Chemical Characterization, In-silico Evaluation, and Molecular Docking Analysis of Antiproliferative Compounds Isolated from the Bark of Anthocephalus cadamba Miq.

AIMS: The present study aimed to isolate and characterize chemical compounds from Anthocephalus cadamba Miq. bark and evaluate their anticancer activity by in silico, molecular docking, and in vitro studies. BACKGROUND: Anthocephalus cadamba is a traditionally used Indian medicinal plant. The anticancer and phytochemical properties of this plant remain unexplored except for a few studies. OBJECTIVES: The objective of the study was to evaluate the antiproliferative activity of extract and fractions against breast cancer and prostate cancer cell lines and isolate and characterize active compounds from bio-active guided fractions. Moreover, the anticancer activity of isolated compounds against breast and prostate cancer cell lines was also evaluated, in addition to in silico and molecular docking interactions of isolated compounds with VEGFR2 and PDGFRα target proteins. METHODS: The compounds were isolated and purified with the help of repeated column chromatography, and spectral techniques, such as 1D, 2D NMR, and GC-MS/MS, were used to identify and elucidate the structure of the compounds. Moreover, prediction of activity spectra for substances, physiochemical properties, bioactivity radar prediction, bioactivity score, natural-product likeness, ADME, and toxicity parameters of isolated compounds (AC-1 to AC-4) was performed through various in-silico databases and servers. To evaluate the docking interaction profile and binding energies of compounds, three docking tools were utilized, such as AutoDock, AutoDock Vina, and iGEMDOCK, against two targets VEGFR2 and PDGFRα. MD simulation was performed through ligand and receptor molecular dynamic server (LARMD). RESULTS: It was found that the A. cadamba bark chloroform fraction demonstrated a significant inhibitory effect against MDA-MB-231, MCF-7, and PC-3 cells in a dose-time-dependent manner. The bioassay-guided isolation afforded four molecules AC-1 to AC-4 from chloroform fraction. Moreover, the GC-MS/MS profiling identified fourteen new molecules which were not reported earlier from A. cadamba. The in-silico study showed that the isolated compounds (AC-1 to AC-4) followed Lipinski's rule and had good oral bioavailability. While compound AC-4 had positive bioactivity scores except for kinase inhibitor activity. The ADMET profiling revealed that AC-4 was non-toxic and easily absorbed in the human intestine, and transportable in the blood-brain barrier compared to AC-1, AC-2, AC-3, and standard drug doxorubicin. Molecular docking and MD simulation assessment also signified AC-4 anticancer activity with dual inhibitory action against the target proteins VEGFR2 and PDGFRα amongst the studied compounds. The in vitro cell viability assay of isolated compounds demonstrated that AC-1 showed IC50 (µg/mL) value of 34.96 ±3.91, 47.76±3.80 69.1±4.96, AC-2; 68.26±4.22, 54.03±5.14, >100, AC-3; 35.34±4.14, 51.5±51.5, 70.8±5.25 and AC-4; 44.2±3.57, 24.2±2.67, 51.2±2.54 for MDA-MB-231, MCF-7, and PC-3 cancer cell lines, respectively and compared with standard drug doxorubicin. Moreover, fluorescence microscopy confirmed the apoptogenic property of compounds. We also found that AC-4 exhibited significant intracellular ROS production in breast cancer cells, thereby inducing apoptosis and eventually cell death. CONCLUSION: In conclusion, A. cadamba afforded four pure molecules AC-1 to AC-4 with the identification of fourteen new compounds. The entire in-silico studies concluded that the AC-4 compound had better oral bioavailability, bioactivity score, and ADMET profile among studied molecules. Molecular docking analysis and MD simulation also supported AC-4 dual inhibitory action against both VEGFR2 and PDGFRα receptors. Moreover, the isolated molecules AC-1, AC-2, AC-3, and AC-4 were found to be active against MDA-MB-231, MCF-7, and PC-3 cancer cells. The molecule AC-4 was found to induce ROS-mediated apoptosis in breast cancer cells. It was found that the anticancer inhibitory potentiality of AC-4 is directed to its molecular stereochemistry which specifically binds to the target proteins of breast cancer cells with no toxicological effect. Therefore, AC-4 is suggested to be an effective aspirant for novel drug design and discovery.

