisted mainly of polymorphonuclear leukocytes (PMNs), macrophages and B lymphocytes, while T lymphocytes were less predominant. ELAM-1 and GMP-140 were expressed in the peritubular capillaries. These findings suggest that endothelial activation of the peritubular capillaries may cause interstitial infiltration of PMNs and macrophages, resulting in the development of acute interstitial nephritis. Four months later, he developed severe interstitial pneumonitis, and his symptoms were not improved by high-dose steroid pulse and cyclophosphamide pulse treatment. Eight weeks after the second admission, cyclosporin A (Cy A) was started. Three weeks after starting Cy A, he was free from symptoms and his chest radiograph was normalized. Renal function was also improved by Cy A. These observations suggest that endothelial activation by adhesion molecules may play an important role in RA-related autoimmune diseases and that Cy A might be efficacious in such cases.

Clinicopathological study of IgA nephropathy in patients with congenitally reduced nephron mass.

In experimental animal models, a reduction in the number of functioning nephrons is considered to play a role in the progression of glomerular injury. In human renal diseases, however, whether a superimposed reduction in the number of nephrons causes the exacerbation of preexistent glomerulopathy has not been elucidated. We herein report the results of a clinicopathological study of five patients with IgA nephropathy (IgAN) which occurred in a reduced nephron mass status (four cases of congenital solitary kidney and one case of bilateral hypoplastic kidneys). Four of the five patients had chronic renal failure (CRF) and exhibited a relatively rapid course to CRF as primary IgAN. Renal biopsy revealed that all four of the patients with CRF had glomerular hypertrophy and focal segmental glomerular sclerosis. In addition, two of them had a focal active lesion. In one patient with bilateral hypoplastic kidneys renal biopsies were performed twice in eight years. During this period her creatinine clearance deteriorated from 60.0 ml/min to 20.7 ml/min. Her first renal biopsy showed mild mesangial proliferation without sclerotic lesions, glomerular hypertrophy and mesangial IgA deposition, while all of them were prominent in the second renal biopsy. These observations suggest that IgAN superimposed on a nephron loss status may be frequently associated with a progressive course of disease, and careful follow-up and early treatment should be considered in such a condition.

[Anesthetic management of a patient with pheochromocytoma using autotransfusion].

Autotransfusion was employed for a 51 year-old male patient with intravesical pheochromocytoma and paroxysmal hypertension in an attempt to avoid side-effects of conventional blood transfusion and to minimize the change in intravascular volume after removal of the tumor. Blood was withdrawn twice (980g in all) before surgery. Anesthesia was maintained with N2O-O2-sevoflurane and epidural anesthesia. Sodium nitroprusside was administered when necessary to control blood pressure. Hypotension associated with removal of the tumor could be successfully prevented by autotransfusion. This case demonstrates usefulness of autotransfusion to control hypotension following removal of pheochromocytoma.

Segmental hyperhidrosis in syringomyelia with Chiari malformation.

Segmental hyperhidrosis was seen in three cases of syringomyelia with Chiari malformation confirmed by magnetic resonance imaging. Using the iodine-starch reaction, all three cases showed hyperhidrosis in the areas roughly corresponding to those of sensory disturbances. Two possible modes of pathogenesis are discussed: stimulation of preganglionic neurons and interference to the inhibitory tract.

Biochemical classification of Clostridium botulinum type C and D strains and their nontoxigenic derivatives.

The biochemical properties of 11 toxigenic and 10 nontoxigenic type C and D strains of Clostridium botulinum were studied. All of the strains examined were motile and hemolytic and produced lipase and liquid gelatin. Fermentation of several sugars and the production of lecithinase, indole, and hydrogen sulfide varied with the strain. The strains were classified into four groups based on their sugar fermentation profiles. The resulting classification was identical to the classification which had been proposed from the relationship between toxin production and phages and similar to the grouping based on the nature of toxin antigenic structures. Lecithinase production was negative in the cells belonging to group III, and indole and hydrogen sulfide production was negative in the cells of groups III and IV. Strains belonging to groups III and IV have many characteristics different from those of groups I and II.

Phosphatidylethanolamine methyltransferase activity in developing, demyelinating, and diabetic mouse brain.

The catalysis by phosphatidylethanolamine methyltransferase (PEMT) of phosphatidylcholine (PC) synthesis by the successive methylation of phosphatidylethanolamine in the presence of S-adenosylmethionine (AdoMet) as methyl donor, was detected in actively myelinating mouse brains. PEMT activity in the microsome fraction of fetal mouse brain at 17 days of gestation was 253 mu u/mg protein and that of adult brain after 7 days of remyelination following 6 weeks cuprizone administration was 148 mu u/mg. These figures are much higher than found in normal adult brains (1.7 mu u/mg). An increase in PEMT activity was observed in the brains of genetically transmitted diabetic mice, C57BL/KsJ-db/db, and streptozotocin-induced diabetic mice; 16.3 and 9.2 mu u/mg, respectively. The methyl group of mecobalamin was transferred to homocysteine producing AdoMet and was further metabolized into choline and acetylcholine in brain slices. These results suggest that in the diabetic state, an increase in PC synthesis is probably required in order to replace damaged myelin or to supply choline or acetylcholine essential to for nerve functions. Mecobalamin might serve as the source of the methyl group utilized for PC synthesis.

Mode of action of amiloride in toad urinary bladder. An electrophysiological study of the drug action on sodium permeability of the mucosal border.

The effect of amiloride on the sensitivity to Na of the mucosal border of toad urinary bladder was investigated by recording Na concentration-dependent transepithelial potential difference (Vt) and the intracellular potential. When mucosal Na concentration was normal, amiloride added to the mucosal solution at 10(-4) M markedly reduced the mucosal membrane potential (Vm) and altered the potential profile from a two-step type to a well type. Similar changes were observed when Na was totally eliminated from the mucosal medium. The serosal membrane potential was insensitive to amiloride and elimination of mucosal Na. In the absence of amiloride, the Vt could be described by the Goldman-Hodgkin-Katz equation in the range of mucosal Na concentration from 0 to 16 mM, and amiloride extended this concentration range. By using the Goldman-Hodgkin-Katz equation. Na permeability was calculated from the data of Vt's obtained in the allowed ranges of Na concentration and compared before and after the addition of amiloride. The results show that Na permeability decreases to 1/600 of control when the maximum dose of amiloride (10(-4) M) is applied. The relationship between Na permeability and amiloride concentration is well explained on the basis of assumptions that amiloride binds to the Na site of the mucosal border in one-to-one fashion and in a competitive manner with Na and that Na permeability reduces in proportion to increase in number of the sites bound with amiloride.