Optimization of the spherical integrity for sustained-release alginate microcarriers-encapsulated doxorubicin by the Taguchi method.

This study aimed to develop biodegradable calcium alginate microcarriers with uniform particle size and spherical integrity for sustained-release targeting transarterial chemoembolization. To determine related parameters including the ratio of cross-linking volume (sodium alginate: CaCl2), concentrations of sodium alginate and CaCl2 solutions, collection distance, flow rate, stirring speed, syringe needle diameter and hardening time to fabricate the microcarriers, the Taguchi method was applied. Using different conditions, a total of 18 groups were prepared. The average size of microspheres from different groups was estimated as ~ 2 mm (range 1.1 to 1.6 mm). Signal-to-noise ratio analysis showed the optimal spherical integrity (F1) achieved when the above parameters were designed as 0.1, 2.5 wt%, 6 wt%, 8 cm, 30 mL/h, 150 rpm, 0.25 mm and 2 h, respectively. The best (F1), middle (F2) and worst (F3) groups were used for further experiments. Fourier-transform infrared spectroscopy spectrum showed that F1, F2 and F3 conformations were distinct from original sodium alginate. Drug-loaded calcium alginate microcarriers demonstrated rougher surfaces compared to microspheres without drug under transmission electron microscopy. Compared to pH 7.4, swelling rates in PBS were decreased at pH 6.5. Encapsulation and loaded efficiencies of the Dox-loaded microcarriers were estimated as ~ 40.617% and ~ 3.517%. In vitro experiments indicated that the F1 Dox-loaded microcarriers provide a well sustained-release efficacy for about two weeks at 37 °C in PBS. Treatments of calcium alginate microcarriers without the Dox in two distinct hepatocellular carcinoma-derived cell lines, Huh-7 and Hep-3B, indicated that these microcarriers were non-toxic. The Dox-loaded microcarriers displayed sustained-release capacity and reduced cell viabilities to ~ 30% in both cell lines on Day 12.

Fine control of endothelial VEGFR-2 activation: caveolae as fluid shear stress shelters for membrane receptors.

Recent experimental evidence points to the possibility that cell surface-associated caveolae may participate in mechanotransduction. The particular shape of caveolae suggests that these structures serve to prevent exposure of putative mechanosensors residing within these membrane invaginations to shear stresses at magnitudes associated with initiation of cell signaling. Accordingly, we numerically analyzed the fluid flow in and around caveolae using the equation of motion for flow of plasma at low Reynolds numbers and assuming no slip-condition on the membrane. The plasma velocity inside a typical caveola and the shear stress acting on its membrane are markedly reduced compared to the outside membrane. Computation of the diffusion field in the vicinity of a caveola under flow, however, revealed a rapid equilibration of agonist concentration in the fluid inside a caveola with the outside plasma. Western blots and immunocytochemistry support the role of caveolae as shear stress shelters for putative membrane-bound mechanoreceptors such as flk-1. Our results, therefore, suggest that caveolae serve to reduce the fluid shear stress acting on receptors in their interior, while allowing rapid diffusion of ligands into the interior. This mechanism may permit differential control of flow and ligand activation of flk-1 receptor in the presence of ligands.

Plk1 phosphorylation of Numb leads to impaired DNA damage response.

Although Numb is well-recognized as a cell-fate determinant in stem/progenitor cells, accumulating evidence supports that Numb also has a critical role in adult tissues and cancers, in particular, in the context of regulation of tumor suppressor p53. Herein, we identified Numb as a novel substrate of Polo-like kinase 1 (Plk1). Of significance, we showed that Plk1-mediated phosphorylation of Numb leads to its enhanced proteasomal degradation and impaired Numb/p53 pathway, thus providing another mechanism how Plk1 antagonizes p53 during DNA damage response. In addition, the novel phosphorylation event identified by us further supports the notion that post-translational modifications of Numb uncouple Numb from p53 and lead to p53 destabilization. Finally, our data generated from both human cancer cell lines and mouse xenograft model showed that cancer cells carrying the unphosphorylated form of Numb by Plk1 are more sensitive to doxorubicin, a classical chemotherapeutic drug. Therefore, our work may provide future strategies for improving the efficacy of chemotherapy by targeting Numb phosphorylation by Plk1.

