Nucleic Acid Tests for BK Polyomavirus Is Critical in Renal Transplant Recipients.

This study evaluates the incidence of BK polyomavirus (BKV) and prognosis of BKV infection in kidney transplant recipients (KTRs) who received transplantation in our hospital before and after regular BKV nucleic acid test (NAT) was implemented. METHODS: The study included 74 KTRs who received a single kidney either from standard- or expanded-criteria deceased donor between March 2011 and March 2017. BKV NATs were regularly checked in 26 patients (group 1) in the first posttransplant year in accordance with current guidelines since NAT was implemented in our laboratory in 2014. We retrospectively compared 48 KTRs (group 2) who either received NAT when necessary in another laboratory or were not checked before 2014. RESULTS: There was no significant difference in patient characteristics between groups. BKV viruria were confirmed in 8 of 26 (30.8%) group 1 patients, whereas only 2 of 48 (4.2%) BKV infections were confirmed in group 2. None of the BKV(+) KTRs in group 1 developed BK polyomavirus-associated nephropathy (BKVAN), whereas 2 BKV(+) patients (100%) of group 2 developed BKVAN, which indicates renal function deterioration and biopsy-validated nephropathy. There was no significant difference in graft survival and renal function between the 2 groups. CONCLUSIONS: The risk of BKV infection is considerably higher in KTRs using NAT. Because there is no approval treatment, early diagnosis of BKV infection and early reduction of immunosuppression agents is critical for KTRs. Implementation of regular BKV NAT is mandatory before BKVAN and malignant neoplasms develop.

Rehydration before wet fixation in conventional body fluid cytology - An 18-year experience.

PURPOSE: In conventional cytology, preparation of a specimen by wet fixation for Papanicolaou stain is potentially subject to dry effect or cell loss which may make cytologic interpretation difficult or even impossible. We have been routinely making an additional smear for rehydration with normal saline (rehydration method) before wet fixation to overcome the above shortcomings. METHODS: We reviewed malignant pleural effusion and ascites 15 cases each in our cytology laboratory over the past 1 year. Four slides of each specimen were made. Two were air-dried for Liu's stain (a Romanowsky stain) and the other two were wet-fixed for Papanicolaou stain. The air-dried smears were also served as retained cellularity control. One of the two wet-fixed smears was processed as a control of preservation of nuclear detail whereas the other one stayed air-dried for 10 minutes and then covered with normal saline (rehydration method) for 80 seconds before wet fixation. RESULTS: There was minor cell loss (P = .032). The cells appeared larger with good preservation of nuclear detail (P < .0001 by two-sided Wilcoxon rank sum test) but no red blood cells retained on the slide after rehydration. CONCLUSION: The rehydration method can effectively minimise cell loss, enlarge and preserve the cytological features of malignant cells with haemolysis. This method is simple, practical and good for cytological screening for tumour cells and interpretation especially in a bloody smear. We recommend that the rehydration method be part of traditional cytopreparatory work of wet fixation for Papanicolaou stain in conventional body fluid cytology.

Human Polyomavirus Is Associated With Earlier Onset of Upper Urinary Tract Urothelial Carcinoma in Patients After Kidney Transplantation.

OBJECTIVES: Polyomavirus has been reported to be oncogenic due to viral integration into the human genome. A relatively high prevalence of upper urinary tract urothelial carcinoma (UTUC) was noted after kidney transplantation (KT) in Taiwan. However, little was known about the impact of polyomavirus on the urothelial cancer behavior. Therefore, the aim of this study is to analyze the characteristics of polyomavirus-related UTUC after KT. METHODS: From 2005 to 2014, 27 patients were found to have UTUCs after KT. All the patients underwent standard nephroureterectomy. Detailed perioperative parameters were obtained from chart records. A qualified pathologist who is blinded to the clinical outcome examined large T antigen expression and pathological features. All the patients were divided into two groups according to positive or negative expression of large T antigen. RESULTS: In the patient demography, a significantly younger median age was found in patients with large T antigen-positive UTUCs compared with the negative control group (48.1 ± 8.3 years versus 54.6 ± 4.1 years, respectively, P = .013). As for the pathological features and oncologic outcome, there were no obvious differences between these two groups. Non-organ-confined status and positive lymphovascular invasion are prognostic factors associated with systemic disease recurrence (P = .017 and .001, respectively). CONCLUSIONS: Although UTUC commonly develops in the elderly, earlier onset of post-KT UTUCs was observed especially in patients with positive large T antigen expression in our cohort. This preliminary result provides valuable experience suggesting more frequent upper urinary tract screening for polyomavirus infected patients after KT in Taiwan.

