RaRF confers RA resistance by sequestering RAR to the nucleolus and regulating MCL1 in leukemia cells.

Retinoic acid (RA) has broad clinical applications for the treatment of various cancers, particularly acute promyelocytic leukemia. However, RA-based therapy is limited by relapse in patients associated with RA resistance, the mechanism of which is poorly understood. Here, we suggest a new molecular mechanism of RA resistance by a repressor, named RA resistance factor (RaRF). RaRF suppressed transcriptional activity of the RA receptor (RAR) by directly interacting with and sequestering RAR to the nucleolus in response to RA. RaRF was highly expressed in RA-resistant leukemia cells and its expression was strongly correlated with RA sensitivity. MCL1 was upregulated by RA treatment upon RaRF depletion, accompanying leukemic myeloblast differentiation, which is negatively regulated by ectopic RaRF expression. Collectively, we propose that RaRF may be a factor in the resistance mechanism and thus a potential target for leukemia therapy using RA.

Long-term Clinical Outcomes of First and Second Kidney Transplantation in Patients With Biopsy-Proven IgA Nephropathy.

INTRODUCTION: The recurrence of IgA nephropathy (IgAN) after kidney transplantation (KT) has an effect on graft survival, but there are few reports about long-term clinical outcomes of KT with recurrent IgAN. This study shows the long-term clinical outcomes of KT in patients with IgAN. METHODS: All recipients who had biopsy-proven IgAN were followed from February 1990 to February 2016. We analyzed overall graft and patient survival rates, incidence of recurrent IgAN, factors affecting graft survival, and IgAN recurrence. RESULTS: There were 88 patients with first KT. The mean follow-up duration was 82.5 months. Twenty patients went through graft loss and 1 patient died due to sepsis. IgAN recurred in 15 patients, and 11 patients experienced graft failure. Among the patients who had failed graft after first KT, 7 patients underwent retransplantation. The graft survival period, presence of rejection, and proteinuria were the relevant risk factors for recurrence of IgAN. In the first KT patients, presence of rejection and 1-year serum creatinine were the significant risk factors for graft loss. But recurrence of IgAN was not a relevant risk factor. Overall graft survival rates at 5 and 10 years were 93.8% and 73.1% in the first transplantation group and 100% and 100% in the retransplantation group, respectively. CONCLUSION: Although IgAN recurrence was a significant risk factor for graft failure, the patient who underwent retransplantation showed favorable results. Retransplantation should be considered in patients who lost their first graft after recurrence of IgAN.

Long-term Clinical Outcomes of Kidney Re-transplantation.

BACKGROUND: Kidney re-transplantation is commonly considered to have a higher immunological risk than first kidney transplantation. Because of the organ shortage and increasing waiting lists, long-term outcomes of kidney re-transplantation are being studied. However, reports of re-transplantation outcomes are not common. We have reported our 30 years of experience with second kidney transplantations. METHODS: Of 1210 kidney transplantations between November 1982 and August 2016 performed in our hospital, 105 were second kidney transplantations (2nd KT). Living donor KT was 44; deceased donor KT was 61. RESULTS: Patient survival rates at 1, 5, and 10 years were 100%, 97.2%, and 90.7%, and graft survival rates were 97.0%, 94.6%, and 71.5%, respectively. The leading cause of graft failure in the 2nd KT was chronic rejection (60%). In addition, induction immunosuppressant, maintenance immunosuppressant, delayed graft function, and graft survival time at the 1st KT had a significant impact on graft survival time at the 2nd KT. CONCLUSIONS: Reasonable results in both patient survival and graft survival rates were found in the 2nd KT. Careful monitoring of immunologic risk is needed.

Overall accuracy of cervical cytology and clinicopathological significance of LSIL cells in ASC-H cytology.

