Resistance of MLL-AFF1-positive acute lymphoblastic leukemia to tumor necrosis factor-alpha is mediated by S100A6 upregulation.

Mixed-lineage leukemia (MLL)-AFF1 (MLL-AF4)-positive acute lymphoblastic leukemia (ALL) is associated with poor prognosis, even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The resistance to graft-versus-leukemia (GVL) effects may be responsible for the poor effect of allo-HSCT on MLL-AFF1-positive ALL. Cytotoxic effector mechanisms mediated by tumor necrosis factor-alpha (TNF-α) was reported to contribute to the GVL effect. We showed that MLL-AFF1-positive ALL cell lines are resistant to TNF-α. To examine the mechanism of resistance to TNF-α of MLL-AFF1-positive leukemia, we focused on S100A6 as a possible factor. Upregulation of S100A6 expression and inhibition of the p53-caspase 8-caspase 3 pathway were observed only in MLL-AFF1-positive ALL cell lines in the presence of TNF-α. The effect of S100A6 on resistance to TNF-α by inhibition of the p53-caspase 8-caspase 3 pathway of MLL-AFF1-positive ALL cell lines were also confirmed by analysis using small interfering RNA against S100A6. This pathway was also confirmed in previously established MLL-AFF1 transgenic mice. These results suggest that MLL-AFF1-positive ALL escapes from TNF-α-mediated apoptosis by upregulation of S100A6 expression, followed by interfering with p53-caspase 8-caspase 3 pathway. These results suggest that S100A6 may be a promising therapeutic target for MLL-AFF1-positive ALL in combination with allo-HSCT.

Online CHDF system: excellent cost-effectiveness for continuous renal replacement therapy with high efficacy and individualization.

We developed an online continuous hemodiafiltration (CHDF) system with a central reverse osmosis (RO) fluid delivery system in 1996. This was improved to a system composed of a single-patient dialysis machine and RO module in 2003. This comprises a water treatment system, an RO module, a dialysis machine with 3 endotoxin retentive filters, 2 additional roller pump units, and a disposable special circuit. Dialysate is produced online by a dialysis machine using RO water and dialysate concentrate, which passes through endotoxin retentive filters and is supplied via the machine in the usual manner. A disposable special circuit and additional two roller pumps independently regulate dialysate flow and substitute flow from 0 to 12 in steps of 0.1 l/h. Seventy-seven patients with acute kidney injury (AKI) were treated with online CHDF from December 1996 to June 2004. Patient outcome was compared with the other modality of continuous renal replacement therapy from July 1992 to June 2004. The survival rates of each modality were 68.3, 65.0, 56.6 and 74.0% for conventional CHDF, high-flow continuous hemodialysis, high-flow CHDF and high-flow/high-volume CHDF (online CHDF), respectively. The survival rate of the high-volume modality (online CHDF) group was significantly higher (p = 0.046) than that of the low volume modality group (61.1%). Increases in efficacy and efficiency are a challenge facing blood purification therapy, and, moreover, individualized prescriptions are crucial in AKI patients in ICU. However, the cost of the dialysate and substitution fluid is a limitation of the therapy. The greatest advantage of the system is that a very high dose of delivered dialysate and substitute does not lead to a proportional rise in the cost. The online CHDF system is currently one of the most feasible solutions.

Mutations in the non-structural protein 5A gene in patients with chronic hepatitis C virus 1b infection during repeated interferon treatment.

