Clinical-grade,large-scale,feeder-free expansion of highly active human natural killer cells for adoptive immunotherapy using an automated bioreactor

Background aimsNatural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. Ex vivo expansion and activation of NK cells under good manufacturing practice (GMP) conditions are crucial for facilitating large clinical trials. The goal of this study was to optimize a large-scale, feeder-free, closed system for efficient NK cell expansion.MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors and myeloma patients were cultured for 21 days using flasks, cell culture bags and bioreactors. Final products from different expansions were evaluated comparatively for phenotype and functionality.ResultsSignificant NK cell expansions were obtained in all systems. The bioreactor yielded a final product rich in NK cells (mean 38%) ensuring that a clinically relevant cell dose was reached (mean 9.8 × 10⁹ NK cells). Moreover, we observed that NK cells expanded in the bioreactor displayed significantly higher cytotoxic capacity. It was possible to attribute this partially to a higher expression level of NKp44 compared with NK cells expanded in flasks.ConclusionsThese results demonstrate that large amounts of highly active NK cells for adoptive immunotherapy can be produced in a closed, automated, large-scale bioreactor under feeder-free current GMP conditions, facilitating clinical trials for the use of these cells.     

A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer

BackgroundNatural killer (NK) cells derived from patients with cancer exhibit diminished cytotoxicity compared with NK cells from healthy individuals. We evaluated the tumor response and in vivo expansion of allogeneic NK cells in recurrent ovarian and breast cancerMethodsPatients underwent a lymphodepleting preparative regimen: fludarabine 25 mg/m² × 5 doses, cyclophosphamide 60 mg/kg × 2 doses, and, in seven patients, 200 cGy total body irradiation (TBI) to increase host immune suppression. An NK cell product, from a haplo-identical related donor, was incubated overnight in 1000 U/mL interleukin (IL)-2 prior to infusion. Subcutaneous IL-2 (10 MU) was given three times/week × 6 doses after NK cell infusion to promote expansion, defined as detection of ≥100 donor-derived NK cells/μL blood 14 days after infusion, based on molecular chimerism and flow cytometryResultsTwenty (14 ovarian, 6 breast) patients were enrolled. The median age was 52 (range 30–65) years. Mean NK cell dose was 2.16 × 10⁷cells/kg. Donor DNA was detected 7 days after NK cell infusion in 9/13 (69%) patients without TBI and 6/7 (85%) with TBI. T-regulatory cells (Treg) were elevated at day +14 compared with pre-chemotherapy (P = 0.03). Serum IL-15 levels increased after the preparative regimen (P = < 0.001). Patients receiving TBI had delayed hematologic recovery (P = 0.014). One patient who was not evaluable had successful in vivo NK cell expansionConclusionsAdoptive transfer of haplo-identical NK cells after lymphodepleting chemotherapy is associated with transient donor chimerism and may be limited by reconstituting recipient Treg cells. Strategies to augment in vivo NK cell persistence and expansion are needed.     

Clinical-grade manufacturing of autologous mature mRNA-electroporated dendritic cells and safety testing in acute myeloid leukemia patients in a phase I dose-escalation clinical trial

Background aimsRNA-electroporated dendritic cell (DC)-based vaccines are rapidly gaining interest as therapeutic cancer vaccines. We report on a phase I dose-escalation trial using clinical-grade manufactured mature RNA-electroporated DC in acute myeloid leukemia (AML) patients.MethodsCD14⁺ cells were isolated from leukapheresis products by immunomagnetic CliniMACS separation and differentiated into mature DC (mDC). mDC were electroporated with clinical-grade mRNA encoding the Wilm's tumor (WT1) antigen, and tested for viability, phenotype, sterility and recovery. To test product safety, increasing doses of DC were administered intradermally four times at 2-week intervals in 10 AML patients.ResultsIn a pre-clinical phase, immunomagnetic monocyte isolation proved superior over plastic adherence in terms of DC purity and lymphocyte contamination. We also validated a simplified DC maturation protocol yielding a consistent phenotype, migration and allogeneic T-cell stimulatory capacity in AML patients in remission. In the clinical trial, highly purified CD14⁺ cells (94.5 ± 3.4%) were obtained from all patients. A monocyte-to-mDC conversion factor of 25 ± 10% was reached. All DC preparations exhibited high expression of mDC markers. Despite a decreased cell recovery of mDC after a combination of mRNA electroporation and cryopreservation, successful vaccine preparations were obtained in all AML patients. DC injections were well tolerated by all patients.ConclusionsOur method yields a standardized, simplified and reproducible preparation of multiple doses of clinical-grade mRNA-transfected DC vaccines from a single apheresis with consistent mature phenotype, recovery, sterility and viability. Intradermal injection of such DC vaccines in AML patients is safe.     

