Antiproliferative effect of Solanum nigrum L. water extract on breast can-cer cells: potential roles of apoptosis and oxidative stress.

Breast cancer is the most progressive cancer among women worldwide. The currently available chemotherapeutic agents induce severe unacceptable adverse effects in breast cancer patients. In this context, natural medicinal herbs are gaining importance to find non-toxic effective anticancer drugs. Solanum nigrum is one of the major traditional medicinal plants widely used in Ayurveda for the treatment of various diseases. This study investigated the anticancer effect of Solanum nigrum water extract (SNWE) against MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines. SNWE significantly induced oxidative stress-mediated apoptotic cell death in a concentration-dependent manner. Real-time PCR results illustrated the upregulation of proapoptotic genes and downregulation of antiapoptotic genes after SNWE treatment in MCF-7 and MDA-MB-231 cell lines. Immunofluorescence analysis showed increased expressions of apoptotic markers like p53, Caspase3 and BAX by SNWE treatment. In conclusion, the findings of this study indicate the antiproliferative effect and apoptosis-inducing property of SNWE in both cell lines. Further studies are warranted on testing the anticancer activity of S. nigrum L. using animal models of cancer.

The Role of Mitochondrial Dysfunction in Cytotoxic Effects of Solanum nigrum Water Extract on MCF-7 and MDA-MB-231 Breast Cancer Cells.

BACKGROUND: Recent studies suggest that numerous naturally occurring agents have the potential to kill cancer cells via mitochondrial dysfunction. Solanum nigrum is a herb widely used in alternative medical systems. This study aimed to investigate the cytotoxic effect of Solanum nigrum water extract (SNWE) against Michigan Cancer Foundation-7 (MCF-7) and MD Anderson-Metastatic Breast Cancer-231 (MDA-MB-231) cells. METHODS: We used an MTT reduction assay for cytotoxicity analysis. To explore the mode of action, the cellular adenosine triphosphate (ATP) levels and mitochondrial membrane potential were analyzed using a colorimetric ATP assay and Rhodamine-123 fluorescent staining, respectively, during SNWE treatment for 72 h. RESULTS: The cytotoxic effect was significant in both cell lines, with IC50 values of 4.26 µg/mL and 5.30 µg/mL in MCF-7 and MDA-MB-231 cells, respectively. The 24, 48, and 72 h treatments of 100 µg/mL SNWE showed 0.85 ± 0.07, 0.38 ± 0.1, and 0.20 ± 0.1 nM ATP in MCF-7 cells and 0.94 ± 0.07, 0.84 ± 0.2 and 0.46 ± 0.2 nM in MDA-MB-231 cells, respectively. The SNWE treatment altered the mitochondrial membrane potential (ΔΨm) in a concentration-dependent manner in both the breast cancer cell lines, to 29.6 ± 4.1% in MCF-7 and 28.7 ± 4.17% in MDA-MB-231 cells, when compared with healthy mitochondria (100% ΔΨm). CONCLUSIONS: The cytotoxic effects of Solanum nigrum against breast cancer cells are associated with energy metabolism. Additional studies are warranted to test the anticancer effect of Solanum nigrum using an animal model of breast cancer.

2-(Benzhydryl sulfinyl)-N-sec-butylacetamide) isolated from fig augmented trastuzumab-triggered phagocytic killing of cancer cells through interface with Fcγ receptor.

The objective of the current study was to extract 2-(benzhydryl sulfinyl)-N-sec-butylacetamide), a novel compound from fig, and then determine its role in enhancing trastuzumab-triggered phagocytic killing of SKOV-3 cancer cells. In this study, Soxhlet was used to extract the compound from the mature and air-dried fig fruits. The production of the isolated extracts was enhanced by using polar and non-polar solvents. Several solvents, such as methanol, ethyl acetate, chloroform, and n-hexane, were used to isolate the effective compound 2-(benzhydryl sulfinyl)-N-sec-butylacetamide) from the organic layer. UV-spectroscopy, FT-IR, 1H-NMR, and 13C-NMR were applied to identify the purified compound. The in vitro and in vivo assays demonstrated that the 2-(benzhydryl sulfinyl)-N-sec-butylacetamide) can increase the activity of the phagocytic cells, via the interaction with FcY receptors, along with trastuzumab, and the pathway can use a model for the therapeutic strategy for effective treatment of ovarian cancer cells.