OBJECTIVE: The aims of this study were (i) to investigate the diagnostic accuracy of Papanicolaou (Pap) smears and (ii) to evaluate the clinicopathological significance of the presence of low-grade squamous intraepithelial lesion (LSIL) cells in atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (HSIL) (ASC-H) cytology. METHODS: We retrospectively reviewed paired cytological and histological findings from 3141 patients. ASC-H cytology was classified as either ASC-H or LSIL with some features suggestive of the presence of a concurrent HSIL (LSIL-H). Clinicopathological characteristics were evaluated through a retrospective study and meta-analysis. RESULTS: The accuracy of the cytological diagnosis was 93.7% (2942 of 3141 cases). The positive predictive value (PPV) of ASC-H for cervical intraepithelial neoplasia grade 2 or worse (CIN 2+ ) was 51.4%. In cases of LSIL-H, CIN 2+ histology was more prevalent in the pre-menopausal period (19-44 years) than in peri- and postmenopausal periods (older than 45 years) (P = 0.024). There was no difference in the ability of LSIL-H and ASC-H to predict CIN 2+. CONCLUSION: The Pap smear is a good cervical cancer screening method. Although there was no difference in the predictive value for CIN 2+ between LSIL-H and ASC-H, the presence of definite LSIL cells was more predictive of CIN 2+ in younger patients than in older patients.

ASXL2 promotes proliferation of breast cancer cells by linking ERα to histone methylation.

Estrogen receptor alpha (ERα) has a pivotal role in breast carcinogenesis by associating with various cellular factors. Selective expression of additional sex comb-like 2 (ASXL2) in ERα-positive breast cancer cells prompted us to investigate its role in chromatin modification required for ERα activation and breast carcinogenesis. Here, we observed that ASXL2 interacts with ligand E2-bound ERα and mediates ERα activation. Chromatin immunoprecipitation-sequencing analysis supports a positive role of ASXL2 at ERα target gene promoters. ASXL2 forms a complex with histone methylation modifiers including LSD1, UTX and MLL2, which all are recruited to the E2-responsive genes via ASXL2 and regulate methylations at histone H3 lysine 4, 9 and 27. The preferential binding of the PHD finger of ASXL2 to the dimethylated H3 lysine 4 may account for its requirement for ERα activation. On ASXL2 depletion, the proliferative potential of MCF7 cells and tumor size of xenograft mice decreased. Together with our finding on the higher ASXL2 expression in ERα-positive patients, we propose that ASXL2 could be a novel prognostic marker in breast cancer.

Successful treatment of disseminated adenovirus infection with ribavirin and intravenous immunoglobulin in an adult renal transplant recipient: a case report.

Disseminated adenovirus infection in recipients of renal transplants is a rare but often fatal complication. We present a case of a 32-year-old woman who underwent renal transplantation from a deceased donor. Ten months after transplantation, she presented with dysuria, hematuria, and febrile illness. Despite the use of antibiotics, the patient's symptoms continued and worsened and the serum creatinine level was increased. The results of urine and serum polymerase chain reaction were positive for adenovirus. Renal biopsy revealed viral interstitial nephritis. The patient was treated with ribavirin, intravenous immunoglobulin, and reduction in immunosuppression. Her symptoms progressively improved from 7 days after the treatment. Serum and urine polymerase chain reaction for adenovirus became negative 10 and 21 days after the treatment, respectively. She remained in good health with excellent allograft function 6 months later.

Clinical features and natural history of acquired third, fourth, and sixth cranial nerve palsy.

PURPOSE: Clinical features of acquired third, fourth, and sixth cranial nerve palsy showed variation among previous studies. Evaluation of natural course with objective criteria will establish accurate recovery rates and important factors for recovery. METHODS: Retrospective chart review was performed on 206 patients who visited a neuro-ophthalmic department with acquired third, fourth, and sixth nerve palsy. Aetiology and results of ocular exam on each visit were reviewed, and multivariate logistic regression analysis was performed to identify independent factors affecting recovery. RESULTS: The sixth cranial nerve was affected most frequently (n=108, 52.4%) and vascular disease (n=64, 31.1%) was the most common aetiology. Recovery was evaluated with change of deviation angle for 108 patients, who were first examined within a month of onset and followed up for at least 6 months. Ninety-two (85.2%) patients showed overall (at least partial) recovery and 73 (67.6%) showed complete recovery. In univariate analysis, initial deviation angle was found to be only significant factor associated with complete recovery (P=0.007) and most patients who experienced successful management of treatable underlying disease showed recovery. CONCLUSIONS: With objective criteria based on deviation angle, overall recovery rate from the third, fourth, and sixth nerve palsy was 85.2%. Patients who had smaller initial eyeball deviation or successful management of treatable underlying disease had a high chance of recovery.

