Effect of Middle Hepatic Vein Tributaries Preserving Technique Until Just Before Graft Retrieval on Donors' Surgical Outcomes in Living Donor Liver Transplantation.

BACKGROUND: The technique of preserving the major tributaries of the middle hepatic vein (MHV) (V5 and V8) until just before graft retrieval is beneficial to minimize congestion time of the graft. However, it remains unclear whether this technique exerts a burden on donors in terms of operative time, blood loss, and postoperative hepatic dysfunction. In this study we investigated adverse effects of the MHV tributaries preserving technique until immediately before graft retrieval on donors' surgical outcomes. METHODS: Data from 71 donors who underwent right hepatectomy without MHV for a liver transplantation at our hospital from January 2002 to August 2016 were retrospectively reviewed. Donors were divided into 3 groups as follows: group 1 (n = 12), no MHV tributary reconstruction; group 2 (n = 33), single MHV tributary reconstruction; group 3 (n = 26), 2 or 3 MHV tributaries reconstruction. Donor operation time, blood loss, proportion of the remnant liver, maximum postoperative total bilirubin, aspartate aminotransferase, alanine transaminase, minimum platelets, prothrombin time, albumin level, number of days in hospital from surgery to discharge, and surgical complications were compared. RESULTS: Compared with groups 2 and 3, group 1 exhibited shorter average operational time and less average blood loss, but the difference was not significant. Comparisons of all other factors indicated no significant differences. CONCLUSION: The technique of preserving the major tributaries of the MHV until just immediately before graft retrieval does not appear to impose an apparent burden on donors.

Effects of Subnormothermic Perfusion Before Transplantation for Liver Grafts from Donation After Cardiac Death: A Simplified Dripping Perfusion Method in Pigs.

BACKGROUND: Liver transplantation from donors after cardiac death (DCD) provides a solution to the donor shortage. However, DCD liver grafts are associated with a high incidence of primary graft nonfunction. We investigated the effectiveness of subnormothermic porcine liver perfusion, before transplantation from DCD, on graft viability. METHODS: Landrace pigs (25-30 kg) were randomly allocated to 3 groups (5 per group): heart-beating (HB) graft, transplanted after a 4-hour period of cold storage (CS); DCD graft, retrieved 20 minutes after apnea-induced cardiac arrest (respiratory withdrawal) and transplanted after a 4-hour period of CS; and subnormothermic ex vivo liver perfusion (SELP) graft, retrieved in the same manner as the DCD graft but perfused with a subnormothermic oxygenated Krebs-Henseleit buffer (21-25°C, 10-15 cm H2O) for 30 minutes in a simplified dripping manner, without a machine perfusion system, after the 4-hour period of CS, and subsequently transplanted. RESULTS: Although all animals in the HB group survived for >7 days, all animals in the DCD group died within 12 hours after transplantation. In the SELP group, 2 recipients survived for >7 days and another 2 recipients were killed on day 5. The survival rate was significantly better for SELP than for DCD grafts (P = .0016). The values of tumor necrosis factor α were not significantly different between the SELP and HB groups. Preserved structure of the parenchyma was observed in the SELP group on histologic examination. CONCLUSIONS: A simplified subnormothermic perfusion before liver transplantation is expected to improve graft viability and survival.

The effect of IL-17 on the production of proinflammatory cytokines and matrix metalloproteinase-1 by human periodontal ligament fibroblasts.

OBJECTIVES: To investigate the effects of IL-17 on IL-6, IL-1ß, and matrix metalloproteinase (MMP-1) production, and to compare the MMP-1 production between the individual and combined effects of IL-1ß and IL-6 in human periodontal ligament fibroblasts (HPDLF). MATERIALS AND METHODS: Human periodontal ligament fibroblasts were cultured with IL-17 for 0.5, 1, 4, 24, 48, and 72 h, and were cultured with IL-1ß, IL-6/sIL-6R, or a combination of IL-1ß and IL-6/sIL-6R for 24 h. To measure the mRNA levels of IL-6, IL-1ß, and MMP-1, total RNA was extracted from the cultured HPDLF, and a real-time PCR analysis was performed. The protein levels of IL-6, IL-1ß, and MMP-1 in supernatants were measured using enzyme-linked immunosorbent assays (ELISAs). RESULTS: IL-17 significantly increased the expression of IL-6 and MMP-1 mRNA and protein, while IL-17 transiently increased the expression of IL-1ß mRNA. The combination of IL-1ß and IL-6/sIL-6R induced significantly higher levels of MMP-1 protein than IL-1ß alone. CONCLUSIONS: IL-17 upregulated the production of IL-6 and MMP-1 sequentially in HPDLF. IL-6/sIL-6R may enhance the effects of IL-1ß on MMP-1 production. The present results suggest that IL-17 induces MMP-1 production not only directly, but also indirectly by promoting IL-6 production, thus resulting in the degradation of collagens in the PDL.

