Chlamydia pneumoniae replicates in Kupffer cells in mouse model of liver infection

AIM: To develop an animal model of liver infection with Chlamydia pneumoniae (C.pneumoniae) in intraperitoneally infected mice for studying the presence of chlamydiae in Kupffer cells and hepatocytes.METHODS: A total of 80 BALB/c mice were inoculated intraperitoneally with C. pneumoniae and sacrificed at various time points after infection. Chlamydiae were looked for in liver homogenates as well as in Kupffer cells and hepatocytes separated by liver perfusion with collagenase. C. pneumoniae was detected by both isolation in LLC-MK2 cells and fluorescence in situ hybridization (FISH). The releasing of TNFA-α by C. pneumoniae in vitro stimulated Kupffer cells was studied by enzymelinked immunosorbent assay.RESULTS: C. pneumoniae isolation from liver homogenates reached a plateau on d 7 after infection when 6 of 10 animals were positive, then decreased, and became negative by d 20. C. pneumoniae isolation from separated Kupffer cells reached a plateau on d 7 when 5 of 10 animals were positive, and became negative by d 20.The detection of C. pneumoniae in separated Kupffer cells by FISH, confirmed the results obtained by culture.Isolated hepatocytes were always negative. Stimulation of Kupffer cells by alive C. pneumoniae elicited high TNF-α levels.CONCLUSION: A productive infection by C. pneumoniae may take place in Kupffer cells and C. pneumoniae induces a local pro-inflammatory activity. C. pneumoniae is therefore, able to act as antigenic stimulus when localized in the liver. One could speculate that C. pneumoniae infection, involving cells of the innate immunity such as Kupffer cells, could also trigger pathological immune reactions involving the liver, as observed in human patients with primary biliary cirrhosis.     

Liver-specific HBsAg transgenic mice are over-sensitive to Poly(I:C)-induced liver injury in NK cell- and IFN-gamma-dependent manner

BACKGROUND/AIMS: The role of natural killer (NK) cells in the development of hepatitis B virus (HBV)-associated liver injury remains obscure. In this study, we elucidated the role of NK cells in liver injury of HBsAg transgenic mice (HBs-B6), a mimic of human healthy chronic HBsAg carriers, triggered by polyinosinic:polycytidylic acid [Poly(I:C)]. METHODS: HBs-B6 or wild B6 mice were intraperitoneally injected with Poly(I:C) at different doses. Liver injury was evaluated by serum transaminase activity and histopathologic changes. RESULTS: HBs-B6 mice were over-sensitive to Poly(I:C)-induced liver injury, which was absolutely dependent on the presence of NK cells and IFN-gamma produced by intrahepatic NK cells. Much stronger IFN-gamma receptor expression was observed on hepatocytes of HBs-B6 mice, which was significantly enhanced by Poly(I:C) injection. Treatment with IFN-gammain vitro triggered much higher activation of downstream signals (pSTAT1-IRF-1) in hepatocytes of HBs-B6 mice. Depletion of Kupffer cells and neutralization of endogenous IL-12 did not affect Poly(I:C)-induced over-sensitive liver injury in HBs-B6 mice. CONCLUSIONS: NK cells played a critical role in an IFN-gamma dependent, Kupffer cell- and IL-12-independent manner in over-sensitive liver injury triggered by Poly(I:C) in murine chronic HBsAg carriers.     

Kupffer cells participate in early clearance of syngeneic hepatocytes transplanted in the rat liver

BACKGROUND & AIMS: Kupffer cells are activated shortly after deposition of hepatocytes in liver sinusoids, with clearance of a significant fraction of transplanted cells, especially when cells are entrapped in portal spaces. We determined whether perturbation of Kupffer cells would improve transplanted cell engraftment. METHODS: Dipeptidyl peptidase IV-deficient rats were used as recipients of syngeneic Fischer 344 rat hepatocytes. Kupffer cell function was analyzed by measuring phagocytic activity with carbon particle or (99m)Tc-sulfur colloid incorporation. Transplanted cell survival and integration in the liver parenchyma was determined by histochemical analysis of tissues. Transplanted cell proliferation was analyzed in rats conditioned with retrorsine and partial hepatectomy. RESULTS: Gadolinium chloride significantly impaired Kupffer cell function, especially in periportal areas, where transplanted cells were localized. Transplanted cell survival increased by approximately 2-fold in animals treated with gadolinium chloride 24 hours before cell transplantation. In gadolinium-treated rats, more transplanted cells were observed in portal vein radicles, as well as in liver sinusoids, albeit integration of cells in the liver parenchyma was slower in gadolinium-treated rats and cells separated from other hepatocytes in portal vein radicles that failed to exhibit bile canalicular reconstitution. Finally, hepatocyte transplantation in rats primed with retrorsine and partial hepatectomy showed accelerated kinetics of liver repopulation in animals pretreated with gadolinium chloride. CONCLUSIONS: Perturbation of Kupffer cell activity will benefit liver repopulation with cells and further analysis of clinically suitable approaches to exploit this mechanism will be appropriate.     

