The number of alveoli in the human lung

The number of alveoli is a key structural determinant of lung architecture. A design-based stereologic approach was used for the direct and unbiased estimation of alveolar number in the human lung. The principle is based on two-dimensional topology in three-dimensional space and is free of assumptions on the shape, size, or spatial orientation of alveoli. Alveolar number is estimated by counting their openings at the level of the free septal edges, where they form a two-dimensional network. Mathematically, the Euler number of this network is estimated using physical disectors at a light microscopic level. In six adult human lungs, the mean alveolar number was 480 million (range: 274-790 million; coefficient of variation: 37%). Alveolar number was closely related to total lung volume, with larger lungs having considerably more alveoli. The mean size of a single alveolus was rather constant with 4.2 x 10(6) microm3 (range: 3.3-4.8 x 10(6) microm3; coefficient of variation: 10%), irrespective of the lung size. One cubic millimeter lung parenchyma would then contain around 170 alveoli. The method proved to be very efficient and easy to apply in practice. Future applications will show this approach to be an important addition to design-based stereologic methods for the quantitative analysis of lung structure.     

Studies of liver repopulation using the dipeptidyl peptidase IV-deficient rat and other rodent recipients: Cell size and structure relationships regulate capacity for increased transplanted hepatocyte mass in the liver lobule

The feasibility of liver repopulation with hepatocytes has been shown, although clinical applications demand significant hepatic replacement. To show whether portal vascular bed in large animals could accomodate a greater cell number, we analyzed liver repopulation in syngeneic Fischer 344 rats deficient in dipeptidyl peptidase IV. This system allowed localization of transplanted normal hepatocytes in liver or various ectopic sites, as well as dual studies for analysis of gene expression. Interestingly, the product of a dipeptidyl peptidase IV substrate inactivated bile canalicular adenosine triphosphatase (ATPase) activity in normal but not in dipeptidyl peptidase IV- deficient rats, which allowed localization of dipeptidyl peptidase IV- deficient hepatocytes in normal rat liver for additional reversed transplantation systems. Further studies with genetically marked cells showed that because of the size difference between hepatocytes and portal vein radicles, intrasplenically transplanted cells were distributed in periportal areas (zone 1) in mice, whereas in larger animals (rats or rabbits) cells were also distributed downstream to midlobular (zone 2) or perivenous (zone 3) areas. Transplantation of an escalating number of hepatocytes showed that adult rats tolerated intrasplenic injection of a large cell number in single sessions (up to 1 X 10⁸, approximately 10% to 15% of the host hepatocyte mass). Morphometric analysis of recipient livers showed survival of a significantly greater cell number with incorporation in host liver plates. At 4 weeks, transplantation of 2 x 10⁷ hepatocytes into adult rats led to a survival of 1.4 +/- 1.0 x 10⁶ transplanted cells/cm3 liver, whereas after transplantation of 5 x 10⁷ cells or 7.5 x 10⁷ cells, the number of surviving transplanted cells in the liver significantly increased to 4.1 +/- 1.4 x 10⁶ transplanted cells/cm3 liver (mean, 2.9-fold; P<.003) and 5.5 +/- 1.3 x 10⁶ transplanted cells/cm3 liver (mean, 3.9-fold; P<.003), respectively. When cells were injected in greater numbers, transplanted hepatocytes retained normal function and produced more serum albumin or hepatitis B surface antigen in deficient hosts. These data indicate the feasibility in larger animals of significant liver repopulation with hepatocyte transplantation. Use of dipeptidyl peptidase IV-deficient rats should help further analysis of mechanisms in liver repopulation. (Hepatology 1996 Mar;23(3):482-96)     

Skin tests with native, depigmented and glutaraldehyde polymerized allergen extracts.

INTRODUCTION: Dose-response skin prick tests are an important tool to standardise allergen extracts and to evaluate changes in skin test response as a consequence of allergen modifications. OBJECTIVES: To evaluate in vivo and in vitro characteristics of 3 different types of extracts of Phleumpratense, Olea europaea, Parietaria judaica and Dermatophagoides pteronyssinus. MATERIAL AND METHODS: Three types of extracts were used: native unmodified extracts (N), depigmented extracts (DP) (extracts subjected to a mild acid treatment under controlled conditions and dialysis), and a depigmented glutaraldehyde polymerised extract (DPP). Adult patients were skin tested in duplicate with the 3 types of extracts. The dose-response relationship between the geometric mean of the wheal areas and the allergen concentrations was calculated for each patient using regression line analysis. The amount of freeze-dried allergen preparation needed to produce the same wheal size as histamine was calculated in each patient (individual 10 HEP) and for each of the 3 types of extracts. In vitro analysis consisted of major allergen determinations and specific IgE and IgG inhibitions. RESULTS: The respective 10 HEP values for N, DP and DPP preparations were 0.20 mg, 0.15 and 2.11 for D. pteronyssinus. For P. pratense, these values were 0.02 mg, 0.02 and 0.99; for O. europaea 0.15, 0.44 and 4.9; and for P. judaica 0.01, 0.008 and 1.78 mg. CONCLUSIONS: The polymerised depigmented extracts are significantly less allergenic than the corresponding native and depigmented extracts. This could provide a greater safety margin for the administration of higher doses of immunotherapy in a shorter period of time.     