Adhesion of Streptococci to various orthodontic composite resins.

BACKGROUND: This investigation aimed to determine quantitatively the adhesion of Streptococcus mutans and Streptococcus sobrinus to orthodontic composite resins that were tested simultaneously using radio-markers. METHODS: Seven orthodontic composite resins were classified into seven groups: BeautyOrtho Bond (GI), Blugloo (GII), Enlight (GIII), Grengloo (GIV), Kurasper F (GV), Transbond CC (GVI) and Turbo Bond II (GVII). Thirty 4 x 4 x 1 mm blocks of each orthodontic composite resin were made (a total of 210 blocks). Both Streptococcus species were cultivated independently. For the quantitative analysis, radioactive markers were used to codify the bacteria ((3) H for Streptococcus mutans and (14) C for Streptococcus sobrinus). The blocks were submerged in a solution with microorganisms previously radiolabelled for 2 hours at 37 °C in constant movement. The blocks were placed in a combustion system to quantify the Streptococcus adhering to the surface of the materials by capturing the residues and measuring the radiation. RESULTS: Significant differences in bacterial adhesion were found among the groups. The lowest significant scores for both microorganisms were observed in GIII. CONCLUSIONS: The orthodontic composite resin evaluated in GIII exhibited the lowest adhesion of Streptococcus mutans and Streptococcus sobrinus, which may reduce enamel demineralization and the risk of white spot lesion formation.

Microsomal prostaglandin E synthase-1 is involved in multiple steps of colon carcinogenesis.

Accumulating evidence indicates that cyclooxygenase (COX)-2-derived prostaglandin (PG) E(2) is involved in the development of various tumors, including colorectal cancer. However, the precise contribution of microsomal PGE synthase (mPGES)-1, a terminal enzyme that acts downstream of COX-2 in the PGE(2)-biosynthetic pathway, to multiple processes of tumor development is not yet fully understood. Here, we show the pro-tumorigenic role of mPGES-1 in chemical carcinogen-induced colon carcinogenesis and intrasplenic tumor transplantation models. Genetic deletion of mPGES-1 significantly reduced both the total number and size of colorectal polyps at 18 weeks after azoxymethane administration with reduced nuclear translocation of ß-catenin, altered expression profiles of chemokines/cytokines and increased production of antitumorigenic PGs, prostaglandin D(2) and prostacyclin in tumor tissues. At an early stage (6 weeks), mPGES-1 deficiency significantly reduced the number of aberrant crypt foci, while its transgenic overexpression increased the number. Furthermore, the growth of intrasplenically transplanted tumor cells was suppressed in mPGES-1 knockout (KO) mice. Co-culture of tumor cells with bone marrow-derived macrophages (BM-MΦs) isolated from wild-type (WT) mice resulted in the induction of mPGES-1 in BM-MΦs and increased the growth of tumor cells in vitro, whereas mPGES-1-null BM-MΦs failed to facilitate tumor growth. The adoptive transfer of WT BM-MΦs into mPGES-1 KO mice restored the growth of transplanted tumor cells, indicating that mPGES-1 in MΦs is important for the growth of adjacent tumor cells. Taken together, our findings suggest that the inhibition of mPGES-1 is an alternative therapeutic target for colorectal and possibly other cancers.

Genetic deletion of mPGES-1 accelerates intestinal tumorigenesis in APC(Min/+) mice.

