Proteomic alterations of fibroblasts induced by ovarian cancer cells reveal potential cancer targets.

The common spread pattern of ovarian cancer is peritoneal implantation. The growth of the shed ovarian cancer cells in the peritoneal cavity is closely related to the tumor microenvironment. Cancer-associated fibroblasts are vital in the tumor microenvironment. It is not clearly defined that the protein expression alters during the activating process of fibroblasts. This study detected the protein alterations in fibroblasts induced by ovarian cancer cells and explored the potential biological relevance through two-dimensional gel electrophoresis and mass spectrometry. Our data showed that the level of CENPE, BAG2, SOD2, GDI2, CORO1C, CFL1, DSTN, CALD1, PHGDH, PDHA1, AKR1B1, TST and TBCA proteins were significantly up-regulated in the fibroblasts co-cultured with ovarian cancer cells, whereas HSPB1, P4HB and VIM were significantlydown-regulated. However, only BAG2, SOD2 and CORO1C proteins were confirmed to be significantly increased by western blot analysis. The differentially expressed proteins were mainly involved in metabolic processes, cellular component organization, responses to stimulus, multicellular organismal processes, localization, protein depolymerization, cellular senescence and the mitotic pathway. These data demonstrated that fibroblasts had an altered protein expression pattern after being induced by ovarian cancer cells, and participated in multiple cell processes resulting in tumor progression. The differentially expressed proteins should be considered as targets for cancer treatment.

Transition care readiness among patients in a tertiary paediatric department.

INTRODUCTION: A smooth transition of healthcare for young people with chronic illnesses from paediatric to adult healthcare services is important to ensure optimal outcome. At the moment, there are no standard guidelines to assess a patient's readiness to transfer care. METHODS: A cross-sectional study using a self-administered questionnaire, adapted from UNC (University of North Carolina) TRxANSITION self-assessment tool was conducted to evaluate patients' transition care readiness in paediatric haematology and paediatric diabetes clinic. RESULTS: A total of 80 patients (37 thalassaemia and 43 diabetes) with the mean age of 21.2 (SD±4.3) years, were recruited during the 3-month study period. Majority of the patients have basic knowledge regarding their medications, and were able to comply with their follow-up. The mean total score obtained by the respondents on this questionnaire was 15.3 (SD±3.59). Self-management skills and knowledge on disease were the two poorly scored section; with mean score of 3.78 (SD±1.38) and 4.28 (SD±1.20) respectively. Overall, only 21 (26.2%) respondents obtained high score (score above 75th percentile). Seventy-five percent of the respondents admitted that they were not ready for transfer to an adult healthcare service yet at the time of the study. CONCLUSION: We suggest that patients with high score should be prepared for transition to adult facility whereas those with a low score need to be identified to ensure provision of continuous education.

Co-targeting of EGF receptor and neuropilin-1 overcomes cetuximab resistance in pancreatic ductal adenocarcinoma with integrin ß1-driven Src-Akt bypass signaling.

Pancreatic ductal adenocarcinoma (PDAC) cells usually overexpress the epidermal growth factor receptor (EGFR); however, most are resistant to the anti-EGFR monoclonal antibody, cetuximab. In this study, we report that the molecular mechanism of resistance to cetuximab in PDAC cells is mediated by the overexpression of active integrin ß1 with downstream Src-Akt activation; this triggers an EGFR ligand-independent proliferation signaling, bypassing EGFR-blocking effect. Knockdown of integrin ß1 or inhibition of Src or Akt sensitized cetuximab-resistant (CtxR) PDAC cells to cetuximab. We found that neuropilin-1 (NRP1) physically interacts with active integrin ß1, but not inactive one, on the cell surface. To inhibit active integrin ß1-driven signaling by targeting NRP1, while suppressing EGFR signaling, we generated an EGFR and NRP1 dual targeting antibody, Ctx-TPP11, by genetic fusion of the NRP1-targeting peptide, TPP11, to the C terminus of the cetuximab heavy chain (Ctx-TPP11). We demonstrate that Ctx-TPP11 efficiently inhibited the growth of CtxR PDAC cells, in vitro and in vivo. The sensitization mechanism involved downregulating active integrin ß1 levels through NRP1-coupled internalization mediated by the TPP11 moiety, leading to the inhibition of active integrin ß1-driven bypass signaling. Our findings identify aberrant active integrin ß1-driven Src-Akt hyperactivation as a primary resistance mechanism to cetuximab in PDAC cells and offer an effective therapeutic strategy to overcome this resistance using an EGFR and NRP1 dual targeting antibody.