It has been previously reported that the non-structural region 5A (NS5A) of the hepatitis C virus (HCV) includes an interferon sensitivity determining region (ISDR) and that amino acid substitutions in this region are closely associated with the response to interferon (IFN) treatment. We assessed the clinical significance of serial changes of amino acid sequences in the ISDR during repeated IFN treatment in patients with chronic hepatitis C (genotype 1b), related to serum HCV RNA load. During treatment, additional amino acid substitutions in the ISDR were observed in four of eight patients (50% 2/5 of complete responders (CR); 2/3 of non-responders (NR). However, comparing these amino acid substitutions to wild-type ISDR, the number of amino acid mutations was limited to only one amino acid identified in two CRs. The virus load changed regardless of the amino acid substitutions in the ISDR during treatment, and the wild-type and intermediate type (with less than three amino acid substitutions) showed wide variations in virus load. These data indicate that amino acid mutations in the ISDR, which indicate the switch to mutant-type do not occur easily even during repeated IFN treatment, and the additional amino acid substitutions in the ISDR are not a sensitive marker during repeated IFN treatment. In cases where virus load is used as a marker of response to repeated UN treatment, serial examinations are necessary to determine the precise virus load levels.

Amino acid mutations in the interferon sensitivity determining region and serum virus load in hepatitis C virus carriers with long-term normal ALT levels.

The amino acid mutations in a part of the non-structural region 5A (NS5A) of the hepatitis C virus (HCV) genome, called the interferon sensitivity determining region (ISDR), can affect the response to interferon (IFN) treatment. We analyzed the serial changes of the amino acid substitutions in the ISDR during the natural course of patients with sustained long-term normal alanine aminotransferase (ALT) levels in relation to the changes in virus load, and assessed the clinical significance of ISDR in the natural course and IFN treatment. The subjects were nine patients infected with HCV (genotype 1b) who had been examined for serum ALT levels every month for more than 1 year and had well-sustained normal levels. The amino acid sequence of the ISDR was determined by the direct sequencing method, and the number of amino acid mutations was evaluated in comparison with the prototype (HCV-J). Quantitation of serum HCV RNA levels was conducted by the Amplicor-monitor method (Nihon Roche). On the initial analysis of the ISDR, six patients were determined to have no mutations, and three patients had one or two mutations. However, an increased number in amino acid mutations compared with the wild type during the follow-up period was confirmed in only one patient, and that increase was limited to within two amino acids. Virus load changed regardless of the changes in amino acid substitutions in the ISDR. The ISDR was therefore inferred to be a stable region unrelated to the virus load in patients with well-sustained normal ALT levels. Additional changes of amino acid sequence in this region were not a sensitive marker for determining whether IFN treatment is indicated.

Congenital hepatic fibrosis with fatal cholestatic liver damage.

A 23-year-old male with congenital hepatic fibrosis died because of progressive cholestatic liver damage. Pathologically, marked extension of fine fibers along the sinusoids in addition to fibrosis in Glisson's sheath, miniaturization and pseudo-glandular formation of hepatocytes as parenchymal damage, and nodular regenerative hyperplasia were considered as the cause of rapid aggravation of liver damage or portal hypertension.

Transcatheter arterial embolization for impending rupture of an isolated internal iliac artery aneurysm complicated with disseminated intravascular coagulation.

A 90-year-old male, with impending rupture of an isolated internal iliac artery aneurysm (IIAA) complicated with disseminated intravascular coagulation (DIC) was successfully treated with transcatheter arterial embolization (TAE). After TAE, enlargement of the aneurysm was arrested and coagulation-fibrinolytic abnormalities induced by DIC improved without severe complications. Although IIAA is relatively rare, the post-operative mortality of patients with ruptures is reportedly high. We assessed the usefulness of this procedure for impending rupture of IIAA, especially for patients in high risk groups.

Increased serum levels and sinusoidal expression of thrombomodulin in acute liver damage.

Thrombomodulin (TM) is a surface glycoprotein of endothelial cells involved in both anticoagulation and antifibrinolysis. In this study, we assessed the clinical significance of TM in acute liver damage by using a rat model induced by intraperitoneal injection of D-galactosamine (Gal-N). Serum TM levels were measured with enzyme immunoassay utilizing rabbit anti-rat TM antibody. Simultaneously, immunohistochemical examination was performed using the same antibody. Serum TM levels increased significantly after the injection of Gal-N compared with preinjection levels, peaking from 48 to 72 hours after injection and normalizing by 168 hours. Changes in parenchymal damage were synchronized with changes of TM, and changes of TM levels mirrored changes of liver weight. In immunohistochemical examination, TM immunoreactivity was observed only on the endothelial surfaces of both the artery and portal vein within Glisson's sheath in controls. After injection of Gal-N, TM immunoreactivity was gradually intensified, especially around the necrotic area and the central veins. These findings disappeared with improvement of parenchymal damage. Both the increase of serum TM levels and intensified TM immunoreactivity in the liver were synchronized with acute liver parenchymal damage induced by Gal-N. These findings on TM are related to endothelial damage with parenchymal necrosis and liver regeneration interacting with both homeostasis of microcirculation and healing of parenchymal damage.