Phytochemical Profiling of Microalgae Euglena tuba and Its Anticancer Activity in Dalton's Lymphoma Cells.

INTRODUCTION: Natural phytochemicals are considered safe to use as therapeutic agents. There is a growing trend toward exploring anticancer effects of crude algal extracts or their active ingredients. Euglena tuba, a microalga, contains excellent antioxidant potential. However, the anticancer property of E. tuba has not been explored. This study investigates the chemical profiling as well as antitumor property of methanolic extract of E. tuba (ETME) against Dalton's lymphoma (DL) cells. MATERIALS AND METHODS: E. tuba, procured from northern part of India, was extracted in 70% methanol, dried at room temperature, and stored at -20 ∘C for future use. A freshly prepared aqueous solution of ETME of different concentrations was employed into each experiment. The ETME mediated anti-tumor response in Dalton's lymphoma was evaluated in the inbred populations of BALB/c (H2d) strain of mice of either sex at 8-12 weeks of age. The cytotoxicity of ETME in cancer cells, effects on morphology of cell and nucleus, alteration in the mitochondrial membrane potential, and level of expression of proapoptotic proteins (Bcl-2, cyt C, Bax and p53) were done using known procedures. RESULTS: The ETME contained high content of total alkaloids (96.02 ± 3.30 mg/100 mg), flavonoids (15.77 ± 2.38 mg/100 mg), carbohydrate (12.71 ± 0.59 mg/100 mg), ascorbic acid (12.48 ± 2.59 mg/100 mg), and phenolics (0.94 ± 0.05 mg/100 mg). Gas chromatography-mass spectrometry (GC-MS) analysis indicated the presence of 23 phytochemicals with known anticancer properties. DL cells treated with ETME exhibited significant and concentration dependent cytotoxicity. Florescent microscopy and flow cytometry of ETME treated DL cells indicated significant repair in cellular morphology and decreased mitochondrial potential, respectively. Western blot analysis displayed up-regulation of proapoptotic proteins (Bax, Cyt-c, p53) and down regulation of anti-apoptotic protein (Bcl2) in DL cells treated with ETME. CONCLUSIONS: The findings of this study clearly indicated that the anticancer property of ETME was mediated via reduction in mitochondrial potential and induction of apoptotic mechanism. Further studies are warranted to explore the anticancer activities of active ingredients present in this microalga of pharmaceutical importance.

Cellular antioxidant potential and inhibition of foodborne pathogens by a sesquiterpene ilimaquinone in cold storaged ground chicken and under temperature-abuse condition.

A sesquiterpene quinone, ilimaquinone, was accessed for its cellular antioxidant efficacy and possible antimicrobial mechanism of action against foodborne pathogens (Staphylococcus aureus and Escherichia coli) in vitro and in vivo. Ilimaquinone was found to be protective against H2O2-induced oxidative stress as validated by the reduction in the ROS levels, including increasing expression of SOD1 and SOD2 enzymes. Furthermore, ilimaquinone evoked MIC against S. aureus and E. coli within the range of 125-250 µg/mL. Ilimaquinone established its antimicrobial mode of action against both tested pathogens as evident by bacterial membrane depolarization, loss of nuclear genetic material, potassium ion, and release of extracellular ATP, as well as compromised membrane permeabilization and cellular component damage. Also, ilimaquinone showed no teratogenic effect against zebrafish, suggesting its nontoxic nature. Moreover, ilimaquinone significantly reduced the S. aureus count without affecting the sensory properties and color values of cold-storaged ground chicken meat even under temperature abuse condition.