Involvement of the Fas system in liver allograft rejection

OBJECTIVE: Recent studies suggest that apoptosis is an important mechanism of cell death in the rejection of liver allografts and that this process is mediated via Fas. The aim of this study was to analyze the expression of the Fas system during the liver allograft rejection and its evolution after treatment. METHODS: We evaluated 14 patients with liver allograft rejection before and after treatment. Fas immunostaining was performed by the labeled streptavidin-biotin peroxidase method using a 200-fold dilution of a monoclonal antibody. Assessment of apoptosis was determined by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) technique on deparaffined liver samples. Serum levels of soluble Fas antigen (sFas) were detected by an enzyme immunoassay procedure. Twelve liver transplant patients without allograft rejection were analyzed as a control group. RESULTS: The number of hepatocytes expressing Fas antigen, the percentage of apoptotic hepatocytes, and the sFas levels were higher in patients with liver allograft rejection than in controls (27.9+/-23.1% vs 1.4+/-1.2%, p < 0.001; 2.2+/-0.9% vs 1.0+/-0.1%, p = 0.02; 24.2+/-39.6 vs 2.8+/-4.0 IU/ml, p = 0.03, respectively). There was a correlation between the levels of sFas, AST (r = 0.86, p < 0.001), ALT (r = 0.78, p = 0.02), and gamma-globulin levels (r = 0.86, p < 0.001). After the rejection treatment we found a significant decrease in the Fas antigen expression (18.6+/-13.3%, p < 0.05), TUNEL index (0.2+/-0.4, p < 0.05), and levels of sFas (9.9+/-30.25 IU/ml, p = 0.005). CONCLUSIONS: 1) The demonstration of hepatocytes with Fas antigen expression and the labeling of the nuclei by the TUNEL assay suggest that apoptosis mediated by the Fas system plays a role in the pathogenesis of liver allograft rejection. 2) The Fas expression and the sFas levels decreased in patients with treatment response.     

Platelet-leucocyte aggregates form in the mesenteric vasculature in patients with ulcerative colitis

BACKGROUND AND AIMS: Inflammation and thrombosis are closely related processes, which may play a role in the pathogenesis, as well as complications, of inflammatory bowel disease (IBD). Platelet activation and platelet-leucocyte aggregation are increased and platelet aggregation is known to occur in the mesenteric vasculature in IBD. The aims of this study were to test the hypotheses that platelet-leucocyte aggregation, platelet activation and neutrophil activation occur in the mesenteric vessels of patients with ulcerative colitis (UC). PATIENTS AND METHODS: Platelet-leucocyte aggregates (PLAs), platelet activation (P-selectin expression) and neutrophil activation (L-selectin expression, which decreases on neutrophil activation) were assessed flow cytometrically in mesenteric arterial, and venous blood sampled in eight patients with UC and eight controls with colonic carcinoma undergoing intestinal resections. RESULTS: In the patients with UC, the number of PLAs in the mesenteric vein exceeded that in the artery, the median rise being 38% (P=0.02). In UC, arterial PLA numbers were 0.17 (0.02-0.32) (median, range) x 10(9)/l versus venous 0.26 (0.09-1.6) x 10(9)/l (P=0.02). The median percentage increase was 45%. Mesenteric PLA formation did not occur in patients with colonic carcinoma [arterial 0.06 (0.03-0.49) x 10(9)/l vs. venous 0.05 (0.02-0.35) x 10(9)/l; P=0.55]. The median percentage change was +45% for UC patients and -5% for controls. No arteriovenous gradient was observed in P-selectin expression, but L-selectin expression (arbitrary units), increased in the mesenteric vasculature of the UC patients [arterial 839 (503-995), venous 879 (477-1035); P=0.03] and fell in those with colonic carcinoma [arterial 900 (660-959), venous 850 (546-957); P=0.04]. The median percentage change was +4% for UC and -7% for controls. CONCLUSION: The finding of increased numbers of PLAs in the venous mesenteric circulation supports the hypothesis that activated vascular endothelium stimulates PLA formation in UC.     

