Suppression of natural killer cell activity in biliary obstruction.

Prior studies regarding immune function in bile-duct ligated rats have illustrated blunted immune function. The present study measures natural killer (NK) cell activity. Rats underwent bile duct ligation (BDL) or sham celiotomy (SC) and were sacrificed at 1, 2, and 3 weeks after surgery. Ficoll-Hypaque density centrifugation was used to obtain a purified preparation of splenocytes. NK cell activity was determined by incubating varying concentrations of splenocytes with chromium-labelled YAC-1 tumor cells for 4 hours. Chromium release was measured by a gamma counter and expressed as per cent activity (compared with 100 per cent activity obtained by complete lysis with detergent). The experiments were repeated after preincubation in tissue culture flasks to remove an adherent cell population. NK cell activity was decreased at all spleen cell:target cell ratios studies at 1 week after BDL. At 2 weeks after BDL, NK cell activity was decreased at all but the lowest two concentrations; and 3 weeks after BDL, NK cell activity was decreased only at the highest concentrations used. Separation of an adherent cell fraction restored NK cell activity. This suppression in NK cell activity one week after BDL may account, in part, for the poor response of BDL animals to bacterial and immune challenge. Restoration of activity after removal of an adherent cell fraction suggests that macrophages may be at least partly responsible for this inhibition.

Effect of aminoguanidine on in vivo expression of cytokines and inducible nitric oxide synthase in the lungs of endotoxemic rats.

Endotoxemia results in the release of multiple mediators such as tumor necrosis factor alpha (TNF-infinity), interleukin-1 beta (IL-1 beta), and nitric oxide (NO), which is thought to be responsible for the hypotension of septic shock. Although there are many reports on the presence of these mediators in serum, in vivo expression of TNF-infinity, IL-1 beta and inducible nitric oxide synthase (iNOS) at the tissue level has not been studied extensively. We investigated in vivo expression of these cytokines and iNOS in the lungs of rats that were injected with saline, endotoxin or endotoxin plus aminoguanidine (AG), an inhibitor of iNOS. Expression of TNF-infinity, IL-1 beta and iNOS was absent in control (saline treated) but was increased in endotoxin treated animals. In animals treated with endotoxin plus AG (400 mg/kg), expression of iNOS was markedly inhibited whereas there was no effect on expression of TNF-infinity and IL-1 beta. The inhibitory effect of AG was probably dose dependent because a lower concentration of AG (50 mg/kg) showed no change in the expression of iNOS.

Light and electron microscopic observations of epithelial shedding in stored canine small intestine.

Simple cold storage of canine small intestine is accompanied by ischemic damage to the intestinal mucosa. Progression of damage observed during cold storage is unique and has not been observed with other organs. The mucosal damage begins within 15 min after the onset of the storage, with progressive involvement of the gut as the storage period lengthens. Cytoplasmic blebs develop from the base of the epithelial cells and detach the epithelium from the basal lamina. While the process begins uniformly along the length of the villus, separation of the epithelium occurs first at the villus tip. The epithelium, which is shed into the intestinal lumen, is otherwise undamaged. Blebbing occurs in enteroendocrine and goblet cells and is not restricted to enterocytes. Early blebs occur in proximity to mucosal mast cells and subepithelial nerves. Tissue damage in cold is possibly related to enzymes that are still active at storage temperatures.

Does level of ligation influence results in a murine biliary obstruction model?

BACKGROUND: Despite advances in perioperative management, patients with extrahepatic biliary obstruction still experience a high rate of complications and death after surgery. The rat is commonly used as an experimental animal for research in obstructive jaundice. Ligation of the rat bile duct high in the liver hilum is assumed to produce a more severe model of biliary obstruction than low ligation. The differences are attributed to the ability of the rat bile duct to dilate. Differences in level of ligation may, thus, explain some discrepancies between studies. MATERIALS AND METHODS: To test this hypothesis, female Lewis rats underwent high ligation (HL), low ligation (LL), and sham celiotomy. Colloidal carbon clearance, bilirubin, total serum bile acids, and hematocrit were measured 12 days later. Liver and spleen weight, presence or absence of ascites, infection, and adequacy of ligation were noted and the liver was processed for routine histology and electron microscopy. RESULTS: Although bilirubin levels were higher after HL than after LL, liver and spleen weight, total serum bile salts, and phagocytic constants K and alpha were not different between these two groups. Gross, histologic, and ultrastructural appearance did not differ between HL and LL groups. CONCLUSION: High ligation causes greater hyperbilirubinemia than low ligation, but does not alter other parameters including phagocytic constants. The present study does not confirm the hypothesis that HL creates a more severe model than LL; therefore, it is unlikely that differences in level of ligation explain variability in results between studies.