Hepatitis C virus core protein transactivates insulin-like growth factor II gene transcription through acting concurrently on Egr1 and Sp1 sites.

The possibility that hepatitis C virus core gene product (HCV-core) acts as a transactivator in insulin-like growth factor II (IGF-II) gene transcription was tested. HCV-core protein increases endogenous IGF-II expression from promoter 4 (P4) of the IGF-II gene through two cis-acting elements: Sp1 and Egr1 binding sites. Sp1 and Egr1 both bind to IGF-II P4 and functionally cooperate in mediating the maximal activity of IGF-II P4. HCV-core protein induced the binding of Sp1 and Egr1 on its binding sites on IGF-II P4. In addition, Sp1 and Egr1 were stimulated to phosphorylate by HCV-core, and its DNA binding activity was up-regulated upon HCV-core transfection. Transfection with HCV-core in HepG2 cells stimulated the membrane translocation of protein kinase C (PKC) and the treatment of HCV-core transfected cells with calphostin C, a PKC inhibitor, blocked induction of Sp1 and Egr1 DNA binding activity, and eventually transcriptional transactivations of the IGF-II gene. Increasing the DNA binding activity of the phosphorylated form of Sp1 and Egr1 might be an important mechanism for regulating IGF-II gene expression and for promoting cell division during hepatic carcinogenesis. These results indicate that HCV-core functions as a positive regulator of IGF-II transcription through the PKC pathway and that Sp1 and Egr1 are direct targets of the transcriptional regulation of the IGF-II gene which plays an important role in hepatitis C virus pathogenesis during the formation of hepatocellular carcinoma (HCC).

Correlation of VEGF with contrast enhancement on dual-phase dynamic helical CT in liver tumors: preliminary study.

The purpose of this preliminary study is to elucidate that vascular endothelial growth factor (VEGF) influences contrast enhancement of hepatic tumors on computed tomography (CT). Fourteen patients with hepatic tumors (11 hepatocellular carcinomas; 3 metastatic cancers) underwent a dual-phase dynamic helical CT or computed tomographic hepatic arteriography. The attenuation of each mass was determined as hyperattenuation, isoattenuation or hypoattenuation with respect to the adjacent nontumorous parenchyma. Gun-needle biopsy was done for each tumor, and paraffin sections were immunostained with anti- VEGF antibody by the avidin-biotin-peroxidase complex method. The pathologic grade was made by intensity (1 +, 2+, 3+) and area (+/-, 1 +, 2+). The tumor ranged 2.0-14.0 cm in size (mean, 5.8 cm). In arterial phase, the intensity was not correlated with the degree of enhancement (p=0.086). However, the correlation between the attenuation value of hepatic arterial phase and the area of positive tumor cells was statistically significant (p=0.002). VEGF may be the factor that enhances the hepatic mass with water-soluble iodinated contrast agent in CT.

Activation of the insulin-like growth factor II transcription by aflatoxin B1 induced p53 mutant 249 is caused by activation of transcription complexes; implications for a gain-of-function during the formation of hepatocellular carcinoma.

Aflatoxin B1 (AFB1) induced mutation of the p53 gene at codon 249 (p53mt249) is critical during the formation of hepatocellular carcinoma (HCC) following hepatitis B virus (HBV) infection. p53mt249 markedly increases insulin-like growth factor II (IGF-II) transcription largely from promoter 4, accumulating the fetal form of IGF-II. Modulation of the transcription factor binding to IGF-II P4 by wild-type p53 and p53mt249 was identified. Wild-type p53 inhibited binding of transcription factors Sp1 and TBP on the P4 promoter, while p53mt249 enhanced the formation of transcriptional complexes through enhanced DNA-protein (Sp1 or TBP) and protein-protein (Sp1 and TBP) interactions. p53mt249 stimulates transcription factor Sp1 phosphorylation which might be a cause of increased transcription factor binding on the P4 promoter while wild-type p53 does not. Transfection of hepatocytes with p53mt249 impaired induction of apoptosis by the HBV-X protein and TNF-alpha. Therefore, the blocking of apoptosis through enhanced production of IGF-II should provide a favorable opportunity for the selection of transformed hepatocytes. These results explain the molecular basis for the genesis of HCC by p53mt249 which was found to be induced by a potent mutagen, AFB1.