Hybrid artificial liver support system for treatment of severe liver failure

AIM: To construct a novel hybrid artificial liver support system (HALSS) and to evaluate its efficacy in patients with severe liver failure. METHODS: Hepatocytes were isolated from suckling pig by the modified Seglen's method. Isolated hepatocytes were cultured in a spinner flask for 24 h to form spheroids before use and the functions of spheroids were detected. HALSS consisted of a plasma separator, a hemo-adsorba and a bioreactor with hepatocytes spheroids in its extra-fiber space. HALSS was applied to 10 patients with severe liver failure. The general condition and the biochemical indexes of the patients were studied just before and after the treatment. RESULTS: The number of cells per liver was about 2-4×1010 (mean, 3.1±1.5×1010). The cell viabilities were more than 95%. After 24 h of spheroid culture, most hepatocytes formed spheroids. The levels of albumin and urea in the medium of spheroid culture were higher than those in supernatant of petri dish culture (P= 0.0015 and 0.0001, respectively). The capacity of albumin production and urea synthesis remained stable for more than one wk and declined rapidly after two weeks in vitro. In HALSS group, the duration of HALSS treatment was 6-10 h each time. All patients tolerated the treatment well without any fatal adverse reaction. After HALSS treatment, the general condition, psychic state, encephalopathy and hepatic function of the patients were improved. The survival rate of the HALSS group, Plasmapheresis group and control group was 30% (3/10), 20% (2/10) and 0% (0/10), respectively (P = 0.024). Two weeks after treatment, Tbil and ALT decreased and the PTA level elevated in HALSS group and pasmapheresis group (Pvalue: 0.015 vs0.020, 0.009 vs 0.012 and 0.032 vs 0.041, respectively). But there was no significant change of blood albumin concentration before and after treatment in HALSS group and Plasmapheresis group. CONCLUSION: The HALSS established by us is effective in supporting liver function of patients with severe liver failure.     

Conserved balance of hepatocyte nuclear DNA content in mononuclear and binuclear hepatocyte populations during the course of chronic viral hepatitis

AIM: To analyze the percentages of hepatocytes with increased nuclear DNA content, i.e., tetraploid (4n) and octoploid (8n) nuclei, and then compared mononuclear and binuclear hepatocyte populations: METHODS: The percentages of mononuclear diploid (2n), 4n, and 8n hepatocytes and those of binuclear 2×2n, 2×4n, and 2×8n hepatocytes were determined with a method that can simultaneously measure hepatocyte nuclear DNA content and binuclearity in 62 patients with chronic hepatitis B or C. The percentage of 4n and 8n hepatocytes in the mononuclear hepatocyte population was compared with the percentage of 2×4n and 2×8n hepatocytes in the binuclear hepatocyte population. RESULTS: The percentages of 4n and 8n hepatocytes in mononuclear hepatocytes and 2×4n and 2×8n hepatocytes in binuclear hepatocytes were similar, regardless of the activity or fibrosis grade of chronic hepatitis and regardless of the infecting virus. CONCLUSION: The distribution of nuclear DNA content within mononuclear and binuclear hepatocyte populations was conserved during the course of chronic viral hepatitis.     

In vivo hepatic HB-EGF gene transduction inhibits Fas-induced liver injury and induces liver regeneration in mice: a comparative study to HGF

BACKGROUND/AIMS: It is unknown whether heparin-binding EGF-like growth factor (HB-EGF) can be a therapeutic agent, although previous studies suggested that HB-EGF might be a hepatotrophic factor. This study explores the potential of hepatic HB-EGF gene therapy in comparison with HGF. METHODS: Mice received an intraperitoneal injection of the agonistic anti-Fas antibody 72 h after an intravenous injection of either adenoviral vector (1x10(11) particles) expressing human HB-EGF (Ad.HB-EGF), human HGF (Ad.HGF) or no gene (Ad.dE1.3), and were sacrificed 24 or 36 h later to assess liver injury and regeneration. RESULTS: Exogenous HB-EGF was predominantly localized on the membrane, suggesting the initial synthesis of proHB-EGF in hepatocytes. The control Ad.dE1.3-treated mice represented remarkable increases in serum ALT and AST levels and histopathologically severe liver injuries with numerous apoptosis, but a limited number of mitogenic hepatocytes. In contrast, the liver injuries and apoptotic changes were significantly inhibited, but the mitogenic hepatocytes remarkably increased, in both the Ad.HB-EGF- and Ad.HGF-treated mice. More mitogenic hepatocytes and milder injuries were observed in the Ad.HB-EGF-treated mice. CONCLUSIONS: HB-EGF has more potent protective and mitogenic effects for hepatocytes than HGF, at least for the present conditions. In vivo hepatic HB-EGF gene transduction is therapeutic for Fas-induced liver injury.     

