RNA-based therapy for Cryptosporidium parvum infection: proof-of-concept studies

Cryptosporidium is a leading cause of moderate-to-severe diarrhea in children. Nitazoxanide, the only FDA-approved treatment for cryptosporidiosis, has limited efficacy in those at highest risk for sequelae. RNA-argonaute (Ago) complexes to Cryptosporidium nucleoside diphosphate kinase (cpNDK) decreased the Cryptosporidium parvum mRNA by 95% in infected cells in vitro. Treatment of mice by oral gavage with ssRNA/Ago complexes encapsulated in lipid nanoparticles led to delivery of the complexes into intestinal epithelial cells. Treatment of C. parvum infected mice with ssRNA/Ago complexes targeting cpNDK led to the resolution of oocyst shedding in 4/5 SCID/beige mice. These results confirm the potential use of antisense therapy as an alternative approach to cryptosporidiosis treatment.

Laboratory Characterization of Unclassified Bleeding Disorders By Non-Conventional Tests

Introduction: Hematologists frequently face a percentage of patients with a mild bleeding tendency due to a haemostatic abnormality that cannot be identified with conventional laboratory techniques. Such patients are termed as having an unclassified bleeding disorder (UBD). A good diagnosis is important in order to prevent bleedings during invasive processes and/or childbirth by choosing the optimal therapeutic treatment. We aimed to investigate hemostatic parameters that may be altered in patients with UBD in order to determine the cause of their bleeding symptoms. In particular, possible defects in the tissue factor (TF)-mediated regulation of coagulation or in the plasmin generation during the fibrinolysis, as well as the possible beneficial effects of treatment with antibodies blockers of TFPI. Methods: This is a single-centre, case-control, non-interventionist, prospective study. During an 8 months-period, 40 patients with bleeding symptoms (evaluated with ISTH-BAT score) were studied. Routine coagulation tests (aPTT and PT) and platelet function testing [aggregometry, PFA-100, flow cytometry and Total Thrombus-formation Analysis System (T-TAS; Zarcos, Japan)] were performed. In 17 patients, no abnormalities were detected in platelet function and/or in coagulation tests; so the following procedures were performed: Thrombin generation test by Calibrated automated thrombography (CAT) in samples of platelet poor plasma with corn trypsin inhibitor (CTI), an inhibitor of contact activation phase, using a low amount of TF (1 pM TF and 4 µM phospholipids) as a trigger to allow the evaluation of the TF-dependent pathway. Plasmin generation (PG) test with a kit from Synapse Research Institute (Maastricht, The Netherlands), using Thrombinoscope software. TFPI activity in plasma, measured with ACTICHROME® TFPI kit (Biomedica Diagnostics, USA). The effects of rFVIIa (Novoseven, NovoNordisk; 90 µg/kg) and of a human Anti-TFPI recombinant Ab (clon mAb2021, Creative Biolabs; 400 ng/ml) were tested in CAT, PG and TFPI activity tests. Results: Those patients with aPTT, PT and a platelet function within normal range were further studied performing thrombin generation, plasmin generation and TFPI activity tests. Table 1 shows the results obtained. Samples from patients 1, 2, 4, 7, 8, 9 and 10 had a diminished generation of thrombin, and in vitro treatment with anti-TFPI and rFVIIa only ameliorated thrombin generation in samples from patients 4, 7, 8 and 9. Plasma from patients 8 and 10 had increased activity of TFPI. Generation of thrombin in samples from patients 3, 5, 6 and 11 was within normal range. Plasmin generation was increased and not modified by in vitro treatment with anti-TFPI and rFVIIa in samples 3 and 11; whereas samples 5 (with normal plasmin generation) and 6 (with no data of plasmin generation due to lack of enough sample) had a high TFPI activity in plasma that was inhibited by anti-TFPI. Normal values in all these parameters evaluated were found in six patients, indicating the involvement of different mechanisms that are still unknown. Conclusions: UBD have a diverse pathological basis for the bleeding. So, a single laboratory test to make a correct diagnosis of this pathology cannot be recommended. In accordance with this fact, a personalized treatment should be applied for each patient. Non-conventional laboratory tests need to be standardized and included for studying possible defects in the regulation of TF and/or plasmin pathways that can be involved in very rare mild bleeding phenotypes. TFPI inhibition might emerge as a good therapy for some of these patients. Failure to detect the bleeding cause in some of these patients, suggests the need to perform further studies in this field. This work was supported by Novo Nordisk Pharma S.A. Table 1- Thrombin and plasmin generation and TFPI activity in samples of patients with UBD. Results out of normal range are shown in red. LT: lagtime; ETP: endogenous thrombin potential; EPP: endogenous plasmin potential; TFPI: Tissue factor pathway inhibitor. Figure 1 Figure 1. Disclosures Alvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Martín: Novo Nordisk: Speakers Bureau; Pfizer: Speakers Bureau. Jiménez-Yuste: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Canales: Eusa Pharma: Consultancy, Honoraria; Sandoz: Honoraria, Speakers Bureau; Sanofi: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Consultancy; Gilead/Kite: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

