Adsorptive properties of albumin, fibrinogen, and gamma-globulin on fluorinated diamond-like carbon films coated on PTFE.

Fluorinated diamond-like carbon (F-DLC) films were deposited on polytetrafluoroethylene (PTFE) using radio frequency (RF) plasma-enhanced chemical vapor deposition (CVD) by changing the ratio of tetrafluoromethane (CF(4)) and methane (CH(4)). To enhance the adhesion strength of the F-DLC film to the PTFE substrate, the PTFE surface was modified with a N(2) plasma pre-treatment. XPS analysis of the films showed that the C-C bond decreased with increases in the CF(4) ratio, whereas the C-F bond increased with the CF(4) ratio. The F/C ratio of the film also increased with the CF(4) ratio. The pull-out test showed that the adhesion strengths of the films (CF(4)-0-60%) were improved with the plasma pre-treatment. In the film without the plasma pre-treatment, adhesion strength increased with the CF(4) ratio. In contrast, in the case with the plasma pre-treatment, the adhesion strength of the F-DLC film decreased with the increased CF(4) ratio. Regarding the adsorption of albumin, fibrinogen, and gamma-globulin, the amount of adsorbed albumin on the film decreased with an increasing CF(4) ratio, and the amount of adsorbed fibrinogen and gamma-globulin increased with the CF(4) ratio. The CF(4)-0% DLC film showed the most adsorbed albumin and the least adsorbed fibrinogen and gamma-globulin. This indicates that the CF(4)-0% DLC film has higher anti-thrombogenicity than the F-DLC film.

Prognostic significance of glutamine synthetase expression in unifocal advanced hepatocellular carcinoma.

BACKGROUND/AIMS: Glutamine synthetase (GS) catalyzes the synthesis of glutamine, a major energy source of cells, and is upregulated in a subset of human hepatocellular carcinomas (HCCs). GS expression may be related to tumor recurrence, since GS-expressing tumors have a growth advantage in that they are independent of the extracellular glutamine supply. However, there are no studies concerning the prognostic value of GS expression in patients with HCC. METHODS: Seventy-three patients with a single advanced HCC nodule who underwent curative hepatectomy were included in the study. GS expression in the HCC nodules was analyzed immunohistochemically and was compared with clinicopathologic features and the behavior of the tumors. Survival curves were assessed according to the Kaplan-Meier product-limit method and multivariate analysis based on the Cox regression model was performed. RESULTS: GS expression was strong in 26 cases (35.6%, high-GS group) and weak or absent in 47 cases (64.4%, low-GS group). Univariate analysis showed that the high-GS group had a significantly shorter disease-free survival time than the low-GS group (p=0.042). Multivariate analysis revealed that GS expression (p=0.021), as well as Child's classification (p=0.005) and portal invasion (p=0.039), was a significant and independent prognostic parameter that affected tumor recurrence. CONCLUSION: The results of this study indicate that GS expression may enhance the metastatic potential in HCC, and GS immunostaining may be helpful in identifying HCC patients at high risk for disease recurrence.

Preparation of (-)-periplanone D and its physical and spectroscopic properties.

(-)-Periplanones C and D were obtained in reproducible yields by modifying reported procedures. Our synthetic sample of (-)-periplanone D showed somewhat different physical and spectroscopic properties from those reported in the literature.

A new bioartificial liver using porcine hepatocyte spheroids in high-cell-density suspension perfusion culture: in vitro performance in synthesized culture medium and in 100% human plasma.

A prototype of a bioartificial liver (BAL) based on suspension perfusion culture of porcine hepatocyte spheroids was developed at 150 ml scale. About 2% (4 x 10(9) cells) of whole human liver cells was immobilized. The cell density in the bioreactor was 2.7 x 10(7) cells/ml, which was almost comparable to that of presently developed packed-bed-type BALs. The bioreactor was perfused with culture medium while retaining spheroids. This was done using a rotating stainless filter (pore size 50 microm). In vitro 8-h perfusion experiments utilizing both synthesized culture medium and 100% human plasma demonstrated the spheroids in the bioreactor had almost the same functions on a unit/cell basis as those in small-scale rotational culture. This indicated that the functional deterioration often associated with scaling up had been minimized. Rapid spheroid aggregation and dysfunction in specific human plasma pool must be eliminated before clinical application, although this phenomenon seemed to be inherent to porcine hepatocyte-based BALs. This prototype shows promise in meeting present clinical demands by achieving maximal metabolic activities even in the short term.