Mesenchymal stromal cells derived from acute myeloid leukemia bone marrow exhibit aberrant cytogenetics and cytokine elaboration.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play a fundamental role in the BM microenvironment (BME) and abnormalities of these cells may contribute to acute myeloid leukemia (AML) pathogenesis. The aim of the study was to characterize the cytokine and gene expression profile, immunophenotype and cytogenetics of BM-MSCs from AML patients compared to normal BM-MSCs from healthy donors. AML BM-MSCs showed decreased monocyte chemoattractant protein-1 levels compared to normal BM-MSCs. AML BM-MSCs expressed similar ß1 integrin, CD44, CD73, CD90 and E-cadherin compared to normal BM-MSCs. Cytogenetic analysis revealed chromosomal aberrations in AML BM-MSCs, some overlapping with and others distinct from their corresponding AML blasts. No significant difference in gene expression was detected between AML BM-MSCs compared to normal BM-MSCs; however, comparing the differences between AML and MSCs from AML patients with the differences between normal hematopoietic cells and normal MSCs by Ingenuity pathway analysis showed key distinctions of the AML setting: (1) upstream gene regulation by transforming growth factor beta 1, tumor necrosis factor, tissue transglutaminase 2, CCAAT/enhancer binding protein alpha and SWItch/Sucrose NonFermentable related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4; (2) integrin and interleukin 8 signaling as overrepresented canonical pathways; and (3) upregulation of transcription factors FBJ murine osteosarcoma viral oncogene homolog and v-myb avian myeloblastosis viral oncogene homolog. Thus, phenotypic abnormalities of AML BM-MSCs highlight a dysfunctional BME that may impact AML survival and proliferation.

Long-term risk of tuberculosis in haematopoietic stem cell transplant recipients: a 10-year nationwide study.

BACKGROUND: Tuberculosis (TB) is a serious problem for patients undergoing haematopoietic stem cell transplantation (HSCT) in TB-endemic areas; however, data on these patients are limited. METHODS: We obtained data on 2040 HSCT recipients from the Registry of Catastrophic Illness in Taiwan from 1997 to 2006. We also obtained data on age-, sex- and enrolment date-matched controls from the Longitudinal Health Insurance Database. The cumulative incidence of active TB in HSCT recipients and controls and risk factors for TB were analysed. RESULTS: Among 2040 HSCT recipients identified, 39 (1.9%) had newly diagnosed TB. The incidence rate was 688 per 100 000 person-years. The 10-year cumulative TB incidence was respectively 3.52% and 0.38% in HSCT recipients and controls (P < 0.001). HSCT was an independent risk factor for TB compared with matched controls. Among post-HSCT patients, independent risk factors for TB included age ⩾18 years and allogeneic recipients with graft-versus-host disease (GVHD). Post-HSCT patients with subsequent TB had a higher mortality rate than those without TB (P < 0.001). CONCLUSION: HSCT is associated with an increased risk of TB in endemic regions. Older age and development of chronic GVHD are independent predictors of late onset active TB in HSCT recipients.

Use of azathioprine for graft-vs-host disease is the major risk for development of secondary malignancies after haematopoietic stem cell transplantation: a nationwide population-based study.

BACKGROUND: As more patients are treated by haematopoietic stem cell transplantation (HSCT), development of secondary malignancy (SM) becomes an increasingly common issue in long-term survivors. METHODS: We conducted a nationwide population-based study of the Taiwanese population to analyse patients who received HSCT between January 1997 and December 2010. Standardised incidence ratios (SIRs) were used to compare the risk of SM in HSCT patients and the general population. Multivariate analysis was performed to identify independent predictors of SM. RESULTS: Patients receiving HSCT had a significantly greater risk of developing SM (SIR 2.00; 95% confidence interval (CI) 1.45-2.69; P<0.001). Specifically, the incidence increased for cancers of the oral cavity (SIR 14.18) and oesophagus (SIR 14.75) after allogeneic HSCT. Multivariate analysis revealed an increased SIR for cancer in patients who received the immunosuppressant azathioprine. The risk of SM also increased with greater cumulative doses of azathioprine. CONCLUSIONS: This study demonstrates an increased incidence of SM in Taiwanese patients who received allogeneic HSCT, especially for cancers of the oral cavity and oesophagus. This finding is different from results in populations of Western countries. Physicians should be cautious about azathioprine use for graft-vs-host disease after HSCT.

In vitro and in vivo anticancer effects of mevalonate pathway modulation on human cancer cells.

BACKGROUND: The increasing usage of statins (the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) has revealed a number of unexpected beneficial effects, including a reduction in cancer risk. METHODS: We investigated the direct anticancer effects of different statins approved for clinical use on human breast and brain cancer cells. We also explored the effects of statins on cancer cells using in silico simulations. RESULTS: In vitro studies showed that cerivastatin, pitavastatin, and fluvastatin were the most potent anti-proliferative, autophagy inducing agents in human cancer cells including stem cell-like primary glioblastoma cell lines. Consistently, pitavastatin was more effective than fluvastatin in inhibiting U87 tumour growth in vivo. Intraperitoneal injection was much better than oral administration in delaying glioblastoma growth. Following statin treatment, tumour cells were rescued by adding mevalonate and geranylgeranyl pyrophosphate. Knockdown of geranylgeranyl pyrophosphate synthetase-1 also induced strong cell autophagy and cell death in vitro and reduced U87 tumour growth in vivo. These data demonstrate that statins main effect is via targeting the mevalonate synthesis pathway in tumour cells. CONCLUSIONS: Our study demonstrates the potent anticancer effects of statins. These safe and well-tolerated drugs need to be further investigated as cancer chemotherapeutics in comprehensive clinical studies.