In situ cellular analysis of alpha-fetoprotein gene expression in regenerating rat liver after partial hepatectomy

Cellular analysis of hepatic alpha-fetoprotein gene expression in normal adult rat and during regeneration induced by partial hepatectomy was performed at the cellular level by in situ hybridization using 35S-labeled complementary DNA probes and immunoperoxidase techniques. In normal adult rat liver sections, a few alpha-fetoprotein mRNA-cDNA hybrids are detected over all hepatocytes. No protein is detected with routine immunoperoxidase methods. However, after in vivo colchicine blockade of alpha-fetoprotein secretion, 10 to 20% alpha-fetoprotein-positive hepatocytes are observed. In regenerating livers, at 2,6 and 24 hr (before and at the time of the peak of DNA synthesis in the periportal zones), a rise of the nuclear signal level is observed selectively in periportal hepatocytes, without modification of the cytoplasmic signal. At 48 hr (when most hepatocytes have completed at least one replicative cycle), almost all hepatocytes throughout the liver lobule display a rise of the nuclear (2- to 3-fold) and cytoplasmic (1.5- to 2-fold) signal level compared to nonoperated rats. These data show that all hepatocytes in the adult liver express a small number of alpha-fetoprotein mRNA sequences; they appear to be translated in protein whose secretion can be blocked by colchicine. The moderate increase in alpha-fetoprotein gene expression induced by liver regeneration takes place in all hepatocytes, in apparently two distinct steps: a very early nuclear accumulation of alpha-fetoprotein mRNA sequences and a late cytoplasmic accumulation of alpha-fetoprotein mRNA molecules.     

Protective effect of estradiol on hepatocytic oxidative damage

AIM: To examine the protective effect of estradiol on the cultured hepatocytes under oxidative stress.METHODS: Hepatocytes of rat were isolated by using perfusion method, and oxidative stress wes induced by a serum-free medium and FeNTA. MDA level was determined with TBA method. Cell damage was assessed by LDH assay. Apoptosis of hepatocytes was assessed with cytoflowmetric analysis. Expression of Bcl-xl in cultured hepatocytes was detected by Western blot.The radicalscavenging activity of estradiol was valued by its ability to scavenge the stable free radical of DDPH.RESULTS: Oxidative stress increased LDH (from 168 ± 25 x10-6 IU. cell 1 to 780 ± 62 x 10-6 IU. cell-1 ) and MDA(from 0.28 ±0.07 x 10-6 nmol. cell-1 to 1. 35 ± 0.12 × 10-6 nmol. cell-1 ) levelsin cultured hepatocyte, and estradiol inhibited both LDH andMDA production in a dose dependent manner. In thepresence of estradiol t0-6 mol. L-1, 107 mol. L-1 and 10-8 mol.L-1 ,the LDH levels are 410 ± 53 × 10-6 IU. cell-1 ( P ＜ 0.01 vsoxidative group), 530 ± 37 × 10-6 IU. cell-1 ( P ＜ 0. 01 vsoxidative group), 687 ± 42 x 10-6 IU. cell-1 ( P ＜ 0.05 vsoxidative group) respectively, and the MDA level are 0.71 ±0.12 x 10-6 nmol. cell-1 ( P ＜ 0.01 vs oxidative group), 0.97 ± 0.11 × 10-6 nmol. cell-1 ( P ＜ 0.01 vs oxidative group) and 1.27 ±0. 19 x 10-6 nmol. cell-1 respectively. Estradiol suppressedapoptosis of hepatocytes induced by oxidative stress,administration of estradiol (10-6 mol/ L)decreased theapoptotic rate of hepatocytes under oxidative stress from 18.6 ± 1.2% to 6.5 ± 2.5%, P ＜ 0.01. Bcl-xl expression wasrelated to the degree of liver cell damage due to oxidativestress, and estradiol showed a protective action.CONCLUSION: Estmdiol protects hepatocytes from oxidativedamage by means of its antioxidant activity.     

An improved ex vivo method of primary porcine hepatocyte isolation for use in bioartificial liver systems