The pathophysiology of biliary obstruction and its effect on phagocytic and immune function.

These studies have direct clinical relevance to the multisystem deficits seen in mechanical biliary obstruction (Fig. 3). Defects in two crucial elements of effective phagocytosis (chemotaxis and intracellular killing) have been demonstrated in obstructive jaundice. At the same time, complete diversion of bile (containing bile salts and s-IgA) from the gut lumen causes changes in the endogenous bacterial flora, loss of mucosal integrity, and decreased endotoxin inactivation, resulting in portal bacteremia, endotoxemia, and increased translocation to mesenteric lymph nodes. This increased load comes at a time when the liver is metabolically impaired and RES function is abnormal. Decreased hepatic clearance of intrabiliary bacteria may contribute to the development of cholangitis (by both ascending and hematogenous routes). Inadequate RES control of portal bacteremia results in "spillover" with subsequent systemic bacteremia and localization of organisms in the lungs where they may contribute to pulmonary dysfunction or pneumonia. Although reversal of jaundice is readily accomplished by either external or internal biliary drainage, chronic biliary obstruction results in functional alterations in the liver which are reversed, generally incompletely, only after weeks or months of decompression. External biliary decompression fails to restore the enterohepatic circulation, preventing bile salts, s-IgA, and other substances from entering the lumen of the gut. It is not as effective as internal biliary drainage in reversing RES dysfunction or restoring immune parameters. Even with internal drainage, restoration of normal function in these systems takes weeks or months. Muramyl dipeptide analogues show some promise. A possible unifying mechanism may provide the clues to further experiments which will suggest better ways of reducing the morbidity and mortality in these patients. All macrophages share common functions which include not only phagocytosis but also antigen processing and the production of cytokines. The immune dysfunction noted in obstructive jaundice may be due to inadequate or inappropriate antigen processing or cytokine production by macrophages or to abnormal hepatocyte-Kupffer cell interactions. Kupffer cells are the largest pool of macrophages. Most numerous in periportal areas, Kupffer cells process significant quantities of enteric-derived antigens and Kupffer cell blockade results in an exaggerated response to these antigens. Kupffer cells also act as important scavengers of endotoxin, which stimulates the release of TNF and IL-6.(ABSTRACT TRUNCATED AT 400 WORDS)

Splenocytes from bile duct ligated rats do not elicit a normal immune response in the intact host.

Previous studies have shown impaired immune function in biliary obstruction, and our earlier investigations have demonstrated impaired response to alloantigens in the jaundiced rat host. The present study uses the graft versus host (GVH) popliteal lymph node assay to assess the ability of lymphocytes from bile duct ligated animals to elicit an immune response in normal rats. Female Lewis rats underwent bile duct ligation and transection (BDL) or sham celiotomy. A third group of rats served as normal controls. The animals were killed at intervals from 1 to 6 weeks after surgery, and spleen cell preparations were made. Splenocytes (5 x 10(6) from BDL, sham celiotomy, or normal control rats were injected into the hind footpads of LBNF1 hybrid rats. The contralateral hind footpads were injected with media as controls. The popliteal lymph nodes were removed and weighted 7 days after injection. The BDL rats were clinically jaundiced. GVH response was normal at 1 week and decreased at 2 weeks, remaining depressed through 6 weeks. Contralateral control lymph node weights were similar in all groups. Obstructive jaundice not only impairs host immune defense, but also significantly decreases splenocyte GVH capability.

Serum and cellular factors in murine obstructive jaundice.