Increased stroke risk in Bell's palsy patients without steroid treatment.

BACKGROUND AND PURPOSE: To investigate the risk of stroke development following a diagnosis of Bell's palsy in a nationwide follow-up study. METHODS AND MATERIALS: Information on Bell's palsy and other factors relevant for stroke was obtained for 433218 eligible subjects without previous stroke who had ambulatory visit in 2004. Of those, 897 patients with Bell's palsy were identified. Over a median 2.9 years of follow-up, 4581 incident strokes were identified. We estimated hazard ratios (HR) and 95% confidence intervals [CI] with Cox proportional hazard models adjusting for age, sex, co-morbidities, and important risk factors. Standardized incidence ratio of stroke amongst patients with Bell's palsy was analyzed. RESULTS: Compared with non-Bell's palsy patients, patients with Bell's palsy had a 2.02-times (95% CI, 1.42-2.86) higher risk of stroke. The adjusted HR of developing stroke for patients with Bell's palsy treated with and without systemic steroid were 1.67 (95% CI, 0.69-4) and 2.10 (95%, 1.40-3.07), respectively. CONCLUSIONS: Patients with Bell's palsy carry a higher risk of stroke than the general population. Our data suggest that these patients might benefit from a more intensive stroke prevention therapy and regular follow-up after initial diagnosis.

Predictors of bloodstream infection associated with permanently implantable venous port in solid cancer patients.

BACKGROUND: The purpose of this study is to characterize the risk factors of bloodstream infection (BSI) associated with the use of permanent implantable venous ports (Port-A) in solid cancer patients. METHODS: Solid cancer patients implanted with a Port-A were prospectively observed for the occurrence of Port-A-associated BSI (PABSI), defined as BSI without other identifiable infection foci. A PABSI risk score was developed using the Cox proportional hazards model. RESULTS: A total of 415 patients were registered; 88 PABSI episodes occurred in 58 patients (incidence1.05 per 1000 catheter-days). All but one patient had stage IV cancer. Independent predictors of PABSI occurrence included neutropenia, total parenteral nutrition (TPN), chronic steroid use, invasive procedures, postoperative antibiotics, and preoperative antibiotics. A PABSI risk score with a cut-off value of 0 (sensitivity 88.5%, specificity 64.3%) was defined for stage IV cancer patients as follows: neutropenia, +1.350; TPN, +1.256; chronic steroid use, +1.947; preoperative antibiotics, -0.970; postoperative antibiotics, +0.959; and invasive procedures, +1.098. The median PABSI-free survival was 4.47 months for patients with scores ≥ 0 but not reached for patients with scores <0 (P < 0.0001). CONCLUSION: The PABSI risk score can assist in identifying high-risk solid cancer patients and may assist in designing future preventive strategies.

Capsaicin may induce breast cancer cell death through apoptosis-inducing factor involving mitochondrial dysfunction.

The majority of breast cancer patients are resistant to chemotherapy or radiotherapy due to the down-regulation or lack of caspase-3 expression. Capsaicin was found to inhibit cancer cell growth in caspase-3-deficient human breast cancer cells. This study aimed to investigate the growth-inhibitive effect of capsaicin and its mechanisms in human breast cancer cell lines, MCF-7 and BT-20. The results showed that cell viability decreased in a dose-dependent manner in both the caspase-3-deficient and non-deficient cells through inducing cell apoptosis and arresting the cell cycle in the S phase. Capsaicin significantly decreased mitochondria membrane potential, induced the cleavage of PARP-1, and decreased procaspase-7 expression in both cells. Apoptosis-inducing factor (AIF) was distinctly released from mitochondria and translocated into the cytoplasm and nucleus in MCF-7 cells (52.9%), but not in BT-20 cells (2%) after treatment with 200 µM of capsaicin for 24 hours. Capsaicin inhibited breast cancer cell growth through inducing cell apoptosis and cell cycle arrest in the S phase. This apoptotic effect could be induced through the mitochondrial pathway, and PARP-1 subsequently cleaved by activation of caspase-7. The application of capsaicin in clinical therapy could be useful for breast cancer patients.

Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.

