Changes in Ricinus communis lectin binding to the cell surface of human liver cells in culture : The relationship between in vitro cellular aging and the age of the donor

Ricinus communis lectin RCA I-binding sites on the surface of cultured human liver cells have been studied with a view to establishing a relationship between the aging of the cells in vitro and the age of the donors. Fetal and new-born liver cells exhibited two classes of lectin-binding sites in the early in vitro passages, whereas adult cells in the early passages and fetal and new-born cells in the late passages had only one class of binding site. The modifications of the cell surface RCA I-binding sites would appear to occur either following masking of one of the classes of lectin-binding site during aging and/or be caused by a post-translational event rather than by glycoprotein mis-synthesis. A striking relationship was found between the in vitro cellular aging and human age in vivo, which would suggest that fetal human liver cells could prove to be a convenient model for studying the changes occurring during aging in vivo.     

Influence of dietary selenium on the mutagenic activity of perfusate and bile from rat liver, perfused with 1,1-dimethylhydrazine

The mutagenic effect of 1,1-dimethylhydrazine (UDMH) was studied in the liver perfusion/cell culture system. Male Wistar rats, fed a selenium-deficient diet with or without selenium supplementation in the drinking water, were used as liver donors. UDMH caused an increased mutation frequency in Chinese hamster V79 cells exposed in the perfusate. The effect was statistically significant with both selenium-deficient and selenium-supplemented livers. With selenium-deficient livers, a significant mutagenic effect was also obtained when V79 cells were treated with bile collected after the administration of UDMH. Bile flow and bile acid excretion were not affected by UDMH treatment of selenium-deficient or selenium-supplemented livers. There was a tendency towards reduced C-oxygenation of N,N-dimethylaniline in microsomes from selenium-deficient livers perfused with UDMH. The lactate/pyruvate ratio in the perfusate was increased by UDMH, the effect being more pronounced with selenium-deficient than selenium-supplemented livers.     

Generation,characterization and potential therapeutic applications of mature and functional hepatocytes from stem cells

Liver cancer is the sixth most common tumor in the world and the majority of patients with this disease usually die within 1 year. The effective treatment for end‐stage liver disease (also known as liver failure), including liver cancer or cirrhosis, is liver transplantation. However, there is a severe shortage of liver donors worldwide, which is the major handicap for the treatment of patients with liver failure. Scarcity of liver donors underscores the urgent need of using stem cell therapy to the end‐stage liver disease. Notably, hepatocytes have recently been generated from hepatic and extra‐hepatic stem cells. We have obtained mature and functional hepatocytes from rat hepatic stem cells. Here, we review the advancements on hepatic differentiation from various stem cells, including hepatic stem cells, embryonic stem cells, the induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, and probably spermatogonial stem cells. The advantages, disadvantages, and concerns on differentiation of these stem cells into hepatic cells are highlighted. We further address the methodologies, phenotypes, and functional characterization on the differentiation of numerous stem cells into hepatic cells. Differentiation of stem cells into mature and functional hepatocytes, especially from an extra‐hepatic stem cell source, would circumvent the scarcity of liver donors and human hepatocytes, and most importantly it would offer an ideal and promising source of hepatocytes for cell therapy and tissue engineering in treating liver disease. J. Cell. Physiol. 228: 298–305, 2013. © 2012 Wiley Periodicals, Inc.     

Effect of transplantation of thymus, bone marrow and spleen cells on the regenerative processes in pathologically changed liver]

In tests on CBAXC57BL mice with experimental hepatitis, induced by carbon tetrachloride, the transplantation of cells from the lymphoid organs of healthy donors intensified the repairing process in the recipient pathologically changed liver. The most pronounced normalization was seen in the liver of the animals given thymocytes which suggests a deficit of these cells in experimental toxic hepatitis and indicates a definite role of the thymus in the repairing processes of the damaged liver.     

Plasmodium berghei: preparation of rat hepatic cell suspensions that include infective exoerythrocytic schizonts.

