Changes in bacterial concentration in the liver correlate with that in the hepaticojejunostomy after bile duct reconstruction: implication in the pathogenesis of postoperative cholangitis

Postoperative cholangitis is a frequent and unpredictable complication of unknown etiology following bile duct reconstruction (BDR), particularly for biliary atresia. This study was undertaken to correlate the growth of bacteria in the hepaticojejunostomy with that in the liver after BDR. Quantitative bacterial culture was done on the specimens taken from the liver and from the hepaticojejunostomy at 1 week (group 1, n = 7), 1 month (group 2, n = 7), and 2 months (group 3, n = 7) following BDR with Roux-en-Y hepaticojejunostomy in piglets after 2 weeks of common bile duct ligation. The histological examination of the liver and the hepaticojejunostomy, as well as serial monitoring of hemogram and liver function tests, were performed to correlate the findings with the bacterial concentration of the liver and the hepaticojejunostomy following BDR. The bacterial concentration of the hepaticojejunostomy, expressed as log10 colony-forming units per gram (log10 CFU/g) of the hepaticojejunostomy, showed a progressive decrease from 8.38 ± 1.36 in group 1, 7.07 ± 2.54 in group 2, to 3.56 ± 1.31 in group 3 (p = 0.001). The log10 CFU/g of the liver also showed a progressive decrease from 5.02 ± 1.59 in group 1, 3.16 ± 1.56 in group 2, to 2.19 ± 1.09 in group 3 (p = 0.006). There was a significant positive correlation of the log10 CFU/g of the liver (n = 21) with that of the hepaticojejunostomy (n = 21) following BDR (r = 0.600, p = 0.004). Most of the infectious pathogens isolated from the liver were also isolated from the hepaticojejunostomy. The changes in hemoglobin, bilirubin, albumin, and ammonia significantly correlated with the changes of the bacterial concentration of the liver. The results of the study suggests that hepatic bacterial proliferation after BDR is significantly affected by microbial overgrowth in the bilioenteric anastomosis and is associated with deteriorated liver function and hemogram.

Expression of the male reproduction-related gene (Mar-Mrr) in the spermatic duct of the giant freshwater prawn, Macrobrachium rosenbergii

Phosphorylated sperm proteins are crucial for sperm maturation and capacitation as a priori to their fertilization with eggs. In the freshwater prawn, Macrobrachium rosenbergii, a male reproduction-related protein (Mar-Mrr) was known to be expressed only in the spermatic ducts as a protein with putative phosphorylation and may be involved in sperm capacitation in this species. We investigated further the temporal and spatial expression of the Mar-Mrr gene using RT-PCR and in situ hybridization and the characteristics and fate of the protein using immunblotting and immunocytochemistry. The Mar-Mrr gene was first expressed in 4-week-old post larvae and the protein was produced in epithelial cells lining the spermatic ducts, at the highest level in the proximal region and decreased in the middle and distal parts. The native protein had a MW of 17 kDa and a high degree of serine/threonine phosphorylation. It was transferred from the epithelial cells to become a major protein at the anterior region of the sperm. We suggest that it is involved in sperm capacitation and fertilization in this open thelycal species and this is being investigated.

Effect of Glucocorticoid pretreatment on oxidative liver injury and survival in jaundiced rats with Endotoxing Cholangitis

Introduction: Biliary tract infection is associated with high mortality. This study investigated the effect of glucocorticoid pretreatment on lipopolysaccharide (LPS)-induced cholangitis. Methods: Rats undergoing either sham operation or ligation of the extrahepatic bile duct (BDL) for 2 weeks were randomly assigned to receive intravenous injections of dexamethasone (DX) or normal saline (NS) prior to infusing LPS into the biliary tract. The plasma levels of tumor necrosis factor-α (TNFα), chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) as well as liver mRNA expression of MCP-1 and MIP-2 were determined. Infiltration of monocytes, Kupffer cells, and neutrophils in rat liver were studied with immunohistochemistry. Oxidative liver injury was measured by the malondialdehyde (MDA) content. Results: Dexamethasone pretreatment resulted in significantly decreased plasma levels of TNFα at 1 hour, MCP-1 and MIP-2 at 2 and 3 hours, and decreased liver MCP-1 mRNA expression at 3 hours following LPS infusion in BDL-DX rats than in BDL-NS rats. The number of inflammatory cells in the liver was significantly different between sham- and BDL-treated rats but was not affected by DX pretreatment. Pretreatment with DX resulted in significantly decreased liver MDA contents in the BDL-DX group than that in the BDL-NS group. Jaundiced rats pretreated with 5 mg DX prior to infusion of 1 g of LPS were 6.8 times more likely to survive than those that were not pretreated. Conclusions: Pretreatment of jaundiced, LPS-treated rats with a  supraphysiological dose of dexamethasone may rescue their lives by suppression of chemokine expression and alleviation of oxidative liver injury.

Pharmacokinetics of recombinant human endostatin in rats

The pharmacokinetics of recombinant human endostatin (rh-Endo) has not been established in the rat, although this species of animal is commonly used in the pharmacological studies of rh-Endo. This study aimed to investigate the pharmacokinetics, tissue distribution, and excretion of rh-Endo in rats. 125I-radiolabeled rh-Endo was administered to healthy rats by intravenous (i.v) bolus injection at 1.5, 4.5 and 13.5 mg/kg. The maximum plasma concentration (Cmax) and area under the plasma concentration versus time curve (AUC) of rh-Endo increased proportionally with the increase of the dosage. There were no significant differences in total body clearance (CL) and elimination half-life (t1/2beta) of rh-Endo among the three dosages used. A 93.5% and 2.2% of the radioactivity was recovered in the urine and feces, respectively, in bile-duct intact rats; whereas only 0.1% of the total radioactivity was excreted into the bile in bile-duct cannulated rats. rh-Endo was rapidly and widely distributed in the liver, kidneys, spleen and lungs. Furthermore, a significant allometric relationship between CL, but not volume of distribution (Vd) and t1/2beta of rh-Endo, and the body weight was observed across mouse, rat and monkey, with the predicted values in humans significantly lower than those observed in cancer patients. rh-Endo exhibited a linear pharmacokinetics in rats and it is mainly excreted through the urine.