Bee Venom-Derived BBB Shuttle and its Correlation with Oligodendrocyte Proliferation Markers in Mice Model of Multiple Sclerosis.

Multiple sclerosis is a chronic demyelinating disease with a functional disturbance in the immune system and axonal damages. It was shown that Apamin as a blood-brain barrier shuttle acts as a Ca2+ activated K+ channels (SK channels) blocker. In this study, the effects of Apamin on oligodendrocyte differentiation markers were evaluated on an induced model of MS. Briefly, C57BL/6 male mice (22 ± 5 g) except the control group were fed with 0.2% (w/w) cuprizone pellets for 6 weeks. After cuprizone withdrawal, mice were divided randomly into six groups. Apamin (100 µg/kg/BW) was administered intraperitoneally as a co-treatment during phase I (demyelination) or post-treatment phase II (remyelination) twice a week. Mice were anesthetized, perfused with phosphate-buffered saline, then fixed brains were coronally sectioned and the changes in oligodendrocytes markers such as Olig2, PDGFR-α, and BrdU incorporation were assessed by immunohistochemistry assay. Apamin administration increased Olig2+ cells in phase I as compared to the control group (p < 0.0001). Also, a decreasing trend in PDGFRa+ cells observed after cuprizone withdrawal (p < 0.001). 5-Bromo-2'-deoxyuridine (BrdU) incorporation test was confirmed stimulation of oligodendrocyte progenitor cell proliferation in phase I in the Apamin exposed group (p < 0.0001), especially at the subventricular zone. This study highlights the potential therapeutic effects of Apamin as a bee venom-derived peptide on oligodendrocyte precursor proliferation and elevation in myelin content in an oxidative induced multiple sclerosis model due to cuprizone exposure.

Desloratadine inhibits heterotopic ossification by suppression of BMP2-Smad1/5/8 signaling.

Heterotopic ossification (HO) is a pathological condition in which ectopic bone forms within soft tissues such as skeletal muscle. Human platelet-derived growth factor receptor α positive (PDGFRα+) cells, which were proved to be the original cells of HO were incubated in osteogenic differentiation medium with Food and Drug Administration-approved compounds. Alkaline phosphatase activity was measured as a screening to inhibit osteogenic differentiation. For the compounds which inhibited osteogenic differentiation of PDGFRα+ cells, we examined dose dependency of its effect using alizarin red S staining and its cell toxicity using WST-8. In addition, regulation of bone morphogenetic proteins (BMP)-Smad signaling which is the major signal of osteogenic differentiation was investigated by Western blotting to elucidate the mechanism of osteogenesis inhibitory effect by the compound. In vivo experiment, complete transverse incision of Achilles tendons in mice was made and mice were fed the compound by mixing with drinking water after operation. Ten weeks after operation, we assessed and quantified HO by micro-computed tomography scan. Intriguingly, we discovered desloratadine inhibited osteogenic differentiation of PDGFRα+ cells using the drug repositioning method. Desloratadine inhibited osteogenic differentiation of the cells dose dependently without cell toxicity. Desloratadine suppressed phosphorylation of Smad1/5/8 induced by BMP2 in PDGFRα+ cells. In Achilles tenotomy mice model, desloratadine treatment significantly inhibited ectopic bone formation compared with control. In conclusion, we discovered desloratadine inhibited osteogenic differentiation using human PDGFRα+ cells and proved its efficacy using Achilles tenotomy ectopic bone formation model in vivo. Our study paved the way to inhibit HO in early clinical use because of its guaranteed safety.

The expression of platelet-derived growth factor receptor alpha-positive cells in the stenotic tissue of ureteropelvic junction obstruction in children
.

