Relationship between real-time monitoring of the graft motility and mucosal histology in swine intestinal transplantation.

We studied the correlation between the motility and the mucosal histology of the small bowel seeking to detect rejection in an early stage by real-time monitoring using a swine model. Intestinal transplantation (ITx) was performed orthotopically using FK506 immunosuppression. The distal about 20 cm segment of the allograft was exteriorized as a Thiry-Vella stoma for biopsies. Strain gauge (SG) force transducers were attached to the graft for real-time monitoring of graft motility. Pigs without ITx were used as controls (group 1). Rejection was classified into four groups by histologic findings: nonrejection (group 2), mild rejection (group 3), moderate rejection (group 4), and severe rejection (group 5). Migrating motor complex (MMC) phase III was analyzed for the following parameters: duration, amplitude, interval, motility index, velocity, and frequency of propagation. In group 2, all parameters were almost the same as those for group 1. In contrast, groups 4 and 5 showed most parameters significantly lower than those in group 1. In group 3, the contractility of the MMC was not significantly altered, but the frequency of the propagation was decreased significantly. In conclusion, graft motility detected by a real-time SG method correlated with the grade of mucosal histology. This method is useful to detect rejection at an early stage by examining the frequency of MMC propagation.

New melanogenesis and photobiological processes in activation and proliferation of precursor melanocytes after UV-exposure: ultrastructural differentiation of precursor melanocytes from Langerhans cells.

Photobiological processes involving new melanogenesis after exposure to ultraviolet (UV) light were experimentally studied in C57 black adult mice by histochemistry, cytochemistry, and autoradiography. The trunk and the plantar region of the foot, where no functioning melanocytes were present before exposure, were exposed to UV-A for 14 consecutive days. Both regions revealed a basically similar pattern for new melanogenesis which involved an activation of precursor melanocytes. Essentially all of "indeterminate" cells appeared to be precursor melanocytes, the fine structure of which could be differentiated even from poorly developed Langerhans cells. New melanogenesis was manifested by 4 stages of cellular and subcellular reactions of these cells as indicated by histochemistry of dihydroxyphenylalanine (dopa) and autoradiography of thymidine incorporation: (a) an initial lag in the activation of precursor melanocytes with development of Golgi cisternae and rough endoplasmic reticulum followed by formation of unmelanized melanosomes (day 0 to 2); (b) synthesis of active tyrosinase accumulated in Golgi cisternae and vesicles with subsequent formation of melanized melanosomes in these cells (day 3 to 5); (c) mitotic proliferation of many of these activated cells, followed by an exponential increase of new melanocytes (day 6 to 7); and (d) melanosome transfer with differentiation of 10 nm filaments and arborization of dendrites, but without any significant change in the melanocyte population (day 8 to 14). The melanosome transfer was, however, not obvious until after 7 days of exposure. The size of newly synthesized melanosomes was similar to that of tail skin where native melanocytes were present before exposure.

The role of Kupffer cell oxidant production in early ethanol-induced liver disease.

Considerable evidence for a role of Kupffer cells in alcoholic liver disease has accumulated and they have recently been shown to be a predominant source of free radicals. Several approaches including pharmacological agents, knockout mice, and viral gene transfer have been used to fill critical gaps in understanding key mechanisms by which Kupffer cell activation, oxidant formation, and cytokine production lead to liver damage and subsequent pathogenesis. This review highlights new data in support of the hypothesis that Kupffer cells play a pivotal role in hepatotoxicity due to ethanol by producing oxidants via NADPH oxidase.

The protective role of Kupffer cells in humoral injury of xenoperfused rat livers.