Pro-Apoptotic and Immunotherapeutic Effects of Carbon Nanotubes Functionalized with Recombinant Human Surfactant Protein D on Leukemic Cells.

Nanoparticles are efficient drug delivery vehicles for targeting specific organs as well as systemic therapy for a range of diseases, including cancer. However, their interaction with the immune system offers an intriguing challenge. Due to the unique physico-chemical properties, carbon nanotubes (CNTs) are considered as nanocarriers of considerable interest in cancer diagnosis and therapy. CNTs, as a promising nanomaterial, are capable of both detecting as well as delivering drugs or small therapeutic molecules to tumour cells. In this study, we coupled a recombinant fragment of human surfactant protein D (rfhSP-D) with carboxymethyl-cellulose (CMC) CNTs (CMC-CNT, 10-20 nm diameter) for augmenting their apoptotic and immunotherapeutic properties using two leukemic cell lines. The cell viability of AML14.3D10 or K562 cancer cell lines was reduced when cultured with CMC-mwCNT-coupled-rfhSP-D (CNT + rfhSP-D) at 24 h. Increased levels of caspase 3, 7 and cleaved caspase 9 in CNT + rfhSP-D treated AML14.3D10 and K562 cells suggested an involvement of an intrinsic pathway of apoptosis. CNT + rfhSP-D treated leukemic cells also showed higher mRNA expression of p53 and cell cycle inhibitors (p21 and p27). This suggested a likely reduction in cdc2-cyclin B1, causing G2/M cell cycle arrest and p53-dependent apoptosis in AML14.3D10 cells, while p53-independent mechanisms appeared to be in operation in K562 cells. We suggest that CNT + rfhSP-D has therapeutic potential in targeting leukemic cells, irrespective of their p53 status, and thus, it is worth setting up pre-clinical trials in animal models.

A Rare Cause of Recurrent Lower Gastrointestinal Bleed: Colonic Dieulafoy's Lesion.

Dieulafoy's lesion accounts for 1%-2% of acute gastrointestinal (GI) bleeding cases, and approximately 2% of Dieulafoy's lesions are present in the colon. We report the case of an 83-year-old female who presented with recurrent gastrointestinal bleeding from colonic Dieulafoy's lesion located at the hepatic flexure. She initially presented four weeks prior with melena in the setting of Eliquis use for venous thrombosis, coronary artery disease, and end-stage renal disease. Upper endoscopy revealed esophagitis, gastritis, and duodenitis. Diagnostic colonoscopy and video capsule endoscopy both revealed blood in the colon without an identifiable source. During the second admission for recurrent melena with hemoglobin of 3.9 g/dL, Eliquis was discontinued, and the patient was resuscitated with three units of packed red blood cell transfusions. Repeat colonoscopy revealed a pulsating vessel with active oozing located at the hepatic flexure, consistent with a Dieulafoy's lesion. Hemostatic endoclips and bipolar electrocautery were applied to achieve complete hemostasis. Colonic Dieulafoy's lesions, albeit rare, should be considered in patients presenting with an acute obscure lower GI bleed. Primary hemostasis can be achieved with several endoscopic modalities including epinephrine, hemoclipping, thermocoagulation, or sclerotherapy.

Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells.