AIM: By observing the expression and distribution of platelet-derived growth factor receptor α-positive (PDGFRα+) cells in ureteropelvic junction obstruction (UPJO), to explore their role in the pathogenesis of children with congenital hydronephrosis. MATERIALS AND METHODS: The control group involved specimens of the normal ureter (nephrectomy for tumor; n = 10), and the UPJO group contained specimens of ureteropelvic junction (UPJ) segment excised during pyeloplasty (n = 30). The specimens were investigated using immunofluorescence for the expression and distribution of PDGFRα+ cells in each group by light microscopy with computerized image analysis. Real-time PCR (RT-PCR) was used to study PDGFRα gene expression levels. In addition, small conductance calcium-activated potassium channel 3 (SK3) and closely associated cells consisting of smooth muscle cells (SMCs), interstitial cells of Cajal (ICCs), and nerve fibers were investigated. RESULTS: PDGFRα+ cells were in close proximity to SMCs, ICCs, and nerve fibers. PDGFRα+ cells expressed SK3 channels, which are found to regulate purinergic inhibitory neurotransmission in SMCs. Regarding the expression of PDGFRα+ cells no significant difference was seen between the two groups, while the expression of SK3 channels in PDGFRα+ cells was significantly decreased in the UPJO group versus the control group. CONCLUSION: This study identified the expression of PDGFRα+ cells in the human UPJ. Our results demonstrate the expression of SK3 channels in PDGFRα+ cells was decreased in UPJO, and SK3 channels may be involved in the pathogenesis of UPJO by perturbing the UPJ peristalsis.

The Autonomic Innervation and Uterine Telocyte Interplay in Leiomyoma Formation.

The autonomic innervation of the uterus is involved in multiple pathophysiological processes in both humans and animals. Pathological conditions such as adenomyosis or inflammatory pelvic disease are usually accompanied by significant alterations in uterine innervation. In the current study, we focused on autonomic innervation of uterine fibroids, the identification of recently described interstitial cells, telocytes, and the possible interplay between these structures. In this work, uterine telocytes were identified by immunopositivity for c-kit, CD34, and PDGFRα. Nerves were revealed by immunolabeling for neuronal markers: protein gene product PGP 9.5, inducible nitric oxide synthase (iNOS), choline acetyltransferase (ChAT), and tyrosine hydroxylase (TH). The gross organization of myometrial tissue has been analyzed by routine histology. The results demonstrated that the density of iNOS and ChAT-immunopositive neurons in the uterine fibroids was higher than that in the control samples. The density of telocytes in the fibrosis foci was lower than that in the normal myometrium. Our results suggest that autonomic innervation and telocytes are involved in the microenvironment imbalance characteristic of uterine leiomyoma. Since NOS-positive nerves play an important role in oxidative stress modulation, they might lead to a decrease in the number of telocytes, which are crucial components in the pathogenesis of leiomyoma formation.

Association of Dasatinib With Progression-Free Survival Among Patients With Advanced Gastrointestinal Stromal Tumors Resistant to Imatinib.

Importance: Gastrointestinal stromal tumors (GISTs) are life-threatening when metastatic or not amenable to surgical removal. In a few patients with advanced GISTs refractory to imatinib mesylate, treatment with sunitinib malate followed by regorafenib provides tumor control; however, additional active treatments are needed for most patients. Objective: To evaluate the 6-month progression-free survival (PFS), tumor objective response, and overall survival rates in patients with GISTs treated with dasatinib. Design, Setting, and Participants: This single-arm clinical trial used a Bayesian design to enroll patients 13 years or older with measurable imatinib-refractory metastatic GISTs treated at 14 sarcoma referral centers from June 1, 2008, through December 31, 2009. A control group was not included. Patients were followed up for survival for a minimum of 5 years from date of enrollment. Tumor imaging using computed tomography or magnetic resonance imaging was performed every 8 weeks for the first 24 weeks and every 12 weeks thereafter. Tumor response was assessed by local site using the Choi criteria. Treatment was continued until tumor progression, unacceptable toxic effects after reduction in drug dose, or patient or physician decision. Archival tumor tissue was evaluated for expression of the proto-oncogene tyrosine-protein kinase Src (SRC), phosphorylated SRC (pSRC), and succinate dehydrogenase complex iron sulfur subunit B (SDHB) proteins and for mutation in the V-Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) and platelet-derived growth factor receptor α (PDGFRA) genes. Data analysis was performed from May 19, 2017, through December 20, 2017. Interventions: Dasatinib, 70 mg orally twice daily. Main Outcomes and Measures: The primary end point was the 6-month PFS estimate using greater than 30% as evidence of an active drug and less than 10% as evidence of inactive treatment. Results: In this study, 50 patients were enrolled (median age, 60 years; age range, 19-78 years; 31 [62%] male and 19 [38%] female; 41 [82%] white), and 48 were evaluable for response. The estimated 6-month PFS rate was 29% in the overall population and 50% in a subset of 14 patients with pSRC in GISTs. Objective tumor response was observed in 25%, including 1 patient with an imatinib-resistant mutation in PDGFRA exon 18. Conclusions and Relevance: Dasatinib may have activity in a subset of patients with imatinib-resistant GISTs. Further study is needed to determine whether pSRC is a prognostic biomarker.

