Isolation and Mass Production of Entomopathogenic Nematode Steinernema saimkayai using Semi Solid Dog Feed Medium

Entomopathogenic nematodes (EPNs) were collected from soil using Galleria mellonella as bait and propagated on Wouts medium as well as new semi-solid feed developed from dog feed. The EPN was isolated from the cucumber soil from Kadayar, Thadiyoor, Kerala for the Mass production. The strains collected were identified as Steinernema siamkayai KUT1 and the symbiont bacteria identified as Xenorhabdus stockiae using advanced DNA sequencing method. Nematodes were grown on two different sets of medium viz., Wouts medium on sponge and Semi-solid dog feed in T-flasks. The Steinernema siamkayai KUT1 grown increasingly in semi-solid dog feed medium than other medium. This research suggests that EPN can be used to for the inhibition of malarial parasites in open stagnant water bodies. Breeding grounds for this species could be controlled efficiently using EPNs. Our research concludes the use of newly modified semi solid Dog feed medium containing essential nutrients can be safe and cheap enough for the mass production of EPN

Development and validation of a flow cytometry method to examine circulating leukocyte subpopulations in barramundi (Lates calcarifer)

Bacterial infections are a major contributor to losses in aquaculture production. Diagnosis of these bacterial diseases is based on sacrificing fish and isolating the infectious agent from the internal organs. Infections lead to an inflammatory response in the host that is characterized by changes in leukocyte profile and activity. We hypothesized that the profile of circulating peripheral blood leukocytes and markers of their activity, such as reactive oxygen species production, would change following an event of bacterial infection in fish. We therefore developed a flow cytometry-based methodology for analyzing blood leukocytes in barramundi (Lates calcarifer), then used it to compare healthy fish to those infected with a common pathogenic bacterium, Vibrio harveyi. The initial step of the analysis relied on the effective separation of erythrocytes and leukocytes, and two methods were tested: one based on hypotonic lysis of red blood cells, and the other involving stain-based differentiation using carboxyfluorescein succinimidyl ester (CFSE). Fluorescence-activated cell sorting analysis revealed significant differences in leukocyte parameters of infected and control fish using the stain-based approach vs. hypotonic lysis. Changes in blood leukocytes included an increase in the proportion of monocytes and decrease in that of lymphocytes in infected fish. Furthermore, cell size and granularity of the different leukocyte populations changed over time in infected fish as compared to controls. Monitoring peripheral blood leukocytes provides a non-lethal analysis of inflammation that allows repeated sampling of individual fish over time. This is beneficial given the high variability between individual fish. The use of CFSE-staining for leukocyte isolation proved to be advantageous over a method based on lysis of erythrocytes

Tumor Cell-Associated IL-1α Affects Breast Cancer Progression and Metastasis in Mice through Manipulation of the Tumor Immune Microenvironment

IL-1α is a dual function cytokine that affects inflammatory and immune responses and plays a pivotal role in cancer. The effects of intracellular IL-1α on the development of triple negative breast cancer (TNBC) in mice were assessed using the CRISPR/Cas9 system to suppress IL-1α expression in 4T1 breast cancer cells. Knockout of IL-1α in 4T1 cells modified expression of multiple genes, including downregulation of cytokines and chemokines involved in the recruitment of tumor-associated pro-inflammatory cells. Orthotopical injection of IL-1α knockout (KO) 4T1 cells into BALB/c mice led to a significant decrease in local tumor growth and lung metastases, compared to injection of wild-type 4T1 (4T1/WT) cells. Neutrophils and myeloid-derived suppressor cells were abundant in tumors developing after injection of 4T1/WT cells, whereas more antigen-presenting cells were observed in the tumor microenvironment after injection of IL-1α KO 4T1 cells. This switch correlated with increased infiltration of CD3+CD8+ and NKp46+cells. Engraftment of IL-1α knockout 4T1 cells into immunodeficient NOD.SCID mice resulted in more rapid tumor growth, with increased lung metastasis in comparison to engraftment of 4T1/WT cells. Our results suggest that tumor-associated IL-1α is involved in TNBC progression in mice by modulating the interplay between immunosuppressive pro-inflammatory cells vs. antigen-presenting and cytotoxic cells