BACKGROUND: The present experiments were performed to determine whether serum or cellular factors are responsible for the immune suppression observed in biliary obstruction. METHODS: Male Lewis strain rats underwent bile duct ligation and division (BDL) or sham celiotomy (SC). Spleen cells (splenocytes) and lymph node cells (lymphocytes) were isolated 3 to 14 days later. Levels of direct and total serum bilirubin and total conjugated bile acids and response of BDL and SC splenocytes and lymphocytes to concanavalin A (Con A) and phytohemagglutinin were measured. Splenocytes from normal Lewis rats were then added to medium containing 20 microliters, 40 microliters, and 100 microliters BDL or SC serum and incubated with Con A. BDL or SC splenocytes were injected into normal Lewis rats, and splenocytes from those rats were studied 24 hours later (adoptive transfer). Splenocytes from BDL and SC rats were separated into nylon wool adherent and nonadherent fractions and incubated with Con A and phytohemagglutinin. RESULTS: The mitogenic response to Con A and phytohemagglutinin was depressed in BDL splenocytes but normal in BDL lymphocytes. BDL serum suppressed the proliferative response of normal splenocytes, and adoptive transfer of cells produced immunosuppression. Removal of a nylon wool adherent fraction of cells restored the lymphoproliferative response in BDL splenocytes. CONCLUSIONS: The blastogenic response to phytohemagglutinin and Con A was decreased in BDL splenocytes. This effect was transferrable by both serum and cells. Removal of a subpopulation of splenocytes corrected the defect.

Impaired bacterial killing in early obstructive jaundice.

Sprague-Dawley rats were challenged with intraperitoneal injection of 10(7) Streptococcus pneumoniae 10 days after common duct ligation (BDL) or sham celiotomy (SC). Quantitative bacterial cultures were performed on liver, spleen, lung, kidney, and heart blood samples obtained 4 hours after injection. All 13 (100%) BDL animals had positive heart blood cultures, but only 6 of 12 (50%) SC animals remained bacteremic (p < 0.05). Significantly more viable organisms were recovered from lung, liver, spleen, and kidney of BDL animals compared with SC controls. BDL impaired the host's ability to kill this encapsulated gram-positive organism. Viable bacteria remained in all organs studied, which was associated with continuing bacteremia.

Impaired response to alloantigens in murine biliary obstruction.

Male Lewis strain rats underwent bile duct ligation and division (BDL), selective hepatic duct ligation (SHL), simple ligation and recanalization (RCN), or sham celiotomy (SC). Unoperated rats served as normal controls (NC). At intervals of 1, 2, and 3 weeks postoperatively, the popliteal lymph node assay was used to study host versus graft (HVG) response. LBN-F1 splenocytes (5 x 10(6)) were injected into the hind foot pads, and the contralateral foot pad was injected with medium as a control. The popliteal lymph nodes were removed and weighed 7 days later. In the BDL group, HVG response was significantly impaired at 1 (BDL, 12.9 +/- 5.1 mg; SC, 21.6 +/- 2.6; NC, 22.4 +/- 9.4; P < 0.005, BDL vs SC or NC), 2 (BDL, 12.6 +/- 5.6; SC, 19.1 +/- 3.0; NC, 15.8 +/- 5.8; P < 0.001, BDL vs SC), and 3 weeks (BDL, 8.9 +/- 3.9; SC, 21.7 +/- 6.3; NC, 16.7 +/- 3.8; P < 0.001, BDL vs SC or NC). SHL did not cause hyperbilirubinemia or impair the HVG response at 2 weeks (SHL, 17.2 +/- 4.5; NC, 16.7 +/- 7.4). The serum bilirubin was normal 2 and 3 weeks after RCN, and the HVG response was normal in both groups; however, the HVG response was somewhat lower at 2 weeks (RCN, 12.1 +/- 2.1) than at 3 weeks (RCN, 18.2 +/- 4.4; P < 0.01, RCN 2 weeks vs RCN 3 weeks). BDL causes significant impairment in the murine response to alloantigens as measured by the popliteal lymph node assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Peritoneal neutrophil chemotaxis is impaired in biliary obstruction.