Hepatocarcinogenesis in transgenic mice carrying hepatitis B virus pre-S/S gene with the sW172* mutation.

Hepatitis B virus (HBV) carrying the rtA181T/sW172* mutation conferred cross-resistance to adefovir and lamivudine. Cell-based and clinical studies indicated that HBV carrying this mutation had an increased oncogenic potential. Herein, we created transgenic mouse models to study the oncogenicity of the HBV pre-S/S gene containing this mutation. Transgenic mice were generated by transfer of the HBV pre-S/S gene together with its own promoter into C57B6 mice. Four lines of mice were created. Two of them carried wild-type gene and produced high and low levels of HBV surface antigen (HBsAg) (TgWT-H and L). The other two carried the sW172* mutation with high and low intrahepatic expression levels (TgSW172*-H and L). When sacrificed 18 months after birth, none of the TgWT mice developed hepatocellular carcinoma (HCC), whereas 6/26 (23.1%) TgSW172*-H and 2/24 (8.3%) TgSW172*-L mice developed HCC (TgWT vs TgSW172*; P=0.0021). Molecular analysis of liver tissues revealed significantly increased expression of glucose-regulated protein 78 and phosphorylated extracellular signal-regulated kinases 1 in TgSW172* mice, and decreased expression of B-cell lymphoma-extra large in TgSW172*-H mice. Higher proportion of apoptotic cells was found in TgSW172*-H mice, accompanied by increased cyclin E levels, suggesting increased hepatocyte turnover. Combined analysis of complimentary DNA microarray and microRNA array identified microRNA-873-mediated reduced expression of the CUB and Sushi multiple domains 3 (CSMD3) protein, a putative tumor suppressor, in TgSW172* mice. Our transgenic mice experiments confirmed that HBV pre-S/S gene carrying the sW172* mutation had an increased oncogenic potential. Increased endoplasmic reticulum stress response, more rapid hepatocyte turnover and decreased CSMD3 expression contributed to the hepatocarcinogenesis.

TC-N19, a novel dual inhibitor of EGFR and cMET, efficiently overcomes EGFR-TKI resistance in non-small-cell lung cancer cells.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) show a clinical benefit when used to treat patients with EGFR-mutated non-small-cell lung cancer (NSCLC), but this treatment unfortunately fails in patients with TKI-resistant tumors. We here provide evidence that TC-N19 (N19), a novel dual inhibitor of EGFR and cMET, efficiently overcomes the EGFR-TKI resistance in EGFR-mutated NSCLC cells via simultaneous degradation of both proteins by ubiquitin proteasomes. Comparison with HSP90 inhibitor treatment and knockdown of EGFR and cMET by small hairpin RNAs reveal that the reduction of EGFR and cMET expression by N19 is responsible for overcoming the intrinsic TKI resistance mediated by paxillin (PXN) in high PXN-expressing cells, PXN-overexpressing PC9 cells (PC9-PXN), the EGFR-T790M-mediated TKI resistance in H1975 and CL97 cells, and the acquired resistance to gefitinib in gefitinib-resistant PC9 cells (PC9GR). Annexin V-PI staining assay showed that the induction of apoptosis in NSCLC cells by N19 depended on the reduction in levels of both proteins. Xenograft tumor formation in nude mice induced by a PC9-PXN-stable clone and by PC9GR cells was nearly completely suppressed by N19 treatment, with no changes in animal body weight. MTT assays of normal lung cells and reticulocytes showed no cytotoxicity responses to N19. In summary, N19 may act as a novel dual inhibitor of EGFR and cMET that induces apoptosis in TKI-resistant EGFR-mutated NSCLC cells and suppresses xenograft tumor formation. We suggest that N19 may be a potential new-generation TKI or HSP90 inhibitor used for treatment of NSCLC patients who show resistance to current TKI-targeting therapies.

Mesothelin-specific cell-based vaccine generates antigen-specific immunity and potent antitumor effects by combining with IL-12 immunomodulator.