Clonotypic analysis of T cells in patients with autoimmune and viral hepatitis.

The immunopathogenesis of autoimmune hepatitis (AIH), and the role of T cells in the onset and maintenance of this disease, are still unclear. Since T cells expand clonally after stimulation by an antigen, it is important to analyze the behavior of T cells at a clonal level. We have established recently a novel system, using reverse transcriptase-polymerase chain reaction (RT-PCR) and subsequent single-strand conformation polymorphism (SSCP) that allows the identification of clonal accumulation of T cells in a lymphocyte population. Using this system, we demonstrated that oligoclonal T cells were accumulated in the liver of patients with AIH, and that identical T-cell clonotypes were detected in two different regions of the liver, although these features were also observed in cases with viral hepatitis. Only in cases with AIH, however, nearly all identical T cells were found to belong to CD8+ subset and there were very few CD4+ T cells in this population. Our results suggest that common antigens presented to CD8+ T cells in the context of HLA class I molecule are distributed diffusely in the liver of AIH. These findings also suggest that antigens recognized by CD4+ T cells may be relatively heterogeneous in the liver with AIH.

[Cryoglobulinemia associated with hepatitis C-related chronic liver disease in Japan].

The association between the actual status of cryoglobulins (CGs) expression in hepatitis C-related chronic liver disease (C-CLD) and different types of liver pathology, HCV-RNA titers and genotypes was investigated. Sixteen out of 1340 ordinary clinical specimens were CGs-positive (1%), and in 8 of them (50%) the patients blood was HCV-RNA positive. CGs was detected in 63% of C-CLD patients as a whole, but when compared with the histological findings in the liver, it was 88% in F3, and 92% in A3, and thus high percentages were detected in patients with progression of liver fibrosis and patients with strong activity. Serum IgG, IgM, transaminase and gamma-GTP levels were significantly higher in the patients with CGs, and their RA test and C3dCIC levels tended to be higher, but the differences were not significant, and no association was found with the anti-nuclear antibody positive level, or the HCV-RNA titers or genotypes. Based on the above, there was a clear involvement of HCV infection, especially the activity and histological progression of hepatitis in CGs formation in Japan as well, but there were few extrahepatic manifestations, suggesting differences in CGS levels and immune response to CGs from cases in Western countries.

[Outcome of the cases of chronic liver disease type C who exhibited long term response to interferon therapy; comparison of the cases with or without serum HCV-RNA].

We evaluated the outcome of 71 Japanese patients with chronic hepatitis C who exhibited a sustained normal level of serum ALT for one to five years after the cessation of interferon (IFN) therapy. Patients were classified into two groups: 9 (13%) cases with a sustained positive for serum HCV-RNA (incomplete responders: ICR) and 62 (87%) cases with a sustained negative for serum HCV-RNA (complete responders: CR). The anti-C100-3 titer was similarly decreased in both groups especially up to one year after cessation of IFN becoming negative in 50% of each group over a 4-year period. The anti-C-22 titer was also decreased in the CR group and stabilized at about 100U (RIA). The titer of anti-C-22 was either increased or maintained at pretreatment levels in most cases of ICR. Monitoring of anti-C-22 may be useful in distinguishing the presence of HCV viremia from a previous HCV infection. According to the backgrounds between both groups, ICR cases were significantly older and more often of genotype 1b (67%) than CR cases (1b; 32%), however there were no significant differences in sex, liver histology or pretreatment levels of HCV-RNA. Serum levels of HCV-RNA in ICR cases after IFN therapy either increase or remained similar to pretreatment levels however most patients with ICR showed an improvement in liver histology. Thus if the serum ALT level remained normal in case of ICR the objective of administering IFN to treat chronic hepatitis C was considered to have been achieved despite the persistence of HCV viremia.