Induction of anaphylatoxin C5a receptors in rat hepatocytes by lipopolysaccharide in vivo: mediation by interleukin-6 from Kupffer cells

BACKGROUND & AIMS: In normal rat liver, anaphylatoxin C5a induces glucose output from hepatocytes indirectly via prostanoids released from Kupffer cells. Correspondingly, it was found that hepatocytes, in contrast to Kupffer cells, did not express C5a receptors. Lipopolysaccharide (LPS) has been reported to enhance C5a receptor expression in murine livers. This might be the result of de novo expression in hepatocytes. METHODS: C5a receptor expression was investigated in hepatocytes after in vivo treatment of rats with LPS and in vitro stimulation of isolated cells with LPS and proinflammatory cytokines on messenger RNA (mRNA) and protein level, and functionally in isolated hepatocytes and perfused liver. RESULTS: In vivo treatment of rats with LPS induced C5a receptor mRNA and protein in hepatocytes with a maximum after 8-10 hours. At this time-point, C5a directly activated glycogen phosphorylase in isolated hepatocytes and enhanced glucose output in perfused livers without the involvement of prostanoids. LPS failed to induce C5a receptors in cultured hepatocytes in vitro, whereas interleukin (IL) 6 and IL-1beta, which are known to be released from Kupffer cells on stimulation with LPS, did so. In cocultures of hepatocytes with Kupffer cells, LPS induced C5a receptors in hepatocytes in an IL-6-dependent manner. CONCLUSIONS: Thus, IL-6 from Kupffer cells appears to be the main mediator of LPS-induced de novo expression of C5a receptors in hepatocytes.     

Hepatocyte-derived cysteinyl leukotrienes modulate vascular tone in experimental cirrhosis

BACKGROUND & AIMS: The leukotrienes C(4)/D(4)/E(4) (cysteinyl-LTs) are 5-lipoxygenase (5-LO)-derived eicosanoids with potent vasoconstrictor, proliferative, and profibrogenic properties that may participate in key pathophysiologic events in liver cirrhosis. We examined the cysteinyl-LT biosynthetic pathway in liver tissue and purified liver cells isolated from rats with carbon tetrachloride-induced cirrhosis, and assessed the vasoactive properties of LTD(4) in hepatic stellate cells (HSCs) and anesthetized rats. METHODS & RESULTS: Liver homogenates from cirrhotic rats had increased 5-LO mRNA and cysteinyl-LT content, as determined by Northern blot and enzyme immunoassay, respectively. In isolated rat liver cells, 5-LO mRNA expression was found to be restricted to Kupffer cells. However, among the liver cells (i.e., hepatocytes, Kupffer cells, HSCs, and sinusoidal endothelial cells), hepatocytes exhibited the highest ability to generate cysteinyl-LTs from the unstable intermediate LTA(4). Hepatocytes from cirrhotic rats showed an enhanced baseline generation of cysteinyl-LTs, but their ability to synthesize cysteinyl-LTs from exogenous LTA(4) was found to be similar to that of hepatocytes from normal animals. Both LTD(4) and hepatocyte-conditioned medium increased intracellular Ca(2+) concentration and induced contraction in HSCs, suggesting that hepatocyte-derived cysteinyl-LTs could act in a paracrine fashion on nearby nonparenchymal liver cells. The relevance of these in vitro findings was further established in vivo by the observation that LTD(4) significantly increased portal pressure in anesthetized rats. CONCLUSIONS: These data suggest a role for hepatocyte-derived cysteinyl-LTs in mediating hepatic vascular tone abnormalities in cirrhosis.     