Antimelanogenic effects of luteolin 7-sulfate isolated from Phyllospadix iwatensis Makino.

BACKGROUND: Abnormal deposition of melanin may cause an aesthetic skin problem; therefore, the control of unwanted excessive melanin synthesis is the major goal of cosmetic research. OBJECTIVES: To identify novel tyrosinase (TYR) inhibitors from marine plants and examine their cellular antimelanogenic effects. METHODS: The extracts of 50 marine plants endemic to Korea were screened against human TYR. Active constituents were then isolated from the selected plant extracts that showed potential and their chemical structures elucidated. Furthermore, their antimelanogenic effects were examined using murine melanoma B16/F10 cells and human epidermal melanocytes (HEM). RESULTS: Among the tested extracts, that of Phyllospadix iwatensis Makino exhibited the strongest human TYR inhibitory activity. The active constituents were purified from the butanol fraction of the P. iwatensis extract and identified as hispidulin 7-sulfate and luteolin 7-sulfate. Luteolin 7-sulfate inhibited human TYR more strongly than hispidulin 7-sulfate, luteolin, hispidulin and arbutin. Furthermore, luteolin 7-sulfate showed lower cytotoxicity than luteolin in both B16/F10 cells and HEM. Luteolin 7-sulfate attenuated cellular melanin synthesis more effectively in B16/F10 cells and HEM stimulated by α-melanocyte-stimulating hormone and l-tyrosine than arbutin. CONCLUSIONS: This study demonstrates that luteolin 7-sulfate isolated from P. iwatensis is a human TYR inhibitor with advantageous antimelanogenic properties, and would be useful for development as a therapeutic agent for the control of unwanted skin pigmentation.

HLA-B*59:09, a novel allele identified by sequence-based typing in a cord blood donated by a Korean woman.

The new allele B*59:09 showed two nucleotide differences with B*59:01:01:01 in exon 3.

Pelvic congestion syndrome diagnosed using endoscopic ultrasonography.

Pelvic congestion syndrome (PCS) presents with a non-cyclic pelvic pain lasting more than six months in duration in premenopausal women. Pelvic ultrasonography or computed tomography is usually the first imaging modality used to evaluate patients with suspected PCS. PCS is confirmed by visible congestion of the pelvic veins on selective ovarian venography. To our knowledge, the role of endoscopic ultrasonography (EUS) has not been reported. EUS showed multiple dilated structures especially on left side around the uterus and ovaries, which are compatible with other radiological investigations of PCS Although PCS is not typical areas within the scope of practice of endosonographers, it is useful to be familiar with the findings. We report a case of PCS that was diagnosed with the aid of EUS.

A carcinoid tumour arising within a tailgut cyst: a diagnostic challenge.

A 49-year-old man was referred with constipation that had lasted for a few months. On colonoscopy, a subepithelial tumour more than 4 cm in size was seen in the rectum. He underwent endoscopic ultrasound and pelvic magnetic resonance imaging. He was preoperatively diagnosed with a rectal duplication cyst based on imaging studies. However, the final histopathologic diagnosis after transanal excision of the rectal mass was rectal carcinoid tumour with tailgut cyst. Tailgut cysts are very rare congenital lesions in the presacral area and are most often discovered incidentally in middle-aged women. It is difficult to distinguish the imaging appearance of tailgut cysts from that of many other retrorectal cysts. Malignant transformation of tailgut cysts has been estimated to occur in 2 to 13% of cases. We report the diagnostic difficulties encountered in a case of carcinoid tumour arising from a tailgut cyst in a male patient.