Previous studies have shown impaired reticuloendothelial function in biliary obstruction. The chemotactic response of polymorphonuclear leukocytes from peripheral blood and peritoneal fluid of jaundiced rats (Group 1) was compared to that of sham operated controls (Group 2) and normal rats (Group 3). Male Sprague-Dawley rats underwent bile duct ligation or sham celiotomy. Studies were performed from 1 to 3 weeks after surgery. Mean serum bilirubin was 6.8 mg percent in Group 1 and normal in Groups 2 and 3. Peritoneal neutrophils were induced by intraperitoneal injection of 10 ml of 10 percent peptone broth 16 hours before the study, harvested from peritoneal fluid and peripheral blood, and isolated on Ficoll-Hypaque. F-met-leu-phe (FMLP) chemoattractant (10(-7) M) was used to induce migration of neutrophils across 3 mu filters. The filters were removed, mounted on slides, stained, and counts averaged for five oil immersion fields for each of three wells. Data were expressed as number of neutrophils per oil immersion field. Peritoneal neutrophil chemotaxis was significantly decreased in Group 1 (10.3 +/- 8.1) compared with Groups 2 (17.0 +/- 7.3) and 3 (20.2 +/- 6.4). A similar trend was noted in polymorphonuclear leukocytes from peripheral blood (Group 1: 13.1 +/- 7.8, Group 2: 18.2 +/- 6.7, Group 3: 17.4 +/- 5.9; P = 0.1). This impairment in neutrophil chemotaxis may contribute to the high rate of septic complications observed in the jaundiced host.

Bacterial phagocytosis in obstructive jaundice. A microbiologic and electron microscopic analysis.

Reticuloendothelial system dysfunction has been suggested as an explanation for the increased susceptibility to infection in patients with obstructive jaundice. In the present study, the response of cholestatic rats to a bacterial challenge was investigated and the uptake of bacteria by their Kupffer cells was examined with the electron microscope. Rats underwent bile duct ligation (BDL, n = 8) of sham celiotomy (SC, n = 8) and were allowed to recover for 10 days. They were then injected with 10(9) Staphylococcus aureus IV and killed at intervals of 15, 30, 60, and 180 minutes after injection. Two from each group were killed at each interval. Quantitative blood cultures were performed, and specimens of liver and lung were obtained for quantitative bacterial culture and processed for electron microscopy. Bacteria were rapidly cleared from the bloodstream of SC animals but persisted in BDL rats. Electron microscopy consistently demonstrated bacteria within Kupffer cell phagocytic vesicles of both SC and BDL animals at each interval selected. There was no morphologic difference in these vesicles between the two groups. Bacteremia persists in BDL rats subjected to a bacterial challenge despite rapid uptake of bacteria in apparently normal phagocytic vesicles. This study suggests a defect in intracellular killing of bacteria, an impairment of delivery of bacteria to RE cells, or a combination of these factors.

Host immunosuppression following orthotopic bowel allotransplantation in rats using low-dose cyclosporine.

This study shows that Le rats bearing total bowel allografts treated with CyA (5 mg/kg for 14 days) demonstrated within one month immunological unresponsiveness to donor and third-party alloantigens as determined by MLR responses. Assays performed in long-term survivors (greater than 100 days) showed a continued depressed MLR response to antigen-specific (BN) alloantigens. The response to third-party (ACI) alloantigens was not significantly depressed. After the long-term bowel recipients rejected donor skin grafts, the MLR responses were increased over control, but the transplanted bowel remained intact.

Assessment of the lymphocyte response to silicone.