Localization of hepatitis A virus in marmoset liver tissue during the acute phase of experimental infection

Electron microscopic and virological studies of marmoset liver tissue with acute infection of hepatitis A virus (HAV), especially in the earlier stages of infection, were carried out to characterize the maturation process of HAV. Four marmosets were inoculated intravenously with HAV suspension and sacrificed 1 week, 2 weeks, 3 weeks and 4 weeks after inoculation respectively. Hepatitis A antigen (HAAg) in 10% liver homogenates of marmosets was examined by radioimmunoassay and a large amount of HAAg was detected in the liver homogenate of two marmosets sacrificed 2 weeks and 3 weeks after inoculation respectively. The histodiagnosis of the marmoset sacrificed 2 weeks after HAV inoculation was normal. However, many clusters of virus-like particles about 27 nm in diameter, in both "solid" and "empty" forms were found, mainly in vesicles of Kupffer cells by electron microscopy. In the animal that developed mild hepatitis 3 weeks after inoculation HAV-like particles were found in vesicles of hepatocytes by electron microscopy. By immune electron microscopy using peroxidase-conjugated anti-hepatitis A antibody, HAAg was detected on the particles present within the cytoplasmic vesicles of Kupffer cells or hepatocytes and on the surrounding membrane of the vesicles which contained HAV-like particles.     

The role of nonparenchymal and parenchymal liver cells in the catabolism of extracellular purines

Adenosine-degrading enzymes within the liver lobule can modulate both vascular and metabolic effects of circulating adenosine in the liver. Since it has not been fully established whether nonparenchymal cells participate in the elimination of sinusoidal purines, isolated Kupffer cells and endothelial cells were tested for their capacity to degrade extracellular purines. After perfusion and digestion of rat livers by collagenase, the resulting mixed cell population was separated by centrifugal elutriation. The isolated parenchymal and nonparenchymal cells were incubated for up to 2 hr in the presence of [8(-14)C]adenosine, [8(-14)C]guanosine and [8(-14)C]hypoxanthine (50 mumoles per liter). In the deproteinized medium, adenosine, guanosine, inosine, adenine, guanine, xanthine, hypoxanthine, uric acid and allantoin were separated by reversed-phase high-performance liquid chromatography. Radioactive peaks were collected and counted. Nonparenchymal cells catalyzed the degradation of adenosine into inosine and hypoxanthine. However, the formation of xanthine, uric acid or allantoin from adenosine could only be detected in hepatocyte suspensions. Within 15 min, adenosine was completely eliminated from the medium by Kupffer cells, whereas endothelial cells catabolized only less than half of the initial amount of the adenine nucleoside during this time period. Accordingly, incubation of nonparenchymal cells in the presence of hypoxanthine did not result in the formation of further breakdown products of the purine, whereas its catabolites slowly accumulated in the medium of hepatocytes. Guanosine conversion into guanine and xanthine was much slower in endothelial cells as compared to Kupffer cells and hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Augmenter of liver regeneration promotes hepatocyte proliferation induced by Kupffer cells

AIM: To observe the effects of augmenter of liver regeneration (ALR) on Kupffer cells and to determine whether ALR promotes hepatocyte proliferation induced by Kupffer cells. METHODS: Kupffer cells and hepatocytes were cultured in vitro and various concentrations of recombinant rat ALR (rrALR) were added. 3H-thymidine, BrdU and 3H-leucine incorporation was determined in cultured Kupffer cells and hepatocytes, in hepatocytes conditioned by Kupffer cells, and in associated medium. rrALR was labeled by iodination and used to determine its binding activity by Scatchard analysis in Kupffer cells and primarily cultured rat hepatocytes. RESULTS: rrALR stimulated DMA replication in Kupffer cells and protein synthesis both in cells and in medium in a non-concentration-dependent manner. The effect was significant at the concentration of 1μg/L ALR. However, rrALR had no effect on primarily cultured hepatocytes, when hepatocytes were cultured with the Kupffer cell medium conditioned by ALR, DNA replication and protein synthesis in hepatocytes increased significantly at the concentration of 1μg/L ALR. When the ALR concentration was increased, its effect on hepatocyte proliferation decreased to the basal level. Scatchard analysis indicated the presence of a single class of high affinity receptors with a dissociation constant (Kd) of 0.883 nmol/L and a maximum binding capacity (Bmax) of 126.1 pmol/g protein in the rat Kupffer cells. CONCLUSION: ALR can promote hepatocyte proliferation induced by Kupffer cells, which is associated with the concentration of ALR, suggesting that Kupffer cells play a dual role in liver regeneration.     

