Peripheral Donor-specific Antibodies Are Associated With Histology and Cellular Subtypes in Protocol Liver Biopsies of Pediatric Recipients.

BACKGROUND: The cellular infiltrate in protocol liver biopsies (PB) following pediatric liver transplantation remains mostly uncharacterized, yet there is increasing concern about the role of inflammation and fibrosis in long-term liver allografts. We aimed to define cell types in PB and to analyze their relationship with donor-specific antibodies (DSA) and histological phenotype. METHODS: PB were performed at least 1 year after transplantation. We identified 4 phenotypes: normal, fibrosis, inflammation, inflammation with fibrosis. Cell types were counted after immunostaining for CD3, CD4, CD8, CD68, CD20, MUM1, and FoxP3. RESULTS: Forty-four patients underwent 1 PB between 2000 and 2015. Eleven percent (5/44) of PB displayed normal histology, 13.6% (6/44) fibrosis, 34.1% (15/44) inflammation, and 40.9% (18/44) inflammation and fibrosis. The main cell types in the portal tracts and lobules were CD3+ and CD68+ cells. Frequency of de novo DSA was 63% (27/44). The presence of CD8+ cells in the lobules was associated with fibrosis. Inflammation and fibrosis in PB were associated with the presence of circulating de novo DSA, number of de novo DSA, and C1q binding activity when compared to other phenotypes. CONCLUSIONS: T cells (CD3+) and macrophages (CD68+) were the most prevalent cell-types in PB. In the presence of inflammation, portal tracts were enriched in CD3+, CD20+ but displayed fewer CD68+. This coincided with the presence and number of de novo DSA. How these cellular and humoral actors interact is unclear, but peripheral DSA may be a marker of immune cellular activity in the seemingly quiescent allograft.

Endoplasmic reticulum stress induces inverse regulations of major functions in portal myofibroblasts during liver fibrosis progression.

Portal myofibroblasts (PMF) form a sub-population of highly proliferative and proangiogenic liver myofibroblasts that derive from portal mesenchymal progenitors. Endoplasmic reticulum (ER) stress was previously shown to modulate fibrogenesis, notably in the liver. Our aim was to determine if ER stress occurred in PMF and affected their functions. PMF were obtained after their expansion in vivo from bile duct-ligated (BDL) rats and referred to as BDL PMF. Compared to standard PMF obtained from normal rats, BDL PMF were more myofibroblastic, as assessed by higher alpha-smooth muscle actin expression and collagen 1 production. Their proangiogenic properties were also higher, whereas their proliferative and migratory capacities were lower. CHOP expression was detected in the liver of BDL rats, at the leading edge of portal fibrosis where PMF accumulate. BDL PMF displayed ER dilatation and an overexpression of the PERK pathway downstream targets, Chop, Gadd34 and Trb3, in comparison with standard PMF. In vitro, the induction of ER stress by tunicamycin in standard PMF, caused a decrease in their proliferative and migratory activity, and an increase in their proangiogenic activity, without affecting their myofibroblastic differentiation. Conversely, the treatment of BDL PMF with the PERK inhibitor GSK2656157 reduced ER stress, which caused a decrease in their angiogenic properties, and restored their proliferative and migratory capacity. In conclusion, PMF develop ER stress as they expand with the progression of fibrosis, which further increases their proangiogenic activity, but also inhibits their proliferation and migration. This phenotypic switch may restrict PMF expansion while they support angiogenesis.

Establishment and characterization of rat portal myofibroblast cell lines.

The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC) and portal fibroblasts (PF). In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5'-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myo)fibroblasts and their contribution to the progression of liver fibrosis.

The portal fibroblast: not just a poor man's stellate cell.