A novel frameshift mutation in exon 6 (the site of Asn 291) of the lipoprotein lipase gene in type I hyperlipidemia.

A new heterozygous lipoprotein lipase gene defect has been identified in a type I hyperlipidemic patient at the position of notable amino acid Asn 291. The patient is a 33-year-old male. His body mass index (BMI) was 18.5 kg/m2. The total cholesterol (TC), triglycerides (TG) and high density lipoprotein-cholesterol (HDL-C) concentration from his fasting plasma were 4.8, 11.9 and 0.4 mmol/l, respectively. The lipoprotein lipase (LPL) activity and mass in the postheparin plasma (PHP) from the patient were 0.58 mmol/ml/h (normal range: 7.7+/-2.6) and 244 ng/ml (normal range: 192+/-30), respectively. The hepatic lipase activity of the PHP from the patient was 10.6 mmol/ml/h (normal range: 9.9+/-3.6). DNA analysis of the LPL gene revealed that this patient had a heterozygous one nucleotide deletion of A coding Asn 291, resulting in a premature termination of the LPL protein at amino acid residue 303. The other abnormality in the LPL gene of the proband was an amino acid residue 194 defect (Ile194-->Thr), which is known to cause a defective enzyme. A medium-chain triglyceride (MCT) loading test was conducted to find how this triglyceride affects plasma lipoprotein metabolism in this patient in a short term (Fig. 3). The plasma total cholesterol (TC) or high density lipoprotein (HDL)-C levels did not change significantly after oral administration of a fatty meal containing long chain triglycerides (LCT) or MCT. The plasma TG level, on the other hand, increased from 11.9 to 19.2 mmol/l (+61%) at 6 h after loading a fatty meal containing LCT, whereas the plasma TG levels tended to even decrease at 6 h after oral administration of an MCT, tricaprin (from 11.6 to 10.5 mmol/l (-9.4%)). These results suggest that MCT, as opposed to LCT, is useful for treatment of type I hyperlipidemia with a novel mutation at the notable amino acid Asn 291 of the LPL gene.

Effect of troglitazone on plasma lipid metabolism and lipoprotein lipase.

AIMS: To clarify how troglitazone, an insulin-sensitizing agent, affects lipid metabolism and postheparin plasma lipoprotein lipase (LPL). METHODS: Fifteen patients (3 male, 12 female) (the average age 62+/-7 years; the mean body mass index (BMI) 25+/-3 kg/m2 ) were recruited for this study. The serum lipids and postheparin plasma lipoprotein lipase (LPL) mass before and 4 weeks after oral administration of troglitazone (200 mg day-1 ) were measured. A mouse preadipocyte cell line, 3T3-L1, was incubated with troglitazone and LPL enzyme protein mass in the culture media was measured by an enzyme linked immunosorbent assay. A reverse transcription polymerase chain reaction (RT-PCR) using primers specific for the carboxyl terminal 135 amino acid of mouse LPL cDNA was used to evaluate the effect of troglitazone on expression of LPL and Northern blot analysis carried out to determine expression of LPL. RESULTS: The average levels before treatment of fasting serum total cholesterol, triglycerides, high density lipoprotein cholesterol, plasma glucose and glycohaemoglobin A1c were 5.6+/-0.9, 1.8+/-1.0, 1.5+/-0.5, 8.1+/-1.7 mmol l-1 and 7.8+/-1.6% respectively. Four weeks after treatment, those levels were 5.4+/-0.9, 1.2+/-0.3 (P=0.004), 1.6+/-0.5 (P=0.02) mmol l-1, 7.7+/-2.3 mmol l-1 and 7. 3+/-0.6% (P=0.01), respectively. The postheparin plasma LPL mass increased from 226+/-39 to 257+/-68 ng ml-1 (P=0.03) during that period. The LPL mass in the media of 3T3 L1 cells cultured in the presence of 10, 20 or 30 microm of this compound increased in a dose dependent manner. RT-PCR revealed that the area of the bands of the RT-PCR products on 1.5% agarose gel analyzed with NIH image from the cell extracts cultured in the presence of 10 microm troglitazone was significantly larger (P=0.0069) than that in the absence of this compound. Northern blot analysis revealed that in the cultured 3T3-L1 cells, the expression of LPL was enhanced in the presence of 10 microm troglitazone. CONCLUSIONS: Troglitazone improves plasma triglyceride-rich lipoproteins metabolism by enhancing the expression of LPL in adipocytes.

Histopathological prognostic factors influencing long-term prognosis after surgical resection for hepatic metastases from colorectal cancer.

