Diagnostics for Rift Valley fever virus

Master of ScienceDepartment of Diagnostic Medicine/PathobiologyA. Sally DavisRift Valley fever virus (RVFV) is a mosquito-borne, zoonotic Phlebovirus that is a significant threat to ruminants and humans. RVFV is categorized as an overlap Select Agent by the Department of Health and Human Services and US Department of Agriculture. Therefore, the study of RVFV’s pathogenesis and the development of novel diagnostic tools for the prevention and control of outbreaks and virus spread is crucial. RVF is endemic to sub-Saharan Africa but has spread beyond the continent to the Arabian Peninsula indicating the competence of the virus to emerge in new areas. Thus, the high likelihood of RVF’s spread to other non- endemic countries also spurs the need for development and implementation of rapid diagnostic tests and surveillance programs. In the US, RVFV is a Select Agent, requiring BSL-3 enhanced containment practices for research work. First, we developed a method for the detection of RVFV RNA by reverse transcriptase real-time PCR (RT-qPCR) using non-infectious, formalin- fixed, paraffin-embedded tissues (FFPET). The results from FFPET RT-qPCR were compared to prior results for fresh-frozen tissues (FFT) RT-qPCR, as well as immunohistochemistry and histopathology completed on the same FFPET blocks. We developed a novel technique using a rapid and low cost magnetic bead extraction method for recovery of amplifiable RVFV RNA from FFPET. FFPET RT-qPCR can serve as an alternative tissue-based diagnostic test, which does not require a BSL-3 research facility. Second, we assessed the diagnostic accuracy and precision of a recombinant RVFV nucleoprotein based competitive ELISA (cELISA) assay to detect RVFV antibodies. The cELISA results were compared to the virus neutralization test, the gold standard serological assay for RVFV. This prototype cELISA is easy to implement, sensitive, specific, and safe test for the detection of antibodies to RVFV in diagnostic and surveillance applications. RVF is an important transboundary disease that should be monitored on a regular basis. The diagnostic tests developed and validated in this thesis could be used in endemic or non-endemic countries for the early detection of RVF and assist with the implementation of countermeasures against RVFV

Rift Valley fever viral RNA detection by in situ hybridization in formalin-fixed, paraffin-embedded tissues

Sporadic outbreaks of Rift Valley fever virus (RVFV), a zoonotic, mosquito-borne Phlebovirus, cause abortion storms and death in sheep and cattle resulting in catastrophic economic impacts in endemic regions of Africa. More recently, with changes in competent vector distribution, growing international trade, and its potential use for bioterrorism, RVFV has become a transboundary animal disease of significant concern. New and sensitive techniques that determine RVFV presence, while lessening the potential for environmental contamination and human risk, through the use of inactivated, noninfectious samples such as formalin-fixed, paraffin-embedded (FFPE) tissues are needed. FFPE tissue in situ hybridization (ISH) enables the detection of nucleic acid sequences within the visual context of cellular and tissue morphology. Here, we present a chromogenic pan-RVFV ISH assay based on RNAscope® technology, which is able to detect multiple RVFV strains in FFPE tissues, enabling visual correlation of RVFV RNA presence with histopathologic lesions.This work was done in partial completion of Izabela Ragan’s PhD dissertation.The USDA Agricultural Research Service and the Department of Diagnostic Medicine, College of Veterinary Medicine, Kansas State University’s startup funds for Dr. Davis. This work was also supported through the Department of Homeland Security (grant no. 2010-ST061-AG0001), Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD).http://online.liebertpub.com/VBZ2020-06-26hj2019Paraclinical Science