Hepatitis B virus-X protein upregulates the expression of p21waf1/cip1 and prolongs G1-->S transition via a p53-independent pathway in human hepatoma cells.

Progression through the cell cycle is controlled by the induction of cyclins and activation of cognate cyclin-dependent kinases. The human hepatitis B virus-X (HBV-X) protein functions in gene expression alterations, in the sensitization of cells to apoptotic killing and deregulates cell growth arrest in certain cancer cell types. We have pursued the mechanism of growth arrest in Hep3B cells, a p53-mutant human hepatocellular carcinoma (HCC) cell line. In stable or transient HBV-X transformed Hep3B cells, HBV-X increased protein and mRNA levels of the cyclin-dependent kinase inhibitor (CDKI) p21(waf1/cip1) increased binding of p21(waf1/cip1) with cyclin-dependent kinase 2 (CDK2), markedly inhibited cyclin E and CDK2 associated phosphorylation of histone H1 and induced the activation of a p21 promoter reporter construct. By using p21 promoter deletion constructs, the HBV-X responsive element was mapped to a region between -1185 and -1482, relative to the transcription start site. Promoter mutation analysis indicated that the HBV-X responsive site coincides with the ets factor binding sites. These data indicate that in human hepatocellular carcinoma cells, HBV-X can circumvent the loss of p53 functions and induces critical downstream regulatory events leading to transcriptional activation of p21(waf1/cip1). As a consequence, there is an increased chance of acquisition of mutations which can enhance the genesis of hepatomas. Our results also emphasize the chemotherapeutic potential of p21(waf1/cip1) inhibitors, particularly in the HBV-X infected hepatoma which lacks functional p53.

Mutations in the p53 tumor suppressor gene in tree shrew hepatocellular carcinoma associated with hepatitis B virus infection and intake of aflatoxin B1.

Infection with hepadnaviruses and exposure to aflatoxin B1 (AFB1) are considered to be major risk factors in the development of hepatocellular carcinoma (HCC) in humans. A high rate of p53 mutations at codon 249 has been reported in these tumors. The tree shrew (Tupaia belangeri chinensis) is a useful animal model for the development of HCC after human hepatitis B virus (HBV) infection or AFB1 treatment. Therefore, it was of particular interest to determine whether the p53 gene in tree shrew HCCs associated with HBV infection and/or with exposure to AFB1 is affected in the same manner as in human HCCs. We determined the tree shrew p53 wild-type nucleotide sequences by RT-PCR and automatic DNA-sequencing. Tree shrew wild-type p53 sequence showed 91.7 and 93.4% homologies with human p53 nucleotide and amino acids sequences, respectively, while it showed 77.2 and 73.7% homologies in mice. One HCC and normal liver tissue from AFB1 treated and one HCC from AFB1- and HBV-treated tree shrew showed no change in p53 sequences, while three HCCs from AFB1- and HBV-treated tree shrews showed point mutations in p53 sequences. One HCC showed point mutations at codon 275, which is on the DNA-binding domain of p53 gene, which might be a cause of gain-of-function during the development of HCC. As a result, our finding indicates that tree shrews exposed to AFB1 and/or HBV had neither codon 249 mutations nor significant levels of other mutations in the p53 gene, as is the case with humans.

A radioimmunoassay method for detection of DNA based on chemical immobilization of anti-DNA antibody.

High selectivity provided by biomolecules such as antibodies and enzymes has been exploited during the last two decades for development of biosensors. Of particular importance are efficient immobilization methods for biomolecules in order to preserve their biological activities. In this study, we have evaluated immobilization strategies for an anti-DNA antibody on a self-assembled monolayer of omega-functionalized thiols. The antibody was immobilized via peptide bond formation between the primary amines in the antibody and the carboxyl groups on the self-assembled monolayer. The peptide bond coupling was achieved by activating COOH groups on the surface through N-Hydroxysuccimide (NHS)-ester formation, followed by acylation of NH2 group in the antibody. DNA binding activity of the immobilized antibody was examined by counting beta emission from 35S-labeled DNA.