Loeffler endocarditis as a rare cause of heart failure with preserved ejection fraction: A case report and review of literature.

RATIONALE: Hypereosinophilic syndrome (HES) is a rare disease characterized by hypereosinophilia and its ensuing organ damage. Cardiac involvement is divided into 3 chronological stages: an acute necrotic stage; a thrombus formation stage; and a fibrotic stage. Infiltration of the myocardium by eosinophilic cells followed by endomyocardial fibrosis is known as "Loeffler endocarditis." PATIENT CONCERNS: We report a case of a 60-year-old man diagnosed with left-sided restrictive cardiomyopathy. DIAGNOSIS: The patient experienced heart failure with preserved ejection fraction. The cardiac MRI showed intense, linear, delayed gadolinium enhancement of the endocardium of the lateral wall of the left ventricle, and obliteration of the LV apex. He was ultimately identified as Loeffler endocarditis. INTERVENTION: A bone marrow smear and biopsy revealed the FIP1L1-PDGFRA fusion gene was positive in 82% of segmented nucleated cells. OUTCOME: Our patient responded well to prednisone at 1 mg/kg/d. LESSONS: HES is a rare disease that often afflicts the heart. Cardiac involvement in hypereosinophilia, especially Loeffler endocarditis, carries a poor prognosis and significant mortality. Early detection and treatment of the disease is therefore essential. Further studies are needed to ascertain therapeutic corticosteroid dosages and develop targeted gene therapies, both important steps to ameliorate the effects of Loeffler endocarditis and improve patient outcomes.

Myocardial Telocyte-Like Cells: A Review Including New Evidence.

Telocytes (TCs) are a controversial cell type characterized by the presence of a particular kind of prolongations, known as telopodes, which are long, thin, and moniliform. A number of attempts has been made to establish the molecular phenotype of cardiac TCs (i.e., expression of c-kit, CD34, vimentin, PDGRFα, PDGRFß, etc.). We designed an immunohistochemical study involving cardiac tissue samples obtained from 10 cadavers with the aim of determining whether there are TC-like interstitial cells that populate the interstitial space other than the mural microvascular cells. We applied the markers for CD31, CD34, PDGRFα, CD117/c-kit, and α-smooth muscle actin (α-SMA). We found that, in relation to two-dimensional cuts, the endothelial tubes could be misidentified as TC-like cells, the difference being the positive identification of endothelial lumina. Moreover, we found that cardiac pericytes express PDGRFα, CD117/c-kit, and α-SMA, and that they could also be misidentified as TCs when using light microscopy. We reviewed the respective values of the previously identified markers for achieving a clear-cut identification of cardiac TCs, highlighting the critical lack of specificity.

Premature contractions of the bladder are suppressed by interactions between TRPV4 and SK3 channels in murine detrusor PDGFRα+ cells.

During filling, urinary bladder volume increases dramatically with little change in pressure. This is accomplished by suppressing contractions of the detrusor muscle that lines the bladder wall. Mechanisms responsible for regulating detrusor contraction during filling are poorly understood. Here we describe a novel pathway to stabilize detrusor excitability involving platelet-derived growth factor receptor-α positive (PDGFRα+) interstitial cells. PDGFRα+ cells express small conductance Ca2+-activated K+ (SK) and TRPV4 channels. We found that Ca2+ entry through mechanosensitive TRPV4 channels during bladder filling stabilizes detrusor excitability. GSK1016790A (GSK), a TRPV4 channel agonist, activated a non-selective cation conductance that coupled to activation of SK channels. GSK induced hyperpolarization of PDGFRα+ cells and decreased detrusor contractions. Contractions were also inhibited by activation of SK channels. Blockers of TRPV4 or SK channels inhibited currents activated by GSK and increased detrusor contractions. TRPV4 and SK channel blockers also increased contractions of intact bladders during filling. Similar enhancement of contractions occurred in bladders of Trpv4 -/- mice during filling. An SK channel activator (SKA-31) decreased contractions during filling, and rescued the overactivity of Trpv4 -/- bladders. Our findings demonstrate how Ca2+ influx through TRPV4 channels can activate SK channels in PDGFRα+ cells and prevent bladder overactivity during filling.