[Risk factors for pulmonary hypertensive crisis (PHC) following VSD repair in infants].

We examined 100 patients who had undergone VSD repair from 1988 to 1991 to determine the risk factors to induce PHC postoperatively. We analyzed age, weight, preoperative Pp/Ps, Rp/Rs, Qp/Qs, and postoperative Pp/Ps using discriminant analysis. We had 34 cases of pulmonary hypertension (PH: Pp/Ps > 0.7) preoperatively. Among 100 cases, PHC developed in 6 patients and 2 of them died postoperatively. Incidence of PHC in all patients was 6%, and that in patients with PH was 18%. PHC fatality rate was 33%. The analysis revealed that the occurrence of PHC was significantly higher among those whose ages were below 2.1 years, and with weight of under 9.85 kg, preoperative Pp/Ps > 0.73, Rp/Rs < 0.34, and postoperative Pp/Ps > 0.43. In those with preoperative PH, the risk for PHC increased significantly when their postoperative Pp/Ps exceeded 0.44.

[Study of hepatocellular carcinoma type C by quantitation of serum HCV-RNA using branched DNA probe assay].

Serum HCV-RNA levels were determined by newly developed branched DNA probe (bDNA) assay in 87 patients with hepatocellular carcinoma (HCC), compared with 73 patients with chronic hepatitis active and 30 patients with liver cirrhosis. Patients with decompensated liver cirrhosis (LC-d) had a significant lower viremia (mean 3.2Meg.eq./ml, bDNA positive rate; 40%) than chronic hepatitis active (18.0; 64%) and compensated LC (LC-c, 17.9; 80%) (p < 0.05). Those data indicates that HCV replication may decrease as progression of LC. In contrast, there was no difference between the levels of HCC with LC-c (8.2; 85%) and LC-d (6.1; 89%), and their positive rate of bDNA assay were significantly higher than LC-d without HCC (p < 0.01). Therefore, patients undergoing LC in whom serum HCV-RNA sustained high level may correspond to the high risk group of HCC. In HCC of heavy drinker, serum HCV-RNA levels (11.3; 91%) were significantly higher than the levels (4.6; 79%) of HCC without heavy drink (p < 0.05). The result indicates that heavy drink may induced an increase in HCV replication.

[Serum levels of HCV-RNA determined by branched DNA (bDNA) probe assay in chronic hepatitis C: method and clinical significance on interferon (IFN) therapy].

Recently serum levels of HCV RNA (s-HCV RNA) in chronic hepatitis C has been regarded as one of an important indicator for the activity of disease and outcome of IFN therapy. Quantitative analysis by competitive RT-PCR, however, requires special skills and is too expensive for clinical use. We attempted to determine serial sample of s-HCV RNA and genotypes in 117 cases treated with IFN using bDNA probe assay which has lately been developed by Chiron corporation. Cases with complete response to IFN therapy showed 62% (49/79) in lower than 10(6) levels, 27% (8/30) in 10(6)-10(7) and only 5% (2/41) in higher than 10(7). Genotype III, IV had higher response than type II on the same level of s-HCV RNA. These data indicates that determination of s-HCV levels by bDNA assay may be useful for prediction of the outcome of IFN therapy and making adequate schedule of the therapy in each cases.

[Long-term interferon (IFN), neominophagen C (SNMC) combination therapy of active liver cirrhosis type C].