Gadoxetic acid-enhanced MRI for T-staging of gallbladder carcinoma: emphasis on liver invasion.

OBJECTIVE: To evaluate the diagnostic performance of gadoxetic acid-enhanced MRI with an emphasis on the usefulness of the hepatobiliary phase (HBP) in T-staging of gallbladder carcinoma. METHODS: 66 patients with surgically confirmed gallbladder carcinoma underwent MRI. Two radiologists independently reviewed two sets of gadoxetic acid-enhanced MRI without and with the HBP. Local tumour spread was evaluated according to T-staging, and the results were compared with pathological findings. The diagnostic performance of two image sets to differentiate each T-stage was compared. RESULTS: The sensitivities of MRI with the HBP to differentiate T1 vs ≥ T2 lesions, ≤ T2 vs ≥ T3 lesions and ≤ T3 vs T4 lesions were 96.3%, 85.7% and 100% for Observer 1 and 92.6%, 95.2% and 100% for Observer 2, respectively (p<0.0001). By adding the HBP, the sensitivities to differentiate ≤ T2 vs ≥ T3 lesions were increased from 66.7% to 85.7% for Observer 1 and from 81.0% to 95.2% for Observer 2, although there was no significant difference (p>0.05). The overall accuracies for T-staging were increased from 80.3% to 86.4% for Observer 1, a statistically significant degree (p=0.046), and from 83.8% to 87.9% for Observer 2 (p>0.05). The k-value for the two observers indicated excellent agreement. CONCLUSION: Gadoxetic acid-enhanced MRI provided acceptable diagnostic performance for T-staging of gallbladder carcinoma. Addition of the HBP aids in the detection of liver invasion. ADVANCES IN KNOWLEDGE: In the T-staging of gallbladder carcinoma, gadoxetic acid-enhanced MRI with the HBP may enhance detection of liver invasion.

HLA-A*02:328, a novel allele identified by sequence-based typing in cord blood from a Korean woman.

The novel allele A*02:328 showed one nucleotide difference with A*02:06:01 in exon 3 resulting in an amino acid change at codon 120 from Gly to Arg.

Correlation of the apparent diffusion coefficiency values on diffusion-weighted imaging with prognostic factors for breast cancer.

OBJECTIVE: The aim of this study was to correlate the apparent diffusion coefficient (ADC) value of breast cancer with prognostic factors. METHODS: 335 patients with invasive ductal carcinoma not otherwise specified (IDC NOS) and ductal carcinoma in situ (DCIS) who underwent breast MRI with diffusion-weighted imaging were included in this study. ADC of breast cancer was calculated using two b factors (0 and 1000 s mm(-2)). Mean ADCs of IDC NOS and DCIS were compared and evaluated. Among cases of IDC NOS, mean ADCs were compared with lymph node status, size and immunochemical prognostic factors using Student's t-test. ADC was also correlated with histological grade using the Kruskal-Wallis test. RESULTS: Mean ADC of IDC NOS was significantly lower than that of DCIS (p<0.001). However, the mean ADC of histological grade of IDC NOS was not significantly different (p=0.564). Mean ADC of oestrogen receptor (ER)-positive or progesterone receptor (PR)-positive cancer was significantly lower than that of ER-negative or PR-negative cancer (p=0.003 vs p=0.032). Mean ADC of Ki-67 index-positive cancer was significantly lower than that of Ki-67 index-negative cancer (p=0.028). Mean ADC values of cancers with increased microvascular density (MVD) were significantly lower than those of cancer with no MVD increase (p=0.009). No correlations were observed between mean ADC value and human growth factor receptor 2 expression, tumour size and lymph node metastasis. CONCLUSION: Low ADC value was correlated with positive expression of ER, PR, increased Ki-67 index, and increased MVD of breast cancer.

The study of new vacuum pressure measurement technique using ultrasonic acoustic impedance transducers.