DNA binding acridine–thiazolidinone agents affecting intracellular glutathione

Three new acridine–thiazolidinone derivatives (2a–2c) have been synthesized and their interactions with calf thymus DNA and a number of cell lines (leukemic cells HL-60 and L1210 and human epithelial ovarian cancer cell lines A2780) were studied. The compounds 2a–2c possessed high affinity to calf thymus DNA and their binding constants determined by spectrofluorimetry were in the range of 1.37 × 10⁶–5.89 × 10⁶ M^?1. All of the tested derivatives displayed strong cytotoxic activity in vitro, the highest activity in cytotoxic tests was found for 2c with IC₅₀ = 1.3 ± 0.2 μM (HL-60), 3.1 ± 0.4 μM (L1210), and 7.7 ± 0.5 μM (A2780) after 72 h incubation. The cancer cells accumulated acridine derivatives very fast and the changes of the glutathione level were confirmed. The compounds inhibited proliferation of the cells and induced an arrest of the cell cycle and cell death. Their influence upon cells was associated with their reactivity towards thiols and DNA binding activity.     

Immunosuppressant FK506 decreases the intracellular magnesium in the human osteoblast cell by inhibiting the ERK1/2 pathway

AimsPrevious studies reported that FK506 influences bone mineralizing and hypomagnesemia, and also has immune modifying properties. This study examined whether or not the function of Mg²⁺ in bone metabolism plays a role in the loss of bone volume caused by immunosuppressants.Main methodsThe effects of the FK506 treatment on the intracellular magnesium and lactate dehydrogenase (LDH) activity were examined in cultured human osteoblasts (HOB) cells. The magnesium concentration was determined using microfluorescence techniques and atomic absorption spectrophotometry. Western blotting was used to measure the level of extracellular signal-regulated kinases 1/2 (ERK 1/2) activation.Key findingsFK506 (0.1 μM) did not affect cell death in HOB cells after a 24 hour treatment but decreased the level of ERK 1/2 activation. In HOB cells, the mean [Mg²⁺]i after exposure to a 1 mM extracellular Mg²⁺ ([Mg²⁺]o) buffer was 0.53 ± 0.01 mM (n = 25). Exposure to 100 nM FK506 produced a significant decrease in [Mg²⁺]i (0.41 ± 0.01 mM). The ERK inhibitor (PD98059) and FK506 produced similar effects but they were not cumulative.SignificanceThis study examined the role of ERK1/2 activation on the regulation of magnesium in HOB. These results suggest that the inhibition of ERK phosphorylation is an essential intermediate in the effects of FK506 on magnesium. Overall, FK506 causes bone disorders partly by decreasing [Mg²⁺]i accompanied by the inhibition of ERK 1/2.     

Immune mobilization of autologous blood progenitor cells: direct influence on the cellular subsets collected

Background aimsA phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells.MethodsInterleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte–macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin's lymphoma n = 2).ResultsToxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1–3) to collect 3.2 × 10⁶ CD34⁺ cells/kg (median; range 1.9–6.6 × 10⁶/kg). Immune mobilization increased the number of CD3⁺ CD8⁺ T cells (P = 0.002), CD56⁺ natural killer (NK) cells (P = 0.0001), CD8⁺ CD56⁺ T cells (P = 0.002) and CD4⁺ CD25⁺ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 × 10⁶ IU/m²/day.ConclusionsImmune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.     

GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion

Background aimsBone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque? PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance.MethodsBM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential.ResultsNo differences were noticed in cell recovery and viability between the groups. Fluorescence-activated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45⁺ fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage.ConclusionsBoth GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications.     