Human surfactant protein D (SP-D) belongs to the family of collectins that is composed of a characteristic amino-terminal collagenous region and a carboxy-terminal C-type lectin domain. Being present at the mucosal surfaces, SP-D acts as a potent innate immune molecule and offers protection against non-self and altered self, such as pathogens, allergens, and tumor. Here, we examined the effect of a recombinant fragment of human SP-D (rfhSP-D) on a range of breast cancer lines. Breast cancer has four molecular subtypes characterized by varied expressions of estrogen (ER), progesterone (PR), and epidermal growth factor (EGF) receptors (HER2). The cell viability of HER2-overexpressing (SKBR3) and triple-positive (BT474) breast cancer cell lines [but not of a triple-negative cell line (BT20)] was reduced following rfhSP-D treatment at 24 h. Upregulation of p21/p27 cell cycle inhibitors and p53 phosphorylation (Ser15) in rfhSP-D-treated BT474 and SKBR3 cell lines signified G2/M cell cycle arrest. Cleaved caspases 9 and 3 were detected in rfhSP-D-treated BT474 and SKBR3 cells, suggesting an involvement of the intrinsic apoptosis pathway. However, rfhSP-D-induced apoptosis was nullified in the presence of hyaluronic acid (HA) whose increased level in breast tumor microenvironment is associated with malignant tumor progression and invasion. rfhSP-D bound to solid-phase HA and promoted tumor cell proliferation. rfhSP-D-treated SKBR3 cells in the presence of HA showed decreased transcriptional levels of p53 when compared to cells treated with rfhSP-D only. Thus, HA appears to negate the anti-tumorigenic properties of rfhSP-D against HER2-overexpressing and triple-positive breast cancer cells.

Design of expert guided investigation of native L-asparaginase encapsulated long-acting cross-linker-free poly (lactic-co-glycolic) acid nanoformulation in an Ehrlich ascites tumor model.

Present study explores native L-asparaginase encapsulated long-acting cross-linker-free PLGA-nanoformulation in an Ehrlich ascites tumor model. L-asparaginase-PLGA nanoparticles for tumor were prepared using a double emulsion solvent evaporation technique, optimized and validated by Box-Behnken Design. L-ASN-PNs showed a particle size of 195 nm ± 0.2 nm and a PDI of 0.2. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) techniques revealed its smooth morphology and elicited an in-vitro release of 80% of the drug, following the Higuchi drug release model. In-vivo studies of L-ASN-PNs on an Ehrlich ascites tumor (EAT) model were completed and compared with the standard medication of 5-fluorouracil (5-FU) treatment. L-ASN-PN treated mice showed a 51.15% decrease in tumor volume and 100% survival rate with no reduction in body weight, no haemotoxicity and no hepatotoxicity, as evident from the hematological parameters, and liver enzyme parameters that were well within the prescribed limits. Chemotherapy has severe side effects and restricted therapeutic success. Henceforth, the purported L-Asparaginase PLGA nanoparticles are a suitable entity for better tumor regression, intra-tumor accumulation and no hematological side-effects.

Neuroprotective Effects of Dried Tubers of Aconitum napellus.

The present study was designed to explore the neuroprotective properties of Aconitum napellus (Ranunculaceae). The plant detoxification was done using either water, or cow or goat milk as per the Ayurvedic shodhana method. The evaluation of the neuroprotective role of A. napellus was performed on diabetic neuropathy induced by streptozotocin in Sprague Dawley (SD) rats. Body mass, blood sugar level, oral glucose tolerance test, hyperalgesia, cold allodynia, motor co-ordination test, and locomotor activity, oxidative biomarkers (TBARS, reduced glutathione, catalase and superoxide dismutase) and sciatic nerve histomorphology were assessed. The in vitro studies were done on human neuroblastoma cell line SHSY-5Y and used an MTT assay to assess the antiproliferative activity of different extracts. Results suggest that the goat milk treated chloroform extract has less percentage of aconitine. After administration of the detoxified chloroform extract to the diabetic animals, there was a significant improvement in the myelination and degenerative changes of the nerve fibers along with behavioral changes (p < 0.05 as compared with diabetic control group). The findings of the in vitro research show an effective neuroprotective role of A. napellus. This suggests that A. napellus should be further investigated for its effect in diabetic pathology.

Prognostic Value of Complement Properdin in Cancer.