INTRODUCTION: Primary porcine hepatocytes are commonly, used in bioartificial liver devices and for in vitro studies of hepatocyte function. Although in vivo isolation of porcine hepatocytes can give high yield and viability, such methods are time-consuming and expensive, requiring specialist surgical facilities. AIM: To develop a simple, low-cost, high viability, high yield, reproducible ex vivo method for obtaining functional porcine hepatocytes for use in bioartificial liver systems. METHODS: Weanling piglets (12 kg) were killed with pentobarbitone sodium, the infra-hepatic inferior vena cava was clamped and the supra-hepatic inferior vena cava cannulated. The whole liver was retrogradely perfused in situ with cold saline and excised, followed by an ex vivo open-loop and re-circulating perfusion method (at 37 degrees C) in five steps. The liver was disrupted, sequentially filtered in washing buffer, purified by centrifugation and resuspended in Williams E medium. Viability and cell number were assessed using trypan blue exclusion. The cells were subsequently cultured in serum-free chemically-defined medium and function was assessed. RESULTS: The time interval from when the animals were killed to the final cell wash was 105+/-5 min (n = 20). Cell viability was 85+/-6% with a yield of (2.4+/-0.5) x 10(10) from 12+/-1 kg piglets using 0.03% (w/v) collagenase (n = 20). Hepatocytes from all isolations were successfully plated and grown in monolayer culture. In freshly isolated hepatocytes (day 0) total protein content (TP) was 1.2+/-0.1 mg/10(6) cells (n = 5) and 1.2+/-0.3 mg/10(6) cells (n = 5) for day 2 monolayer cultures, corresponding to approximately 9x10(6) hepatocytes per dish. The percentage of total LDH released into the medium was 13+/-4% for day 0 and 8+/-4% at day 2; conversely, intracellular LDH activities were 87+/-4% and 92+/-4% of the total, respectively. The urea synthesis rate was 196+/-36 nmol/h/mg total protein at day 0 (n = 5) and 292+/-62 nmol/h/mg protein (n = 9) at day 2. The total P450 content was 99+/-11 pmol/mg total protein for fresh cells (n = 5) and maintained at 89+/-35 pmol/mg total protein in day 2 cultures. CONCLUSIONS: This ex vivo method provides a high viability, high yield, cost-effective and rapid technique for isolating functional porcine hepatocytes with high plating efficiency, which compares favourably with results obtained using complex in vivo techniques.     

Mobilization of peripheral blood hematopoietic stem cells following liver resection surgery

BACKGROUND/AIMS: Stem cells are characterized by plasticity, namely the ability of interchanging between various tissue and organs. In this regard, many studies have demonstrated the presence of antigenic structures relevant to the hematopoietic stem cell on hepatocytes, thus suggesting that in certain conditions liver cells may derive from the hematopoietic compartment. The aim of this study has been to verify whether surgical liver resection can activate bone marrow, by mobilizing peripheral blood hematopoietic stem cells (CD34+ cells) putatively able to induce liver repopulation. METHODOLOGY: White blood cell and CD34+ cell count was determined at baseline (before surgery) and then monitored in the postoperative period in 13 patients undergoing liver resection (in most cases because of malignant, primary or secondary liver diseases) and, as a control group, in 12 patients affected with other diseases requiring abdominal surgery, but not liver resection. Moreover, to assess the basal value of circulating CD34+ cells, 50 healthy blood donors were included in the study. The CD34+ cell count has been carried out by flow cytometry, by applying conventional protocols. RESULTS: Patients, as altogether considered, showed at baseline a significantly higher white blood cell count as compared to healthy controls (7.41+/-2.89 x 10(3)/microL vs. 6.00+/-1.37 x 10(3)/microL, P<0.01), as opposed to the CD34+ cell count, the results of which were significantly lower (2.8+/-1.8/microL vs. 4.1+/-1.9/microL, P<0.01). The increase of CD34+ cells was significantly higher in patients following liver resection as compared to others (+6.5+/-4.1/microL vs. +0.7+/-1.4/microL, P<0.001), whereas the variation of white blood cell count was not statistically significant (+1.87+/-3.76 x 10(3)/microL vs. + 1.51+/-2.87 x 10(3)/microL). CONCLUSIONS: Our results indicate that hepatic injury caused by extensive liver resection may constitute a trigger to the mobilization of hematopoietic stem cells putatively able to differentiate into hepatocytes, thus starting the recovery process of liver. These data could open innovative views to the treatment of certain liver diseases (e.g. fulminant hepatic failure), in particular by the administration of hematopoietic growth factors, such as G-CSF or GM-CSF, after the hepatic damage, to contribute, through the activation of the hematopoietic compartment, to a more efficient liver regeneration.     