Kupffer cell function in chronic ethanol-fed rats

In an attempt to clarify the Kupffer cell function in alcoholism, chronic ethanol-fed rats were investigated. The clearance of latex particles in the rat was analysed to estimate the function of the reticuloendothelial system in the liver, and the phagocytic function of Kupffer cells was measured by counting particles in the cell after isolation of non-parenchymal cells by collagenase digestion of the liver following an injection of latex particles and subsequently by staining of endogenous peroxidase activities. In addition, the number of Kupffer cells and their phagocytic function were examined histologically in fresh frozen sections of liver after an injection of particles. Serum ethanol concentration in the ethanol-fed rats was 10-60 mumol/l. The clearance of latex particles was markedly reduced in the ethanol-fed rats as compared with the paired controls (P less than 0.01). Markedly decreased-phagocytic function was found in 20% of Kupffer cells in the chronic ethanol-fed rats. The number of Kupffer cells in the ethanol-fed rats was increased as compared with the paired control rats. Chemotaxis analysis revealed that hepatocytes when incubated with ethanol, produced chemotactic factor for Kupffer cells and polymorphonuclear cells. These abnormal Kupffer cell functions may contribute to the pathogenesis of alcoholic liver disease.     

High frequency of chimerism in transplanted livers

Recent studies have shown that primitive stem cells can mobilize and differentiate into hepatocytes. We investigated the time and extent in which cells of recipient origins could differentiate into hepatocytes and other cells in human liver allografts. Microsatellite analysis, which can assess quantitatively the proportions of recipient and donor DNA, was performed in posttransplantation liver biopsy specimens from 17 patients at various times. Combined fluorescence in situ hybridization (FISH) for Y chromosome and immunofluorescence for different cell types was also performed in 10 of these cases with sex mismatch. Organ chimerism in the transplanted livers was found to be of variable extent, and the recipients' DNA in the posttransplantation liver biopsy specimens (excluding portal tracts) amounted up to 50%. The recipient DNA in the posttransplantation liver biopsy specimens increased after liver transplantation by as early as 1 week, peaked at around 30 to 40 weeks, and could be shown 63 weeks after transplantation. Most (64%-75%) of the recipient-derived cells showed macrophage/Kupffer cell differentiation. Only up to 1.6% of the recipient-derived cells in the liver grafts showed hepatocytic differentiation in the liver grafts and made up 0.62% of all hepatocytes of both donor and recipient origins. These livers had mild or minimal injury histologically. In conclusion, our results show that most of the recipient-derived cells in the liver allografts were macrophages/Kupffer cells and only a small proportion of hepatocytes was recipient derived. However, with regard to recipient-derived hepatocytes, our data cannot distinguish between transdifferentiation and cell fusion.     

Activation of Kupffer cells and caspase-3 involved in rat hepatocyte apoptosis induced by endotoxin.

BACKGROUND/AIMS: Sepsis and lipopolysaccharides (LPS) cause mild to severe hepatic dysfunction. In this study, Kupffer cell activation, involvement of TNFalpha and caspases downstream of the TNFalpha receptor were examined in hepatocyte apoptosis induced by LPS. METHODS: In in vivo experiments, male Sprague-Dawley rats were injected intravenously with LPS, and small amounts of the blood and liver were sampled to evaluate apoptosis. Kupffer cells were inactivated by pretreatment with gadolinium chloride for 2 days. In in vitro experiments, hepatocytes and Kupffer cells were separately isolated from rat livers using collagenase perfusion. RESULTS: LPS induced time-dependent and dose-dependent increases in the number of TUNEL-positive cells, which coincided with the apoptotic features of hepatocytes demonstrated by electron microscopy and DNA ladder. Activation of caspase-3-like proteases was observed with an increase in the number of apoptotic hepatocytes. Immunostaining with activated caspase-3-specific antibody showed that caspase-3 was activated only in the cytoplasm of TUNEL-positive hepatocytes. Inactivation of Kupffer cells by gadolinium chloride was concomitantly accompanied by the prevention of caspase-3 activation, hepatocyte apoptosis and liver injury induced by LPS. The co-culture system of hepatocytes and Kupffer cells, but neither cell culture system, individually, showed LPS-induced hepatocyte apoptosis. Kupffer cell-conditioned medium induced hepatocyte apoptosis, whereas addition of anti-TNFalpha antibody to Kupffer cell-conditioned medium did not. Additions of acetyl-DEVD-CHO, acetyl-YVAD-CHO, and acetyl-IETD-CHO to Kupffer cell-conditioned medium decreased the number of apoptotic hepatocytes. CONCLUSIONS: These results suggest that the activation of Kupffer cells, TNFalpha and caspases downstream of TNFR1 were involved in hepatocyte apoptosis induced by LPS.     