The induced synthesis of bioactive prostanoids downstream of cyclooxygenase-2 (COX-2) and prostaglandin H(2) (PGH(2)) exerts a critical event in colorectal carcinogenesis. Here we demonstrate that APC(Min/+) mice with genetic deletion of microsomal prostaglandin E synthase-1 (mPGES-1), which catalyses the terminal conversion of PGH(2) into PGE(2), surprisingly develop more and generally larger intestinal tumors than do mPGES-1 wild type littermates (mean number of tumors/intestine 80 vs. 38, p<0.0005, mean tumor diameter 1.64 vs. 1.12 mm, p<0.0005). No deviation regarding the expression of other PGE(2) related enzymes (COX-1, COX-2, mPGES-2, cPGES, and 15-PGDH) or receptors (EP1-4) was obvious among the mPGES-1 deficient mice. PGE(2) levels were suppressed in tumors of mPGES-1 deficient animals, but the concentrations of other PGH(2) derived prostanoids were generally enhanced, being most prominent for TxA(2) and PGD(2). Thus, we hypothesise that a redirected synthesis towards other lipid mediators might (over)compensate for loss of mPGES-1/PGE(2) during intestinal tumorigenesis. Nevertheless, our results question the suitability for mPGES-1 targeting therapy in the treatment or prevention of colorectal cancer.

Inorganic polyphosphate: a possible stimulant of bone formation.

Inorganic polyphosphates [Poly(P)] are often distributed in osteoblasts. We undertook the present study to verify the hypothesis that Poly(P) stimulates osteoblasts and facilitates bone formation. The osteoblast-like cell line MC 3T3-E1 was cultured with Poly(P), and gene expression and potential mineralization were evaluated by reverse-transcription polymerase chain-reaction. Alkaline phosphatase activity, von Kossa staining, and resorption pit formation analyses were also determined. The potential role of Poly(P) in bone formation was assessed in a rat alveolar bone regeneration model. Poly(P) induced osteopontin, osteocalcin, collagen 1alpha, and osteoprotegerin expression and increased alkaline phosphatase activity in MC 3T3-E1 cells. Dentin slice pit formation decreased with mouse osteoblast and bone marrow macrophage co-cultivation in the presence of Poly(P). Promotion of alveolar bone regeneration was observed locally in Poly(P)-treated rats. These findings suggest that Poly(P) plays a role in osteoblastic differentiation, activation, and bone mineralization. Thus, local poly(P) delivery may have a therapeutic benefit in periodontal disease.

TLR engagement prevents transplantation tolerance.

In many experimental models, heart, pancreas and kidney allografts are accepted long-term following costimulation-targeting therapies, whereas skin, lung and intestine resist the induction of tolerance under the same regimens. We noted that a common feature of the resistant organs is their constant exposure to commensal microbes and hypothesized that these microorganisms may stimulate Toll-like receptors (TLRs), promote alloresponses and prevent tolerance induction. This hypothesis prompts the predictions that TLR engagement at the time of transplantation should avert tolerance to heart allografts in animals treated with costimulation-targeting therapies, whereas inhibition of TLR signaling should promote tolerance to skin allografts under the same conditions. Indeed, engagement of a single TLR was sufficient to prevent anti-CD154-mediated long-term cardiac allograft acceptance and correlated with abolished intragraft recruitment of CD4+/FoxP3+ regulatory T cells and the development of linked-suppression. Conversely, a lack of donor and recipient MyD88-dependent signaling led to successful skin allograft acceptance in anti-CD154-treated animals. Thus, the status of TLR signaling contributes to the resistance versus susceptibility of organs to transplantation tolerance.

MyD88-dependent pathways mediate resistance to Cryptosporidium parvum infection in mice.

Cryptosporidium spp. cause diarrheal disease worldwide. Innate immune responses mediating resistance to this parasite are not completely understood. To determine whether MyD88-dependent pathways play a role in resistance to Cryptosporidium parvum, we compared the course of infection in MyD88(-/-) mice to that in their wild-type (WT) littermate controls. Three- to 4-week-old mice were infected with C. parvum, and infection was monitored by quantifying fecal oocyst shedding. Twelve days postinfection, the histology of the intestines was examined to quantify intestinal parasite burden and to determine if there were any pathological changes. Fecal oocyst shedding and intestinal parasite burden were significantly greater in MyD88(-/-) mice than in littermate controls. Nonetheless, both WT and MyD88(-/-) mice cleared the infection within 3 weeks. These results indicate that MyD88-dependent pathways are involved in mediating initial resistance to C. parvum. Since gamma interferon (IFN-gamma) is known to mediate resistance to C. parvum, we also studied infection in MyD88(-/-) mice and WT controls in which this cytokine was temporarily neutralized. Fecal oocyst shedding, as well as intestinal parasite burden, intestinal inflammation, and mortality, was significantly greater in MyD88(-/-) mice in which IFN-gamma was neutralized than in IFN-gamma-neutralized WT mice or in MyD88(-/-) mice in which this cytokine was active. These results suggest that MyD88 and IFN-gamma had an additive effect in conferring protection from C. parvum infection. While this study confirms the importance of IFN-gamma in conferring resistance to infection with C. parvum, it suggests that MyD88-mediated pathways also play a role in innate immunity to this parasite.