Management of carbon tetrachloride-induced acute liver injury in rats by syngeneic hepatocyte transplantation in spleen and peritoneal cavity

AIM: Acute hepatitis may seldom have a fulminant course.In the treatment of this medical emergency, potential liver support measure must provide immediate and sufficient assistance to the hepatic function. The goal of our study was to study the adequacy of hepatocyte transplantation (HCTx) in two different anatomical sites, splenic parenchyma and peritoneal cavity, in a rat model of reversible acute hepatitis induced by carbon tetrachloride (CCl4).METHODS: After CCl4 intoxication, 84 male Wistar rats used as recipients were divided in to four experimental groups accordingly to their treatment: Group A (n=24): intrasplenic transplantation of 10&#177;10^6 isolated hepatoo/tes, Group B (n=24):intrapedtoneal transplantation of 20&#215;10^6 isolated hepatocytes attached on plastic microcarriers, Group C (n= 18): intrasplenic injection of 1 mL normal saline (sham-operated controls),Group D (n=18): intraperitoneal injection of 2.5 mL normal saline (sham-operated controls). Survival, liver function tests (LIT) and histology were studied in all four groups, on d 2,5 and 10 post-HCTx.RESULTS: The ten-day survival (and mean survival) in the 4 groups was 72.2% (8.1&#177;3.1), 33.3% (5.4&#177;3.4), 0%(3.1&#177;1.3) and 33.3% (5.4&#177;3.6) in groups A, B, C, D,respectively (PAB＜0.05, PAc＜0.05, P80=NS). In the final survivors, LIT (except alkaline phosphatase) and hepatic histology retumed to normal, independently of their previous therapy. Viable hepatocytes were identified within splenic parenchyma (in group A on d 2) and both in the native liver and the fatty tissue of abdominal wall (in group B on d 5).CONCLUSION: A significantly better survival of the intrasplenically transplanted animals has been demonstrated,Intraperitoneal hepatocytes failed to promptly engraft. A different timing between liver injury and intraperitoneal HCTx may give better results and merits further investigation.     

Presence of disease specific autoantibodies against liver sinusoidal cells in primary biliary cirrhosis

AIM: To investigate the presence of autoantibodies directed against liver sinusoidal cells in primary biliary cirrhosis (PBC).METHODS: Liver biopsies from 21 PBC patients were studied and compared with 12 liver biopsies from disease controls [3 patients with hepatitis B (HBV) virus, 3 patients with hepatitis C virus (HCV), 3 patients with non-alcoholic steatohepatitis and 3 patients with acute alcoholic hepatitis (AAH)]. As healthy controls, we used tissue specimens adjacent to metastatic liver adenocarcinoma. Normal serum was taken from staff members of the unit. The determination of the cell type targeted by autoantibodies present in the patients sera was performed by indirect immunofluorescence (IIF) analysis using paraffin-embedded liver sections as a substrate. Sera from homologous or heterologous PBC patients or sera from the disease control group were used as primary antibodies. The presence of autoantibodies was identified using confocal microscopy.RESULTS: In total, 18/21 (85.7%) PBC patients exhibited positive staining in the sinusoidal cells, 10/21 (47.6%) in lymphocytes, 8/21 (38%) in cholangiocytes and 7/21 (33.3%) in hepatocytes, when homologous serum and fluorescein isothiocyanate-conjugated immunoglobulin type G (IgG) secondary antibody were used. PBC sections incubated with heterologous PBC serum showed reduced staining (20% for sinusoidal cells, 20% for lymphocytes, 20% for cholangiocytes and 13.3% for hepatocytes). When IgM immunoglobulin, instead of IgG, was used as secondary antibody, positive staining was observed in 75% of lymphocytes, 62.5% of cholangiocytes, 37.5% of hepatocytes and 50% of the sinusoidal cells with a much stronger staining intensity. No staining was observed when either normal or PBC sera were used as a primary antibody on liver sections from the disease control group. When PBC sera were incubated with healthy control sections, weak positive staining of cholangiocytes was observed in 3/21 (14.3%) PBC serum samples. Steatohepatitis serum on PBC sections gave a positive staining of some hepatocytes and lymphocytes but no staining on viral hepatitis sections. Incubation with HBV sera stained some hepatocytes, cholangiocytes and intra-sinusoidal or portal lymphocytes of PBC, HBV and AAH patients but not HCV patients.CONCLUSION: In this study, for the first time in diseased liver tissue, we have demonstrated that a large proportion of PBC patients have disease specific autoantibodies against liver sinusoidal cells.     