OBJECTIVE: We aimed to present new histopathological features of metastatic liver nodules as more reliable prognostic factors after surgical resection for colorectal metastatic cancer. METHODS: Clinicopathological features, including newly proposed histopathological ones, of 63 consecutive patients were reviewed retrospectively to determine which most strongly correlated with long-term prognosis after hepatectomy for metastatic tumors from colorectal cancers, using univariate and multivariate analysis. RESULTS: The 1-, 3-, and 5-year cancer-related survival rates after hepatectomy were 87.8%, 55.2%, and 47.3%, respectively. New histopathological features we proposed, which are expansive growth, marginal fibrosis, and peritumorous lymphocytic infiltration of hepatic tumor, were significant prognostic factors for cancer-related survival after hepatectomy in an univariate analysis. Also in a multivariate analysis, i.e., a stepwise Cox regression analysis, infiltrative, i.e., not expansive, growth of hepatic tumor was one of significant and independent poor prognostic factors for survival after hepatectomy, with moderate to severe lymphatic vessel involvement of the primary colorectal lesion, microscopic cancer invasion at the surgical margin of hepatectomy, and extrahepatic distant metastases. CONCLUSIONS: Our results suggest that our proposed new histopathological features of hepatic metastases were good predictors of prognosis after surgical resection for hepatic metastases from colorectal cancer. Especially, infiltrative growth of hepatic tumor is strongly correlated with a poor prognosis after hepatectomy.

Efficacy of a bioreactor filled with porcine hepatocytes immobilized on nonwoven fabric for ex vivo direct hemoperfusion treatment of liver failure in pigs.

We developed a new bioreactor for a bioartificial liver filled with porcine hepatocytes immobilized on polyester nonwoven fabric (NWF) and in our previous study showed that this NWF bioreactor has promising in vitro efficiency. In the present study, we investigated the efficacy of the NWF bioreactor in a direct hemoperfusion experiment conducted to treat pigs with liver failure. Porcine hepatocytes were isolated from the whole liver of a Sangen strain pig. They were immobilized in a 200 ml column containing NWF via perfusion in a closed circuit for 24 h to prepare a NWF bioreactor. The following day an operative liver failure model was produced by creating a portocaval shunt and ligating the entire hepatoduodenal ligament in the porta hepatis. Perfusion treatment was initiated 4 h after operative induction of liver failure and continued for about 1 h. The pigs which underwent perfusion treatment showed significant improvements in survival and blood data, including ammonia, total bile acid, glucose, and prothrombin time, attributed to significant improvements in the post- as compared to the prebioreactor levels in the perfused blood of the treated pigs. These beneficial effects of the NWF bioreactor were based on its excellent composition which allows the accommodation of adequate numbers of hepatocytes and direct contact between hepatocytes and perfused blood.

Auxiliary heterotopic partial liver transplantation in pigs with acute ischemic liver failure.

BACKGROUND/AIMS: Fulminant hepatic failure (FHF) is usually fatal without liver transplantation. Auxiliary heterotopic partial liver transplantation (AHPLT) may offer advantages over orthotopic liver transplantation (OLT) or any other heterotopic procedure for the treatment of patients with fulminant liver failure. We studied AHPLT in a severe acute hepatic failure model in pigs. METHODOLOGY: Group A (control: n = 5) underwent portal vein and hepatic artery ligation and side-to-side portocaval shunting. Group B (AHPLT: n = 15) underwent host portal vein and hepatic artery ligation and AHPLT. RESULTS: All of the pigs in group A died within 48 hours from massive liver necrosis. Ten of the 15 pigs (67%) in group B had well-functioning grafts. Five of these ten died between 8 and 17 days postoperatively due to various complications. The remaining five survived for sixty days postoperatively in healthy condition. At the time of sacrifice, four of these five had well-functioning grafts weighing 739 +/- 52 g (mean +/- SEM) and regenerated, but still atrophied, host livers weighing 262 +/- 23 g (p < 0.0002). On the other hand, the one remaining pig had an atrophied graft weighing 310 g and a well-regenerated host liver weighing 470 g, probably due to a late, poorly functioning graft associated with severe rejection. CONCLUSION: AHPLT may result in survival despite host hepatic failure, and the host liver may recover within two months, despite total interruption of blood inflow.

[Development of a bioartificial liver using porcine hepatocyte spheroids].