BACKGROUND: The role of Kupffer cells in a hepatic xenograft rejection is still unclear. We investigated the effect of blocking Kupffer cells on xenogeneic humoral injury using rat livers as the xenoperfusion models. METHODS: Rat livers were perfused with fresh human blood after pretreatment either with normal saline (group 1; n = 8) or with gadolinium chloride (GdCl3) solution (group 2; n = 8). Tissue injury was evaluated by alanine aminotransferase release and histological examination. Tumor necrosis factor-alpha (TNF-alpha) production from rat livers was measured by enzyme-linked immunosorbent assay and also examined by immunohistochemistry. In addition, Kupffer cells were isolated after pretreatment either with normal saline or with GdCl3 solution and incubated with human serum. Localization of human C3 and IgM was examined by immunofluorescence. RESULTS: Alanine aminotransferase release in group 2 was significantly higher than in group 1 (P = 0.015). Histological examination revealed more severe tissue injury in group 2. The mean TNF-alpha level was not significantly different between the two groups. In immunohistochemistry, TNF-alpha was positive primarily on vascular endothelial cells in both groups. Immunofluorescence of saline-treated Kupffer cells showed an uptake of human C3 in the cytoplasm, whereas no uptake was observed in GdCl3-treated cells. The uptake of human IgM did not differ between the two groups. CONCLUSIONS: These results suggest that Kupffer cells have a protective role in preventing xenogeneic humoral injury. Their ability to absorb xenogeneic complements may contribute to this protective mechanism.

Humoral injury in porcine livers perfused with human whole blood.

BACKGROUND: We investigated the influence of humoral injury during xenoperfusion of porcine livers by human blood. METHODS: The porcine livers were perfused under physiological conditions for 9 hr. The perfusates consisted of porcine whole blood in group 1, human whole blood in group 2, and human whole blood with soluble complement receptor type 1 (300 microg/ml) in group 3. RESULTS: Liver enzyme release and serum hemoglobin in group 2 increased significantly after 3 hr of xenoperfusion, compared with those in group 1 and group 3 (P<0.05). Severe histological damage with minimal cellular infiltration was observed in group 2 after 6 hr of xenoperfusion, but was present only at trace levels in group 1 and group 3. In group 2, von Willebrand factor, a possible target of natural antibodies, was induced on sinusoidal endothelial cells after 3 hr of xenoperfusion, correlating with diffuse deposition of human IgM and membrane attack complex. In group 3, von Willebrand factor, human IgM, and membrane attack complex staining in the intralobular region were present at trace levels. In group 3, the indocyanine green removal capacity, representing hepatocyte function, was significantly higher than in group 2 (P<0.05). CONCLUSIONS: Based on these results, we suggest that humoral injury is a major cause of liver damage during liver xenoperfusion. The pattern of humoral injury in xenoperfused livers may be attributed to anatomical features of the liver and unique responses of sinusoidal endothelial cells to xenoperfusion.

Evaluation of ammonia and lidocaine clearance, and galactose elimination capacity of xenoperfused pig livers using a pharmacokinetic analysis.

BACKGROUND: We introduced the pharmacokinetic method into the functional evaluation of xenogeneic extracorporeal liver perfusion as an artificial liver assist device, and examined the influence of xenogeneic humoral injury on the metabolic function of xenoperfused pig livers. METHODS: Isolated pig livers were perfused with fresh porcine blood (group 1; n=5) or fresh human blood (group 2; n=5) for 9 hr. Clearance (CL) of ammonia and lidocaine, and galactose elimination capacity (Vmax) were determined at three points during the perfusion using a one-compartment pharmacokinetic model. RESULTS: Concentrations of ammonia and lidocaine decreased exponentially and those of galactose decreased linearly after a bolus injection in both groups. A one-compartment model provided satisfactory curve fittings for these test substances. No decreases of ammonia CL, lidocaine CL, or galactose Vmax were observed until 9 hr in either group. No differences were observed between the two groups with respect to these metabolic functions. In group 1, only slight interlobular edema was observed at 9 hr. In group 2, membrane attack complex was diffusely deposited at 3 hr and severe interlobular damage was histologically observed at 9 hr, although hepatocellular damage was minimal even at 9 hr. Alpha glutathione S-transferase and mitochondrial aspartate aminotransferase were comparable between the two groups. CONCLUSIONS: Pharmacokinetic analysis allowed the evaluation of ammonia CL, lidocaine CL, and galactose Vmax of the perfused pig livers. Despite xenogeneic humoral injuries, the xenoperfused livers maintained these metabolic functions at the same levels as the alloperfused livers for 9 hr.