Regulation of IL-1-induced gingival collagenase gene expression by activator protein-1 (c-Fos/c-Jun).

Matrix metalloproteinase-1 is probably involved in the progression of periodontal disease. The aim of this study was to investigate whether IL-1beta stimulates the expression of the activator protein 1 (AP-1) transcription factor and, consequently, if the AP-1 transcription factor participates in the regulation of collagenase gene expression in human gingival fibroblast cells. In this study, we demonstrate that the concentration of the protein components of AP-1 transcription factor, c-Fos and c-Jun, is enhanced by IL-1beta both at mRNA and protein levels, utilizing Northern blot analysis, electrophoretic mobility gel shift assay and Western blot analysis. The IL-1beta stimulated the collagenase-CAT and AP-1-CAT activities in a dose dependent manner with respect to the amount of DNA used in transfections. Further, overexpression of c-Fos and c-Jun proteins revealed a dose-dependent transcriptional activation of the collagenase promoter. These findings, coupled with the existence of AP-1 consensus DNA binding sites on the collagenase gene promoter, show that regulation of collagenase gene expression by IL-1beta involves the transcription factor AP-1 in gingival fibroblasts.

Expression of membrane cofactor protein (MCP, CD46) in human liver diseases.

Membrane cofactor protein (MCP, CD46) is one of the complement regulatory proteins, and is widely distributed in human organs and protects cells from complement-mediated cytotoxicity. We analysed the distribution and the intensities of MCP in liver diseases and evaluated the role of MCP during hepatocarcinogenesis. Western blot analysis revealed that relative densities (density of the sample/density of the standard sample) of MCP in 27 HCC, 18 liver cirrhosis, nine chronic hepatitis and 12 normal liver were 0.63+/-0.23, 0.21+/-0.07, 0.25+/-0.10 and 0.11+/-0.03 (mean+/-s.d.) respectively. MCP expression in hepatocellular carcinoma (HCC) was significantly higher than that in both liver cirrhosis and chronic hepatitis (P < 0.01). The difference in the tumour sizes, the grades of differentiation and viral marker status did not affect the expression. Immunohistological analysis revealed that MCP was distributed mainly in the basolateral membrane of the hepatic cord in non-cancerous liver, along with endothelial cells and bile duct cells. In HCC, the protein was observed on the membrane in a non-polarized fashion. These data suggest that HCC cells acquire the increased MCP expression in a development of HCC and may escape from tumour-specific complement-mediated cytotoxicity.

Expression of alpha-fetoprotein and albumin genes in human hepatocellular carcinomas: limitations in the application of the genes for targeting human hepatocellular carcinoma in gene therapy.

For an approach of gene therapy for hepatocellular carcinoma (HCC), transcriptional regulatory sequence (TRS) of either alpha-fetoprotein (AFP) or albumin has been used for targeting cancer cells. To examine the feasibility of using TRSs of these genes for possible gene therapy of HCCs, the cellular distribution of AFP and albumin gene transcripts was studied in 25 cases of surgically removed human HCCs. AFP gene expression was observed in HCC nodules of 13 cases. The expression in HCC was heterogeneous, and the distribution of the transcripts was mostly sparse and spotty. The higher the serum AFP levels, the larger population of the AFP-expressing HCC cells tended to reflect. In noncancerous liver, a slight AFP expression was found by Northern blot analysis, but the transcripts were not detected in the liver sections. In contrast, albumin expression was found in all HCCs as well as in noncancerous hepatocytes. In HCC, the transcripts for albumin were distributed in cancer cells, and the expression varied with nodules. There were more albumin-expressing cancer cells than the AFP-expressing cells. Albumin expression was retained even in poorly differentiated HCC, although the intensity of the signal was not as strong as in more-differentiated HCCs. Metastatic HCC nodules revealed transcripts for both AFP and albumin genes, and those were clearly recognized in the lung tissue. These results suggest that, for gene therapy for HCCs, neither AFP nor albumin are ideal options for targeting HCC cells. AFP-TRS may be used as a transcriptional regulator in selected cases in which AFP gene expression is observed in the cancer nodules. The serum AFP level appears to be an important indicator in selecting cases. Albumin-TRS in conjunction with retroviral vector might be used in limited cases such as HCCs with no AFP expression. However, careful consideration must be taken, because albumin is constitutively expressed in normal hepatocytes, and AFP-expressing nonmalignant progenitor cells possibly exist.