The biocompatibility of silicone is once again the focus of increased interest. Long considered inert, silicone has now been reported to be responsible for macrophage inhibition in rats and to possibly cause adjuvant disease in humans, and the related compound silica has elicited an antibody response in mice. The present study evaluates lymphocytic response to silicone as expressed by the demonstration of immunologic memory, or changes in specific lymphocyte subpopulations. Thirty-six female Lewis rats (250 gm body weight) were used as test animals. Group 1 (n = 12) was injected subcutaneously with 2.5 ml Freund's Complete Adjuvant (FCA) alone. Group 2 (n = 12) was injected with 2.5 ml FCA sonicated with silicone gel. Group 3 (n = 6) was injected with 2.5 ml FCA, and at 4 weeks, gel-filled silicone implants were placed subcutaneously. Group 4 (n = 6) was injected with 2.5 ml FCA sonicated with silicone gel, and gel-filled silicone implants were placed at 4 weeks. An additional group of six rats (group 5) served as control for the experimental animals, and a group of four rats (group 6) served as naive control. Groups 1 and 2 were sacrificed at 4 weeks, and splenic lymphocytes were obtained for lymphocyte transformation assays performed against silicone. Assays also were run with the addition of the known mitogens Con A, PHA, LPS, and pokeweed. Cytofluorographic analysis of pan-T, T-helper, T-suppressor, and B-cell populations was performed. Groups 3, 4, 5, and 6 were harvested at 8 months, and splenic lymphocytes were subjected to lymphocyte transformation assay.(ABSTRACT TRUNCATED AT 250 WORDS)

A tribute to the late Dr. Ryo Shimazu: a chronic bowel allograft model in the rat.

A chronic model of bowel allotransplantation is described. End-to-end microvascular anastomosis between superior mesenteric vessels was utilized. The recipient splenic vein was preserved to avoid postoperative pancreatitis. Euro-Collins solution was used to flush the vasculature in the lumen of the transplant. Low-dose cyclosporine was used for immunosuppression. With experience, 89% long-term survival was achieved.

Organ preservation injury in small bowel transplantation.

Histological features of preservation injury were studied in a dog model of total small bowel transplantation. It was remarkable that substantial microscopic injury was evident during cold ischemia, unlike in other organ systems. This was early in onset and was related to the duration of cold storage. There was further progression of injury during reperfusion, as expected. Nevertheless, the small bowel was noted to have substantial ability to recover from this storage- and reperfusion-related injury. Histological features of damage and recovery are described in detail.

Experimental small-bowel transplantation: alternative strategies for graft prolongation.

Alternative combined immunosuppressive therapy was tested in canine orthotopic bowel transplantation. Despite sporadic long-term survival, cyclosporine is still questionably effective. Triple-drug therapy (cyclosporine, azathioprine, and prednisone) combined with antilymphocyte serum or with a short segment graft was effective in reducing the early postoperative mortality due to acute rejection but did not alter the long-term survival rate. There was no apparent relationship between the serum cyclosporine levels and survival. The long-term survivors (longer than 100 days) maintained relatively low serum trough levels of cyclosporine. These suggest that orthotopic bowel transplantation in the dog, and probably in the human as well, requires improved immunosuppressive regimens.

Long-term nutritional function of orthotopic small bowel autotransplants.

Nutritional function following autografting of the entire small bowel was followed in 11 dogs for 12 months. Although positive nitrogen balance was recovered within a few weeks, the animals did not achieve their preoperative body weight for up to 6 months. Fat and D-xylose absorption remained depressed and never fully recovered. Hematocrit and serum iron did not normalize until the second postoperative month or later. Abnormal serum albumin and albumin/globulin ratio persisted for 12 months. When the mesenteric vein was anastomosed to the vena cava rather than to the portal vein, this group had more severe abnormalities in body weight, hematocrit, total protein, and serum albumin, in addition to a significant rise in liver enzymes. These findings represent a discouraging portent for the functional utility of small bowel transplants.

Impaired clearance of Escherichia coli bacteremia in early biliary obstruction.

Adult male rats underwent common bile duct ligation or sham celiotomy. At intervals of 7 and 14 days postoperatively, bacteremia was induced by intravenous injection of 10(9) Escherichia coli or intraperitoneal injection of 10(6) E. coli. Serial quantitative blood cultures and quantitative whole organ cultures were obtained. One week after surgery, clearance of bacteremia was impaired in all of the animals. Clearance of intraperitoneally injected E. coli was less efficient in the duct ligation rats. Fourteen days postoperatively, clearance of bacteremia induced by intravenous or intraperitoneal injection had improved in the sham celiotomy rats but was still significantly impaired in the duct ligation rats. An increased number of viable E. coli were recovered from the lungs of duct ligation rats after intravenous administration. We found that rats with obstructive jaundice do not respond normally to a bacteremia challenge. This impairment in reticuloendothelial function can be noted as early as 1 week after common duct ligation.