Alteration of PDGFRß-Akt-mTOR pathway signaling in fibrosarcomatous transformation of dermatofibrosarcoma protuberans.

Dermatofibrosarcoma protuberans (DFSP) is a cutaneous mesenchymal tumor of intermediate malignancy and fibroblastic/myofibroblastic differentiation. Fibrosarcomatous (FS) component is a high-grade component of DFSP. The detailed oncogenic difference between DFSP and FS components is not clear. We thus investigated the Akt-mTOR pathway in both components. We used 65 tumor samples obtained from 65 patients. The phosphorylation of Akt-mTOR pathway proteins (Akt, mTOR, 4EBP1, and S6RP) and PDGFRα/ß was assessed by immunohistochemical staining, the results of which were confirmed by Western blotting. The immunohistochemical results were as follows: in ordinary DFSP components, p-PDGFRα-positive tumors were 41.9% (18/43 cases), p-PDGFRß 55.8% (24/43 cases), p-Akt 51.2% (22/43 cases), p-mTOR 39.5% (17/43 cases), p-4EBP1 46.5% (20/43 cases), and p-S6RP 41.8% (18/43 cases); in DFSP components of FS-DFSP, 52.6% (10/19 cases), 47.4% (9/19 cases), 52.6% (10/19 cases), 36.8% (7/19 cases), 52.6% (10/19 cases), and 52.6% (10/19 cases); and in FS components, 45.5% (10/22 cases), 36.4% (8/22 cases), 72.7% (16/22 cases), 54.5% (12/22 cases), 72.7% (16/22 cases), and 68.2% (15/22 cases), respectively. There were significant positive correlations of the phosphorylation of most of the Akt-mTOR pathway proteins (p-Akt, p-mTOR, p-4EBP1, and p-S6RP) with each other (P < .05). Phospho-PDGFRß was well correlated with the phosphorylation of Akt-mTOR pathway proteins in DFSP components of ordinary and FS-DFSPs, but these correlations were weaker in FS components. This study suggested the association of activation of Akt-mTOR pathway proteins and PDGFR with the progression of DFSP to FS. The Akt-mTOR pathway is thus a potential therapeutic target in imatinib-resistant DFSP/FS.

The effects of perinatal steroid therapy on growth factor levels during different stages of the developing brain.

OBJECTIVE: Excess glucocorticoid (GC) exposure on the fetal brain during critical stages of development has considerable effects on the development of the central nervous system (CNS). This study thus aimed to evaluate the differential effects of GC exposure on critical growth factor levels during different stages of brain maturation. METHODS: For this purpose, forty-two rat pups were divided into six groups based on the timing of betamethasone administration. Rats in the treatment groups were exposed to intraperitoneal betamethasone injections beginning at different time points (postnatal days 1, 2, and 3). Rats in the placebo group received the same volume of 0.9% saline via the same fashion. Pups were sacrificed at 24 h following the final injection for determining the neuronal density and immunohistochemical evaluation of critical growth factors. RESULTS: In the groups treated with betamethasone on postnatal day 1 (P1) and P2, which correspond to 22-24 and 24-28 gestational weeks in humans, the neuronal count in the hippocampal regions was significantly lower than their control groups. However, if steroid therapy was administered on P3, corresponding to 28-32 weeks in humans, no difference was observed between the two groups. Growth factors were affected in different ways depending on the steroid administration time and evaluated region. CONCLUSIONS: The results suggest that the modulating effect of steroids on neuron count and growth factor response depends on the stage of brain development at the time of exposure. Therefore, this may be one of the key determinants affecting the deleterious and beneficial effects of GCs on the CNS.