We have discussed hereby about the latest studies on bioartificial liver systems including those examined in human clinical trials. Based on the discussion and in order to meet the present requirement of clinical sides to the system, that is, maximal metabolic capacities up to 6 hours of plasma perfusion treatment, we have developed a new bioartificial liver based on high-cell density suspension perfusion culture of porcine hepatocyte spheroids, which have two or three times higher functional expressions that those of conventional cultures. The module developed can retain a large number of hepatocytes(4 x 10(9)) in 150 mL volume without any functional deterioration usually accompanied by high-cell density cultivation, even when compared with small-scale dish-level cultures.

Comparisons of porcine hepatocyte spheroids and single hepatocytes in the non-woven fabric bioartificial liver module.

We previously developed a new bioreactor of the bioartificial liver composed of non-woven fabric. We have also experimented with hepatocyte spheroids, with the aim of improving the efficiency of this NWF bioreactor. In this study, we compared the efficiencies of NWF bioreactors employing hepatocyte spheroids versus single hepatocytes. Hepatocytes were isolated from a whole pig liver by Seglen's method. 1.0 x 10(10) single hepatocytes were immobilized in the NWF bioreactor. Another 1.0 x 10(10) hepatocytes were allowed to form spheroids by 24 hr suspension culture in a 4-L culture vessel, before being immobilized in the bioreactor. Hepatocyte spheroids were found to be functionally superior, on a per-cell basis, to single hepatocytes in the NWF bioreactor. However, the NWF bioreactor employing hepatocyte spheroids exhibited lower efficiency than thaT employing single hepatocytes, because the total number of the hepatocytes had decreased during the 24 hr suspension culture.

In vitro function of porcine hepatocyte spheroids in 100% human plasma.

To assess their functional stability, porcine hepatocyte spheroids, which have great potential in bioartificial liver systems and/or xenogeneic liver transplantation, were cultured in vitro in 100% human plasma, and their ammonium detoxification capacity was measured. In a short-term functional study, collagen-gel entrapped and rotationally cultured porcine hepatocyte spheroids showed almost the same functional expression, which was approximately twice that of monolayers. No significant functional deterioration was observed up to day 10 as compared with those in a synthesized medium without collagen gel entrapment. In a longer term study (4 wk) using rotational culture, morphological and functional deterioration presumably due to immunorejection involving complement proteins and natural antibodies progressed gradually with culture time. On the termination of the culture, the detoxification ability for the residual cells was approximately two-thirds that on day 2.

Short-term hypothermic preservation of porcine hepatocyte spheroids using UW solution.

The feasibility of University of Wisconsin (UW) solution in short-term hypothermic preservation of porcine hepatocyte spheroids was investigated, because they have great potential in bioartificial liver (BAL) systems. Porcine hepatocyte spheroids preserved for 3 days expressed almost comparable levels of albumin secretion as those without preservation, during 8 subsequent days of recultivation in continuous rotational culture, whereas isolated single cells did not reorganize into spheroids and completely lost their function in recultivation. Although for 3-day-preserved spheroids, the albumin secretion was lowered immediately after recultivation (Days 0-2), it was completely restored to that of nonpreserved ones. The function was completely lost in recultivation for 7-day-preserved ones. These results demonstrate that reorganization into spheroids is effective in preventing the functional loss of porcine hepatocytes occurring in hypothermic preservation, and that spheroid formation should precede the preservation as long as spheroid culture is finally used in BAL systems. Also, porcine hepatocyte spheroids are shown to be satisfactory stored in UW solution up to 3 days without significant cellular or functional loss.

Development of a new bioartificial liver module filled with porcine hepatocytes immobilized on non-woven fabric.

We developed a new bioartificial liver module, a non-woven fabric (NWF) module, which was filled with hepatocytes immobilized on non-woven polyester fabric, and compared its efficacy with a suspension culture vessel. Porcine hepatocytes were isolated from whole pig liver, which formed spheroids in a 1L suspension culture vessel. Some of these spheroids were allowed to construct a 50 mL NWF module by inoculation onto 12 sheets of disk-shaped non-woven fabric then stuffed into a 50 mL column. The remaining spheroids were transferred to a 100 mL spinner flask and placed in suspension. The 50 mL NWF module maintained a much better metabolic and synthetic function than the 100 mL suspension culture vessel throughout the study. Based on these results, we developed a 200 mL radial-flow-type NWF module. This module filled with single hepatocytes showed promising efficiency.

Large-scale preparation and function of porcine hepatocyte spheroids.