Augmenter of liver regeneration promotes hepatocyte proliferation induced by Kupffer cells

AIM: To observe the effects of augmenter of liver regeneration (ALR) on Kupffer cells and to determine whether ALR promotes hepatocyte proliferation induced by Kupffer cells. METHODS: Kupffer cells and hepatocytes were cultured in vitro and various concentrations of recombinant rat ALR (rrALR) were added. 3H-thymidine, BrdU and 3H-leucine incorporation was determined in cultured Kupffer cells and hepatocytes, in hepatocytes conditioned by Kupffer cells, and in associated medium. rrALR was labeled by iodination and used to determine its binding activity by Scatchard analysis in Kupffer cells and primarily cultured rat hepatocytes. RESULTS: rrALR stimulated DNA replication in Kupffer cells and protein synthesis both in cells and in medium in non-concentration-dependent manner. The effect was significant at the concentration of 1 μg/L ALR. However, rrALR had no effect on primarily cultured hepatocytes, when hepatocytes were cultured with the Kupffer cell medium conditioned by ALR, DNA replication and protein synthesis in hepatocytes increased significantly at the concentration of 1 μg/L ALR. When the ALR concentration was increased, its effect on hepatocyte proliferation decreased to the basal level. Scatchard analysis indicated the presence of a single class of high affinity receptors with a dissociation constant (Kd) of 0.883 nmol/L and a maximum binding capacity (Bmax) of 126.1 pmol/g protein in the rat Kupffer cells. CONCLUSION: ALR can promote hepatocyte proliferation induced by Kupffer cells, which is associated with the concentration of ALR, suggesting that Kupffer cells play a dual role in liver regeneration.     

Virus-liver cell interactions in duck hepatitis B virus infection. A study of virus dissemination within the liver

Thirty-five 1-day-old Pekin-Aylesbury ducks were inoculated intravenously or intraperitoneally with duck hepatitis B virus, and the time-course of infection was examined by Southern-blot, dot-blot, and in situ hybridization and by immunohistochemistry. Randomly scattered single infected hepatocytes were first seen on days 1 and 2 after inoculation and by day 3 occurred as single cells, pairs, and groups of 5-10 adjoining cells. From day 4 after inoculation all hepatocytes were positive for duck hepatitis B surface antigen and deoxyribonucleic acid. Duck hepatitis B virus deoxyribonucleic acid levels in liver extracts and serum increased logarithmically from days 2 to 3 to a plateau by days 4 to 5 after inoculation. Infected and control birds showed no significant differences during the first 7 days in terms of liver histology, hepatocyte morphology, or mitotic activity. It was concluded that (a) virus gains access to randomly distributed hepatocytes without first replicating in other cell types, and then begins disseminating to adjacent cells following anatomic boundaries; (b) markers of infection in liver and serum show reproducible kinetics, thus making this in vivo system amenable to further quantitative study; and (c) hepatocytes in this system are highly permissive to virus replication without the development of significant cytopathology.     

Stimulation of DNA synthesis in hepatocytes by Kupffer cells after partial hepatectomy

The role of Kupffer cells during reparative regeneration of rat liver was investigated with an in vitro experimental model. Conditioned media from primary cultures of Kupffer cells isolated from intact and regenerating liver were added to primary cultures of hepatocytes, and [3H]thymidine incorporation into DNA was studied. Kupffer cell-conditioned media from intact liver and regenerating remnant liver significantly stimulated DNA synthesis in hepatocytes as compared with control media (p less than 0.05). Moreover, the stimulating activity of Kupffer cells prepared from regenerating liver at 6 and 12 hr after partial hepatectomy was significantly higher than that of Kupffer cells from untreated rats (p less than 0.05). The activity was found in serum-free conditioned media. This stimulating activity exponentially increased as the increase of the number of the cultured cells, indicating that the stimulating activity was released directly by cultured Kupffer cells. These results suggest that Kupffer cells stimulate DNA synthesis in hepatocytes by producing and releasing certain factor(s) at an early stage of liver regeneration after partial hepatectomy.     