Portal fibroblasts, the resident fibroblasts of the portal tract, are found in the mesenchyme surrounding the bile ducts. Their roles in liver homeostasis and response to injury are undefined and controversial. Although portal fibroblasts almost certainly give rise to myofibroblasts during the development of biliary fibrosis, recent lineage tracing studies suggest that their contribution to fibrogenesis is limited compared with that of hepatic stellate cells. Other functions of portal fibroblasts include participation in the peribiliary stem cell niche, regulation of cholangiocyte proliferation, and deposition of specific matrix proteins. Portal fibroblasts synthesize elastin and other components of microfibrils; these may serve structural roles, providing stability to ducts and the vasculature under conditions of increased ductal pressure, or could regulate the bioavailability of the fibrogenic transforming growth factor ß in response to injury. Viewing portal fibroblasts in the context of fibroblast populations throughout the body and studying their niche-specific roles in matrix deposition and epithelial regulation could yield new insights into their contributions in the normal and injured liver. Understanding the functions of portal fibroblasts will require us to view them as more than just an alternative to hepatic stellate cells in fibrosis.

Isolation of rat portal fibroblasts by in situ liver perfusion.

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis. Portal fibroblasts have been isolated using various techniques including outgrowth and liver perfusion with enzymatic digestion followed by size selection. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Laser captured hepatocytes show association of butyrylcholinesterase gene loss and fibrosis progression in hepatitis C-infected drug users.

UNLABELLED: Chronic hepatitis C virus (HCV) infection is complicated by hepatic fibrosis. Hypothesizing that early fibrogenic signals may originate in cells susceptible to HCV infection, hepatocyte gene expression was analyzed from persons with chronic HCV at different stages of liver fibrosis. Four HCV-infected subjects with precirrhosis liver fibrosis (Ishak fibrosis 3-5) were matched for age, race, and gender to five HCV-infected subjects with no evidence of fibrosis (Ishak fibrosis 0). Hepatocytes from each subject were isolated from liver biopsies using laser capture microdissection. Transcriptome profiling was performed on hepatocyte RNA using hybridization arrays. We found that hepatocytes in precirrhosis fibrosis were depleted for genes involved in small molecule and drug metabolism, especially butyrylcholinesterase (BCHE), a gene involved in the metabolism of drugs of abuse. Differential expression of BCHE was validated in the same tissues and cross-sectionally in an expanded cohort of 143 HCV-infected individuals. In a longitudinal study, serum BCHE activity was already decreased at study inception in 19 fibrosis progressors compared with 20 fibrosis nonprogressors (P < 0.05). Nonprogressors also had decreased BCHE activity over time compared with initial values, but these evolved a median (range) 8.6 (7.8-11.4) years after the study period inception (P < 0.05). Laser captured portal tracts were enriched for immune related genes when compared with hepatocytes but precirrhosis livers lost this enrichment. CONCLUSION: Chronic HCV is associated with hepatocyte BCHE loss years before hepatic synthetic function is impaired. These results indicate that BCHE may be involved in the pathogenesis of HCV-related fibrosis among injection drug users.

p75 Neurotrophin receptor is a marker for precursors of stellate cells and portal fibroblasts in mouse fetal liver.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) and portal fibroblasts (PFs) are 2 distinct mesenchymal cells in adult liver. HSCs in sinusoids accumulate lipids and express p75 neurotrophin receptor (p75NTR). HSCs and PFs play pivotal roles in liver regeneration and fibrosis. However, the roles of mesenchymal cells in fetal liver remain poorly understood. In this study, we aimed to characterize mesenchymal cells in mouse fetal liver. METHODS: We prepared an anti-p75NTR monoclonal antibody applicable for flow cytometry and immunohistochemistry. p75NTR(+) cells isolated from fetal liver by flow cytometry were characterized by reverse-transcription polymerase chain reaction, immunohistochemistry, and cell cultivation. Lipid-containing cells were visualized by Oil-red O staining. RESULTS: p75NTR(+) cells in fetal liver were clearly distinct from endothelial cells and showed characteristics of mesenchymal cells. At embryonic day (E) 10.5, p75NTR(+) cells were present at the periphery of the liver bud in close contact with endothelial cells, and spread over the liver at E11.5. With the formation of the liver architecture, they began to localize to 2 distinct areas, parenchymal and portal areas, and lipid-containing p75NTR(+) cells increased accordingly. p75NTR(+) cells around portal veins were adjacent to cholangiocytes and expressed Jagged1, a crucial factor for the commitment of hepatoblasts to cholangiocytes. By cultivation, p75NTR(+) cells showed features of adult HSCs with markedly increased expression of glial fibrillary acidic protein and alpha-smooth muscle actin. CONCLUSIONS: p75NTR(+) mesenchymal cells in fetal liver include progenitors for HSCs and PFs, and the anti-p75NTR monoclonal antibody is useful for their isolation.