The complement system is readily triggered by the presence of damage-associated molecular patterns on the surface of tumor cells. The complement alternative pathway provides rapid amplification of the molecular stress signal, leading to complement cascade activation to deal with pathogens or malignant cells. Properdin is the only known positive regulator of the alternative pathway. In addition, properdin promotes the phagocytic uptake of apoptotic T cells by macrophages and dendritic cells without activating the complement system, thus, establishing its ability to recognize "altered-self". Dysregulation of properdin has been implicated in substantial tissue damage in the host, and in some cases, chronic unresolved inflammation. A corollary of this may be the development of cancer. Hence, to establish a correlation between properdin presence/levels in normal and cancer tissues, we performed bioinformatics analysis, using Oncomine and UALCAN. Survival analyses were performed using UALCAN and PROGgeneV2 to assess if properdin can serve as a potential prognostic marker for human lung adenocarcinoma (LUAD), liver hepatocellular carcinoma (LIHC), cervical squamous cell carcinoma (CESC), and pancreatic adenocarcinoma (PAAD). We also analyzed levels of tumor-infiltrating immune cells using TIMER, a tool for characterizing immune cell composition in cancers. We found that in LUAD and LIHC, there was a lower expression of properdin in the tumors compared to normal tissues, while no significant difference was observed in CESC and PAAD. Survival analysis demonstrated a positive association between properdin mRNA expression and overall survival in all 4 types of cancers. TIMER analysis revealed that properdin expression correlated negatively with tumor purity and positively with levels of infiltrating B cells, cytotoxic CD8+ T cells, CD4+ helper T cells, macrophages, neutrophils and dendritic cells in LUAD, CESC and PAAD, and with levels of B cells, CD8+ T cells and dendritic cells in LIHC. Immunohistochemical analysis revealed that infiltrating immune cells were the most likely source of properdin in the tumor microenvironment. Thus, complement protein properdin shows promise as a prognostic marker in cancer and warrants further study.

Secretion of functionally active complement factor H related protein 5 (FHR5) by primary tumour cells derived from Glioblastoma Multiforme patients.

The complement system is an important humoral immune surveillance mechanism against tumours. However, many malignant tumours are resistant to complement mediated lysis. Here, we report secretion of complement factor H related protein 5 (FHR5) by primary tumour cells derived from Glioblastoma multiforme (GBM) patients. We investigated whether the secreted FHR5 exhibited functional activity similar to factor H, including inhibition of complement mediated lysis, acting as a co-factor for factor I mediated cleavage of C3b, and decay acceleration of C3 convertase. Immunoblotting analysis of primary GBM cells (B30, B31 and B33) supernatant showed the active secretion of FHR5, but not of Factor H. ELISA revealed that the secretion of soluble GBM-FHR5 by cultured GBM cells increased in a time-dependent manner. Primary GBM-FHR5 inhibited complement mediated lysis, possessed co-factor activity for factor I mediated cleavage and displayed decay acceleration of C3 convertase. In summary, we detected the secretion of FHR5 by primary GBM cells B30, B31 and B33. The results demonstrated that GBM-FHR5 shares biological function with FH as a mechanism primary GBM cells potentially use to resist complement mediated lysis.

Human SP-D Acts as an Innate Immune Surveillance Molecule Against Androgen-Responsive and Androgen-Resistant Prostate Cancer Cells.

Surfactant Protein D (SP-D), a pattern recognition innate immune molecule, has been implicated in the immune surveillance against cancer. A recent report showed an association of decreased SP-D expression in human prostate adenocarcinoma with an increased Gleason score and severity. In the present study, the SP-D expression was evaluated in primary prostate epithelial cells (PrEC) and prostate cancer cell lines. LNCaP, an androgen dependent prostate cancer cell line, exhibited significantly lower mRNA and protein levels of SP-D than PrEC and the androgen independent cell lines (PC3 and DU145). A recombinant fragment of human SP-D, rfhSP-D, showed a dose and time dependent binding to prostate cancer cells via its carbohydrate recognition domain. This study, for the first time, provides evidence of significant and specific cell death of tumor cells in rfhSP-D treated explants as well as primary tumor cells isolated from tissue biopsies of metatstatic prostate cancer patients. Viability of PrEC was not altered by rfhSP-D. Treated LNCaP (p53+/+) and PC3 (p53 -/-) cells exhibited reduced cell viability in a dose and time dependent manner and were arrested in G2/M and G1/G0 phase of the cell cycle, respectively. rfhSP-D treated LNCaP cells showed a significant upregulation of p53 whereas a significant downregulation of pAkt was observed in both PC3 and LNCaP cell lines. The rfhSP-D-induced apoptosis signaling cascade involved upregulation of Bax:Bcl2 ratio, cytochrome c and cleaved products of caspase 7. The study concludes that rfhSP-D induces apoptosis in prostate tumor explants as well as in androgen dependent and independent prostate cancer cells via p53 and pAkt pathways.