Ovarian cancer is a gynecologic malignancy with a high mortality rate. In the present study, we developed a novel cell-based vaccine, Meso-VAX, to generate mesothelin antigen-specific immune responses and immunotherapy against ovarian cancer. Mesothelin, a secreted protein anchored at the cell membrane, has recently been identified as a potential new tumor antigen for ovarian cancer. In this study, mice vaccinated with Meso-VAX and adeno-associated virus (AAV)-IL-12 exhibited dramatic increases in the number of mesothelin-specific CD4(+) helper and CD8(+) cytotoxic T-cell precursors, higher titers of anti-mesothelin Abs and in vitro tumor killing activity, and all of these mice were tumor-free after 60 days of tumor challenge. In addition, a significant reduction in peritoneal tumors and longer survival were noted in the mice vaccinated with Meso-VAX combined with AAV-IL-12. CD4(+) helper and CD8(+) cytotoxic T lymphocytes were essential for the antitumor effect generated by Meso-VAX combined with AAV-IL-12. The post-vaccination sera of the mice vaccinated with Meso-VAX and AAV-IL-12 also showed mesothelin-specific complement-dependent cell-mediated cytotoxicity. Our results suggest that a Meso-VAX cell-based vaccine combined with AAV-IL-12 can generate antigen-specific immunological responses and antitumor effects on ovarian cancer.

Pulsed radiofrequency inhibited activation of spinal mitogen-activated protein kinases and ameliorated early neuropathic pain in rats.

Background: Pulsed radiofrequency (PRF) has been widely used to treat chronic pain, but the effectiveness and mechanisms in preventing early neuropathic pain have not been well explored. Even fewer knowledge is available in its impact on glia-mediated nociceptive sensitization. This study aims to elucidate the modulation of PRF on nerve injury-induced pain development and activation of spinal mitogen-activated protein kinases (MAPKs). Methods: In a rat spinal nerve ligation (SNL) model, a low-volt PRF treatment was applied to the L5 dorsal root ganglion after nerve injury. Nociceptive behaviours were measured by von Frey and heat withdrawal tests at multiple time points. MAPK activations, including p-ERK and p-p38, as well as TNF-á level in the spinal dorsal horn were assessed and the cell types that expressed MAPK activation were identified by double immuno fluorescence staining.Results: We found that SNL promptly induced neuropathic pain in the affected hind limb for over 1 week as well as increased p-ERK and p-p38 in the spinal dorsal horn. PRF significantly attenuated SNL-induced mechanical allodynia and thermal hyperalgesia for 5­7 days. PRF also inhibited ERK and p38 activations, which were found majorly located within neurons and microglia, respectively. Besides, PRF significantly suppressed expression of TNF-á in the spinal dorsal horn throughout the course. Conclusions: Low-volt PRF significantly ameliorated SNL-induced acute pain. Inferentially, PRF may inhibit spinal sensitization by down-regulating spinal MAPK activations and activation-mediated cytokine release.We demonstrated that early PRF treatment in acute nerve injury helps to ameliorate neuropathic pain development.

Dynamics of ASXL1 mutation and other associated genetic alterations during disease progression in patients with primary myelodysplastic syndrome.

Recently, mutations of the additional sex comb-like 1 (ASXL1) gene were identified in patients with myelodysplastic syndrome (MDS), but the interaction of this mutation with other genetic alterations and its dynamic changes during disease progression remain to be determined. In this study, ASXL1 mutations were identified in 106 (22.7%) of the 466 patients with primary MDS based on the French-American-British (FAB) classification and 62 (17.1%) of the 362 patients based on the World Health Organization (WHO) classification. ASXL1 mutation was closely associated with trisomy 8 and mutations of RUNX1, EZH2, IDH, NRAS, JAK2, SETBP1 and SRSF2, but was negatively associated with SF3B1 mutation. Most ASXL1-mutated patients (85%) had concurrent other gene mutations at diagnosis. ASXL1 mutation was an independent poor prognostic factor for survival. Sequential studies showed that the original ASXL1 mutation remained unchanged at disease progression in all 32 ASXL1-mutated patients but were frequently accompanied with acquisition of mutations of other genes, including RUNX1, NRAS, KRAS, SF3B1, SETBP1 and chromosomal evolution. On the other side, among the 80 ASXL1-wild patients, only one acquired ASXL1 mutation at leukemia transformation. In conclusion, ASXL1 mutations in association with other genetic alterations may have a role in the development of MDS but contribute little to disease progression.