Blood surveillance and detection on platelet bacterial contamination associated with septic events.

The objective of this study was to evaluate the risk of transfusion-associated septic events in Taiwan. In Taiwan, most blood components are provided from blood centres; so platelet (PLT) bacterial contaminations are rarely reported. The study's aim is to investigate the prevalence of PLT bacterial contamination in Kaoshiung Armed Forces General Hospital by using BacT/ALERT system for routine screening. A total of 82 apheresis and 2256 whole blood-derived PLT units were tested. A measured quantity of 1 mL aliquots were taken as samplings from all blood bag tubing of PLT units, and then further incubated in a bacterial detection system (BacT/ALERT). The subcultures of true-positive bottles underwent bacterial identification by using Vitek system, microscopic observation and culture-based methods. Eight units (0.34%, 8 of 2338) were found to have bacterial contamination. The true-positive rate of the whole blood-derived and apheresis PLTs was 0.31% (7 of 2256) and 1.22% (1 of 82), respectively. Six microorganisms were identified with the most dominate being Staphylococcus epidermidis. One case of transfusion-associated sepsis was confirmed; in addition, the holding period of PLTs (F = 4.522, P = 0.034) and positive detection of PLT bacterial contamination (F = 46.605,P < 0.001) were associated with post-transfusion sepsis. Thus, in this study, although the transfusion-associated septic event was rarely found and PLT units were provided from blood centres, bacterial screening was necessary to safely quarantine the transfusion. The holding period of PLT units should be no more than 4 days in order to avoid possible bacterial contamination.

Craniosynostosis and congenital tracheal anomalies in an infant with Pfeiffer syndrome carrying the W290C FGFR2 mutation.

Pfeiffer syndrome (OMIM 101600) is an autosomal dominant disorder characterized by craniosynostosis, midface hypoplasia, ocular proptosis and digital malformations. We report on a type II Pfeiffer female infant with craniosynostosis, hydrocephalus, and characteristic craniofacial and digital abnormalities. The patient had a history of airway difficulty. Bronchoscopy at age four months revealed low tracheal stenosis and fibrous cartilaginous rings. She underwent tracheostomy for the treatment of cyanotic episodes. Molecular analysis revealed a de novo missense mutation c.870 G>T (TGG>TGT) in the FGFR2 gene that predicts a substitution of cysteine for tryptophan at the codon 290, (W290C). There is phenotypic heterogeneity of tracheal anomalies due to FGFR2 mutations. A review of the literature shows that Pfeiffer patients with the similar tracheal abnormalities can be caused by different FGFR2 mutations and, likewise, the patients with the same FGFR2 mutation may manifest different kinds of tracheal anomalies. Tracheal anomalies may occur in Pfeiffer patients and cause morbidity and mortality because of airway obstruction. Recognition and detailed evaluation of tracheal anomalies should be included in the early diagnostic workup for severe Pfeiffer patients.

Prenatal diagnosis of Dandy-Walker malformation associated with distal limb deficiencies.

We report the perinatal findings of a 23 gestational-week fetus with Dandy-Walker malformation (DWM), ventriculomegaly, symmetrical transverse limb deficiencies, hypertelorism, frontal bossing, low-set ears, and a depressed nasal bridge. The karyotype was 46,XX. We believe that this combination is significant. Concomitant DWM and symmetrical distal limb deficiencies may represent a new entity that awaits more new cases for further delineation.

Bipaddled radial forearm flap for the reconstruction of bilateral buccal defects in oral submucous fibrosis.

Oral submucous fibrosis can result in progressive closure of the mouth. A total of 10 patients with advanced oral submucous fibrosis were surgically treated. The procedure consisted of (1) release of all the intraoral fibrotic tissue, (2) masticatory muscle myotomy and coronoidotomy, and (3) reconstruction with a bipaddled radial forearm flap. Preoperative mouth opening was 0-5mm (mean 2.3mm). The intraoperative mouth opening was 12-20mm (mean 16 mm) after submucous release, and 32-42 mm (mean 35.5mm) after further release via myotomy and coronoidotomy. The proximal flap included one perforator in four patients and two perforators in the remaining six patients. The flaps were 8-9 cm in length and 2-2.5 cm in width. Nine flaps survived uneventfully. Arterial thrombosis was noted in one flap, which was successfully salvaged. Temporomandibular joint subluxation developed in one patient. Two patients needed flap revision due to bulkiness. The postoperative mouth opening was 18-38 mm (mean 28.2mm) after a mean of 21 months' follow-up, and the mean increase was 25.9 mm. A bipaddled radial forearm flap, using a single donor site, can cover two separate buccal defects after release of oral submucosal fibrosis and obviate the need for a second free flap.