A compound chimeric antigen receptor strategy for targeting multiple myeloma.

Current clinical outcomes using chimeric-antigen receptors (CARs) against multiple myeloma show promise in the eradication of bulk disease. However, these anti-BCMA (CD269) CARs observe relapse as a common phenomenon after treatment due to the reemergence of either antigen-positive or -negative cells. Hence, the development of improvements in CAR design to target antigen loss and increase effector cell persistency represents a critical need. Here, we report on the anti-tumor activity of a CAR T-cell possessing two complete and independent CAR receptors against the multiple myeloma antigens BCMA and CS1. We determined that the resulting compound CAR (cCAR) T-cell possesses consistent, potent and directed cytotoxicity against each target antigen population. Using multiple mouse models of myeloma and mixed cell populations, we are further able to show superior in vivo survival by directed cytotoxicity against multiple populations compared to a single-expressing CAR T-cell. These findings indicate that compound targeting of BCMA and CS1 on myeloma cells can potentially be an effective strategy for augmenting the response against myeloma bulk disease and for initiation of broader coverage CAR therapy.

Effect of different concentrations of soybean lecithin and virgin coconut oil in Tris-based extender on the quality of chilled and frozen-thawed bull semen.

AIM: The objective of this study was to evaluate the effects of different concentrations of soybean lecithin (SL) and virgin coconut oil (VCO) in Tris-based extender on chilled and frozen-thawed bull semen quality parameters. MATERIALS AND METHODS: A total of 24 ejaculates were collected from four bulls via an electroejaculator. Semen samples were diluted with 2% VCO in Tris-based extender which consists of various concentrations of SL (1, 1.25, 1.5, and 1.75%). A 20% egg yolk in Tris used as a positive control (C+). The diluted semen samples were divided into two fractions; one for chilling which were stored at 4°C for 24, 72, and 144 h before evaluated for semen quality parameters. The second fraction used for freezing was chilled for 3 h at 4°C, packed into 0.25 mL straws and then cryopreserved in liquid nitrogen. The samples were then evaluated after 7 and 14 days. Chilled and frozen semen samples were thawed at 37°C and assessed for general motility using computer-assisted semen analysis, viability, acrosome integrity and morphology (eosin-nigrosin stain), membrane integrity, and lipid peroxidation using thiobarbituric acid reaction test. RESULTS: The results showed that all the quality parameters assessed were significantly (p<0.05) improved at 1.5% SL concentration in chilled semen. Treatment groups of 1, 1.25, 1.5, and 1.75% SL were higher in quality parameters than the control group (C+) in chilled semen. However, all the quality parameters in frozen-thawed semen were significantly higher in the C+ than the treated groups. CONCLUSION: In conclusion, supplementation of 1.5% SL in 2% VCO Tris-based extender enhanced the chilled bull semen. However, there was no marked improvement in the frozen-thawed quality parameters after treatment.

Effect of different concentrations of egg yolk and virgin coconut oil in Tris-based extenders on chilled and frozen-thawed bull semen.