Alteration of the pharmacokinetics of COL-3, a matrix metalloproteinase inhibitor, due to actue gastrointestinal tosicity of doxorubicin

Purpose Combination of COL-3, a matrix metalloproteinase inhibitor, and doxorubicin (DOX) might be a promising anticancer regimen. The present study was to examine the potential pharmacokinetic interactions and toxicity profile following their coadministration in rats.
Methods Normal rats were treated with single agent or different combinations with oral or intravenous COL-3 and DOX, and the bile-duct cannulated (BDC) rats received oral COL-3 plus DOX. In a separate disposition study, the effects of DOX on the biliary, urinary, and fecal excretion of COL-3 were examined. In addition, the effects of DOX on in vitro protein binding, metabolism, and transport of COL-3 across Caco-2 monolayers were investigated.
Results COL-3 did not affect the pharmacokinetics of DOX in rats. However, treatment with DOX significantly decreased the oral absorption, and prolonged the elimination, of COL-3 in the normal rats, but not in the BDC rats. DOX did not alter the biliary and urinary excretion of COL-3, but significantly decreased the fecal excretion of COL-3. DOX significantly enhanced the basolateral to apical flux of COL-3 across Caco-2 monolayers, but had no apparent effects on the protein binding and metabolism of COL-3. The combination of DOX with oral COL-3 did not significantly (p > 0.05) increase the acute diarrhea score and intestinal damage compared to rats receiving DOX alone.
Conclusions These results indicated that DOX altered the oral absorption and elimination of COL-3, largely resulting from gastrointestinal toxicity caused by biliary excretion of DOX. Further studies are required to explore the efficacy and optimized dosage regimen of this promising combination.

Biliary intervention augments chemotactic reaction and aggravates cholestatic liver injury in rats

Background
Intervention of the biliary system is frequently done in patients with obstructive jaundice and is associated with significant morbidity and mortality. The pathogenesis is unknown.
Materials and methods
A rat model of bile duct ligation (BDL) for 2 weeks was established in which biliary intervention was feasible by injection of normal saline through an indwelling catheter in the bile ducts. Plasma levels of C-C chemokine MCP-1 and C-X-C chemokine MIP-2 were measured by using ELISA. Blood monocytes, Kupffer cells, and neutrophils in the liver were characterized with antibodies to ED1, ED2, and myeloperoxidase (MPO). Lipid peroxidation was measured by malondialdehyde contents and apoptosis by TUNEL stain of the liver.
Results
Biliary intervention resulted in an increase of plasma MCP-1 and MIP-2 proteins by 1 h, which declined to normal level by 3 h in both sham and BDL rats. The levels in BDL rats were significantly higher than in sham at most points. There was a transient increase of ED1- and ED2-positive cells and MPO-staining cells in sham rat liver by 1 h after intervention. ED2-positive cells increased significantly by 1 h, while ED1- and MPO-positive cells decreased, yet insignificantly after intervention in BDL rats. The cell counts in BDL were constantly higher than in sham. Malondialdehyde increased precipitously in BDL by 3 h and was significantly higher than in sham throughout the study period. Parenchymal liver injury, manifested by elevated ALT, as well as apoptosis and necrosis of liver cells, was significantly increased in BDL rats, but not in sham rats.
Conclusion
Biliary intervention augments chemokine expression, precipitates lipid peroxidation, and aggravates liver injury in cholestatic rats.

pH-sensitive expression of calcium-sensing receptor (CaSR) in type-B intercalated cells of the cortical collecting ducts (CCD) in mouse kidney

Background The localization and role of the calcium-sensing receptor (CaSR) along the nephron including the collecting ducts is still open to debate. Methods Using the quantitative, highly sensitive in situ hybridization technique and a double-staining immunohistochemistry technique, we investigated the axial distribution and expression of CaSR along the nephron in mice (C57B/6J) treated for 6 days with acid or alkali diets. Results Under control condition, CaSR was specifically localized in the cortical and medullary thick ascending limb of Henle’s loop (CTAL and MTAL), macula densa (MD), distal convoluted tubule (DCT), and CCD (TALs, MD > DCT, CCD). Along the CCD, CaSR was co-localized with an anion exchanger type 4 (AE4), a marker of the basolateral membrane of type-B intercalated cell (IC-B) in mice. On the contrary, CaSR was not detected either in principal cells (PC) or in type-A intercalated cell (IC-A). CaSR expression levels in IC-B significantly (P < 0.005) decreased when mice were fed NH4Cl (acid) diets and increased when animals were given NaHCO3 (alkali) diets. As expected, cell heights of IC-A and IC-B significantly (P < 0.005) increased in the above experimental conditions. Surprisingly, single infusion (ip) of neomycin, an agonist of CaSR, significantly (P < 0.005) increased urinary Ca excretion without further increasing the hourly urine volume and significantly (P < 0.05) decreased urine pH. Conclusion CaSR, cloned from rat kidney, was localized in the basolateral membrane of IC-B and was more expressed during alkali-loading. Its alkali-sensitive expression may promote urinary alkali secretion for body acid–base balance.