A light and transmission electron microscope study of hepatic portal tracts in the rhesus monkey (Macacus rhesus).

This study reports on morphological features of hepatic portal tracts in the liver of a rhesus monkey. The light microscope shows that the number of each type of principal component comprising a portal tract varies but that there are usually one to five lymphatics, one bile ductule, one bile duct, one arteriolar and one arterial branch of the hepatic artery, and one hepatic portal vein. Bile ductules, in cross section, have 6-10 cells (mostly low pyramidal, but with a few cuboidal) bordering the lumen, an outside diameter of from about 20 to 25 microm, and a luminal diameter of from 2 to 10 microm. Bile ducts, in cross section, have more than 10 cells (about equal numbers of low pyramidal and cuboidal) bordering the lumen, an outside diameter greater than 25 microm and a luminal diameter of greater than 10 microm. The term "pyramidal" has not previously been applied to the cells of the ductules and ducts. The monkey tracts show several cytological features previously undescribed, viz., abortive cilia and basal bodies in the duct cells, abortive cilia in the ductule cells, and an occasional aggregation of ribosomes in arterial endothelial cells. They also show a major histological feature previously mentioned but not illustrated, viz., bundles of nerve processes which exhibit a preferential location, i.e., proximity to the arterioles and arteries.

Effect of microcapsule composition and short-term immunosuppression on intraportal biocompatibility.

With higher nutrient and oxygen supply and close contact to blood, the portal vein is a possible alternative to the peritoneal cavity for transplantation of encapsulated cells. Data regarding intraportal biocompatibility of microcapsules are lacking. Microcapsules were built from five alginate types differing in their molar mass and mannuronic/guluronic acid ratios by complex formation with divalent cations (barium or calcium) or mixtures of divalent cations and polycations. They were injected in the portal vein of rats, and cellular and fibrotic pericapsular infiltration thickness was measured 3 and 7 days after implantation. Overgrowth was characterized using various stainings or immunohistochemistry (hematoxylin and eosin, Giemsa, ED-1 for monocyte/macrophage, alpha-actin for myofibroblasts, CD31 for endothelial cells). The impact of short-term immunosuppression (gadolinium-chloride IV 20 mg/kg/day on days--1 and 4 as well as 10 days of rapamycin PO 1 mg/kg/day, tacrolimus PO 3 mg/kg/day, or combinations of rapamycin/tacrolimus or gadolinium/tacrolimus) was further assessed 3, 7, and 42 days after implantation. Overall, overgrowth increased from day 3 to day 7 (p < 0.05). Three and 7 days after implantation, polycation-containing microcapsules induced more reaction than microbeads (p < 0.0001 and p < 0.01). Considering polycation-free beads, barium-alginate induced the weakest reaction. Biocompatibility of microbeads was independent of mannuronic/guluronic acid ratio and molar mass of the alginate. Infiltration was mainly a monocyte/macrophage-rich foreign body reaction, but an eosinophil-containing immunoallergic reaction was also observed. Short-term immunosuppression significantly reduced infiltration in all conditions and up to 42 days after implantation. Biocompatibility after intraportal infusion was best for barium-alginate microbeads and poorest for polycation-containing microcapsules. Short- and long-term overgrowth could be significantly reduced by short-term immunosuppression.

The correlation between portal myofibroblasts and development of intrahepatic bile ducts and arterial branches in human liver.