The Relevance of SOCS1 Methylation and Epigenetic Therapy in Diverse Cell Populations of Hepatocellular Carcinoma

The suppressor of cytokine signaling 1 (SOCS1) is a tumor suppressor gene found to be hypermethylated in cancers. It is involved in the oncogenic transformation of cirrhotic liver tissues. Here, we investigated the clinical relevance of SOCS1 methylation and modulation upon epigenetic therapy in diverse cellular populations of hepatocellular carcinoma (HCC). HCC clinical specimens were evaluated for SOCS1 methylation and mRNA expression. The effect of 5-Azacytidine (5-AZA), a demethylation agent, was assessed in different subtypes of HCC cells. We demonstrated that the presence of SOCS1 methylation was significantly higher in HCC compared to peri-HCC and non-tumoral tissues (52% vs. 13% vs. 14%, respectively, p < 0.001). In vitro treatment with a non-toxic concentration of 5-AZA significantly reduced DNMT1 protein expression for stromal subtype lines (83%, 73%, and 79%, for HLE, HLF, and JHH6, respectively, p < 0.01) compared to cancer stem cell (CSC) lines (17% and 10%, for HepG2 and Huh7, respectively), with the strongest reduction in non-tumoral IHH cells (93%, p < 0.001). 5-AZA modulated the SOCS1 expression in different extents among the cells. It was restored in CSC HCC HepG2 and Huh7 more efficiently than sorafenib. This study indicated the relevance of SOCS1 methylation in HCC and how cellular heterogeneity influences the response to epigenetic therapy.

Biocontrol Delivery System of Trichoderma harzianum Formulated in Graphite and Silica Nano Particles to Control Rhizoctonia solani on Tomato Plants

This paper reports the performance of a graphite and silica nanoparticles-based delivery system for T. harzianum in controlling the in vitro growth of R. solani and damping-off disease on tomato plants. The in vitro and in vivo experiments were arranged in the randomized complete block design. The in vitro treatment was a dual culture of R. solani and T. harzianum in the various components of formulation on PDA, i.e., T. harzianum + 5 wt.% graphite, T. harzianum + 1wt.% silica NPs., T. harzianum + 5 wt.% graphite + 1 wt.% silica nanoparticles, T. harzianum, 5 wt.% graphite, 1 wt.% silica nanoparticles, fungicide (mancozeb), and a control. The in vivo treatment included the application of T. harzianum in the same compositions as the in vitro treatment, except that there were two controls i.e., inoculated and noninoculated tomato plants with R. solani. T. harzianum by soaking tomato seeds in the formulation suspensions before planting. The results showed that all formulation compositions were able to inhibit the in vitro growth of R. solani. The inhibitions of the colony growth of R. solani caused by formulated and non-formulated T. harzianum were the same. This proved that graphite and silica NPs did not resist to the ability of T. harzianum in controlling R. solani, indicated that the formulation was promising to develop. However, the inhibition of damping-off disease incidence on tomato plants caused by formulated T. harzianum was the same as the non-formulated one only on day 7 after treatments. On days 14, 21, and 28, the inhibitions were lower than the non-formulated ones. It was suggested to reapply the formulation of T. harzianum in the soil at planting and several days after.