The aim of this study was to evaluate the effects of 8% virgin coconut oil (VCO) combined with different percentages of egg yolk in Tris extender on the quality of chilled and frozen-thawed bull semen. A total of 24 ejaculates from four bulls were collected using an electroejaculator. Semen samples were diluted with 8% VCO in Tris extender which contained different concentrations 0% (control), 4%, 8%, 12%, 16% and 20% egg yolk. The diluted semen samples were divided into two fractions: one was chilled and stored at 4°C until evaluation after 24, 72, and 144h; the second fraction was processed by chilling for 3h at 4°C to equilibrate, then packaged in 0.25ml straws and frozen and stored in liquid nitrogen at -196°C until evaluation after 7 and 14 days. Both chilled and frozen semen samples were then thawed at 37°C and assessed for general motility using computer-assisted semen analysis (CASA), viability, acrosome integrity, and morphology (eosin-nigrosin), membrane integrity (hypo-osmotic swelling test) and lipid peroxidation (thiobarbituric acid-reactive substances (TBARS)). The results indicate treatments with 8%, 12%, 16% and 20% egg yolk with 8% VCO had greater sperm quality (P<0.05) as compared with the control. The treatment with 20% egg yolk had the greatest sperm quality (P<0.05) among the treated groups for both chilled and frozen-thawed semen. In conclusion, the use of 8% VCO combined with 20% egg yolk in a Tris-based extender enhanced the values for chilled and frozen-thawed quality variables of bull sperm.

A phase 1b/2b multicenter study of oral panobinostat plus azacitidine in adults with MDS, CMML or AML with ⩽30% blasts.

Treatment with azacitidine (AZA), a demethylating agent, prolonged overall survival (OS) vs conventional care in patients with higher-risk myelodysplastic syndromes (MDS). As median survival with monotherapy is <2 years, novel agents are needed to improve outcomes. This phase 1b/2b trial (n=113) was designed to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D) of panobinostat (PAN)+AZA (phase 1b) and evaluate the early efficacy and safety of PAN+AZA vs AZA monotherapy (phase 2b) in patients with higher-risk MDS, chronic myelomonocytic leukemia or oligoblastic acute myeloid leukemia with <30% blasts. The MTD was not reached; the RP2D was PAN 30 mg plus AZA 75 mg/m2. More patients receiving PAN+AZA achieved a composite complete response ([CR)+morphologic CR with incomplete blood count+bone marrow CR (27.5% (95% CI, 14.6-43.9%)) vs AZA (14.3% (5.4-28.5%)). However, no significant difference was observed in the 1-year OS rate (PAN+AZA, 60% (50-80%); AZA, 70% (50-80%)) or time to progression (PAN+AZA, 70% (40-90%); AZA, 70% (40-80%)). More grade 3/4 adverse events (97.4 vs 81.0%) and on-treatment deaths (13.2 vs 4.8%) occurred with PAN+AZA. Further dose or schedule optimization may improve the risk/benefit profile of this regimen.

Preclinical targeting of aggressive T-cell malignancies using anti-CD5 chimeric antigen receptor.

The outlook for T-cell malignancies remain poor due to the lack of effective therapeutic options. Chimeric antigen receptor (CAR) immunotherapy has recently shown promise in clinical trials for B-cell malignancies, however, designing CARs for T-cell based disease remain a challenge due to the shared surface antigen pool between normal and malignant T-cells. Normal T-cells express CD5 but NK (natural killer) cells do not, positioning NK cells as attractive cytotoxicity cells for CD5CAR design. Additionally, CD5 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas (PTCLs). Here, we report a robust anti-CD5 CAR (CD5CAR) transduced into a human NK cell line NK-92 that can undergo stable expansion ex vivo. We found that CD5CAR NK-92 cells possessed consistent, specific, and potent anti-tumor activity against a variety of T-cell leukemia and lymphoma cell lines as well as primary tumor cells. Furthermore, we were able to demonstrate significant inhibition and control of disease progression in xenograft mouse models of T-ALL. The data suggest that CAR redirected targeting for T-cell malignancies using NK cells may be a viable method for new and complementary therapeutic approaches that could improve the current outcome for patients.

Fevipiprant, a prostaglandin D2 receptor 2 antagonist, in patients with persistent eosinophilic asthma: a single-centre, randomised, double-blind, parallel-group, placebo-controlled trial.