Eye lens dose correlations with personal dose equivalent and patient exposure in paediatric interventional cardiology performed with a fluoroscopic biplane system

PurposeTo analyse the correlations between the eye lens dose estimates performed with dosimeters placed next to the eyes of paediatric interventional cardiologists working with a biplane system, the personal dose equivalent measured on the thorax and the patient dose.MethodsThe eye lens dose was estimated in terms of H_p(0.07) on a monthly basis, placing optically stimulated luminescence dosimeters (OSLDs) on goggles. The H_p(0.07) personal dose equivalent was measured over aprons with whole-body OSLDs. Data on patient dose as recorded by the kerma-area product (P_KA) were collected using an automatic dose management system. The 2 paediatric cardiologists working in the facility were involved in the study, and 222 interventions in a 1-year period were evaluated. The ceiling-suspended screen was often disregarded during interventions.ResultsThe annual eye lens doses estimated on goggles were 4.13 ± 0.93 and 4.98 ± 1.28 mSv. Over the aprons, the doses obtained were 10.83 ± 0.99 and 11.97 ± 1.44 mSv. The correlation between the goggles and the apron dose was R² = 0.89, with a ratio of 0.38. The correlation with the patient dose was R² = 0.40, with a ratio of 1.79 μSv Gy⁻¹ cm⁻². The dose per procedure obtained over the aprons was 102 ± 16 μSv, and on goggles 40 ± 9 μSv. The eye lens dose normalized to P_KA was 2.21 ± 0.58 μSv Gy⁻¹ cm⁻².ConclusionsMeasurements of personal dose equivalent over the paediatric cardiologist’s apron are useful to estimate eye lens dose levels if no radiation protection devices are typically used.     

Cell proliferation and survival in the mating circuit of adult male hamsters: Effects of testosterone and sexual behavior

The transient actions of gonadal steroids on the adult brain facilitate social behaviors, including reproduction. In male rodents, testosterone acts in the posterior medial amygdala (MeP) and medial preoptic area (MPOA) to promote mating. Adult neurogenesis occurs in both regions. The current study determined if testosterone and/or sexual behavior promote cell proliferation and survival in MeP and MPOA. Two experiments were conducted using the thymidine analog BrdU. First, gonad-intact and castrated male hamsters (n = 6/group) were compared 24 h or 7 weeks after BrdU. In MeP, testosterone-stimulated cell proliferation 24 h after BrdU (intact: 22.8 ± 3.9 cells/mm², castrate: 13.2 ± 1.4 cells/mm²). Testosterone did not promote cell proliferation in MPOA. Seven weeks after BrdU, cell survival was sparse in both regions (MeP: 2.5 ± 0.6 and MPOA: 1.7 ± 0.2 cells/mm²), and was not enhanced by testosterone. In Experiment 2, gonad-intact sexually-experienced animals were mated weekly to determine if regular neural activation enhances cell survival 7 weeks after BrdU in MeP and MPOA. Weekly mating failed to increase cell survival in MeP (8.1 ± 1.6 vs. 9.9 ± 3.2 cells/mm²) or MPOA (3.9 ± 0.7 vs. 3.4 ± 0.3 cells/mm²). Furthermore, mating at the time of BrdU injection did not stimulate cell proliferation in MeP (8.9 ± 1.7 vs. 8.1 ± 1.6 cells/mm²) or MPOA (3.6 ± 0.5 vs. 3.9 ± 0.7 cells/mm²). Taken together, our results demonstrate a limited capacity for neurogenesis in the mating circuitry. Specifically, cell proliferation in MeP and MPOA are differentially influenced by testosterone, and the birth and survival of new cells in either region are not enhanced by reproductive activity.     

Interleukin (IL)-15 in combination with IL-2, fms-like tyrosine kinase-3 ligand and anti-CD3 significantly enhances umbilical cord blood natural killer (NK) cell and NK-cell subset expansion and NK function