Human interleukin 6 gene is activated by hepatitis B virus-X protein in human hepatoma cells.

Interleukin 6 (IL-6) is a pleiotropic cytokine that induces many biological activities, including some aspects of the immune reaction and inflammatory responses. In the liver, IL-6 regulates the synthesis of a broad spectrum of acute-phase proteins. IL-6 is also known to be a factor involved in the immunoregulatory perturbations in patients with chronic liver diseases (CLDs). Here, we report that IL-6 can be induced by hepatitis B virus (HBV)-X protein, as evidenced by high levels of serum IL-6 in patients with CLD with HBV infection, IL-6 productions observed in HBV-X-transfected cells, and transcriptional transactivations of the IL-6 gene by HBV-X. We determined serum levels of IL-6 in patients with chronic hepatitis B (CH-B), chronic hepatitis C (CH-C), liver cirrhosis (LC) caused by hepatitis B, and LC with hepatocellular carcinoma (HCC) caused by hepatitis B (LC+HCC). Mean serum levels of IL-6 in all CLD patients were higher than those in normal controls, and the difference was statistically significant (P < 0.05). Mean IL-6 levels of LC and LC+HCC patients were significantly higher than those of CH-B patients (P < 0.05). Because the etiological factor in all cases except CH-C (CH-B, LC, and LC+HCC) was HBV, we checked the possibility of HBV-transactivator-X activation of IL-6 promoter. Using deletion constructs of 5'-flanking regulatory regions of the IL-6 gene linked to the chloramphenicol acetyltransferase gene as a reporter, we found that the binding of nuclear factor-kappaB to a cis element is essential and sufficient for the induction of the IL-6 gene by HBV-X. We also found that HBV-X enhances the binding of two subunits of nuclear factor-kappaB (p65 and p52) to their target DNA binding sequences. These observations are relevant, in that HBV-X might play an important role in hepatic inflammation and diseases by up-regulating IL-6 production, which can eventually lead to LC and HCC.

Establishment and characterization of cell lines constitutively expressing hepatitis B virus X-protein.

We prepared human hepatoma cell lines, which expressed the human hepatitis B virus-X gene product. The plasmid pMAMneo-X, containing an HBV-X gene promoter, an enhancer and a structural gene was constructed. Transfected HBV-X gene integration and expression were detected by Southern and Northern blotting, as well as by chloramphenicol acetylase transferase (CAT) assay using various kinds of promoter-CAT reporter systems. HBV-X protein expression in stable transfectants was confirmed by immunofluorescence microscopy. Transfected cell lines showed permanent expression of HBV-X proteins. The HBV-X transfectant activated its target promoters in promoter-CAT constructs as reporters. The HBV-X transfectant enhanced AP-1 transcription factor binding to its target DNA. Therefore, X-transfectants are not only stable, but also have specific biological functions. Cell cycle analysis by flow cytometry showed that the majority of the transfectant cells are arrested in the G1 or G2 phase of the cell cycle. These cell lines may be useful in analyzing the biological functions of HBV-X and its functional role in the formation of hepatocellular carcinomas.

Outcome and predictors of success of biofeedback for constipation.

BACKGROUND: A study was undertaken to determine outcome and to identify predictors of success for biofeedback for constipation. METHODS: Patients who had at least one biofeedback session were evaluated whether or not they completed a treatment course. Parameters assessed included use of cathartics, number of spontaneous bowel movements per week, presence of rectal pain, number of biofeedback sessions and results of anorectal physiology. RESULTS: A total of 194 patients (59 male, 135 female) of median age 71 (range 11-96) years, including 30 with concomitant rectal pain, were treated. The median number of spontaneous bowel movements per week before treatment was 0. Some 35 per cent of patients had complete success (three or more spontaneous bowel movements per week with discontinuation of cathartics), 13 per cent had partial success (fewer than three spontaneous bowel movements per week with continued use of cathartics) and 51 per cent had no improvement. Neither patient age, sex nor duration of symptoms significantly affected outcome. Only 18 per cent of patients who had between two and four sessions had complete success, compared with 44 per cent of those who had five or more (P < 0.001). A total of 63 per cent of patients who completed the treatment protocol experienced complete success, compared with 25 per cent of those who self-discharged (P < 0.0001). CONCLUSIONS: This large study indicates that the success rate of biofeedback for patients with constipation is less than previously reported. However, the success rate improves significantly after five or more sessions and is significantly related to the patient's willingness to complete treatment.