Why would you remove half a brain? The outcome of 58 children after hemispherectomy-the Johns Hopkins experience: 1968 to 1996.

PURPOSE: To report the outcomes of the 58 hemispherectomies performed at Johns Hopkins between 1968 and January 1996. METHODS: Charts were reviewed of the 58 hemispherectomies performed at Johns Hopkins Medical Institutions by the Pediatric Epilepsy Group during the years 1968 to 1996. Twenty-seven operations were done for Rasmussen's syndrome, 24 operations for cortical dysplasias/hemimegalencephalies, and 7 for Sturge-Weber syndrome or other congenital vascular problems. Seizure control alone did not seem to adequately describe the outcomes of the procedure. Therefore, a score was constructed that included seizure frequency, motor disability, and intellectual handicap. This burden of illness score better described the child's handicap before and after surgery. RESULTS: Perioperative death occurred in 4 out of 58 children. Of the 54 surviving children, 54% (29/54) are seizure-free, 24% (13/54) have nonhandicapping seizures, and 23% (12/54) have residual seizures that interfere to some extent with function. Reduction in seizures was related to the etiology of the unilateral epilepsy. Eighty-nine percent of children with Rasmussen's, 67% of those with dysplasias, and 67% of the vascular group are seizure-free, or have occasional, nonhandicapping seizures. All operations were considered by the parents and the physicians to have been successful in decreasing the burden of illness. In 44 the procedure was very successful, in 7 it was moderately successful, and in 3 it was minimally successful. Success was related to the etiology, and early surgery was preferable. CONCLUSION: Hemispherectomy can be a valuable procedure for relieving the burden of seizures, the burden of medication, and the general dysfunction in children with severe or progressive unilateral cortical disease. Early hemispherectomy, although increasing the hemiparesis in children with Rasmussen's syndrome, relieves the burden of constant seizures and allows the child to return to a more normal life. In children with dysplasias, early surgery can allow the resumption of more normal development.

Pancreatic lymph nodal and plexus micrometastases detected by enriched polymerase chain reaction and nonradioisotopic single-strand conformation polymorphism analysis: a new predictive factor for recurrent pancreatic carcinoma.

K-ras point mutations have been observed in approximately 90% of pancreatic carcinomas. We genetically analyzed cases of pancreatic regional lymph nodal and plexus micrometastases in invasive ductal carcinoma of the pancreas who were node negative or had metastases limited histopathologically to pancreaticoduodenal lymph nodes. These cases underwent curative resection in our institute. The utility of genetic analysis was compared with that of histopathological study, in terms of postoperative clinical outcome, as a predictive factor for recurrent pancreatic carcinoma. Samples for DNA extraction were obtained from formalin-fixed, paraffin-embedded specimens. A 0.5-microg quantity of DNA was subjected to enriched PCR and nonradioisotopic single-strand conformation polymorphism analysis. K-ras codon 12 mutations were detected in 83% (10 of 12) of invasive ductal carcinomas. In four cases, the genetic analysis of regional lymph nodal metastases and pancreatic plexus invasion of the pancreatic carcinoma yielded results concordant with those of histopathological analysis. In six cases, however, the metastases detected by genetic analysis were more advanced than was indicated by the histopathological examination. The survival rate of cases with metastases beyond the pancreaticoduodenal lymph nodes was significantly lower than that of cases with metastases limited to the pancreaticoduodenal lymph nodes or with no nodal involvement based on genetic analysis (P < 0.05). Intraoperative analysis of point mutations at K-ras codon 12 in the regional lymph nodes and the pancreatic plexus by enriched PCR/nonradioisotopic single-strand conformation polymorphism analysis is a highly accurate predictive factor for recurrent pancreatic carcinoma.

Cathepsin B in the growth of colorectal cancer: suppressive effect of leupeptin on the growth of DMH-induced rat colon neoplasm.