Prognostic significance of the number of examined lymph nodes in node-negative gastric adenocarcinoma.

AIM: In this study, we investigated the prognostic significance of the number of examined lymph nodes in node-negative gastric adenocarcinoma (GC). PATIENTS AND METHODS: A total of 1194 node-positive and 1030 node-negative GC patients undergoing potentially curative gastrectomy was enrolled in this study. Patients were stratified into 3 groups according to the number of examined lymph nodes: group 1, ≤ 15; group 2, 16-25; group 3, >25. RESULTS: Patients with node-negative GC had significantly favorable survival compared with those with node-positive. Among patients with node-negative T2-T4 disease, the percentage of locoregional relapse was higher in those with <25 examined lymph nodes than in those with ≥ 25 examined lymph nodes. The number of examined lymph nodes affected the overall survival rates for patients with node-negative T2-T4 GC but not for patients with T1 lesions. Tumor size, tumor location, the number of examined lymph nodes, T status, and the presence of perineural invasion were significant prognostic factors as determined by multivariate analysis in node-negative GC. CONCLUSIONS: No survival benefit of examining ≥ 15 lymph nodes was noted for patients with node-negative T1 GC. Extensive lymphadenectomy in patients with node-negative T2-T4 lesions in whom the number of examined lymph nodes was >25 had favorable survival.

Moxifloxacin modifies corneal fibroblast-to-myofibroblast differentiation.

BACKGROUND AND PURPOSE: Fibroblast-to-myofibroblast differentiation is associated with scarring, an important issue in corneal surgery. Moxifloxacin (MOX), commonly applied to prevent post-operative infection, would benefit more if it modifies fibroblast-to-myofibroblast differentiation other than antimicrobial activity. Our purpose was to explore whether MOX has anti-fibrotic effect in human corneal fibroblasts (HCFs). EXPERIMENTAL APPROACH: HCFs were incubated in MOX-containing medium concurrently with TGF-ß1 (co-treatment), before (pretreatment) or after (post-treatment) adding TGF-ß1. HCF contractility was evaluated with a type I collagen gel contraction assay. Expression of α-smooth muscle actin (α-SMA), Smad2, phospho-Smad2-Ser467, Smad4 and Smad7 was determined by immunoblotting. Formation of α-SMA-positive filaments and distribution of active Smad2 were observed under confocal microscopy. Expression of TGF-ß receptor types I (TGFBR1) and II (TGFBR2) was assessed with flow cytometry. KEY RESULTS: MOX did not affect gel contractility or α-SMA filament formation in HCFs without TGF-ß1 stimulation. MOX did, however, retard HCF-containing gel contractility and α-SMA filament formation following TGF-ß1 stimulation in the pretreatment and co-treatment groups but not in the post-treatment group. MOX blocked the expression of Smad2, phospho-Smad2-Ser467 and TGFBR1 under TGF-ß1 incubation. Additionally, MOX enhanced Smad7 expression in TGF-ß1-incubated HCFs, but did not interfere with TGF-ß-triggered Smad2 nuclear translocation or Smad4 expression. CONCLUSIONS AND IMPLICATIONS: MOX inhibited TGF-ß-induced fibroblast-to-myofibroblast differentiation via blocking TGFBR1 and enhancing Smad7 expression. MOX should be used before or during surgery to achieve these effects. These results suggest a de novo mechanism by which MOX participates in corneal wound healing.

Use of a novel vitamin D bioavailability test demonstrates that vitamin D absorption is decreased in patients with quiescent Crohn's disease.