Are interpretations of video defecographies reliable and reproducible?

Video defecography is a dynamic investigation which can influence surgical decision making in constipated patients. A study was therefore undertaken to assess the inter and intraobserver variability in video defecography. Specifically, we sought to assess the interpretation of video defecographies by a group of observers with the same training, guidelines and standards. To determine interobserver variation, four independent observers, two blinded to the patient's history, reviewed 100 randomly sequenced video defecographies performed in constipated patients. The presence or absence of sigmoidocele, rectocele, intussusception or prolapse was noted. Adequate or improper function of the puborectalis, anal canal opening, anorectal angle (ARA) and grade of emptying of the rectum were also assessed. Two weeks after the initial assessment, intraobserver variation was determined by a repeat blinded review of unlabelled randomly sequenced studies. The results of interobserver accuracy for sigmoidoceles, rectoceles, intussusception, rectal prolapse, rectal emptying, opening of the anal canal, puborectalis contraction and straightening of the ARA and rectal emptying were 89.5%, 46.0%, 87.5%, 97.5%, 86.5%, 88.5%, 83.0%, and 80.0%, respectively. The intraobserver variations were 88.5%, 83.8%, 80.5%, 94.5%, 77.0%, 84.8%, 80.5% and 85.5%, respectively. Prior knowledge of the patient's history did not significantly influence the outcome. In summery, video defecography has an overall accuracy of 83.3% and as such is a valid tool in assessing constipated patients.

Patterns of anismus and the relation to biofeedback therapy.

PURPOSE: A study was undertaken to assess physiologic characteristics and clinical significance of anismus. Specifically, we sought to assess patterns of anismus and the relation of these findings to the success of therapy. METHODS: Sixty-eight patients were found to have anismus based on history and diagnostic criteria including anismus by defecography and at least one of three additional tests: anorectal manometry, electromyography, or colonic transit time study. Interpretation of defecography was based on the consensus of at least three of four observers. Anal canal hypertonia (n = 32) was defined when mean and maximum resting pressures were at least 1 standard deviation higher than those in 63 controls. There were two distinct defecographic patterns of anismus: Type A (n = 26), a flattened anorectal angle without definitive puborectalis indentation but a closed anal canal; Type B (n = 42), a clear puborectalis indentation, narrow anorectal angle, and closed anal canal. Outcomes of 57 patients who had electromyographybased biofeedback therapy were reported as either improved or unimproved at a mean follow-up of 23.7 (range, 6-62) months. These two types of anismus were compared with biofeedback outcome to assess clinical relevance. RESULTS: Patients with Type A anismus showed greater perineal descent at rest (mean, 5.1 vs. 3.5 cm; P < 0.01), greater dynamic descent between rest and evacuation (mean, 2.7 vs. 1.4 cm; P < 0.01), greater difference of anorectal angle between rest and evacuation (mean, 14.6 vs. -3.1 degrees; P < 0.001), higher mean resting pressure (mean, 77.1 vs. 62.8 mmHg; P < 0.05), lower mean squeeze pressure (58.8 vs. 80.7 mmHg; P < 0.05), and a higher incidence of anal canal hypertonia (69.2 vs. 33.3 percent; P < 0.01) than did patients with Type B anismus. Only 25 percent of patients who had Type A anismus with anal canal hypertonia were improved by biofeedback therapy. Conversely, 86 percent of patients with Type B anismus without anal canal hypertonia were successfully treated with biofeedback (P < 0.001; Fisher's exact test). CONCLUSIONS: These two distinct physiologic patterns of anismus correlate with the success of biofeedback treatment. Therefore, knowledge of these patterns may help direct therapy.