Pirital virus (Arenaviridae) infection in the syrian golden hamster, Mesocricetus auratus: a new animal model for arenaviral hemorrhagic fever.

Adult Syrian golden hamsters inoculated intraperitoneally with Pirital virus, a recently discovered member of the Tacaribe complex of New World arenaviruses, developed a progressively severe, fatal illness with many of the pathologic features observed in fatal human cases of Lassa fever and other arenaviral hemorrhagic fevers. Most of the animals became moribund by Day 5 and were dead by Day 7 after inoculation. The most consistent histopathologic changes included interstitial pneumonitis, splenic lymphoid depletion and necrosis, and multifocal hepatic necrosis without significant inflammatory cell infiltration. The liver changes ranged from single cell death by apoptosis to coagulative necrosis of clusters of hepatocytes. Immunohistochemical studies of the liver demonstrated the presence and accumulation ot Pirital virus antigen within hepatocytes as well as Kupffer cells. An in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay showed progressively increasing apoptotic activity in the liver of infected hamsters. A human hepatoblastoma cell line (Hep G2/C3A) inoculated with Pirital virus also developed progressive cell destruction and accumulation of viral antigen, as demonstrated by immunofluorescence. Results of this pilot study suggest that the Pirital virus-hamster model is a very promising new small animal model for studying the pathogenesis of arenavirus infections, particularly, the mechanism of direct virus-induced hepatic injury. It may also be useful for testingantiviral agents for treatment of arenaviral hemorrhagic fevers.     

Dietary glycine blunts liver injury after bile duct ligation in rats

AIM： To investigate the effects of （dietary） glycine against oxidant-induced injury caused by bile duct ligation （BDL）.
METHODS： Either a diet containing 5% glycine or a standard diet was fed to male Sprague-Dawley （SD） rats. Three days later, BDL or sham-operation was performed. Rats were sacrificed 1 to 3 d after BDL. The influence of deoxycholic acid （DCA） in the presence or absence of glycine on liver cells was determined by measurement of calcium and chloride influx in cultivated Kupffer cells and lactate dehydrogenase （LDH） activity was determined in the supernatant of cultivated hepatocytes.
RESULTS： Serum alanine transaminase levels increased to about 600 U/L 1 d alter BDL. However, enzyme release was blunted by about two third in rats receiving glycine. Release of the alkaline phosphatase and aspartate aminotransferase was also blocked significantly in the group fed glycine. Focal necrosis was observed 2 d after BDL. Glycine partially blocked the histopathological changes. Incubation of Kupffer cells with DCA led to increased intracellular calcium that could be blocked by incubation with glycine. However, systemic blockage of Kupffer cells with gadolinium chloride had no effects on transaminase release. Incubation of isolated hepatocytes with DCA led to a significant release of LDH after 4 h. This release was largely blocked when incubation with glycine was performed.
CONCLUSION： These data indicate that glycine significantly decreased liver injury, most likely by a direct effect on hepatocytes. Kupffer cells do not appear to play an important role in the pathological changes caused by cholestasis.     

Sustained telomere length in hepatocytes and cholangiocytes with increasing age in normal liver

Telomeres, a validated biomarker of aging, comprise multiple nucleotide repeats capping chromosomes that shorten with each cell cycle until a critical length is achieved, precipitating cell senescence. Only two previous studies focused on the effect of aging in "normal" liver tissue, but these studies were compromised by small sample size, limited age range, tissue derived from individuals with an increased risk of senescence, and the use of liver homogenates. We developed a robust large-volume, four-color quantitative fluorescent in situ hybridization technique to measure telomere length in large numbers of hepatocytes, Kupffer cells, hepatic stellate cells, CD4-positive and CD8-positive lymphocytes, and cholangiocytes. Following validation against the gold standard (Southern blotting), the technique was applied to normal archived paraffin-embedded liver tissue obtained following reperfusion of implanted donor liver. We studied 73 highly selected donors aged 5-79 years with a short medical illness preceding death and no history of liver disease, reperfusion injury, or steatosis and normal graft function 1-year posttransplantation. Cholangiocytes had significantly longer telomeres compared with all other intrahepatic lineages over a wide age range (P < 0.05). Age-related telomere attrition was restricted to sinusoidal cells (i.e., Kupffer cells [P = 0.0054] and stellate cells [P = 0.0001]). Cholangiocytes and hepatocytes showed no age-related telomere shortening. Conclusion: In normal liver and over a broad age range, cholangiocytes have longer telomeres than all other intrahepatic lineages. Age-related telomere length decline is restricted to Kupffer cells and stellate cells. (HEPATOLOGY 2012).     