Employing an enzymatic method to dissociate rat liver, we prepared suspensions of liver cells from rats infected with sporozoites of Plasmodium berghei 3 to 10, 18 to 28, or 29 to 36 hr prior to liver dissociation. These suspensions of liver cells included hepatocytes, Kupffer cells, fibroblasts, and unidentified cells, as well as hepatocytes infected with exoerythrocytic schizonts (HEX) of P. berghei. These HEX were infective for recipient rodents when inoculated intraperitoneally into the recipients. The number of infective HEX present in the liver cell suspensions was quantitated by varying the number of HEX inoculated into recipients. This infectivity assay made it possible to compare the numbers of HEX in suspensions of liver cells from different donor rats. Infective HEX were obtained from donor rats in 35 of 41 experiments. The greatest number of infective HEX was obtained from donors injected with sporozoites 18 to 28 hr prior to liver dissociation. For morphological observation of mature HEX in cell suspensions, hepatic cells were prepared from donors infected with sporozoites 48 hr prior to liver dissociation. For experimental purposes, the preparation of infective HEX in suspensions of liver cells is superior to the preparation of infective HEX in liver fragments, because it is possible to quantitate the number of HEX which are present either visually or by means of the infectivity assay.     

Effect of the spleen cells from partially splenectomized and partially hepatectomized mice on the proliferation of hepatocytes in the regenerating liver of syngeneic recipients

Cells of mouse spleen obtained 48 h after foster splenectomy, foster hepatectomy, resection of 2/3 of spleen or 36 h after resection of 2/3 of liver were introduced intravenously into partially hepatectomized (resection of 2/3 or 1/4 of liver) syngeneic recipients. Cells of regenerating spleen sharply inhibited the mitotic activity of cells of the recipient liver following resection of 1/4 of liver 48 h after the operation and introduction of cells. Inhibition proved to be dose-dependent: it became apparent when 30 million cells were introduced, increased at a dose of 60 million cells and remained at the same level at higher doses. Division of hepatocytes after resection of 1/4 of liver was inhibited by spleen cells taken in the donors 36 h after partial hepatectomy. Spleen cells of intact and pseudo-operated donors had no such ability. Introduction of 60 million of cells of the regenerating spleen and of the spleen of partially hepatectomized animals into recipients with resection of 2/3 of liver did not inhibit reliably the division of hepatocytes, thus indicating the dependence of inhibition on the level of suppressors in the organism. Resection of a major part of liver was accompanied by a greater decrease in the activity of endogenous suppressors which could not be recovered by the introduced cells. Inhibition of cell division by suppressors was not organ specific. Suppressors inhibited proliferation in liver irrespective of the site of operation.     

Impact of Donation Mode on the Proportion and Function of T Lymphocytes in the Liver

Background

Liver T-cells respond to the inflammatory insult generated during organ procurement and contribute to the injury following reperfusion. The mode of liver donation alters various metabolic and inflammatory pathways but the way it affects intrahepatic T-cells is still unclear.

Methods

We investigated the modifications occurring in the proportion and function of T-cells during liver procurement for transplantation. We isolated hepatic mononuclear cells (HMC) from liver perfusate of living donors (LD) and donors after brain death (DBD) or cardiac death (DCD) and assessed the frequency of T-cell subsets, their cytokine secretion profile and CD8 T-cell cytotoxicity function, responsiveness to a danger associated molecular pattern (High Mobility Group Box1, HMGB1) and association with donor and recipient clinical parameters and immediate graft outcome.

Results

We found that T-cells in healthy human livers were enriched in memory CD8 T-cells exhibiting a phenotype of non-circulating tissue-associated lymphocytes, functionally dominated by more cytotoxicity and IFN-γ-production in DBD donors, including upon activation by HMGB1 and correlating with peak of post-transplant AST. This liver-specific pattern of CD8 T-cell was prominent in DBD livers compared to DCD and LD livers suggesting that it was influenced by events surrounding brain death, prior to retrieval.

Conclusion

Mode of liver donation can affect liver T-cells with increased liver damage in DBD donors. These findings may be relevant in designing therapeutic strategies aimed at organ optimization prior to transplantation.     

Specificities of heterologous antisera against human leukaemia cells. 2. Reactions against fetal liver cells and absorption studies with fetal tissue.

Rabbit or goat antisera directed to ALL and AML cells were investigated in cytotoxicity tests with fetal liver cells as targets. After absorption with erythrocytes and spleen cells from allogenic donors the antisera killed fetal liver cells. There was no reaction with remission leukocytes or blood leukocytes from normal donors. Treatment with fetal tissue removed the activity of the AML and ALL antisera against ALL cells but not of the AML antisera against AML cells. This indicates the existence of at least two antigens on the surface of AML cells, one antigen is common with ALL cells and of fetal origin and another one seems to be characteristic of AML cells and not of fetal origin. Because treatment with fetal tissue removed all activity of the ALL antisera it can be assumed that leukaemia-associated antigens on ALL cells are of fetal origin.     