Variability in Platelet-Derived Growth Factor Receptor Alpha Antibody Specificity May Impact Clinical Utility of Immunohistochemistry Assays.

Aberrant regulation of the receptor tyrosine kinase platelet-derived growth factor alpha (PDGFRα) is implicated in several types of cancer. Inhibition of the PDGFRα pathway may be a beneficial therapy, and detection of PDGFRα in tumor biopsies may lead to insights about which patients respond to therapy. Exploratory or clinical biomarker use of PDGFRα IHC has been frequently reported, often with polyclonal antibody sc-338. An sc-338-based assay was systematically compared with anti-PDGFRα rabbit monoclonal antibody D13C6 using immunoblot profiling and IHC in formalin-fixed and paraffin-embedded human tumor cell lines. Application of sc-338 to blots of whole cell lysates showed multiple bands including some of unknown origin, whereas application of D13C6 resulted in a prominent band at the expected molecular mass of PDGFRα. The IHC assay using D13C6 showed appropriate staining in cell lines, whereas the assay using sc-338 suggested nonspecific detection of proteins. An optimized IHC assay using D13C6 showed a range of staining in the tumor stromal compartment in lung and ovarian carcinomas. These observations suggest that use of clone sc-338 produced unreliable results and should not be used for an IHC research grade assay. In addition, this precludes its use as a potential antibody for a clinical diagnostic tool.

Patient-derived glioblastoma stem cells respond differentially to targeted therapies.

The dismal prognosis of glioblastoma is, at least in part, attributable to the difficulty in eradicating glioblastoma stem cells (GSCs). However, whether this difficulty is caused by the differential responses of GSCs to drugs remains to be determined. To address this, we isolated and characterized ten GSC lines from established cell lines, xenografts, or patient specimens. Six lines formed spheres in a regular culture condition, whereas the remaining four lines grew as monolayer. These adherent lines formed spheres only in plates coated with poly-2-hydroxyethyl methacrylate. The self-renewal capabilities of GSCs varied, with the cell density needed for sphere formation ranging from 4 to 23.8 cells/well. Moreover, a single non-adherent GSC either remained quiescent or divided into two cells in four-seven days. The stem cell identity of GSCs was further verified by the expression of nestin or glial fibrillary acidic protein. Of the two GSC lines that were injected in immunodeficient mice, only one line formed a tumor in two months. The protein levels of NOTCH1 and platelet derived growth factor receptor alpha positively correlated with the responsiveness of GSCs to γ-secretase inhibitor IX or imatinib, two compounds that inhibit these two proteins, respectively. Furthermore, a combination of temozolomide and a connexin 43 inhibitor robustly inhibited the growth of GSCs. Collectively, our results demonstrate that patient-derived GSCs exhibit different growth rates in culture, possess differential capabilities to form a tumor, and have varied responses to targeted therapies. Our findings underscore the importance of patient-derived GSCs in glioblastoma research and therapeutic development.

Immunohistochemical markers of advanced basal cell carcinoma: CD56 is associated with a lack of response to vismodegib.

Vismodegib is an effective treatment for advanced basal cell carcinoma (BCC), but primary resistance to vismodegib remains to be elucidated. Alternative approaches are warranted to help selecting patients most likely to be responsive to treatment. The identification of immunohistochemical markers may support this perspective, as well as better understanding of resistance mechanisms. To determine the level of expression of CD56, PDGF-R, CD117, MMP9, TIMP3, and CXCR4 in advanced BCC, and explore whether expression levels are associated with non-response to vismodegib. A cross-sectional study was conducted. Immunohistochemical markers were selected based on their roles in tumour proliferation and/or migration in skin tumours. Tissue samples included pretreatment advanced BCC samples from patients treated with vismodegib, with an available response after six months of treatment. Regression optimised models were used to build hypotheses regarding a possible association between expression levels and non-response to vismodegib, which was then tested by logistic regression. Twenty-three patients were included. The percentage of samples expressing markers ranged from 43.5% (CD117) to 91.3% (CXCR4). CD56 expression was significantly associated with an increased risk of non-response to vismodegib (OR = 5.5; CI 95%: 3.4-29.8; p = 0.0488); a similar association was suggested for CXCR4 (p = 0.066), but not identified for other markers. These results provide a better understanding of the expression of immunohistochemical markers in advanced BCC. Further detailed analysis of CD56 expression may provide insights into guiding further investigation of the correlation between this marker and non-response to vismodegib.