BACKGROUND: Eosinophilic airway inflammation is often present in asthma, and reduction of such inflammation results in improved clinical outcomes. We hypothesised that fevipiprant (QAW039), an antagonist of prostaglandin D2 receptor 2, might reduce eosinophilic airway inflammation in patients with moderate-to-severe eosinophilic asthma. METHODS: We performed a single-centre, randomised, double-blind, parallel-group, placebo-controlled trial at Glenfield Hospital (Leicester, UK). We recruited patients with persistent, moderate-to-severe asthma and an elevated sputum eosinophil count (≥2%). After a 2-week single-blind placebo run-in period, patients were randomly assigned (1:1) by the trial pharmacist, using previously generated treatment allocation cards, to receive fevipiprant (225 mg twice per day orally) or placebo, stratified by the use of oral corticosteroid treatment and bronchoscopy. The 12-week treatment period was followed by a 6-week single-blind placebo washout period. The primary outcome was the change in sputum eosinophil percentage from baseline to 12 weeks after treatment, analysed in the intention-to-treat population. All patients who received at least one dose of study drug were included in the safety analyses. This trial is registered with ClinicalTrials.gov, number NCT01545726, and with EudraCT, number 2011-004966-13. FINDINGS: Between Feb 10, 2012, and Jan 30, 2013, 61 patients were randomly assigned to receive fevipiprant (n=30) or placebo (n=31). Three patients in the fevipiprant group and four patients in the placebo group withdrew because of asthma exacerbations. Two patients in the fevipiprant group were incorrectly given placebo (one at the mid-treatment visit and one throughout the course of the study). They were both included in the fevipiprant group for the primary analysis, but the patient who was incorrectly given placebo throughout was included in the placebo group for the safety analyses. Between baseline and 12 weeks after treatment, sputum eosinophil percentage decreased from a geometric mean of 5·4% (95% CI 3·1-9·6) to 1·1% (0·7-1·9) in the fevipiprant group and from 4·6% (2·5-8·7) to 3·9% (CI 2·3-6·7) in the placebo group. Compared with baseline, mean sputum eosinophil percentage was reduced by 4·5 times in the fevipiprant group and by 1·3 times in the placebo group (difference between groups 3·5 times, 95% CI 1·7-7·0; p=0·0014). Fevipiprant had a favourable safety profile, with no deaths or serious adverse events reported. No patient withdrawals were judged by the investigator to be related to the study drug. INTERPRETATION: Fevipiprant reduces eosinophilic airway inflammation and is well tolerated in patients with persistent moderate-to-severe asthma and raised sputum eosinophil counts despite inhaled corticosteroid treatment. FUNDING: Novartis Pharmaceuticals, AirPROM project, and the UK National Institute for Health Research.

Comparative evaluation of immunohistochemistry and real-time PCR for measuring proinflammatory cytokines gene expression in livers of rats treated with gold nanoparticles.

Gold nanoparticles (GNPs) possess promising applications in targeted drug delivery and controlled release of a variety of chemical agents. However, the immunocompatibility of GNPs is poorly understood. After exposure, GNPs preferentially tend to accumulate is liver, where they induce an acute phase proinflammatory response. We therefore compared the two techniques, immunohistochemistry and real-time PCR for measuring the protein and mRNA expressions of IL-1ß, IL-6 and TNF-α in liver of rats after intraperitoneal injections (5µg/animal) of 10 and 50nm diameter GNPs for 1 and 5days. The results showed that both 10nm and 50nm GNPs induced an acute phase expression of proinflammatory cytokines that receded on day 5. The proinflammatory response on day 1 was comparatively more severe with 50nm GNPs than 10nm GNPs. A comparative evaluation between immunostaining and real-time PCR showed that the latter technique is more sensitive as it could detect the cytokines mRNA expression in control samples as well. This could be partly attributed to the amplification strategy used in real-time PCR and partly to the variations in the half lives of cytokines mRNA and their resulting proteins.