Background aimsInterleukin (IL)-15 and fms-like tyrosine kinase-3 (FLT-3) are crucial factors for the development of human and murine natural killer (NK) cells. Previously, we have demonstrated significant ex vivo expansion and activation of unrelated cord blood (UCB) NK cells with an antibody/cytokine cocktail consisting of anti-CD3 + IL-2 + IL-12 + IL-7 and anti-CD3 + IL-2 + IL-12 + IL-18.MethodsIn the current experiments, we investigated the effects of short-term culture with anti-CD3 + IL-2 + FLT-3 + IL-15 on cord blood (CB) NK cell and NK-cell subset expansion and function. CB mononuclear cells were cultured for 48 h in AIM-V media or AIM-V + IL-2 (5 ng/mL) + anti-CD3 (50 ng/mL) + FLT-3 (50 ng/mL) ± escalating doses of IL-15 (1, 10 or 100 ng/mL). Flow cytometric analysis was performed using various fluorescent-conjugated monoclonal antibodies. In vitro cytotoxicity was determined with a standard europium assay against K562 and Daudi cells.ResultsThere was a 4.8-fold significant increase in NK-cell population (CD3^?/16⁺/56⁺; P < 0.03), 21-fold significant increase in CD3^?/56⁺/158a⁺ (KIR2DL1/S1; P < 0.002), 46-fold significant increase in CD3^?/56⁺/158b⁺ (KIR2DL1/S2; P < 0.002) and 11.5-fold significant increase in CD3^?/56⁺/NKB1⁺ (KIR3DL1; P < 0.01). We also noted a significant increase in both NK and lymphokine-activated killer (LAK) cytotoxicity with IL-2 + anti-CD3 + FLT-3 + IL-15 (100 ng/mL) compared with IL-2 + anti-CD3 + FLT-3 and media alone against K562 (P < 0.01) and Daudi (P < 0.001), respectively.ConclusionsWe have demonstrated a significant increase in UCB NK cells and NK cells expressing a variety of killer immunoglobulin-like receptor (KIR) receptors after short-term culture with anti-CD3, IL-2, FLT-3 and IL-15. Furthermore, there was a significant increase in in vitro NK/LAK cell cytotoxicity.     

Minimally manipulated whole human umbilical cord is a rich source of clinical-grade human mesenchymal stromal cells expanded in human platelet lysate

Background aimsMesenchymal stromal cells (MSC) have recently been identified as a therapeutic option in several clinical conditions. Whereas bone marrow (BM) is considered the main source of MSC (BM-MSC), the invasive technique required for collection and the decline in allogeneic donations call for alternative sources. Human umbilical cord (UC) represents an easily available source of MSC (UC-MSC).MethodsSections of full-term UC were transferred to cell culture flasks and cultured in 5% human platelet lysate (PL)-enriched medium. Neither enzymatic digestion nor blood vessel removal was performed. After 2 weeks, the adherent cells were harvested (P1), replated at low density and expanded for two consecutive rounds (P2 and P3).ResultsWe isolated and expanded MSC from 9/9 UC. UC-MSC expanded with a mean fold increase (FI) of 42 735 ± 16 195 from P1 to P3 in a mean of 29 ± 2 days. By processing the entire cord unit, we theoretically could have reached a median of 9.5 × 10¹⁰ cells (ranging from 1.0 × 10¹⁰ to 29.0 × 10¹⁰). UC-MSC expressed standard surface markers; they contained more colony-forming unit (CFU)-fibroblast (F) and seemed less committed towards osteogenic, chondrogenic and adipogenic lineages than BM-MSC. They showed immunosuppressive properties both in vitro and in an in vivo chronic Graft versus Host disease (cGvHD) mouse model. Both array-Comparative Genomic Hybridization (CGH) analysis and karyotyping revealed no chromosome alterations at the end of the expansion. Animal studies revealed no tumorigenicity in vivo.ConclusionsUC constitute a convenient and very rich source of MSC for the production of third-party ‘clinical doses’ of cells under good manufacturing practice (GMP) conditions.     

Synthesis,radiosynthesis and first in vitro evaluation of novel PET-tracers for the dopamine transporter: [11C]IPCIT and [18F]FE@IPCIT

IntroductionPresent data indicate that merging beneficial structural elements from previously published DAT-ligands highest DAT affinity, selectivity and a suitable metabolic profile should be achieved. This combination led to the development of IPCIT and FE@IPCIT.MethodsPrecursor synthesis was done starting from cocaine in a six step reaction. O-[¹¹C]-methylation was established using [¹¹C]methyl iodide, optimized and subsequently automated. Small scale ¹⁸F-fluroroethylation as well as optimization of reaction parameters and automation were performed. Affinity and selectivity of the candidate substances were tested in standard binding experiments on human membranes. Metabolic stability and blood–brain-barrier (BBB) penetration were determined.ResultsPrecursor compound, IPCITacid, and reference compounds, IPCIT and FE@IPCIT, were obtained in 4.9%, 12.7% and 4.1% yield, respectively. Automated radiosynthesis of [¹¹C]IPCIT yielded 1.9 ± 0.7 GBq (12.5 ± 4%, corrected for decay). Optimum parameters for ¹⁸F-fluoroethylation were 110 °C for 15 min under TBAH catalysis, yielding 67 ± 16% radiochemical incorporation. Affinity was determined as 1.7 ± 0.6 nM for IPCIT, 1.3 ± 0.2 nM for FE@IPCIT and 37 ± 13 nM for the precursor molecule, IPCIT-acid. Results from in vitro and in silico evaluations revealed high stability but also high lipophilicity.ConclusionPresent data indicate high affinity and stability of both IPCIT and FE@IPCIT. Radiolabelling, optimization of reaction parameters and automation succeeded. On the other hand, data concerning BBB-penetration are not promising.     