A quantitative analysis of the liver following ligation of the common bile duct

A quantitative analysis of the liver was performed at intervals of 24, 48, 72 and 96 h and 7, 21 and 50 days following total biliary obstruction (TBO) in the Sprague-Dawley rat. During this period, the liver weight increased from 7.72 +/- 0.51 g (mean +/- SEM) in controls to 24.57 +/- 1.66 g (p less than 0.0005) at 50 days. There was a concomitant reduction in the volume proportion of the liver occupied by liver cells, from 71.37 +/- 1.36% to 21.54 +/- 3.27% (p less than 0.0005), but there were increases in the volume proportions of biliary epithelial cells (BEC) from 0.14 +/- 0.02% to 16.39 +/- 1.12% (p less than 0.0005), of other cell and tissue types from 5.50 +/- 4.89% to 30.73 +/- 2.42% (p less than 0.0005) and of vascular and biliary channel spaces from 22.99 +/- 1.17% to 31.35 +/- 0.87% (p less than 0.0025). In control animals, the liver cell and BEC volume was estimated to be 6240 +/- 360 microns 3 and 100 +/- 10 microns 3, respectively. Following TBO, the liver cell volume was significantly greater than control only from 48-96 h, whereas the BEC increased significantly in volume from 24 to 72 h and then remained approximately 6 times the control value until the end of the period of study. Contrary to the histological appearance and decrease observed in volume proportion, the total liver cell population did not significantly differ from the control value of 8.83 x 10(8) +/- 0.80 x 10(8) cells, other than at 21 days when it increased to 14.90 x 10(8) +/- 1.04 x 10(8) cells (p less than 0.05). When expressed as the number of liver cells/100 g b.wt., an increase from the control value of 4.37 x 10(8) +/- 0.32 x 10(8) cells was observed only at 7 (5.66 x 10(8) +/- 0.42 x 10(8) cells; p less than 0.05) and 21 days (6.94 x 10(8) +/- 0.48 x 10(8) cells; p less than 0.05). This maintenance of liver cell population, following biliary obstruction, at or above the control values matches the clinical observation of preserved liver cell function. The total BEC population in control livers was 1.11 x 10(8) +/- 0.20 x 10(8) cells. A significant increase in this population was observed at 7 days (3.82 x 10(8) +/- 0.62 x 10(8) cells; p less than 0.05) with further increases to 57.90 x 10(8) +/- 6.42 x 10(8) cells (p less than 0.05) at 50 days, 52 times the control value. When expressed as cells/100 g b.wt., similar changes were observed. The results reported here indicate the importance of taking into account the change in the entire organ size and total mass of the cells in question when assessing alterations in their number.     

A monoclonal antibody reactive with human hepatocytes

A monoclonal antibody, RL23/36, reacting preferentially with a determinant expressed on normal human hepatocytes is described. Use of an immunohistochemical technique on frozen sections from a range of 75 human liver biopsy specimens revealed that the determinant detected by RL23/36 was not expressed on hepatocytes from a number of patients with biopsy-proven liver disease. Although a normal staining pattern was observed in 28 of 29 biopsy specimens from patients with no evidence of liver disease, the antibody did not bind to hepatocytes in some cases of chronic active hepatitis (2/13), alcoholic liver disease (2/9), haemochromatosis (1/1), cirrhosis (1/2) and liver metastases (2/8). Furthermore, as in a previous study undertaken in the rat, the antibody failed to bind to tumour cells in the single human hepatoma observed in this study. These results suggest that further studies using RL23/36 may shed light on the pathogenesis of a number of liver diseases, including primary hepatocellular carcinoma.     

phosphatase and tensin homolog is a differential diagnostic marker between nonalcoholic and alcoholic fatty liver disease

AIM: To investigate the protein expression of phosphatase and tensin homolog(PTEN) in human liver biopsies of patients with alcoholic and non-alcoholic liver disease.METHODS: PTEN protein expression was assessed by immunohistochemistry in formalin-fixed, paraffinembedded liver sections of patients with non-alcoholic fatty liver disease(NAFLD)(n = 44) or alcoholic liver disease(ALD)(n = 25). Liver resections obtained from 3 healthy subjects candidate for partial liver donation served as controls. Histological evaluations were performed by two experienced pathologists, and diagnoses established based on international criteria. The intensity of the PTEN staining in nuclei was compared between steatotic and non-steatotic areas of each liver fragment analyzed. For each liver specimen, the antibody-stained sections were examined and scored blindly by three independent observers, who were unaware of the patients' clinical history.RESULTS: In healthy individuals, PTEN immunostaining was intense in both the cytoplasm and nuclei of all hepatocytes. However, PTEN was strongly downregulated in both the nucleus and the cytoplasm of hepatocytes from steatotic areas in patients with NAFLD, independently of the disease stage. In contrast, no changes in PTEN protein expression were observed in patients with ALD, regardless of the presence of steatosis or the stage of the disease. The degree of PTEN downregulation in hepatocytes of patients with NAFLD correlated with the percentage of steatosis(r = 0.3061, P = 0.0459) and the BMI(r = 0.4268, P = 0.0043). Hovewer, in patients with ALD, PTEN expression was not correlated with the percentage of steatosis with or without obesity as a confounding factor(P = 0.5574). Finally, PTEN expression level in steatotic areas of ALD patients was significantly different from that seen in steatotic areas of NAFLD patients(P 0.0001).CONCLUSION: PTEN protein expression is downregulated early in NAFLD, but not in ALD. PTEN immunohistochemical detection could help in the differential diagnosis of NAFLD and ALD.     