Study of lung cancer prevention by Euglena gracilis extracts

Doctor of PhilosophyDepartment of Anatomy and PhysiologyMajor Professor Not ListedLung cancer is the leading cause of cancer-related deaths. Because traditional treatment measures are associated with several limitations such as severe side effects and complications, natural products could be explored to develop novel preventative and therapeutic strategies against lung cancer. Euglena gracilis (E. gracilis) is a unicellular micro-alga used as a nutritional and functional dietary supplement because of its several health benefits. E. gracilis has long been known to have medicinal properties for multiple diseases including cancer. However, its anti- tumor properties against lung cancer are unknown. This dissertation examined the effect of water extract of Euglena gracilis (EWE) on the growth of lung cancer and demonstrated the mechanisms of its antitumor properties. In the first part of the dissertation, the antitumor and immunomodulatory effects of partially purified water extract from E. gracilis (EWE) against lung cancer were examined. Oral pretreatment with EWE three weeks before the Lewis lung carcinoma (LLC) cell inoculation significantly attenuated the growth of LLC tumors in immunocompetent syngeneic mouse lungs. EWE administration significantly reduced the granulocyte population in mouse peripheral blood and also directly attenuated granulocytic MDSCs (gMDSCs) in primary cell culture with mouse bone marrow cells, which was attributed to the gMDSC-specific apoptosis. These findings indicate that partially purified water extract from E. gracilis contains significant bioactive materials that, when consumed, attenuate the lung tumor growth by stimulating host antitumor immunity. Further, we prepared other fractions by boiling the EWE (bEWE) and then, evaluated the antitumor effects of both EWE and bEWE using orthotopic lung cancer syngeneic mouse models with LLC cells. The treatment with bEWE further attenuated the growth of LLC tumors in the lungs. Since Euglena water extracts were administrated daily via drinking water, there is a likelihood of alteration of intestinal microbiota and/or their metabolites. Therefore, we explored the fecal microbial communities from the mice treated with EWE, bEWE, and PBS using 16S rRNA gene sequencing. A more diverse intestinal microbiota composition in both extract-treated groups than that in the PBS group was observed. Particularly, a decrease in the ratio of Firmicutes to Bacteroidetes and significant increases in Akkermansia and Muribaculum were observed in both EWE- and bEWE-treated groups. The fecal microbiota transplantation (FMT) studies established that the attenuation of lung carcinoma growth in mice by Euglena water extracts is associated with the alteration of the gut microbiota. We also showed the attenuation of lung carcinoma growth using chemical-carcinogen- induced lung tumorigenesis in mice that closely mimics human lung cancer development. The fecal metabolites between the PBS and bEWE treated mice were different. The metabolites such as succinate, malate, triethanolamine, acetylserine, aspartate, glutamate, and threonine that can be metabolized to short-chain fatty acids (SCFAs) were increased with bEWE treatment. More importantly, the levels of fecal SCFAs such as acetate, butyrate, and propionate were increased upon Euglena water extract treatment. Further, an in vitro study showed inhibition of lung cancer growth with SCFAs treatment via apoptosis. Together, these studies elucidate the understanding of the anti-tumor efficacy of the water extracts from E. gracilis on the prevention and treatment of lung carcinoma growth. Further, these studies highlight the role of gut microbiota and its metabolites with the oral administration of E. gracilis water extracts in the attenuation of lung carcinoma growth using different lung carcinoma model. These findings may facilitate the usage of water extracts from E. gracilis for the development of novel prevention and therapeutic strategies against cancers

Oral Administration of Water Extract from Euglena gracilis Alters the Intestinal Microbiota and Prevents Lung Carcinoma Growth in Mice

The antitumor effects of a partially purified water extract from Euglena gracilis (EWE) and EWE treated by boiling (bEWE) were evaluated using orthotopic lung cancer syngeneic mouse models with Lewis lung carcinoma (LLC) cells. Daily oral administration of either EWE or bEWE started three weeks prior to the inoculation of LLC cells significantly attenuated tumor growth as compared to the phosphate buffered saline (PBS) control, and the attenuation was further enhanced by bEWE. The intestinal microbiota compositions in both extract-treated groups were more diverse than that in the PBS group. Particularly, a decrease in the ratio of Firmicutes to Bacteroidetes and significant increases in Akkermansia and Muribaculum were observed in two types of EWE-treated groups. Fecal microbiota transplantation (FMT) using bEWE-treated mouse feces attenuated tumor growth to an extent equivalent to bEWE treatment, while tumor growth attenuation by bEWE was abolished by treatment with an antibiotic cocktail. These studies strongly suggest that daily oral administration of partially purified water extracts from Euglena gracilis attenuates lung carcinoma growth via the alteration of the intestinal microbiota

Local immune checkpoint blockade therapy by an adenovirus encoding a novel PD-L1 inhibitory peptide inhibits the growth of colon carcinoma in immunocompetent mice

•A novel inhibitory peptide interfering with the PD-L1/PD-1 immune checkpoint pathway, dubbed PD-L1ip3, was designed.•The affinity of PD-L1ip3 for PD-L1 was only a few times weaker than that of its natural ligand, PD-1.•Direct treatment with PD-L1ip3 enhanced the ability of CD8+ T cells primed with cancer antigens to kill cancer cells in culture.•A combination treatment including transduction into cancer cells of a gene encoding PD-L1ip3 coupled with direct administration of PD-L1ip3 in peptide form significantly attenuated the growth of murine colon carcinoma in mice. A novel peptide that interferes with the PD-1/PD-L1 immune checkpoint pathway, termed PD-L1 inhibitory peptide 3 (PD-L1ip3), was computationally designed, experimentally validated for its specific binding to PD-L1, and evaluated for its antitumor effects in cell culture and in a mouse colon carcinoma syngeneic murine model. In several cell culture studies, direct treatment with PD-L1ip3, but not a similar peptide with a scrambled sequence, substantially increased death of CT26 colon carcinoma cells when co-cultured with murine CD8+ T cells primed by CT26 cell antigens. In a syngeneic mouse tumor model, the growth of CT26 tumor cells transduced with the PD-L1ip3 gene by an adenovirus vector was significantly slower than that of un-transduced CT26 cells in immunocompetent mice. This tumor growth attenuation was further enhanced by the coadministration of the peptide form of PD-L1ip3 (10 mg/kg/day). The current study suggests that this peptide can stimulate host antitumor immunity via blockade of the PD-1/PD-L1 pathway, thereby increasing CD8+ T cell-induced death of colon carcinoma cells. The tumor site-specific inhibition of PD-L1 by an adenovirus carrying the PD-L1ip3 gene, together with direct peptide treatment, may be used as a local immune checkpoint blockade therapy to inhibit colon carcinoma growth