Expression and significance of Ku80 and PDGFR-α in nasal NK/T-cell lymphoma.

BACKGROUND: To investigate the clinical and prognostic significance of Ku80 and PDGFR-α in nasal type NK/T cell lymphoma (NKTCL). METHODS: Immunohistochemistry for Ku80 and PDGFR-α was performed on tissue sections from 35 patients diagnosed with NKTCL. We analyzed the relationship between Ku80 and PDGFR-α expression and the clinicopathological features of NKTCL. We further performed multivariate analyses to identify prognostic factors for progression free survival (PFS) of patients. RESULTS: Ku80 expression rate in NKTCL was 94.3% compared with that in reactive lymphoid hyperplasia of nasopharynx (P=0.003). The positive expression rate of PDGFR-α in the group of NKTCL was 91.4% compared with that in control group (P=0.004). The positive correlation between Ku80 and PDGFR-α was also found (r=0.496, P=0.002). Ku80 and PDGFR-α expressions were not correlated with patient's gender, age, B symptoms, LDH, Ann Arbor stage, IPI score and treatment (P>0.05). High expression of Ku80 and PDGFR-α was shown to be correlated with worse PFS (P=0.003 and 0.034, respectively). Multivariate analysis with a Cox proportional hazards model further suggested that Ku80 high expression rate (HR, 11.495; P=0.009), PDGFR-α high expression rate (HR, 4.836; P=0.031) and International Prognostic Index (IPI) score of 3-4 (HR, 7.308; P=0.001) were statistically independent prognostic factors for patients' PFS. Our results suggest that Ku80 and PDGFR-α may be valuable indicators for predicting the survival of NKTCL patients. CONCLUSION: Ku80 and PDGFR-α might be effective predictive indicators for the prognosis of NKTCL. A large prospective study is required to confirm the prognostic significance of Ku80 and PDGFR-α in NKTCL.

Imatinib Treatment in PDGFRA-Negative Childhood Hypereosinophilic Syndrome.

We report a 4-year-old female who presented with severe hypereosinophilia (215.7 K/µl) and end-organ dysfunction. Extensive evaluation including whole exome sequencing was performed, revealing no causative mutation. Initial treatment with corticosteroids, leukapheresis, and hydroxyurea decreased her absolute eosinophil count (AEC), although it remained elevated. Despite the absence of a PDGFRA mutation, an imatinib trial resulted in normalization of her AEC. Imatinib was discontinued after sustained normal counts for 1 month. AECs have remained normal for more than 1 year off therapy. This provides support for consideration of imatinib in the treatment of hypereosinophilia even in the absence of a known tyrosine kinase mutation.

Mesoderm Differentiation from hiPS Cells.

Human induced pluripotent stem (hiPS) cells are very attractive tools for modeling diseases and regenerative medicine. However, to achieve them, the efficient differentiation methods of hiPS cells into aimed cell type in vitro are necessary. Because mesoderm cells are useful in particular, we have developed the differentiation of mouse embryonic stem (mES) cells into mesoderm cells previously. In this time, these methods were improved for hiPS cells and now human mesoderm cells are able to be obtained efficiently. It is certain that the new methods are applicable to various studies and therapies.

Single CD271 marker isolates mesenchymal stem cells from human dental pulp.

Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow (BMSCs), adult MSCs are isolated from craniofacial tissues including dental pulp tissues (DPs) using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cells (DMSCs). In this study, we used different combinations of surface markers (CD51/CD140α, CD271, and STRO-1/CD146) to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells (DPCs) obtained from DP and compared their capacity to undergo multilineage differentiation. Fluorescence-activated cell sorting revealed that 27.3% of DPCs were CD51(+)/CD140α(+), 10.6% were CD271(+), and 0.3% were STRO-1(+)/CD146(+). Under odontogenic conditions, all three subsets of isolated DMSCs exhibited differentiation capacity into odontogenic lineages. Among these isolated subsets of DMSCs, CD271(+) DMSCs demonstrated the greatest odontogenic potential. While all three combinations of surface markers in this study successfully isolated DMSCs from DPCs, the single CD271 marker presents the most effective stem cell surface marker for identification of DMSCs with high odontogenic potential. Isolated CD271(+) DMSCs could potentially be utilized for future clinical applications in dentistry and regenerative medicine.