To obtain a large number of porcine hepatocyte aggregates (spheroids) that have great potential in a bioartificial liver (BAL), we performed spheroid formation at a high cell density in a 1-L-scale spinner flask fitted with a silicon tubing apparatus for oxygen supply. We thereby obtained, within 24 hours, approximately fifty times more porcine hepatocyte spheroids as compared with the results of previous reports. The amount obtained corresponds to 2.5x10(9) cells and to roughly one-sixth of the cell number required for a BAL for a human patient. When we cultured spheroids continuously in suspension, they expressed three times more albumin secretion and twice the ammonium removal as compared with conventional monolayers during 10 days culture. Collagen gel entrapment of spheroids particularly lowered albumin secretion. We therefore conclude that the supension culture vessel of porcine hepatocyte spheroids is one of the most promising module types.

Functional stability of porcine hepatocyte spheroids in various culture systems under 100% porcine and human plasma conditions.

To select an immobilization method suitable for bioartificial liver (BAL) modules utilizing porcine hepatocyte spheroids, functional activities were compared in various systems in 100% porcine and human plasma together with a synthesized medium. The spheroids, continuously suspended in rotating dishes or entrapped in collagen (CN) gel, expressed approximately two times higher ammonium detoxification abilities over conventional monolayers during 8 days of direct contact with 100% human or porcine plasma with a standardized inoculum cell number. No significant deterioration was observed in the abilities as compared with that in a synthesized medium. Although the cell number gradually decreased in rotational culture, the abilities per cells remaining on Day 10 were two times higher than in the CN-gel entrapped spheroids in all the media examined, presumably due to the diffusion limitation by the gel. Thus, in utilizing porcine hepatocyte spheroids in BAL modules, immobilization allowing direct contact of spheroids with perfused patient plasma was concluded to be possible and suitable.

Surgical resection for small hepatocellular carcinoma.

BACKGROUND: Surgical resection for hepatocellular carcinoma (HCC) can be curative in selected patients, particularly in those with a solitary small HCC (s-sHCC; 2 cm or less in diameter). However, even these patients often have a risk of tumor recurrence or death from underlying liver dysfunction. Therefore it is important to determine which clinicopathologic features are related to the long-term prognosis after resection of s-sHCC. METHODS: Fifty patients with s-sHCC underwent partial hepatectomy at our department between 1977 and 1992. Six (12%) died of liver failure in hospital after operation. Eight clinicopathologic features were examined in the remaining 44 patients with regard to their long-term prognosis by use of univariate and multivariate analyses. RESULTS: The 1-, 3-, and 5-year survival rates were 90%, 75%, and 53%, respectively. The corresponding disease-free survival rates were 80%, 53%, and 30%, respectively. None of the following parameters was significantly related to survival rate or disease-free survival rate: presence of vascular invasion or capsular formation, the distance of free surgical margin (1 cm or more or not), serum alpha-fetoprotein level, positive hepatitis B surface antigen, and preoperative transarterial embolization. Complicated liver function was the only significant factor related to survival rate and disease-free survival rate. CONCLUSIONS: A good hepatic reserve is an important factor in treating patients with s-sHCC by surgical resection, even for a long-term prognosis. Liver transplantation should be considered for patients with severe cirrhosis and s-sHCC, even though a curative resection might be possible.

How can we share the portal blood inflow in auxiliary partial heterotopic liver transplantation without portal hypertension?

BACKGROUND: Auxiliary partial heterotopic liver transplantation (APHLT) may be an attractive treatment for fulminant hepatic failure in which recovery of the host liver is expected. We investigated the functional relationship between an auxiliary heterotopic partial liver graft and the host liver with or without portal hypertension in pigs. METHODS: Sixteen pigs underwent hepatic artery ligation and APHLT; in addition, various degrees of portal hypertension were created by banding the host portal vein. The pigs were randomly divided into three groups: group A (n = 5), the host portal vein was not banded; group B (n = 6), the host portal vein was banded to make the host portal pressure higher than that of the graft by 2 mm Hg; and group C (n = 5), the host portal vein was banded to make the host portal pressure the same as that of the graft. RESULTS: All of the pigs in group A had necrotic and atrophied grafts with graft portal vein thrombosis and well-hypertrophied host livers. All of the pigs in group B had well-functioning grafts and necrotic and atrophied host livers. Three of the five pigs in group C had well-functioning grafts and host livers, although the remaining two pigs exhibited the same results as those in group A. CONCLUSIONS: APHLT could be a valuable alternative treatment for temporary support in fulminant hepatic failure. However, if the recipient does not exhibit portal hypertension, the host portal vein should be banded to make the host portal pressure at least the same as that of the graft.