Ultrasonic sensors of 500 kHz frequency have been used to study the measuring of gas pressure in the range 1.33 × 10(3)-2.026 × 10(5) Pa. From the experimental data it was observed that amplitude of the transmitted ultrasound was dependent on gas pressure. The results confirm that by using appropriate instruments including sensors and electronic devices, pressure of the gas can be successfully measured by measuring the magnitude of the received signal. The proposed method is expected to be applied to develop as new vacuum pressure measurement instrument.

Development of a new dynamic gas flow-control system in the pressure range of 1 Pa-133 Pa.

A new flow-control system (FCS-705) has been developed at Korea Research Institute of Standards and Science. The system is intended for calibration of vacuum gauges in the pressure range of 1 Pa-133 Pa by comparison method. This paper describes some basic characteristics of the system including; (1) the design and construction of the system, (2) the generation of stable pressures in the chamber, (3) achieving high upstream pressure limit by installing a short duct in the by-pass pumping line, and (4) investigation of the gas flow regimes within the short duct.

Influence of chimeric human-bovine fibers on adenoviral uptake by liver cells and the antiviral immune response.

Human adenoviruses (HAdV) are widely used for in vitro and in vivo gene transfer. Viral hepatotropism, inflammatory responses and neutralization by pre-existing antibodies (NAbs) are obstacles for clinical applications of HAdV vectors. Although the multifactorial events leading to innate HAdV toxicity are far from being elucidated, there is a consensus that the majority of intravenously injected-HAdV vectors is sequestered by Kuppfer cells, probably independently of coagulation factors. In this study, we show that the adenoviral-associated humoral and innate cytokine immune responses are significantly reduced when HAdV-5 vector carrying human bovine chimeric fibers (HAdV-5-F2/BAdV-4) is intravenously injected into mice. Fiber pseudotyping modified its interaction with blood coagulation factors, as FIX and FX no longer mediate the infection of liver cells by HAdV-5-F2/BAdV-4. As a consequence, at early time points post-infection, several cytokines and chemokines (IFN-gamma, IL-6, IP-10, MCP-1, RANTES and MP1beta) were found to be present at lower levels in the plasma of mice that had been intravenously injected with HAdV-5-F2/BAdV-4 compared with mice injected with the parental vector HAdV-5. Moreover, genetic modification of the fiber allowed HAdV-5-F2/BAdV-4 to partially escape neutralization by NAbs.

Characterization of naturally occurring parainfluenza virus type 2 (hPIV-2) variants.

BACKGROUND: Human parainfluenza viruses (hPIV) are respiratory pathogens responsible for upper and lower respiratory tract infections. In most labs, the clinical diagnosis of hPIV is routinely done using techniques based on the detection of viral antigens such as immunofluorescence assay or/and viral isolation. STUDY DESIGN: Five hPIV-2 isolated from respiratory samples exhibited unusual phenotypic and antigenic characteristics. These isolates showed important syncytial cytopathic effect and failed to react with one specific monoclonal antibody. These variant strains were subsequently compared with hPIV-2 prototype strain by cellular and molecular techniques. RESULTS: Both variant and prototype strains showed similar growth kinetics. Observation of plaque formation and syncytia assay indicated a more important fusogenic activity for the variant strains. Sequencing of fusion (F) and hemagglutinin-neuraminidase (HN) genes showed differences between the "atypical" hPIV-2 isolates and the Greer hPIV-2 prototype strain. These differences were analyzed with molecular modelling and structure prediction soft wares. A potential new glycosylation site in HN, in addition to minor changes observed in the predicted structure for the variant strains could explain their antigenic variation. Genetic changes in the fusion peptide and the cleavage site of F could also explain the difference observed in the fusion activity. CONCLUSIONS: Continuous global viral surveillance is essential to monitor antigenic changes that may occur in nature particularly with regards to the implementation of diagnostic assays. The differences observed in F and HN between the prototype strain and clinical hPIV-2 variants could also provide new data for the analysis of Paramyxovirus fusion mechanisms and their pathogenesis.

Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu.