BACKGROUND/AIMS: The development of the intrahepatic bile ducts most likely requires interactions between epithelial and mesenchymal cells. In view of the epithelial-mesenchymal interactions between portal myofibroblasts (pMFs) and biliary epithelial cells in adult diseases of the bile ducts, we investigated the presence and function of pMFs during the development of intrahepatic bile ducts, as well as the development of intrahepatic branches of the hepatic artery. METHODS: We performed haematoxylin-eosin-stainings and immunohistochemistry for alpha-smooth-muscle actin, cytokeratin 19 and vimentin on serial sections of 45 fetal and postnatal liver biopsies. RESULTS: The mesenchyme of portal tracts in the ductal plate stage devoid of a hepatic artery branch, contained numerous and diffusely scattered pMFs. Portal tracts with a hepatic artery branch were always larger than those without and showed a decreasing number of pMFs. In the remodeling stage, all portal tracts contained a hepatic artery branch, and pMFs were restricted to the periductal mesenchyme. These periductal pMFs disappeared after full incorporation of the bile duct. CONCLUSION: Our findings strongly suggest interactions between pMFs and epithelial cells of the developing bile ducts. The development of the intrahepatic arterial branches always precedes the incorporation of the tubular segments of the ductal plate.

Peribiliary myofibroblasts in biliary type liver fibrosis.

Biliary type liver fibrosis develops as part of the wound healing response to bile duct injury in chronic cholestatic liver diseases. The origin of myofibroblasts accumulating together with extracellular matrix around proliferating bile duct structures (referred to as ductular reaction) in the setting of cholestatic injury, has been investigated mostly in the rat bile duct ligation model. Evidence indicates that hepatic stellate cells undergo a myofibroblastic transition following bile duct ligation and that myofibroblastic hepatic stellate cells disclose chemoattraction towards bile duct structures in cholestatic liver. On the basis of morphological studies, nevertheless, the origin of peribiliary myofibroblasts has also been attributed to the activation and proliferation of portal fibroblasts. Bile duct epithelial cells of the ductular reaction actively contribute to the promotion and regulation of biliary type liver fibrogenesis. They synthesize and release a number of paracrine mediators such as transforming growth factor-beta, connective tissue growth factor, platelet-derived growth factor-BB, and endothelin-1 that target different liver cell types, including hepatic stellate cells and portal fibroblasts. Through these interactions, bile duct epithelial cells and peribiliary myofibroblasts cause periportal fibrosis in cholestatic and also probably other types of liver diseases.

Intrahepatic mast cell population correlates with clinical outcome in biliary atresia.

PURPOSE: To determine if mast cells influence the clinical outcome in biliary atresia (BA), the authors examined the intrahepatic mast cell population in BA. METHODS: Mast cells were identified histochemically using Toludin Blue and immunohistochemically using antimast cell tryptase antibody in formalin-fixed paraffin-embedded sections from 21 cases of BA. Patients were divided into 3 groups; group I (n = 8) with good liver function, group II (n = 8) with moderate liver dysfunction, and group III (n = 5) with severe liver dysfunction. Liver biopsies from patients with choledochal cysts (CDC, n = 5), and normal liver (NL, n = 4) served as controls. The results were compared among the groups. RESULTS: Both histochemical and immunohistochemical methods showed similar data. Mast cells were seen mostly in the portal tracts. Mast cell numbers per medium power field (20 x magnification) were higher in BA than in the controls (15. 03 +/- 2.25 v 3.85 +/-.65, [mean +/- SEM], P <.05, BA v CDC; 15.03 +/- 2.25 v 1.73 +/-.06, [mean +/- SEM], P <.05, BA v NL, immunohistochemical data). Clinical correlation showed an association between higher mast cell number and liver dysfunction (32.62 +/-.80 v 8.52 +/-.87 [mean +/- SEM], group III v group I; P <. 05, immunohistochemical data). CONCLUSION: Increased mast cell population in BA adversely affects liver function and raises the possibility that type I allergic reaction may play role in the pathology of BA.