BACKGROUND: Vitamin D deficiency is a common problem in patients with Crohn's disease (CD). The aim of this study was to determine the ability of normal subjects and patients with quiescent CD to absorb vitamin D(2) using a novel vitamin D bioavailability test. In addition, we evaluated whether the location of disease or previous surgery had any influence on the bioavailability of vitamin D(2) in CD patients. METHODS: Ten normal subjects (50% female) and 37 CD patients with quiescent disease (51% female) were included in this study. Subjects who recently received any vitamin D(2) were excluded. The vitamin D bioavailability test was performed in all subjects. After a baseline blood draw, all subjects were then given a single 50,000 IU oral dose of vitamin D(2) in a capsule formulation and had their blood drawn 12 hours later to determine serum vitamin D(2), which reflected their vitamin D(2) absorption capacity. RESULTS: Forty-two percent and 29% of CD patients were found to be either vitamin D-deficient (25-hydroxyvitamin D [25(OH)D] ≤20 ng/mL] or insufficient [25(OH)D 21-29 ng/mL], respectively. Twelve hours after ingesting 50,000 IU vitamin D(2) , vitamin D(2) levels rose from a baseline of 0.7 ± 0.7 ng/mL (mean ± SEM) to 49.8 ± 3.0 ng/mL in normal subjects. In CD patients, baseline vitamin D(2) levels rose from 0 ng/mL to 34.8 ± 2.8 ng/mL. CD patients had on average a 30% decrease in their ability to absorb vitamin D(2) (P = 0.01). Moreover, we found a wide variability of vitamin D(2) bioavailability in CD patients. Analysis of variance (ANOVA) revealed no statistical difference of vitamin D(2) bioavailability between patients in the CD subgroup stratified by the location of disease, the type of surgery, and receiving or not receiving surgery. CONCLUSIONS: More than 70% of the patients with quiescent CD were vitamin D-deficient or insufficient. The ability to absorb vitamin D(2) in CD patients is unpredictable and the only way to determine this is to perform a vitamin D bioavailability test. Use of this test may guide clinicians in administering the appropriate therapeutic dose of vitamin D for treating vitamin D deficiency in patients with CD.

Prognostic effect of steatosis on hepatocellular carcinoma patients after liver resection.

AIMS: Overweight/obesity is currently a common health issue that may cause many diseases, even malignancies. The influence of steatosis on long-term results of surgical treatment for hepatocellular carcinoma (HCC) is not well known. The aim of this study is to analyze the results of hepatectomy for HCC patients with steatosis. METHODS: The study included 1048 patients who underwent hepatectomy for HCC from 1999 to 2005. The patients were divided into two groups; group A patients without steatosis (n = 693) and group B patients with steatosis (n = 355). The clinicopathological data and long-term survival were analyzed. RESULTS: Mean tumor size in group B patients was smaller than that in group A patients (4.61 ± 3.40 vs. 5.91 ± 4.36 cm, p < 0.01). Group B patients showed lower tumor differentiation grade, lower vascular invasion rate and better 5-year overall survival compared to group A patients (61.2% vs. 50.1%, p = 0.001). By multivariate analysis, steatosis was found to be associated with well-differentiated, small-sized, and less α-fetoprotein productive tumors. When focusing on the tumors >5 cm in diameter, group B patients had better survival rate than group A patients (p = 0.041). Vascular invasion and steatosis were independent prognostic factors for the overall survival. CONCLUSION: HCC in steatotic liver was less aggressive than that in non-steatotic liver. HCC patients with steatosis have better surgical outcomes than those without steatosis. Vascular invasion and steatosis were independent prognostic factors for the overall survival if tumors were >5 cm in diameter.

Interleukin 10 and residual kidney function are associated with risk of vascular calcification in patients undergoing peritoneal dialysis.

AIMS: Vascular calcification is a common complication among dialysis patients and its pathogenesis involves a variety of factors. The roles of pro-inflammatory cytokines and residual kidney function (RKF) in peritoneal dialysis (PD) patients with vascular calcification have not been investigated. MATERIALS AND METHODS: 157 stable PD patients were enrolled. All patients had plain X-ray film examination including chest (posterior-anterior view, CXR) and pelvis. Vascular calcification was interpreted as calcified deposit over aortic arch and linear calcification of pelvic arteries. Relevant biochemical data, pro-inflammatory markers, and PD-related factors were measured and collected. RESULTS: Vascular calcification prevalence in CXRs was higher than that in pelvis films (38.2% vs. 22.3%, p < 0.05). Patients with vascular calcification in CXR had higher incidence of calcification in pelvis films (p < 0.05). Only a minor portion (14.6%) had two calcification sites. Regression analysis revealed that age, PD duration, body mass index, and RKF were independent factors associated with vascular calcification in CXR. Age, diabetes, IL-10 and RKF were factors associated in pelvis films. Factors independently related to vascular calcification in both films were age, duration, diabetes, IL-10, and RKF. CONCLUSIONS: Besides traditional risk factors, IL-10 and RKF were important factors associated with vascular calcification in PD patients.