Studies of the T-lymphocytes resident in the spleens of thymectomized, irradiated, bone marrow-reconstituted (TXB) mice.

The T-lymphocytes resident in the spleens of thymectomized, lethally irradiated mice that had been reconstituted with syngeneic bone marrow (TXB) were characterized. Both recently reconstituted N-TXB, (approximately 3 weeks after bone marrow injection) and aged (>6 months after reconstitution) A-TXB animals were studied. The T-lymphocytes from spleens of recently reconstituted N-TXB donors did not respond to PHA but did react significantly to Concanavalin A (Con A). The lack of PHA sensitivity was not due to dilution of reactive cells by other cell types. Removal of adherent cells, likewise, did not restore N-TXB spleen cell PHA responsiveness. N-TXB splenic T-cells were cortisone resistant. N-TXB spleen cells by themselves did not cause a graft vs host response. However, N-TXB spleen cells amplified the graft vs host response of normal lymph node cells but not N-TXB lymph node cells. Addition of cyclic GMP enhanced [³H]thymidine uptake of N-TXB spleen cells caused by Con A. N-TXB spleen cells were exclusively spleen seeking. The Con A reactive cell within N-TXB spleens was demonstrated to be of donor origin. Fetal liver as well as syngeneic bone marrow contained cells capable of reconstituting the Con A response. Spleen cells from aged. (>6 months) A-TXB were found to be PHA sensitive. Competitive inhibition assays measuring θ expression in A-TXB spleen cells indicate a significant increase in the θ positive lymphocyte population occurred with time. The data indicate that considerable reconstitution of θ positive cells had occurred in A-TXB donors. The results also suggest that the T-lymphocyte population of the TXB spleen may be a unique subpopulation of T-lymphocytes that resides exclusively in spleen and bone marrow.     

Uptake of hyaluronan in hepatic endothelial cells is not directly affected by endotoxin and associated cytokines

The uptake of hyaluronan (HYA) labeled with 3H in its acetyl group was measured in cultured liver endothelial cells from normal rats and from rats previously treated with sublethal doses of Escherichia coli endotoxin (ET). Replicate cultures were also exposed to recombinant human tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) or interferon-gamma for 1 to 3 h before the measurement of hyaluronan uptake. Under all conditions, HYA was absorbed by endothelial cells at rates consistent with receptor-mediated absorption. In cells exposed to HYA 20 h after isolation, rate of uptake was less than half the rate in cells exposed 6 or 7 h after isolation. Cellular uptake of HYA was neither reduced nor enhanced by any of the treatments with cytokines. Prior exposure of the cell donors to ET caused a three-fold increase in their plasma HYA but did not alter the subsequent rate of cellular HYA uptake in vitro, either with or without added treatment with TNF-alpha or IL-1. It was concluded that the elevation of plasma HYA caused by septicaemia or by the experimental administration of ET or TNF-alpha cannot be attributed to direct interference with HYA receptors on hepatic endothelial cells.     

Modification of low density lipoprotein by desialylation causes lipid accumulation in cultured cells: discovery of desialylated lipoprotein with altered cellular metabolism in the blood of atherosclerotic patients

Low density lipoprotein (LDL) isolated from the blood of healthy donors was partially desialylated by incubating the lipoprotein with sialidase (neuraminidase). The addition of LDL treated with neuraminidase to cultured human aortic intimal cells of smooth muscle origin caused a substantial increase in intracellular cholesteryl esters, free cholesterol and triglycerides. Cultured cells took up and degraded desialylated LDL much more effectively than untreated (native) LDL. LDL were also isolated from an atherogenic blood plasma of patients with coronary artery disease, i.e. the plasma capable of inducing the accumulation of lipids in cultured cells. Patients' LDL, similarly to the mother plasma, were atherogenic, i.e. stimulated the accumulation of intracellular lipids. LDL isolated from nonatherogenic plasma of healthy donors proved to be nonatherogenic. Atherogenic patients' LDL had a 2- to 5-fold lower level of sialic acid as compared with nonatherogenic LDL of healthy donors. The uptake and degradation of atherogenic patients' LDL were much more effective than in the case of nonatherogenic LDL of healthy donors. We assume that atherogenic properties of LDL obtained from patients' blood plasma are explained exactly by a low sialic acid content.     