Production of reactive oxygen species and expression of inducible nitric oxide synthase in rat isolated Kupffer cells stimulated by Leptospira interrogans and Borrelia burgdorferi

AIM: To evaluate the production of reactive oxygen species (ROS) and the expression of inducible nitric oxide synthase (iNOS) in rat isolated Kupffer cells (KCs) stimulated by Leptospira interrogans and Borrelia burgdorferi. METHODS: Rat Kupffer cells were separated by perfusion of the liver with 0.05% collagenase, and purified by Percoll gradients. Purified Kupffer cells were tested in vitro with alive L. interogans and B. burgdorferi preparations. The production of ROS was determined by chemiluminescence, whereas iNOS protein expression was evaluated by Western blot assay using anti-iNOS antibodies. RESULTS: B. burgdorferi and to a less extent L. interrogans induced ROS production with a peak 35 min after infection. The chemiluminescence signal progressively diminished and was undetectable by 180 min of incubation. Leptospirae and borreliae induced an increased iNOS expression in Kupffer cells that peaked at 6 hours and was still evident 22 h after infection. CONCLUSION: Both genera of spirochetes induced ROS and iNOS production in rat Kupffer cells. Since the cause of liver damage both in leptospiral as well as in borrelial infections are still unknown, we suggest that leptospira and borrelia damage of the liver can be initially mediated by oxygen radicals, and is then maintained at least in part by nitric oxide.     

Targeted and nontargeted liposomes for in vivo transfer to rat liver cells of a plasmid containing the preproinsulin I gene.

A plasmid containing the rat preproinsulin I gene was entrapped in large liposomes and intravenously administered to rats. Four hours after inoculation, the livers were processed for the isolation of hepatocytes. Kupffer cells, and endothelial cells, DNA was purified, and the exogenous DNA was detected in the different cell DNA preparations by Southern blotting. By using liposomes consisting of phospholipids and cholesterol, Kupffer cells were shown to be, on a per cell basis, the primary target for gene incorporation. In an attempt to target the liposomes to other liver cells, a glycolipid, lactosylceramide, was included in the lipid bilayer of the liposomes; this resulted in a substantial increase in the proportion of the exogenous gene in the hepatocytes and mainly in the endothelial cells, with a simultaneous decrease of this proportion in the Kupffer cells. Thus, it is shown that inclusion of a specific glycolipid within the bilayer of the liposomes may direct the DNA-containing vesicles to specific cell types in the liver.     

Production of reactive oxygen species and expression of inducible nitric oxide synthase in rat isolated Kupffer cells stimulated by Leptospira interrogans and Borrelia burgdorfen

AIM: To evaluate the production of reactive oxygen species (ROS) and the expression of inducible nitric oxide synthase (iNOS) in rat isolated Kupffer cells (KCs) stimulated by Leptospira interrogans and Borrelia burgdorferi. METHODS: Rat Kupffer cells were separated by perfu-sion of the liver with 0.05% collagenase, and purified by Percoll gradients. Purified Kupffer cells were tested in vitro with alive L.interogans and B. burgdorferi preparations. The production of ROS was determined by che-miluminescence, whereas iNOS protein expression was evaluated by Western blot assay using anti-iNOS antibodies. RESULTS: B. burgdorferi and to a less extent L interrogans induced ROS production with a peak 35 min after infection. The chemiluminescence signal progressively diminished and was undetectable by 180 min of incubation. Leptospirae and borreliae induced an increased iNOS expression in Kupffer cells that peaked at 6 hours and was still evident 22 h after infection. CONCLUSION: Both genera of spirochetes induced ROS and iNOS production in rat Kupffer cells. Since the cause of liver damage both in leptospiral as well as in borreliai infections are still unknown, we suggest that leptospira and borrelia damage of the liver can be initially mediated by oxygen radicals, and is then maintained at least in part by nitric oxide.