Attenuating effect of a semipurified alcohol extract of soy protein on glomerular injury in spontaneous hypercholesterolemic male Imai rats.

We investigated whether soy protein's alcohol-extractable components (SPEs; mainly consisting of isoflavones) have the ability to attenuate glomerular injury in male Imai rats of a spontaneous focal segmental glomerulosclerosis model. Male Imai rats were fed a casein-based diet with and without SPEs. Group 1 (Cont) was fed a standard diet without additional SPEs, and groups 2 (SPE-1) and 3 (SPE-2) were fed a standard diet supplemented with a semipurified alcohol extract of soy protein, 0.05 and 0.10 g/100 g of diet, respectively. Body weight, urinary protein level, serum constituents, and systolic blood pressure were evaluated every 4 weeks from 12 through 28 weeks of age. At 28 weeks of age, rats were studied morphologically. Growth rates were not different among the three groups throughout the experiment. SPE-supplemented diets resulted in less proteinuria and less hyperlipidemia. The decline in renal function shown by blood urea nitrogen and creatinine clearance was less marked in the animals fed the SPE-supplemented diets. Each SPE-supplemented diet equally induced less glomerular hypertrophy and less renal histological damage compared with nonsupplemented diets. The present study showed a beneficial effect of a semipurified alcohol extract of soy protein on glomerular disease.

Organic anion transport study in mutant rats with autosomal recessive conjugated hyperbilirubinemia.

The EHBR is a mutant rat strain with congenital conjugated hyperbilirubinemia bred from a Sprague-Dawley rat. Transport of conjugated bilirubin, indocyanine green, and tetrabromosulfophtalein from liver to bile is severely impaired in these rats. Serum bilirubin amounts to 6.0 +/- 0.05 mg/dl (n = 4) in adult rats, with 97% conjugates. The bile flow is reduced to about 65% of the control group, whereas total bile acid in 10-min bile samples is similar. Liver histology of 10 week-old rats revealed neither intracellular pigmentation nor architectural abnormalities.

The effect of Sudan III on drug metabolizing enzymes.

We have examined the induction of drug metabolizing enzymes in rat liver microsomes by azo dye, 1-(p-phenylazophenylazo)-2-naphthol (Sudan III). Marked increases were observed in the levels of cytochrome P-448 as well as in p-nitroanisole O-demethylase (p-NAD), amaranth (AR) and neoprontosil reductases (NPR) and 7-ethoxycoumarin O-deethylase (ECD) activities. On the other hand, aminopyrene N-demethylase activity was not significantly increased. Further, induced ECD activity was inhibited 90% by a specific antibody against cytochrome P-448 while the inhibition observed with an antibody against cytochrome P-450 was less than 25%. Simultaneous administration of Sudan III and 3-methylcholanthene (3-MC) induced cytochrome P-448 up to a level brought about by either Sudan III or 3-MC treatment alone. In contrast, Sudan III did not induce cytochrome P-448 in the 3-MC insensitive DBA/2 mouse. Solubilized microsomes from Sudan III-treated rats showed an identical sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) pattern with those from 3-MC-treated animals. It is concluded that the cytochrome P-448 induced in liver by Sudan III is very similar to that induced by 3-MC. Sudan III also induced UDP-glucuronyltransferase activity towards 1-naphthol and estradiol. It did not induce NADPH-cytochrome c reductase, nor any of the enzymes which constitute the microsomal electron transport chain except for cytochrome P-448.

Effects of cilostazol on heart rate and its variation in patients with atrial fibrillation associated with bradycardia.