Therapeutic Effects of The As-Synthesized Polylactic Acid/Chitosan Nanofibers Decorated With Amphotricin B For In Vitro Treatment of Leishmaniasis

Leishmaniasis as a third most important vector-borne disease caused by different species of a Leishmania flagellum protozoan. Leishmaniasis as a one of the biggest concerns of the World Health Organization due to the severe side effects, the emergence of resistance, secondary bacterial infections, reports as epidemics especially in immunocompromised individuals. The main purpose of this study was synthesis and characterization of the polylactic acid/chitosan nanofibers decorated with amphotricin B as a new delivery system for in vitro treatment of Leishmania wounds. The prepared nanofibers were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), Dynamic light scattering (DLS), X-ray diffraction (XRD) and in vitro drug release test. The anti leishmaniasis effect of nanofibers decorated with amphotricin B against Leishmania major promastigotes and its cytotoxicity on macrophages were determined respectively with flow cytometry test. The in-vitro drug release assay indicates 84% of the amphotricin B loaded in nanofibers after 400 minutes. The average concentration of the amphotricin B loaded in polylactic acid/chitosan nanofibers and conventional form of amphotricin B that prevented the growth of 50% of the promastigotes of L.major was 1.29 and 4.34 µg/ml, respectively.

Ovarian Cancer-Associated Ascites Have High Proportions of Cytokine-Responsive CD56bright NK Cells

Ovarian cancer is the most lethal gynecological malignancy, with serous histotype as the most prevalent epithelial ovarian cancer (EOC). Peritoneal ascites is a frequent comorbidity in advanced EOC. EOC-associated ascites provide a reliable sampling source for studying lymphocytes directly from tumor environment. Herein, we carried out flow cytometry-based analysis to readdress issues on NK and T lymphocyte subsets in women with advanced EOC, additionally evaluating phenotypic modulation of their intracellular pathways involved in interleukin (IL)-2 and IL-15 signaling. Results depicted ascites as an inflammatory and immunosuppressive environment, presenting significantly (p < 0.0001) higher amounts of IL-6 and IL-10 than in the patients’ blood, as well as significantly (p < 0.05) increased expression of checkpoint inhibitory receptors (programmed death protein-1, PD-1) and ectonucleotidase (CD39) on T lymphocytes. However, NK lymphocytes from EOC-associated ascites showed higher (p < 0.05) pS6 phosphorylation compared with NK from blood. Additionally, in vitro treatment of lymphocytes with IL-2 or IL-15 elicited significantly (p < 0.001) phosphorylation of the STAT5 protein in NK, CD3 and CD8 lymphocytes, both from blood and ascites. EOC-associated ascites had a significantly (p < 0.0001) higher proportion of NK CD56bright lymphocytes than blood, which, in addition, were more responsive (p < 0.05) to stimulation by IL-2 than CD56dim NK. EOC-associated ascites allow studies on lymphocyte phenotype modulation in the tumor environment, where inflammatory profile contrasts with the presence of immunosuppressive elements and development of cellular self-regulating mechanisms.

lncRNA GAU1 Induces GALNT8 Overexpression and Potentiates Colorectal Cancer Progression

lncRNA is a key epigenetic regulator in biological processes. In the human cancer transcriptome library MiTranscriptome, we identified GAU1 as the top upregulated lncRNA in colorectal cancer (CRC) by sample set enrichment analysis (overexpression ranking percentile = 99.75 %, P < 10 − 50 ), which is coexpressed with the potential oncogene GALNT8 (Spearman rho = 0.67 , P = 2.44 × 10 − 23 , TCGA dataset n = 184 ). Experimental data revealed that GAU1 regulates the expression of GALNT8. The overexpression of either GAU1 or GALNT8 significantly promotes the cell cycle and proliferation of CRC cell lines and correlates with poor prognosis in patients with CRC ( P = 3.04 × 10 − 2 ), while silencing of GAU1 or GALNT8 suppressed the cancer cell proliferation and induced the CRC cell line resistance to oxaliplatin in vitro treatment. Our results suggested that the previously less studied GAU1 and GALNT8 may play as CRC prognosis markers and potential targets for chemotherapy treatment.