Characterization of the osteogenic potential of mesenchymal stem cells from human periodontal ligament based on cell surface markers.

Mesenchymal stem cell (MSC)-mediated therapy has been shown to be clinically effective in regenerating tissue defects. For improved regenerative therapy, it is critical to isolate homogenous populations of MSCs with high capacity to differentiate into appropriate tissues. The utilization of stem cell surface antigens provides a means to identify MSCs from various tissues. However, few surface markers that consistently isolate highly regenerative MSCs have been validated, making it challenging for routine clinical applications and making it all the more imperative to identify reliable surface markers. In this study, we used three surface marker combinations: CD51/CD140α, CD271, and STRO-1/CD146 for the isolation of homogenous populations of dental mesenchymal stem cells (DMSCs) from heterogeneous periodontal ligament cells (PDLCs). Fluorescence-activated cell sorting analysis revealed that 24% of PDLCs were CD51(+)/CD140α(+), 0.8% were CD271(+), and 2.4% were STRO-1(+)/CD146(+). Sorted cell populations were further assessed for their multipotent properties by inducing osteogenic and chondrogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into osteogenic and chondrogenic lineages but with varying degrees. CD271(+) DMSCs demonstrated the greatest osteogenic potential with strong induction of osteogenic markers such as DLX5, RUNX2, and BGLAP. Our study provides evidence that surface marker combinations used in this study are sufficient markers for the isolation of DMSCs from PDLCs. These results provide important insight into using specific surface markers for identifying homogenous populations of DMSCs for their improved utilization in regenerative medicine.

CXCL12 Gene Therapy Ameliorates Ischemia-Induced White Matter Injury in Mouse Brain.

UNLABELLED: Remyelination is an important repair process after ischemic stroke-induced white matter injury. It often fails because of the insufficient recruitment of oligodendrocyte progenitor cells (OPCs) to the demyelinated site or the inefficient differentiation of OPCs to oligodendrocytes. We investigated whether CXCL12 gene therapy promoted remyelination after middle cerebral artery occlusion in adult mice. The results showed that CXCL12 gene therapy at 1 week after ischemia could protect myelin sheath integrity in the perifocal region, increase the number of platelet-derived growth factor receptor-α (PDGFRα)-positive and PDGFRα/bromodeoxyuridine-double positive OPCs in the subventricular zone, and further enhance their migration to the ischemic lesion area. Coadministration of AMD3100, the antagonist for CXCL12 receptor CXCR4, eliminated the beneficial effect of CXCL12 on myelin sheath integrity and negatively influenced OPC proliferation and migration. At 5 weeks after ischemia, CXCR4 was found on the PDGFRα- and/or neuron/glia type 2 (NG2)-positive OPCs but not on the myelin basic protein-positive mature myelin sheaths, and CXCR7 was only expressed on the mature myelin sheath in the ischemic mouse brain. Our data indicated that CXCL12 gene therapy effectively protected white matter and promoted its repair after ischemic injury. The treatment at 1 week after ischemia is effective, suggesting that this strategy has a longer therapeutic time window than the treatments currently available. SIGNIFICANCE: This study has demonstrated for the first time that CXCL12 gene therapy significantly ameliorates brain ischemia-induced white matter injury and promotes oligodendrocyte progenitor cell proliferation in the subventricular zone and migration to the perifocal area in the ischemic mouse brain. Additional data showed that CXCR4 receptor plays an important role during the proliferation and migration of oligodendrocyte progenitor cells, and CXCR7 might play a role during maturation. In contrast to many experimental studies that provide treatment before ischemic insult, CXCL12 gene therapy was performed 1 week after brain ischemia, which significantly prolonged the therapeutic time window of brain ischemia.