Evaluation of mobilized peripheral stem cells according to CD34 and aldehyde dehydrogenase expression and effect of SSClo ALDHbr cells on hematopoietic recovery

Background aimsWe evaluated hematopoietic stem cells according to CD34 expression and aldehyde dehydrogenase (ALDH) activity in peripheral blood and apheresis product samples from patients after mobilization with granulocyte–colony-stimulating factor (G-CSF) alone or G-CSF after high-dose cyclophosphamide (4 g/m² once daily, intravenously on day 1). We also investigated the relationship between the number of SSC^lo CD45^dim CD34^hi cells, SSC^lo ALDH^br cells and engraftment.MethodsThirty patients (20 males and 10 females), who were candidates for autologous peripheral blood stem cell transplantation, were included in the study. Cyclophosphamide + G-CSF was used for 17 and G-CSF alone for 24 mobilizations. Primary diagnoses were multiple myeloma (n% = 14), Hodgkin's lymphoma (n% = 7), non-Hodgkin's lymphoma (n% = 2), acute myloid leukemia (n% = 2), chronic lymphocytic leukemia (n% = 1) and germ cell testis tumor (n% = 1).ResultsNumbers of SSC^lo CD45^dim CD34^hi cells and SSC^lo ALDH^br cells were highly correlated in both peripheral blood and apheresis products (P < 0.001). We could not find a relationship between the transplanted SSC^lo CD45^dim CD34^hi cell dose or SSC^lo ALDH^br cell dose and platelet or neutrophil recovery. The optimal thresholds for SSC^lo CD45^dim CD34^hi cells were 5.40 × 10⁶/kg for neutrophil recovery and 7.22 × 10⁶/kg for platelet recovery. The optimal thresholds for SSC^lo ALDH^br cells were 6.53 × 10⁶/kg for neutrophil recovery and 8.72 × 10⁶/kg platelet recovery.ConclusionsAccording to our data, numbers of SSC^lo ALDH^br cells are in very good agreement with numbers of SSC^lo CD45^dim CD34^hi cells and can be a predictor of stem cell mobilization.     

Cytotoxic effects of stem bark extracts and pure compounds from Margaritaria discoidea on human ovarian cancer cell lines

Margaritaria discoidea (Baill.) G. L. Webster (Euphorbiaceae) is a well-known medicinal plant in Africa used for the treatment of various diseases. So far, no cytotoxic effects of plant extracts on cancer cell lines have been reported.Aim of the studyTo evaluate the cytotoxicity against human ovarian cancer cells of extracts of M. discoidea and characterize the major bioactive compounds.MethodsBoth organic and aqueous extracts of this plant were obtained by maceration. The sulforhodamine B cell proliferation assay was used for evaluation of their cytotoxic activities and the potential bioactive compounds were characterized by gas chromatography–mass spectrometry.ResultsThe organic extract of M. discoidea showed stronger cytotoxicity than the aqueous extract with IC₅₀ values of 14.4 ± 3.0, 14.2 ± 1.2 and 34.7 ± 0.5 µg/ml on OVCAR-8, A2780 and cisplatin-resistant A2780cis ovarian cancer cells, respectively. The organic extract was further subjected to bioassay-guided fractionation by partitioning with n-hexane, ethyl acetate, and n-butanol in water. The ethyl acetate fraction was the most potent on the three ovarian cancer cell lines. A GC–MS analysis of trimethylsilyl derivatives of this fraction indicated the presence of phenolic compounds such as gallic acid and the alkaloid securinine. The IC₅₀ values of these two compounds were determined to be in the range of 3–16 µM, which indicated that they could contribute to the cytotoxic activity of the extract of M. discoidea.ConclusionsThis study has evaluated the cytotoxicity of stem bark extracts of M. discoidea against ovarian cancer cells and provided a basis of further development of this plant for the treatment of ovarian cancer.     