In order to use adenovirus (Ad) type 5 (Ad5) for cancer gene therapy, Ad needs to be de-targeted from its native receptors and re-targeted to a tumor antigen. A limiting factor for this has been to find a ligand that (i) binds a relevant target, (ii) is able to fold correctly in the reducing environment of the cytoplasm and (iii) when incorporated at an optimal position on the virion results in a virus with a low physical particle to plaque-forming units ratio to diminish the viral load to be administered to a future patient. Here, we present a solution to these problems by producing a genetically re-targeted Ad with a tandem repeat of the HER2/neu reactive Affibody molecule (ZH) in the HI-loop of a Coxsackie B virus and Ad receptor (CAR) binding ablated fiber genetically modified to contain sequences for flexible linkers between the ZH and the knob sequences. ZH is an Affibody molecule specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) that is overexpressed in inter alia breast and ovarian carcinomas. The virus presented here exhibits near wild-type growth characteristics, infects cells via HER2/neu instead of CAR and represents an important step toward the development of genetically re-targeted adenoviruses with clinical relevance.

Decreased immune reactivity towards a knobless, affibody-targeted adenovirus type 5 vector.

In this study, a prototype Adenovirus type 5 (Ad5) vector deleted of the fiber knob domain and carrying an Affibody molecule as the targeting ligand showed decreased susceptibility to human pre-existing antibodies. This vector, Ad5/R7-Z(taq)Z(taq), has short fibers carrying seven shaft repeats, a non-native trimerization signal and an affibody molecule (Z(taq)) reactive to Taq polymerase. Ad5/R7-Z(taq)Z(taq) could be specifically targeted to 293 cells stably expressing membrane-bound anti-Z(taq) idiotypic affibody called Z(ztaq) (293Z(ztaq)). Sera from 50 blood donors were analyzed for neutralization activity (NA) against the parental Ad5/Fiwt vector and knobless Ad5/R7-Z(taq)Z(taq) on 293Z(ztaq) cells. Twenty-three sera had NA titers (> or =1:64) against Ad5/Fiwt (46%) and only two against Ad5/R7-Z(taq)Z(taq) (4%). Characterization of sera with NA titers showed that the knob domain is one of the targets of the antibodies. Neutralization assays using sera pre-adsorbed on knob and hexon proteins showed that the NA of the sera was carried mainly by anti-knob and anti-hexon antibodies, but in certain sera the anti-hexon antibodies represent the major population of the neutralizing antibodies (NAbs). Our results suggested that a combination of knob deletion and hexon switching could be an effective strategy for Ad vectors to better evade the anti-Ad NAbs.

Analysis of colonoscopic findings in the differential diagnosis between intestinal tuberculosis and Crohn's disease.

BACKGROUND AND STUDY AIMS: Intestinal tuberculosis and Crohn's disease are chronic inflammatory bowel disorders that are difficult to differentiate from one another. This study aimed to evaluate the diagnostic value of various colonoscopic findings in the differential diagnosis between intestinal tuberculosis and Crohn's disease. PATIENTS AND METHODS: Colonoscopic findings on initial work-up were prospectively recorded in patients with an initial diagnosis of either intestinal tuberculosis or Crohn's disease. These findings were analyzed after a final diagnosis of intestinal tuberculosis (n = 44) or Crohn's disease (n = 44) had been made after follow-up. RESULTS: Four parameters (anorectal lesions, longitudinal ulcers, aphthous ulcers, and cobblestone appearance) were significantly more common in patients with Crohn's disease than in patients with intestinal tuberculosis. Four other parameters (involvement of fewer than four segments, a patulous ileocecal valve, transverse ulcers, and scars or pseudopolyps) were observed more frequently in patients with intestinal tuberculosis than in patients with Crohn's disease. We hypothesized that a diagnosis of Crohn's disease could be made when the number of parameters characteristic of Crohn's disease was higher than the number of parameters characteristic of intestinal tuberculosis, and vice versa. Making these assumptions, we calculated that the diagnosis of either intestinal tuberculosis or Crohn's disease would have been made made correctly in 77 of our 88 patients (87.5 %), incorrectly in seven patients (8.0 %), and would not have been made in four patients (4.5 %). CONCLUSIONS: A systematic analysis of colonoscopic findings is very useful in the differential diagnosis between intestinal tuberculosis and Crohn's disease.