Possible effects of IFN and SNMC combination therapy for active liver cirrhosis were described. 28 patients with active liver cirrhosis were treated with natural IFN alpha 3-6 MU TIW for 3-17 months (mean, 8 +/- 6). 8 of 28 patients received combination therapy of SNMC (40 ml iv, TIW). 8 (29%) of 28 patients sustained normal ALT levels with no relapse (complete response) which involved 6 patients with persistent lack of serum HCV RNA sequences after IFN therapy. In those 8 patients, 4 had been received combination of SNMC. There were 4 (14%) patients with relative response. Patients with genotype III or IV had significantly higher response to IFN therapy than the patients with genotype II. In active liver cirrhosis, determination of HCV genotype and HCV RNA levels thought to be significant for the indication of IFN therapy, and that combination of SNMC may provide beneficial effect to IFN therapy for active liver cirrhosis.

[Airborne fungi during the last ten years in Sagamihara].

Airborne fungi have been surveyed every week during the 10 years from 1983 to 1992 in Sagamihara. This is a follow up of a 1970 and a 1980 survey at the same sampling station (19.5 m above the ground), using the same gravity potato dextrose agar plate. There were two peak seasons during tsuyu (rainy season; June) and autumn (September to October). The most predominant fungi found, except for yeasts and non-sporulated fungi, were Cladosporium (2 seasons) and Alternaria (more frequently in tsuyu). These were followed by Epicoccum (tsuyu), Aureobasidium, Curvularia (summer), Ulocladium (autumn), Penicillium, Arthrinium, Nigrospora (summer), Fusarium, Trichoderma, Pestalotia in decreasing order of their total frequency throughout the 10 years. The most common fungi found during the most years were different from those found during 1970, particularly in the frequency of Aspergillus and Penicillium, but similar to those during 1980 in Sagamihara as well as in most areas of Japan.

[Cerebral protection during aortic arch replacement: usefulness of continuous O2 saturation monitoring of internal jugular bulb to optimize cerebral perfusion].

How to optimize cerebral perfusion pressure and flow during selective extracorporeal circulation is a crucial problem for cerebral protection in surgical repair of aortic arch aneurysm. Among 47 cases of aortic arch replacement between 1980 and 1992, extracorporeal circulation (ECC) for the first 17 cases [group-1] were hypothermic ECC with selective cerebral perfusion (SCP) and 8 cases [group-2] with hypothermic ECC with hypothermic cardiac arrest. For the latest 16 cases [group-3] we introduced continuous O2 saturation monitoring by oximetry catheter placed in internal jugular bulb (SIJVO2) and maintained SIJVO2 value above 90% to effectively adjust pump flow to optimize cerebral perfusion pressure and flow for cerebral protection. We have retrospectively compared the effectiveness of SIJVO2 monitoring among these three groups. The mortality was 35% (6 cases: group-1), 37% (3 cases: group-2) and 6% (1 case: group-3) respectively. Cerebral dysfunction which were diagnosed in immediate postoperative period were 23% (4 cases: group-1), 0% (0 case: group-2) and 6% (1 case: group-3) respectively. We conclude continuous monitoring of SIJVO2 during selective ECC in aortic arch replacement is useful to optimize cerebral perfusion pressure and flow thereby reducing postoperative cerebral damage by selective ECC.

[Adequate parameters for the diagnosis of disseminated intravascular coagulation (DIC) in patients with liver diseases].

Despite of many investigations addressing the problem on the diagnosis of DIC associated with liver diseases, however, an adequate clinical and laboratory criteria has not yet been established. We attempted to clarify this problem by evaluating the changes of plasma levels of PIC, D dimer, TAT and several endothelial factors in 20 patients with severe liver disease who had the evidence of hemorrhage, and were treated with AT III concentrate and gabexate mesilate (FOY). In patients who show a good response to treatment, plasma levels of PIC and D dimer before treatment were both significantly higher (p < 0.01) than those in patients who did not respond, while there was no significant difference in other coagulation fibrinolysis parameters except for platelet count which showed rather lower in the response group (p < 0.05). We believe that combination assay for both PIC and D dimer will be adequate to differentiate whether the hemostatic abnormalities are induced mainly by DIC or hepatic insufficiency.