Chemical composition and free radical-scavenging,anticancer and anti-inflammatory activities of the essential oil from Ocimum kilimandscharicum

ObjectiveThe essential oil from the leaves of Ocimum kilimandscharicum (EOOK), collected in Dourados-MS, was investigated for anticancer, anti-inflammatory and antioxidant activity and chemical composition.Materials and methodsThe essential oil was extracted by hydrodistillation, and the chemical composition was performed by gas chromatography–mass spectrometry. The essential oil was evaluated for free radical-scavenging activity using the DPPH assay and was tested in an anticancer assay against ten human cancer cell lines. The response parameter (GI₅₀) was calculated for the cell lines tested. The anti-inflammatory activity was evaluated using carrageenan-induced pleurisy in mice.ResultsThe chemical composition showed 45 components with a predominance of monoterpenes, such as camphor (51.81%), 1,8 cineole (20.13%) and limonene (11.23%). The EOOK exhibited potent free radical-scavenging activity by the DPPH assay with a GI₅₀ of 8.31 μg/ml. The major constituents, pure camphor (IC₅₀ = 12.56 μg/ml) and mixture of the limonene: 1, 8 cineole (IC₅₀ = 23.25 μg/ml) displayed a potent activity. The oral administration of EOOK (at 30 and 100 mg kg⁻¹), as well as the pure camphor or a mixture of 1,8 cineole with limonene, significantly inhibited the carrageenan (Cg) induced pleurisy, reducing the migration of total leukocytes in mice by 82 ± 4% (30 mg kg⁻¹ of EOOK), 95 ± 4% (100 mg kg⁻¹ of EOOK), 83 ± 9% (camphor) and 80 ± 5% (mixture of 1,8 cineole:limonene 1:1). In vitro cytotoxicity screening against a human ovarian cancer cell line displayed high selectivity and potent anticancer activity with GI₅₀ = 31.90 mg ml⁻¹. This work describes the anti-inflammatory, anticancer and antioxidant effects of EOOK for the first time.ConclusionsThe essential oil exhibited marked anti-inflammatory, antioxidant and anticancer effects, an effect that can be attributed the presence of majorital compounds, and the response profiles from chemical composition differed from other oils collected in different locales.     

DNA binding and photocleavage properties and apoptosis-inducing activities of a ruthenium porphyrin complex [(Py-3′)TPP-Ru(phen)2Cl]Cl and its heterometallic derivatives

The interactions of a ruthenium porphyrin complex [(Py-3′)TPP-Ru(phen)₂Cl]Cl (phen = 1,10-phenanthroline, (Py-3′)TPP = 5-(3′-pyridyl-10,15,20-triphenylporphyrin) (1) and its heterometallic derivatives, [Ni(Py-3′)TPP-Ru(phen)₂Cl][PF₆] (2) and [Cu(Py-3′)TPP-Ru(phen)₂Cl][PF₆] (3), with calf thymus DNA have been investigated by spectroscopic and viscosity measurements in this study. The results showed that these synthetic complexes can bind to double strand helix DNA in groove binding mode, and the intrinsic binding constants of complexes 1, 2 and 3, as calculated according to the decay of the Soret absorption, are (1.35 ± 0.5) ×10⁵ M^?1 (s = 4.2), (1.29 ± 0.5) × 10⁵ M^?1 (s = 5.6) and (1.22 ± 0.5) × 10⁵ M^?1 (s = 6.2) (s is the binding-site size), respectively, which are consistent with those obtained from ethidium bromide-quenching experiments. Further investigations on the photocleavage properties of these complexes on plasmid pBR 322 DNA showed that complexes 1, 2 and 3 could cleave single chain DNA and convert DNA molecules from supercoiled form to the nicked form. As determined by MTT assay, the complexes were also identified as potent antiproliferative agents against A375 human melanoma cells, MCF-7 human breast adrenocarcinoma cells, Colo201 human colon adenocarcinoma cells and HepG2 human liver cancer cells. Complex 1 inhibits the growth of A375 cells through induction of apoptotic cell death and G0/G1 cell cycle arrest. Further investigation on intracellular mechanisms indicated that Complex 1 induced depletion of mitochondrial membrane potential (ΔΨ_m) in A375 cells through regulating the expression of pro-survival and pro-apoptotic Bcl-2 family members. Our results suggest that ruthenium porphyrin complexes could be candidates for further evaluation as chemopreventive and chemotherapeutic agents for human cancers.     