Early gastric cancer: virtual gastroscopy.

Gastric cancer is one of the most common cancers and one of the most frequent causes of cancer-related deaths worldwide. Early detection and accurate preoperative staging of early gastric cancer (ECG) offers the best prognosis and is essential for planning optimal therapy such as endoscopic mucosal resection or gastric resection. Recent advances in computed tomographic technology and three-dimensional imaging software have enabled more accurate gastric imaging. Virtual gastroscopy (VG) is helpful in the detection and evaluation of EGC in the same way as gastroscopy. VG has a wider field of view than conventional gastroscopy, the angle of the virtual cancer can be adjusted omnidirectionally, and it has no blind point because retrospective reconstruction is available. Thus, VG is a promising method for evaluating gastric lesions despite its limitations. This report describes the clinical usefulness of VG with multidetector row computed tomography for EGC and axial computed tomography.

Tumor cell targeted gene delivery by adenovirus 5 vectors carrying knobless fibers with antibody-binding domains.

Most human carcinoma cell lines lack the high-affinity receptors for adenovirus serotype 5 (Ad5) at their surface and are nonpermissive to Ad5. We therefore tested the efficiency of retargeting Ad5 to alternative cellular receptors via immunoglobulin (Ig)-binding domains inserted at the extremity of short-shafted, knobless fibers. The two recombinant Ad5's constructed, Ad5/R7-Z(wt)-Z(wt) and Ad5/R7-C2-C2, carried tandem Ig-binding domains from Staphylococcal protein A (abbreviated Z(wt)) and from Streptococcal protein G (C2), respectively. Both viruses bound their specific Ig isotypes with the expected affinity. They transduced human carcinoma cells independently of the CAR pathway, via cell surface receptors targeted by specific monoclonal antibodies, that is, EGF-R on A549, HT29 and SW1116, HER-2/neu on SK-OV-3 and SK-BR-3, CA242 (epitope recognized by the monoclonal antibody C242) antigen on HT29 and SW1116, and PSMA (prostate-specific membrane antigen) expressed on HEK-293 cells, respectively. However, Colo201 and Colo205 cells were neither transduced by targeting CA242 or EGF-R nor were LNCaP cells transduced by targeting PSMA. Our results suggested that one given surface receptor could mediate transduction of certain cells but not others, indicating that factors and steps other than cell surface expression and virus-receptor interaction are additional determinants of Ad5-mediated transduction of tumor cells. Using penton base RGD mutants, we found that one of these limiting steps was virus endocytosis.

Genetic modification of adenovirus 5 tropism by a novel class of ligands based on a three-helix bundle scaffold derived from staphylococcal protein A.

The use of adenovirus (Ad) as an efficient and versatile vector for in vivo tumor therapy requires the modulation of its cellular tropism. We previously developed a method to genetically alter the tropism of Ad5 fibers by replacing the fiber knob domain by an extrinsic trimerization motif and a new cellular ligand. However, fibers carrying complex ligands such as single-chain antibody fragments did not assemble into functional pentons in vitro in the presence of penton base, and failed to be rescued into infectious virions because of their inability to fold correctly within the cytoplasm of Ad-infected cells. Here we show that the coding sequence for a disulfide bond-independent three-helix bundle scaffold Z, derived from domain B of Staphylococcal protein A and capable of binding to the Fc portion of immunoglobulin (Ig) G1, could be incorporated into modified knobless Ad fiber gene constructs with seven shaft repeats. These fiber gene constructs could be rescued into viable virions that were demonstrated to enter 293 cells engineered for IgG Fc surface expression but not unmodified 293 cells, via a mechanism that could be specifically blocked with soluble Fc target protein. However, the tropism modified viruses showed a slightly impaired cellular entry and a lower infectivity than wildtype (WT) virus. In addition, we generated recombinant fibers containing an IgA binding Affibody ligand, derived from combinatorial specificity-engineering of the Z domain scaffold. Such fiber constructs also showed the expected target specific binding, indicating that the affibody protein class is ideally suited for genetic engineering of Ad tropism.