BACKGROUND: Heart-rate (HR) variability is an important predictor of mortality in patients with heart disease. We examined the effects of cilostazol, a quinolinone derivative, on HR and HR variability in patients with chronic atrial fibrillation associated with bradycardia episodes. PATIENTS AND METHODS: Thirteen patients with chronic atrial fibrillation associated with bradycardia episodes (minimal HR <40/min and/or pauses, ie, episodes with an RR interval > 2.5 sec) received cilostazol (100 or 200 mg/day) orally for at least 2 months and 24-hour Holter electrocardiography was performed before and after the start of cilostazol administration. RESULTS: Minimal HR was significantly increased, by an average of 14 beats/min (bpm), at 3.3 +/- 0.8 weeks (mean +/- SD) after the start of cilostazol treatment. The number of pauses was significantly decreased. As a consequence, mean HR was increased by an average of 18 bpm. Maximal HR was also increased by an average of 19 bpm. The circadian variation of the HR, determined by cosine fitting, was not changed by cilostazol treatment. The time-domain HR variabilities, ie, the SD of the mean RR interval and the SD of the 5-minute mean RR intervals, were also unchanged. New York Heart Association functional class was significantly improved and the plasma atrial natriuretic polypeptide level was significantly decreased after the initiation of cilostazol treatment. CONCLUSION: Cilostazol improves the slow HR episodes associated with chronic atrial fibrillation and maintains the HR circadian variation and time-domain variability, indicating that cilostazol has therapeutic utility for the treatment of the slow HR associated with chronic atrial fibrillation.

Metabolism of sulphobromophthalein I: positional isomers of sulphobromophthalein monoglutathione conjugate.

Three positional isomers of sulphobromophthalein glutathione monoconjugate (BSP-mGSH) were detected using a paired-ion HPLC method that employs triethylamine phosphate (TEA-H3PO4) as a pairing agent. To confirm that these compounds were glutathione (GSH) conjugates, sulphobromophthalein (BSP) was incubated with a four-fold volume of GSH under alkaline ammonium hydroxide. At least 6 metabolites (3 di-GSH conjugates and 3 isomers of mono-GSH conjugates) were produced under these conditions. The three mono-GSH conjugates were each purified and identified as compounds with a molecular weight of 1,020 according to FAB mass spectrometry results. Positional isomers of BSP-GSH were provisionally distinguished via the addition of the symbols alpha, beta and delta to the end of each abbreviation, to reflect the amount of isomers present. Thus, the isomer present in the largest quantity was termed BSP-mGSH(alpha), the second most abundant isomer was termed BSP-mGSH(beta) and the third was termed BSP-mGSH(delta). Interestingly, a species difference was recognized in that rat cytosol GSH S-transferase (GST) primarily produced BSP-mGSH(alpha), whereas guinea-pig cytosol generated BSP-mGSH(delta), BSP-mGSH(alpha) and BSP-mGSH(beta) equally and rabbit cytosol mainly produced BSP-mGSH(beta).

Immunosuppressive effect of nucleoside-nucleotide-free diet in rat allogeneic small intestinal transplantation.

BACKGROUND: We evaluated the effects of nucleosides (NS) and nucleotides (NT) on the rejection of rat allogeneic small intestinal transplants. METHODS: A 2-cm segment of jejunum from fetal Fischer rats (RT-1(lvl)) was transplanted at day 19 of gestation into the abdominal wall of 7-week-old Lewis rats (RT-1(l)) by a nonvascular technique. Two weeks before transplantation, recipient rats were separated into an NS-NT-free group and an NS-NT-supplemented group. At 2 days after transplantation, histologic study of the grafts was performed with hematoxylin-eosin staining and interleukin-2 (IL-2) production estimated in recipient blood using an ELISA method. The morphologic findings were graded in a blind fashion on a scale of 0 to 4, with 0 indicating an intact intestinal structure. RESULTS: Mean plasma IL-2 levels of the NS-NT-free group were significantly lower than those of the NS-NT-supplemented group. The mean rejection score of the NS-NT-free group was also significantly lower than that of the NS-NT-supplemented group. CONCLUSIONS: Administration of an NS-NT-free diet reduces acute rejection in rat small intestinal transplantations.