Position-dependent activity of alpha -fetoprotein enhancer element III in the adult liver is due to negative regulation.

alpha-Fetoprotein (AFP) transcription is activated early in hepatogenesis, but is dramatically repressed within several weeks after birth. AFP regulation is governed by multiple elements including three enhancers termed EI, EII, and EIII. All three AFP enhancers continue to be active in the adult liver, where EI and EII exhibit high levels of activity in pericentral hepatocytes with a gradual reduction in activity in a pericentral-periportal direction. In contrast to these two enhancers, EIII activity is highly restricted to a layer of cells surrounding the central veins. To test models that could account for position-dependent EIII activity in the adult liver, we have analyzed transgenes in which AFP enhancers EII and EIII were linked together. Our results indicate that the activity of EIII is dominant over that of EII, indicating that EIII is a potent negative regulatory element in all hepatocytes except those encircling the central veins. We have localized this negative activity to a 340-bp fragment. This suggests that enhancer III may be involved in postnatal AFP repression.

The canals of Hering and hepatic stem cells in humans.

Small, extraportal, hepatic parenchymal cells, positive for biliary-type cytokeratins, may represent hepatic stem cells, canals of Hering (CoH), and/or ductal plate remnants. We evaluated these cells 3 dimensionally in normal human liver and massive necrosis. Tissues from normal human livers and from 1 liver with acetaminophen-induced massive necrosis were serially sectioned, immunostained for cytokeratin 19 (CK19), and sequentially photographed. Images were examined to determine 3-dimensional relationships among CK19-positive cells. Immunostains for other hepatocyte and progenitor cell markers were examined. In normal livers, intraparenchymal CK19-positive cells lined up as linear arrays in sequential levels. One hundred of 106 (94.3%) defined, complete arrays within levels examined, most having 1 terminus at a bile duct, the other in the lobule, beyond the limiting plate. In massive necrosis, there were 767 individual CK19-positive cells or clusters around a single portal tract, 747 (97.4%) of which were spatially related forming arborizing networks connected to the interlobular bile duct by single tributaries. C-kit was positive in normal CoH. CK19 co-expressed with HepPar1, c-kit, and alpha-fetoprotein (AFP) in parenchymal cells in massive necrosis. Small, extraportal, biliary-type parenchymal cells represent cross-sections of the CoH that radiate from the portal tract, usually extending past the limiting plate into the proximate third of the hepatic lobule. The 3-dimensional structure of ductular reactions in massive necrosis suggests that these reactions are proliferations of the cells lining the CoH. Therefore, the CoH consist of, or harbor, facultative hepatic stem cells in humans.

Experimental studies on the stem cell concept of liver regeneration. II.

There is increasing evidence in support of the existence of a hepatic stem or progenitor cell system as well as its participation in the physiological as well as reparative regeneration of the liver and in carcinogenesis. In the present paper it will be demonstrated that under the condition of a simultaneous intoxication with allyl alcohol and tetrachlorcarbon a regenerative process occurs, which is composed of two distinct partial processes: 1. a "proliferative wave" of the persisting, non-necrotic differentiated hepatocytes of the acinus, starting at 24 h after intoxication and ending after >48 h, and 2. a proliferation of cells at the rim of the portal fields, which afterwards enter the acinus and differentiate into hepatocytes, starting at 48 h after intoxication. These results support the participation of a stem or progenitor cell system in the reparative regeneration of the liver.

Microvascular organization of sympathetic ganglia, with special reference to small intensely-fluorescent cells.

The vascular organization of sympathetic ganglia has been reviewed in relation to type II small intensely-fluorescent (SIF) cells. These cells are considered to be secretory cells forming large clusters surrounded by fenestrated capillaries. Immunohistochemical studies have revealed the existence of many kinds of peptides, in addition to catecholamines, in type II SIF cells. These transmitters are thought to enter the bloodstream, perfuse the adjacent ganglionic tissue, and modify the synaptic transmission and activity of sympathetic ganglionic neurons. Several authors reported portal-like intraganglionic microcirculation through which type II SIF cells participate in modulation of the principal ganglionic neurons. One large intraganglionic portal sinus located between SIF cells and principal ganglionic neurons was also reported in the inferior mesenteric ganglion. However, some authors claimed that transmitters could be absorbed through numerous capillary anastomoses, without any portal system in the superior cervical ganglion. It is noticed that the number, size, and partition of SIF-cell clusters are variable in different ganglia and different animal species. It is important to interpret the functional and morphological correlates of intraganglionic microcirculation based on the species and location of ganglia.