Expression of the Androgen Receptor,pAkt, and pPTEN in Breast Cancer and Their Potential in Prognostication

BACKGROUNDImportance of androgen receptor (AR) as an independent prognostic marker in Pakistani women with breast cancer (BCa) remains unexplored. Our aim was to identify the expression and potential prognostic value of AR, its upstream regulator (pAkt) and target gene (pPTEN) in invasive BCa.METHODSThis study used a cohort of 200 Pakistani women with invasive BCa diagnosed during 2002-2011. Expression of AR, pAkt and pPTEN was determined on formalin fixed paraffin embedded tissue sections by immunohistochemistry. The association of AR, pAkt and pPTEN with clinicopathological parameters was determined. Survival analyses were undertaken on patients with ≥ 5 years of follow-up (n = 82).RESULTSExpression of AR, pAkt and pPTEN was observed in 47.5%, 81.3% and 50.6% of patients, respectively. AR-expressing tumors were low or intermediate in grade (P < .001) and expressed ER (P = .002) and PR (P = .001). Patients with AR⁺ tumors had significantly higher OS (Mean OS = 10.2 ± 0.465 years) compared to patients with AR^? tumors (Mean OS = 5.8 ± 0.348 years) (P = .047). Furthermore, AR-positivity was associated with improved OS in patients receiving endocrine therapy (P = .020). Patients with AR⁺ /pAkt⁺ /pPTEN^? tumors, had increased OS (Mean OS = 7.1 ± 0.535 years) compared to patients with AR^?/pAkt⁺/pPTEN^? tumors (Mean OS = 5.1 ± 0.738 years).CONCLUSIONAR-expressing tumors are frequently characterized by low or intermediate grade tumors, expressing ER and PR. In addition, expression of AR, pAkt and pPTEN, could be considered in prognostication of patients with invasive BCa.     

Heart rate and arterial pressure variability and baroreflex sensitivity in ovariectomized spontaneously hypertensive rats

AimsThe present study evaluated the effects of ovariectomy on heart rate and arterial pressure variability and cardiac baroreflex sensitivity (BRS) in female spontaneously hypertensive (SHR) and Wistar–Kyoto rats (WKY).Main methodsSham-surgery animals were used as control. Sixteen weeks after ovariectomy or sham-surgery, animals were recorded. Time series of pulse interval (PI) and systolic AP (SAP) were analyzed by means of autoregressive spectral analysis, which quantifies the power of very low (VLF = 0.01–0.25 Hz), low (LF = 0.25–0.75 Hz) and high frequency (HF = 0.75–2.5 Hz) bands. BRS was assessed by means of linear regression between changes of PI and SAP induced by vasoactive drugs or calculation of α-index, a spontaneous BRS index.Key findingsThere was no difference in baseline PI or SAP between ovariectomized and sham SHR. Spectral analysis of heart rate variability suggested a shift of sympatho-vagal balance toward sympathetic predominance in ovariectomized SHR (LF/HF = 1.8 ± 0.2 versus 0.7 ± 0.2 in sham SHR, p < 0.05). Ovariectomy increased total variance and VLF power of SAP in SHR (29.1 ± 9.6 mmHg² and 18.6 ± 6.3 mmHg² versus 9.1 ± 2.1 mmHg² and 4.3 ± 1.4 mmHg², respectively, in sham SHR, p < 0.05). In addition, ovariectomy reduced reflex bradycardia in SHR (0.18 ± 0.03 ms/mmHg versus 0.34 ± 0.06 ms/mmHg in sham SHR, p < 0.05). Ovariectomy did not affect heart rate and SAP variability or BRS in WKY.SignificanceThese data showed that ovarian hormones deprivation induced marked changes on cardiovascular control, increasing SAP variability and cardiac sympatho-vagal balance and blunting BRS in female hypertensive animals, which reinforce the possible protective role of ovarian hormones on the cardiovascular system.