Real-time monitoring for detecting rejection using strain gauge force transducers in porcine small bowel transplantation.

PURPOSE: The clinical results of small bowel transplantation (SBT) have not been satisfactory mainly because of the immunological barrier. It is important to detect the presence of and to perform adequate treatment of rejection as early as possible to improve graft survival. Therefore, we have established a pig model to monitor graft motility as a means to detect rejection in real time. METHODS: Orthotropic SBT was performed in 25 pigs using FK-506 (0.05 to 0.1 mg/kg/d) immunosuppression. The interdigestive motor patterns were evaluated using strain gauge force transducers (SG). Seven pigs without SBT were treated as controls (C). Animals that displayed migrating motor complex (MMC) activity as evidenced by duration, amplitude, and interval in the graft were alive more than 10 days with adequate oral feeding: the functional graft (FG) group. In contrast the rejection (R) group did not show these activities on data recorded within 10 days before death due to rejection. RESULTS: The FG group showed MMC propagated throughout the graft with all parameters almost the same as the control group except for the duration. In contrast, all parameters in the group R were significantly lower than those in group FG, suggesting that group R motility was obviously impaired by rejection. CONCLUSIONS: The SG method may afford real-time monitoring of transplanted bowel motility that could be useful to detect rejection after SBT.

Exponential hyperbolic sine function fitting of heart rate response to constant load exercise.

We attempted to fit heart rate (HR) changes induced by constant exercise loads of different intensities to an exponential hyperbolic sine curve by the least-squares method, and we compared the results with the fitting of the changes to exponential curves. Seven healthy male volunteers performed three different intensities of constant-load exercise on a bicycle ergometer. The exponential hyperbolic sine function adequately fitted the HR responses induced by all three different intensities of loads: low (30 W: correlation coefficient, r = 0.68 +/- 0.13, mean +/- SD), moderate (75 W: r = 0.93 +/- 0.07) and high (125 W: r = 0.97 +/- 0.02). The first-order exponential curve fitted only the moderate load response. Although the second-order exponential equation fitted the HR response for both the moderate and high loads, the equation did not fit the low-load response (r = 0.43 +/- 0.26). In low-load exercise, the sum of the power of the residuals for the exponential hyperbolic sine curve fitting was significantly smaller than that for the first- or second-order exponential curve fitting. In conclusion, the exponential hyperbolic sine function is useful for quantitative analyses of the HR response to exercise loads of various intensities.

In vitro evaluation of metabolic functions of a bioartificial liver.

The purpose of this study is to develop a bioartificial liver (BAL) with such a simple structure that it can be prepared within several hours and through which whole blood can be perfused as in current hemodialyzers. Hepatocytes were isolated from 37 pigs; each liver weighed 300 to 400 g. The average yield of hepatocytes was 2.4 +/- 0.6 x 10(10) cells per liver, with a cell viability of 89.6 +/- 3.9%. To prepare a BAL device, a cartridge, composed of hollow fibers made of cellulose diacetate was used. Nominal cut-off molecular weight of the hollow fibers was 68 kDa, and the internal diameter was 195 microm. One hundred milliliters of hepatocyte suspension, containing 1 x 10(10) cells, was inoculated into the inner space of the hollow fibers, and both the inlet and outlet of the hollow fiber cartridge were closed. It took only 3 hrs from administration of the pig's anesthesia to the start of an in vitro evaluation of the prepared BAL device. To evaluate the functions of this BAL quantitatively, using a pharmacokinetic method, a mixture of fresh human blood and Dulbecco's modified Eagle medium was circulated in the shell space of the hollow fibers at 200 ml/min. Chemicals (lidocaine, ammonia, and galactose) were then loaded into the perfusion medium. The average intrinsic clearance of the BAL device was found to be 46 ml/min for lidocaine and 8.8 ml/min for ammonia. The galactose elimination capacity of the BAL device was 1.34 mg/min. The metabolic function of the BAL device decreased by 81%, 49%, and 64% of the initial function for lidocaine, ammonia, and galactose, respectively, after 10 days of in vitro circulation.