Fibrous trabeculae in the liver of alligator (alligator mississippiensis).

In the mature alligator, fibrous trabeculae run from the portal areas and capsule through the hepatic parenchyma. The extent of these trabeculae becomes clear only after staining for collagen with, for example, Fast green or Picrosirius red. The trabeculae are less well developed in young caiman. The alligator's liver might use the trabeculae to withstand thrashing of the body.

A rapid method to study heterogeneous gene expression in liver by direct assay of messenger RNA from periportal and perivenous cell lysates.

To study zonation of liver gene expression, we obtained periportal or perivenous rat liver cell lysates virtually devoid of nuclear material by site-directed digitonin infusion in situ. Total RNA was isolated, messenger RNAs were reverse transcribed and complementary DNAs were assayed after polymerase chain reaction-mediated amplification. The zonal distribution of messenger RNAs of alcohol dehydrogenase (little zonation), glutamine synthetase (perivenous) and cytochrome P-450 2E1 (perivenous) messenger RNAs, as analyzed by this technique, were found to be similar to the distribution of corresponding apoproteins. Using appropriate primers or complementary DNAs, zonation of many different messenger RNAs can be determined from the same sample by this simple and rapid method.

Morphometric inflammatory cell analysis of human liver allograft biopsies.

To quantitate the inflammatory cell response within the portal tracts of liver allografts during acute rejection, we retrospectively and in a blinded fashion reviewed 431 biweekly, protocol, core allograft biopsies in 58 consecutive adult recipients. Following the determination of the cross-sectional area of each portal tract, the number of eosinophils, neutrophils, and lymphocytes therein was tabulated. The average number, percentage, and density of each type of portal inflammatory cell were calculated for each biopsy. Each biopsy was prospectively and independently classified as either associated (REJ+) or not associated (REJ-) with acute rejection. Acute rejection consisted of simultaneous allograft dysfunction and qualitative pathologic findings of acute rejection. Biopsies obtained during periods of normal allograft function or during episodes of dysfunction due to other causes were classified as not associated with rejection (REJ-). Ninety biopsies were classified as associated with acute rejection (REJ+) while 241 biopsies were classified as not associated with acute rejection (REJ-). In general, the average portal-tract number, percentage, and density of all inflammatory cells were significantly increased in biopsies associated with acute rejection. In contrast, only the portal-tract eosinophil values were consistently predictive of acute rejection following receiver-operating characteristic curve analysis (sensitivity = 82-86%, specificity = 91-92%). This quantitative method of allograft assessment appears to improve the objectivity of the serial biopsy protocol. By using this method, we found the eosinophil's appearance within the portal tracts to be a dependable indicator of acute rejection.

Biochemical and morphological studies on perivenous and periportal liver parenchymal cells from female rats isolated by digitonin-collagenase method.

Periportal (pp) or perivenous (pv) liver parenchymal cells from female adult Uje: WIST rats were isolated after retro- or antegrade digitonin infusion followed by collagenase perfusion in the opposite direction. The morphological results revealed a distinct acinar-related destruction of the pv- or pp-zone by digitonin. The remaining cells of the respective other zone showed a good structural maintenance. After subsequent conventional collagenase perfusion the yield, viability and structural integrity of the isolated hepatocytes were high. The zonal cell separation was indicated by significant differences in the pp marker glucose-6-phosphatase and the pv marker glutamine synthetase found in the isolated pp or pv cell populations. Under our experimental conditions including the use of female rats, the alanine aminotransferase and glutamate dehydrogenase as well as ethylmorphine N-demethylase and ethoxycoumarin O-deethylase activities were evenly distributed in both preparations. Under stimulating conditions the capacity for urea synthesis was similar in both pv and pp cells.