γ-Irradiation facilitates the expression of adoptive immunity against established tumors by eliminating suppressor T cells

It was found that sublethal (550 rad) whole-body gamma-irradiation of mice bearing established immunogenic tumors enabled tumor-sensitized spleen cells infused intravenously 1 h later to cause complete tumor regression in all mice. In contrast, gamma-irradiation alone caused only a temporary halt in tumor growth, and immune cells gave practically no therapeutic effect at all. This result was obtained with the SA1 sarcoma, Meth A fibrosarcoma, P815 mastocytoma, and P388 lymphoma. Additional experiments with the Meth A fibrosarcoma revealed that the spleen cells from tumor-immune donors that caused tumor regression in gamma-irradiated recipients were T cells, as evidenced by their functional elimination by treatment with anti-Thy-1.2 antibody and complement. It was shown next that adoptive T-cell-mediated regression of tumors in gamma-irradiated recipients was inhibited by an intravenous infusion of spleen cells from donors with established tumors, but not by spleen cells from normal donors. The spleen cells that suppressed the expression of adoptive immunity were functionally eliminated by treatment with anti-Thy-1.2 antibody and complement. Moreover, they were destroyed by exposing the tumor-bearing donors to 500 rad of gamma-radiation 24 h before harvesting their spleen cells. The results are consistent with the interpretation that gamma-radiation facilitates the expression of adoptive T-cell-mediated immunity against established tumors by eliminating a population of tumor-induced suppressor T cells from the tumor-bearing recipient.     

Liver Grafts for Transplantation from Donors with Diabetes: An Analysis of the Scientific Registry of Transplant Recipients Database

Patients with a history of diabetes mellitus (DM) have worse survival than those without DM after liver transplantation. However, the effect of liver grafts from DM donors on the post-transplantation survival of recipients is unclear. Using the Scientific Registry of Transplant Recipients database (2004–2008), 25,413 patients were assessed. Among them, 2,469 recipients received grafts from donors with DM. The demographics and outcome of patients were assessed. Patient survival was assessed using Kaplan–Meier methodology and Cox regression analyses. Recipients from DM donors experienced worse graft survival than recipients from non-DM donors (one-year survival: 81% versus 85%, and five-year survival: 67% versus 74%, P<0.001, respectively). Graft survival was significantly lower for recipients from DM donors with DM duration >5 years (P<0.001) compared with those with DM duration <5 years. Cox regression analyses showed that DM donors were independently associated with worse graft survival (hazard ratio, 1.11; 95% confidence interval, 1.02–1.19). The effect of DM donors was more pronounced on certain underlying liver diseases of recipients. Increases in the risk of graft loss were noted among recipients from DM donors with hepatitis-C virus (HCV) infection, whereas those without HCV experienced similar outcomes compared with recipients from non-DM donors. These data suggest that recipients from DM donors experience significantly worse patient survival after liver transplantation. However, in patients without HCV infection, using DM donors was not independently associated with worse post-transplantation graft survival. Matching these DM donors to recipients without HCV may be safe.     

Porphyrins containing nitric oxide donors: Synthesis and cancer cell-oriented NO release

Four novel porphyrins containing nitric oxide (NO) donors were synthesized, and the structures of all the products were characterized by IR, UV–vis, ¹H NMR, and elementary analysis. Interestingly, these new compounds not only were able to release NO, but also showed cancer cell-oriented accumulation. Higher accumulation of these new porphyrins containing NO donors in BEL-7402 liver cancer cells than in L-02 liver normal cells was corroborated by UV–vis spectroscopy. The biological activity of these porphyrins against BEL-7402 liver cancer cells was tested with a MTT assay. The studies indicated that they had more effective killing of BEL-7402 liver cancer cells than that of L-02 liver normal cells, and they had similar activity against MCF-7 breast cancer cells when compared to 5-fluorouracil in the absence of light.     