[Detection of cytomegalovirus (CMV) antigen for rapid diagnosis and monitoring of CMV diseases in AIDS].

Ten to forty percent of the patients with acquired immunodeficiency syndrome (AIDS) develop sight- or life-threatening cytomegalovirus (CMV) infections. In some patients with AIDS, CMV is detected in the bronchoalveolar lavage fluid (BALF), urine, and other specimens, even when there are no symptoms of CMV disease. An indicator of active CMV infection is needed to facilitate the diagnosis of CMV disease in patients with AIDS or HIV infection and the evaluation of the efficacy of subsequent treatment. The present study was conducted during the period from 1993 to 1994. The subjects consisted of three patients with AIDS and a confirmed diagnosis of CMV disease (one case of retinitis, one case of gastrointestinal disease and one case of pneumonia), and five HIV-positive patients in whom CMV associated disease was ruled out. Those patients were monitored occasionally for the following parameters of active CMV infection and disease: expression of CMV antigen in the nucleus of polymorphonuclear leukocyte (CMV antigenemia), as it was determined with a monoclonal antibody against a lower matrix protein (p65); infectious CMV detected by shell vial method; CMV DNA detected by PCR; anti-CMV antibody titer; and histological findings. CMV p65 antigen was detected in the leukocytes of both the peripheral blood and BALF during the early phase of CMV disease in three out of three cases of the CMV disease group, and this antigen became negative in two out of two cases who responded to the therapy. All the five patients in the CMV-related-disease-negative group were negative for CMV antigenemia.(ABSTRACT TRUNCATED AT 250 WORDS)

[A clinicopathological study of 218 patients with bladder carcinoma treated by radical cystectomy].

Between 1986 and 1995, 218 patients with primary carcinoma of the bladder were treated with radical cystectomy at our hospitals. Clinicopathological specimens and the relative value of prognostic factors were analyzed with both the univariate and multivariate method. Univariate analysis indicated that age, sex, pathological tumor stage, grade, vessel involvement, tumor infiltrating type and positive nodes were predictive of poor cancer-specific survival. Multivariate analysis demonstrated that female patients were predictive of poor cancer-specific survival. As for the risk ratio, the prognostic factors in lymphatics involvement and tumor infiltrating type were 2.77 and 2.47, respectively.

Effects of orthognathic surgery on pharyngeal airway and respiratory function during sleep in patients with mandibular prognathism.

The aim of this study was to determine changes in overnight respiratory function and craniofacial and pharyngeal airway morphology following orthognathic surgery. The subjects were 40 patients in whom mandibular prognathism was corrected by orthognathic surgery: a one-jaw operation in 22 patients and a two-jaw operation in 18 patients. Morphological changes were studied using cone beam computed tomography immediately before surgery and at more than 6 months after surgery, and the apnoea-hypopnoea index (AHI) was measured with a portable polysomnography system. Pharyngeal airway volume was decreased significantly after surgery, especially in the one-jaw operation group. AHI was not changed significantly after surgery in either group, although AHI in one patient in the one-jaw operation group was increased to 19 events/h. There was no significant change in pharyngeal airway morphology in that patient, but he was obesity class 1 and was 54 years old. In conclusion, some patients who are obese, have a large amount of mandibular setback, and/or are of relatively advanced age may develop sleep-disordered breathing after mandibular setback; a two-jaw operation should therefore be considered in skeletal class III patients who have such risks because it decreases the amount of pharyngeal airway space reduction caused by mandibular setback surgery.