Cathepsin B, a thiol protease, has been reported to be involved in cancer progression and metastasis. The suppressive effects of two kinds of protease inhibitors, leupeptin and dietary camostate (FOY-305), on tumorigenesis and progression in 1, 2-dimethylhydrazine (DMH)-induced rat colon neoplasm were examined in relation to tissue cathepsin B activity. Male Donryu rats were treated with leupeptin or FOY-305 during or after the administration of DMH. There were no significant differences in average tumor numbers among all DMH-treated groups. However, the percentage of small tumors was significantly higher in the group in which leupeptin was supplied during DMH administration. This trend was not recognized in the FOY-305-treated groups. The ratio of cathepsin B activity in the tumors to that in the tumor-bearing tissue (T/Tb) was significantly increased with increasing tumor size (P = 0.009). The cathepsin B activity levels in the tumor-bearing mucosa in the groups which received leupeptin or FOY-305 following DMH treatment were both significantly lower than that in the group which received neither protease inhibitor (P = 0.046 and P = 0.0067, respectively). The results obtained indicate that leupeptin may have suppressed tumor growth by lowering the tissue cathepsin B activity.

Cathepsin B in the growth of colorectal cancer: increased activity of cathepsin B in human colorectal cancer.

Cathepsin B, a thiol protease, is involved in cancer metastasis. To clarify the role of cathepsin B in tumor progression in human colorectal cancer, the relationship between its activity, immunohistochemical staining, and clinical tumor progression was investigated. Cathepsin B activity in adenocarcinomas was significantly elevated compared with that in the tumor-bearing tissue. Furthermore, the tumor/tumor-bearing tissue (T/Tb) ratio of the activity was significantly higher than that of colorectal adenoma. Immunohistochemical studies demonstrated intense staining in the cancerous tissue. With respect to the clinical stage of tumors, the activity tended to be higher in tumors that had invaded the serosa or subserosa than in those that invaded the proper muscle. The results suggest that cathepsin B participates in the progression of human colorectal cancer, and its increased expression is a sensitive marker of the differentiation between colorectal adenoma and adenocarcinoma.

Telomere length in human liver diseases.

To determine the role of telomere-mediated gene stability in hepatocarcinogenesis, we examined the telomere length of human liver with or without chronic liver diseases and hepatocellular carcinomas (HCC). The mean telomere restriction fragment (TRF) length of normal liver (n = 13), chronic hepatitis (n = 11), liver cirrhosis (n = 24) and HCC (n = 24) was 7.8 +/- 0.2, 7.1 +/- 0.3, 6.4 +/- 0.2 and 5.2 +/- 0.2 kb, respectively (mean +/- standard error). TRF length decreased with a progression of chronic liver diseases and that in HCC was significantly shorter than that in other chronic liver diseases (p < 0.05). The ratios of TRF length of HCC to that of corresponding surrounding liver of well differentiated (n = 7), moderately differentiated (n = 10) and poorly differentiated (n = 4) HCCs were 0.83 +/- 0.06, 0.75 +/- 0.05 and 0.98 +/- 0.09, respectively. The ratio of poorly differentiated HCC was significantly higher than that of moderately differentiated HCC (p < 0.05). A comparison between the size and telomere length ratio of moderately differentiated HCCs revealed a decrease of the ratio with size until it reached 50 mm in diameter. In contrast, the ratio increased as the size enlarged over 50 mm. These findings suggest that the gene stability of the liver cells mediated by the telomere is reduced as chronic liver disease progresses and that telomerase is activated in poorly differentiated HCC and moderately differentiated HCC over 50 mm in diameter.

Telomerase as a tool for the differential diagnosis of human hepatocellular carcinoma.