Cell Wall Membrane Fraction of Chlorella sorokiniana Enhances Host Antitumor Immunity and Inhibits Colon Carcinoma Growth in Mice

A colon cancer growth inhibitor partially purified from the isolated cell wall membrane fraction of Chlorella sorokiniana, here referred to as Chlorella membrane factor (CMF), was evaluated for its antitumor and immunomodulatory effects in cell culture and in a colon carcinoma mouse model. The CMF treatment dose- and time-dependently inhibited colon carcinoma cell growth in 2-dimensional cultures. Treatment with CMF also significantly inhibited the growth of colon carcinoma spheroids in 3-dimensional cell culture in coculture with T lymphocytes. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of CMF (10 or 30 mg dry weight/kg body weight, every other day) dose-dependently and significantly attenuated the growth of tumor nodules via induction of tumor cell apoptosis. Evaluation of immune cell populations in ascites showed that CMF treatment tended to increase T lymphocytes but lower granulocyte populations. The present study suggests that the cell wall membrane fraction of Chlorella sorokiniana contains a bioactive material that inhibits colon carcinoma growth via direct cell growth inhibition and stimulation of host antitumor immunity. Hence, it is suggested that the Chlorella cell wall membrane extract or a bioactive substance in the extract is an attractive complementary medicine for cancer therapy

A Water Extract from Chlorella sorokiniana Cell Walls Stimulates Growth of Bone Marrow Cells and Splenocytes

A water extract derived from the isolated cell walls of Chlorella sorokiniana (C. sorokiniana, Chlorella water extract, CWE) was analyzed for the presence of lipopolysaccharide (LPS)-related material via the Limulus amebocyte lysate (LAL) assay and evaluated for its growth stimulation effect on the bone marrow cells and splenocytes in vitro cell cultures. The extract contained low levels of LPS-related material, and a mass spectrum suggested that the extract contained many components, including a low level of a lipid A precursor, a compound known as lipid X, which is known to elicit a positive response in the LAL assay. Treatment with the CWE dose- and time-dependently stimulated the growth of mouse bone marrow cells (BMCs) and splenocytes (SPLs). Treatment with the CWE also increased specific BMC subpopulations, including antigen-presenting cells (CD19+ B cells, 33D1+ dendritic cells and CD68+ macrophages), and CD4+ and CD8+ T cells, but decreased the number of LY6G+ granulocytes. Treatment with the CWE also increased cytokine mRNA associated with T cell activation, including TNFα, IFNγ, and granzyme B in human lymphoblasts. The present study indicates that the cell wall fraction of C.sorokiniana contains an LPS-like material and suggests a candidate source for the bioactivity that stimulates growth of both innate and adaptive immune cells

Water extract from Euglena gracilis prevents lung carcinoma growth in mice by attenuation of the myeloid-derived cell population

The partially purified water extract from Euglena gracilis (EWE) was evaluated for its antitumor and immunomodulatory effects in cell cultures and in a mouse orthotopic lung carcinoma allograft model. In two-dimensional cell culture, the EWE treatment inhibited cell growth of both murine Lewis lung carcinoma (LLC) and human lung carcinoma cells (A549 and H1299) in a dose- and time-dependent manner. In contrast, the growth of mouse bone marrow cells (BMCs), but not mouse splenocytes (SPLs), was stimulated by the treatment with EWE. In three-dimensional spheroid culture, spheroid growth of LLC cells was significantly attenuated by EWE treatment. In a mouse LLC orthotopic allograft model, pretreatment with EWE (150-200 mg/kg/day, via drinking water) three weeks prior to the LLC cell inoculation, but not post-treatment after LLC cell inoculation, significantly attenuated the growth of LLC tumors in immunocompetent syngeneic mouse lung. This tumor growth attenuation coincided with a significant decrease in the population of myeloid-derived cells, primarily neutrophils. Flow cytometric analysis revealed that the EWE treatment significantly attenuated growth of granulocytic myeloid-derived suppressor cells (gMDSC) in BMCs and that this decrease was due to induction of gMDSC-specific apoptosis and differentiation of monocytic MDSCs (mMDSC) to macrophages. The present study provides evidence that EWE pretreatment inhibits lung carcinoma growth mainly by stimulating host antitumor immunity through attenuation of growth of gMDSCs and decreasing the number of peripheral granulocytes. This study suggests that the partially purified extract derived from Euglena gracilis contains significant bioactive materials that prevent lung carcinoma growth