Computerized tomography in the diagnosis of nonalcoholic fatty liver--comparison with liver biopsy

The frequency of nonalcoholic fatty liver disease (NAFLD) is high, an estimated 10-25% of the population in different countries. Due to the lack of specific symptoms or irregularities in laboratory examinations the major role in diagnosis is played by, apart form liver biopsy, the imaging examinations. AIM: The assessment of the correlation between intensity of fatty liver in computed tomography (CT) and in liver biopsy. INVESTING GROUP AND METHOD: The initial group comprised of 147 patients who had undergone CT of the abdomen. Within this group the range of attenuation coefficient in Hansfield units was measured in certain areas of liver and spleen. Density of the tissues being given (in Hansfield units) the increase of the fat content in the liver above the norm (tKT) was calculated, according to the formula: tKT = [(spleen density in OH units + 4 OH units) - liver density in OH units] x 0.8 = fat above norm in mg/l g of the liver tissue. Out of this group 12 patients (8 men and 4 women, average age 45.9 +/- 14.5 years) with clinical features of NAFLD on clinical indications for liver biopsy were selected. RESULTS: 3 patients the received value of tKT was found to be correct, that is tKT < 0. In these patients the estimated percentage of hepatocytes affected by steatosis (%TB) reached 5-20%. In the subgroup that comprised if the folio wing 6 patients %TB ranged from 30 to 70% and tKT--from 3.2 to 10. In the group of patients with the highest %TB, reaching 100%, the tKT value reached respectively 5, 10.8 and 13.6. The analysis of the relation between %TB and tKT showed a linear correlation of these two parameters. Following the increase of the intensity of the fatty liver in the biopsy the value of tKT rises. CT of the liver with the range of attenuation coefficient marked (in Hansfield units) is a useful tool for quantitative assessment of the fatty pulp.     

Localization of factor VIIIC: antigen in guinea-pig tissues and isolated liver cell fractions

Factor VIIIC:antigen (VIII:CAg) was estimated in guinea-pig tissues by an immunoradiometric assay using a human inhibitor antibody. In homogenized guinea-pig tissues, VIII:CAg was shown to be stable and to be predominantly located in the liver (9 +/- 1.2 units; mean +/- SEM, n = 8). Lesser amounts were detected in spleen (1.3 +/- 0.02 units), lung (0.6 +/- 0.07) and kidney (0.4 +/- 0.06). In isolated liver cell fractions separated by centrifugal elutriation VIII:CAg was mainly detected in the hepatocyte fraction (0.3 +/- 0.07 units/10(8) cells;mean +/- SEM, n = 5) and in lesser amounts in the endothelial (0.02 +/- 0.01 units/10(8) cells) and the Kupffer cell fractions (0.05 +/- 0.02 units/10(8) cells). The liver concentration of VIII:CAg was (0.17 +/- 0.02 units/g) which was 20% of the plasma concentration (0.96 +/- 0.01 units/ml, n = 8) suggesting that VIII:CAg may not be stored in the liver but is rapidly exported following synthesis.     

p53 protein expression in chronic hepatitis C; effect of interferon alpha 2b therapy

BACKGROUND/AIMS: Gene p53 plays an important role in apoptosis or regeneration of damaged tissues. HCV activates gene p53 directly and/or indirectly. In chronic hepatitis, hepatocyte regeneration is regulated by gene p53 and growth factors. The aim of the study was the evaluation of protein p53 expression on hepatocytes in chronic hepatitis C regarding inflammatory activity and fibrosis as well as the influence of interferon alpha 2b (IFNalpha2b) on gene p53 activity. METHODOLOGY: In 24 patients with chronic hepatitis C treated with IFNalpha2b (18MU sc/week/48 weeks), in whom liver biopsies were performed before and after IFNalpha2b treatment. The immunohistochemical method determined protein p53 in formalin-fixed, paraffin-embedded liver tissue sections. The percentage of p53-positive hepatocytes and intensity of protein p53 accumulation were evaluated under a light microscope. RESULTS: IFN treatment caused the increase in the percentage of p53-positive hepatocytes and the intensity of protein p53 accumulation. The highest level of protein p53 expression was observed in IFNalpha2b-treated tissues with the decrease in inflammatory activity and fibrosis. HCV infection elimination did not affect protein p53 expression. We observed the decrease in p53-positive hepatocytes in tissues without improvement of morphological regeneration during IFNalpha2b therapy. CONCLUSIONS: The increase in protein p53 expression can be considered a prognostic marker of the positive effect of IFNalpha2b treatment and HCV elimination. It may result from the effect of the increase in regeneration or apoptosis of HCV infected hepatocytes.