BACKGROUND: Telomerase activation is thought to be essential for the immortality of cancer cells. We measured telomerase activity in human liver samples, including hepatocellular carcinoma (HCC), and evaluated this assay as a tool for the diagnosis of HCC using 21-gauge (21-G)-needle biopsy specimens. METHODS: Ninety-four liver samples (27 HCC, 27 liver cirrhosis, 37 chronic hepatitis, and 3 normal liver) that were surgically resected or biopsied with a 12-gauge Silverman needle and 13 HCC samples that were biopsied with a 21-G needle were analyzed for telomerase activation. RESULTS: Eleven of 29 (38%) tumor-bearing liver samples were weakly telomerase-positive, whereas telomerase activity was observed infrequently in nontumor-bearing liver samples (6 of 35; 17%) and in normal liver samples (0 of 3; 0%). The positivity of surgical samples for well differentiated, moderately differentiated, and poorly differentiated HCC was 88% (7 of 8), 87% (13 of 15), and 0% (0 of 2), respectively. In telomerase-positive HCC, 43% (3 of 7) of well differentiated samples were weakly positive, whereas 92% (12 of 13) of moderately differentiated samples were strongly positive. The difference in the tumor sizes and viral marker status did not affect the activity. The telomerase activity of the 21-G-needle biopsied specimens showed no significant difference from that of the surgical samples. The positive incidence of 21-G specimens was 80% (8 of 10) and 100% (2 of 2) in well differentiated HCC and moderately differentiated HCC, respectively. CONCLUSIONS: An incremental positivity of telomerase was observed during hepatocarcinogenesis. The use of this assay in 21-G-needle biopsy specimens may be useful in clinical examination.

Cellular distribution of transcripts for tissue inhibitor of metalloproteinases 1 and 2 in human hepatocellular carcinomas.

The cellular distribution of tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 was studied by using in situ hybridization in surgically removed human hepatocellular carcinomas (HCCs) and cholangiocellular carcinomas (CCCs). The purpose of this study was to characterize the protein involvement of TIMPs in the development of HCCs and CCCs. All HCCs and CCCs expressed TIMPs. The distribution of transcripts for TIMPs in the tumors was mostly homogeneous. Expression of TIMP in cancer cells was more intense than that in the surrounding noncancerous liver (either, cirrhosis, chronic hepatitis, or normal), and expression of TIMP-1 was stronger than that of TIMP-2. Expression of TIMPs varied among HCC nodules, but there was no obvious association between the expression level of TIMPs and differentiation stages or invasiveness of the HCCs. Transcripts for TIMPs were clearly demonstrated in the metastatic HCC nodules in the lung. Expression of TIMP-1 CCC was strong, and small nodules of CCC were recognized in the liver. Immunohistochemical study for TIMP-1 revealed a consistent staining of the TIMP protein with the transcripts. In the peritumoral histologically normal liver, which was not infected with either hepatitis B or C virus, expression of TIMP-1 was found in various cell types, but that of TIMP-2 was weak. Expression of TIMP-1 in hepatocytes revealed clear zonal distribution. These results suggest that TIMPs may act on modulating the matrix/tumor interaction and may play an important role in growth and invasion of HCCs and CCCs. Expression of TIMP-1 can be a marker of HCC metastasis to the lung, and also that of the extent of CCC invasion. Furthermore, the consistent expression of TIMPs in many cell types of the noncancerous liver suggests some unknown functional role that must be clarified.

Interleukin (IL)-1 beta, IL-6, tumor necrosis factor-alpha, epidermal growth factor, and beta 2-microglobulin levels are elevated in gingival crevicular fluid during human orthodontic tooth movement.

Bone remodeling is a complex process regulated by several mediators. Recent work has revealed that cytokines and growth factors have significant effects on bone cell metabolism. However, little information is available concerning the production of cytokines during orthodontic tooth movement in human subjects, and there is no non-invasive model for determining the production of cytokines. Therefore, the purpose of this study was to identify and quantify the various cytokines in human gingival crevicular fluid (GCF), and to investigate the changes in their levels during orthodontic tooth movement. Twelve patients (mean age, 14.4 years) were used as subjects. An upper canine of each patient having one treatment for distal movement served as the experimental tooth, whereas the contralateral and antagonistic canines were used as controls. The GCF around the experimental and the two control teeth was taken from each subject immediately before activation, and at 1, 24, and 168 hr after the initiation of tooth movement. Cytokine levels were determined by ELISAs. The concentrations of interleukin (IL)-1 beta, IL-6, tumor necrosis factor-alpha, epidermal growth factor, and beta 2-microglobulin were significantly higher in the experimental group than in the controls at 24 hr after the experiment was initiated. All the cytokines remained at baseline levels throughout the experiment for the two control groups. In contrast to cytokine alteration, the amount of total protein in the GCF exhibited a gradual increase, but no significant difference was observed between the control and experimental groups. Since all cytokines in GCF play an important role in the bone remodeling processes in vitro, the present results indicate that the changes in cytokines in GCF are associated with orthodontic tooth movement.