Production of profibrotic cytokines by invariant NKT cells characterizes cirrhosis progression in chronic viral hepatitis

Invariant (inv)NKT cells are a subset of autoreactive lymphocytes that recognize endogenous lipid ligands presented by CD1d, and are suspected to regulate the host response to cell stress and tissue damage via the prompt production of cytokines. We investigated invNKT cell response during the progression of chronic viral hepatitis caused by hepatitis B or C virus infection, a major human disease characterized by a diffused hepatic necroinflammation with scarring fibrotic reaction, which can progress toward cirrhosis and cancer. Ex vivo frequency and cytokine production were determined in circulating and intrahepatic invNKT cells from controls (healthy subjects or patients with nonviral benign or malignant focal liver damage and minimal inflammatory response) or chronic viral hepatitis patients without cirrhosis, with cirrhosis, or with cirrhosis and hepatocellular carcinoma. invNKT cells increase in chronically infected livers and undergo a substantial modification in their effector functions, consisting in the production of the type 2 profibrotic IL-4 and IL-13 cytokines, which characterizes the progression of hepatic fibrosis to cirrhosis. CD1d, nearly undetectable in noncirrhotic and control livers, is strongly expressed by APCs in cirrhotic ones. Furthermore, in vitro CD1d-dependent activation of invNKT cells from healthy donors elicits IL-4 and IL-13. Together, these findings show that invNKT cells respond to the progressive liver damage caused by chronic hepatitis virus infection, and suggest that these cells, possibly triggered by the recognition of CD1d associated with viral- or stress-induced lipid ligands, contribute to the pathogenesis of cirrhosis by expressing a set of cytokines involved in the progression of fibrosis.     

Cytofluorimetric research on the content of glycogen and its fractions in the hepatocytes of patients with liver cirrhosis of different etiologies]

By cytofluorometric method, a study was made of the total glycogen and its two fractions in liver parenchymal cells both in the donors (20 men) and in patients with cirrhosis of different etiology (39 men). The examination was performed on preparations--smears of isolated hepatocytes, obtained from the live functional liver biopsies. The quantitative analysis has shown an increase in the total glycogen content in hepatocytes of patients with cirrhosis by 3 times compared to the norm, and this increase is independent on the etiology of liver cirrhosis. To study the mechanism of the discovered glycogenosis, the activity of key enzymes of glycogenolyses was determined. It was shown that glucose-6-phosphatase and glycogen-phosphorylase activity in the liver with cirrhosis was lower than in the norm. The most considerable changes were shown in hepatocytes of patients with liver cirrhosis in fractional glycogen composition and, even more significant, in the content of a hard soluble fraction. The hard soluble fraction portion was higher in hepatocytes of the patients with liver cirrhosis of alcohol etiology. The quantitative analysis of glycogen fraction contents in liver cells may be the best marker in the differential diagnosis of symptomless elapsing liver cirrhosis.     

LPS-mediated NFkappaB activation varies between activated human hepatic stellate cells from different donors

The activation of hepatic stellate cells (HSC) is recognized as the key event of hepatic fibrosis [Virchows Arch. 430 (1997) 195; Semin. Liver Dis. 21 (2001) 437; Front. Biosci. 7 (2002) d808]. NFkappaB has been associated with the development of the activated phenotype, the expression of proinflammatory genes, and with promoting survival of activated HSC. High levels of circulating endotoxin are observed in liver fibrosis and several lines of evidence indicate that LPS plays an important role in chronic liver disease. Here, we investigated the LPS-induced NFkappaB activation in activated HSC from different human donors. HSC were isolated from liver specimens obtained during surgical liver resection and were activated by culturing on plastic. LPS-induced NFkappaB activity and IL-8 expression revealed a significant correlation but differed significantly comparing HSC from individual donors. These variations seen in LPS mediated NFkappaB activation and chemokine secretion between HSC from different donors in vitro may contribute to differences seen in vivo between patients in the progression of fibrosis and the degree of inflammation during chronic liver disease.     

Mouse fetal liver cells manifest antigen-specific suppressor activity

Liver cells obtained from C57B1/6J mice at different stages of development suppress the primary in vitro induced immune response. Fetal liver cells showed the strongest suppression of the PFC response, an effect which was gradually lost after birth. Thymic or splenic cells were ineffective in suppressing the PFC response under conditions where fetal liver cells from the same donors were highly active. Liver cells from newborn C57B1/6J athymic nude mice were equally suppressive as cells from their normal thymus-bearing littermates. Preculture of liver cells from 18-day-old fetuses with antigen homologous to that used in the indicator system increased suppressor activity severalfold compared with other experimental groups in which cells have been precultured in medium alone or with the addition of a heterologous antigen. The data suggest that antigen-specific suppressor activity is present in fetal liver cells. The possible relevance of these findings in relation to acquisition of self-tolerance is discussed.