Probes for INS

In the 2014⁻2016 West Africa Ebola Virus (EBOV) outbreak, there was a significant concern raised about the potential for secondary bacterial infection originating from the gastrointestinal tract, which led to the empiric treatment of many patients with antibiotics. This retrospective pathology case series summarizes the gastrointestinal pathology observed in control animals in the rhesus EBOV-Kikwit intramuscular 1000 plaque forming unit infection model. All 31 Non-human primates (NHPs) exhibited lymphoid depletion of gut-associated lymphoid tissue (GALT) but the severity and the specific location of the depletion varied. Mesenteric lymphoid depletion and necrosis were present in 87% (27/31) of NHPs. There was mucosal barrier disruption of the intestinal tract with mucosal necrosis and/or ulceration most notably in the duodenum (16%), cecum (16%), and colon (29%). In the intestinal tract, hemorrhage was noted most frequently in the duodenum (52%) and colon (45%). There were focal areas of bacterial submucosal invasion in the gastrointestinal (GI) tract in 9/31 (29%) of NHPs. Only 2/31 (6%) had evidence of pancreatic necrosis. One NHP (3%) experienced jejunal intussusception which may have been directly related to EBOV. Immunofluorescence assays demonstrated EBOV antigen in CD68+ macrophage/monocytes and endothelial cells in areas of GI vascular injury or necrosis.

Abstract

Background and Aims

Regulatory macrophages play a critical role in tissue repair, and we have previously shown that anti-tumour necrosis factor [TNF] antibodies induce these macrophages in vitro and in vivo in IBD patients. The induction of regulatory macrophages can be potentiated using the combination of anti-TNF and thiopurines, consistent with the enhanced efficacy of this combination therapy described in clinical trials. As thiopurines are unfortunately associated with significant side effects, we here aimed to identify alternatives for combination therapy with anti-TNF, using the macrophage induction model as a screening tool.

Methods

Mixed lymphocyte reactions were treated with anti-TNF and a library of 1600 drug compounds. Induction of CD14+CD206+ macrophages was analysed by flow cytometry. Positive hits were validated in vitro and in the T cell transfer model of colitis.

Results

Among the 98 compounds potentiating the induction of regulatory macrophages by anti-TNF were six benzimidazoles, including albendazole. Albendazole treatment in the presence of anti-TNF resulted in alterations in the tubulin skeleton and signalling though AMPK, which was required for the enhanced induction. Combination therapy also increased expression levels of the immunoregulatory cytokine IL-10. In vivo, albendazole plus anti-TNF combination therapy was superior to monotherapy in a model of colitis, in terms of both induction of regulatory macrophages and improvement of clinical symptoms.

Conclusions

Albendazole enhances the induction of regulatory macrophages by anti-TNF and potentiates clinical efficacy in murine colitis. Given its favourable safety profile, these data indicate that the repurposing of albendazole may be a novel option for anti-TNF combination therapy in IBD.

Central nervous system germinomas are characterized by a massive immune cell infiltrate. We systematically characterized these immune cells in 28 germinomas by immunophenotyping and image analysis. mRNA expression was analyzed by Nanostring technology and in situ RNA hybridization. Tumor infiltrating lymphocytes (TILs) were composed of 61.8% ± 3.1% (mean ± SE) CD3-positive T cells, including 45.2% ± 3.5% of CD4-positive T-helper cells, 23.4% ± 1.5% of CD8-positive cytotoxic T cells, 5.5% ± 0.9% of FoxP3-positive regulatory T cells, and 11.9% ±1.3% PD-1-positive TILs. B cells accounted for 35.8% ± 2.9% of TILs and plasma cells for 9.3% ± 1.6%. Tumor-associated macrophages consisted of clusters of activated PD-L1-positive macrophages and interspersed anti-inflammatory macrophages expressing CD163. Germinoma cells did not express PD-L1. Expression of genes encoding immune cell markers and cytokines was high and comparable to mRNA levels in lymph node tissue. IFNG and IL10 mRNA was detected in subfractions of TILs and in PD-L1-positive macrophages. Taken together, the strong immune reaction observed in germinomas involves inflammatory as well as various suppressive mechanisms. Expression of PD-1 and PD-L1 and infiltration of cytotoxic T cells are biomarkers predictive of response to anti-PD-1/PD-L1 therapies, constituting a rationale for possible novel treatment approaches.

Regardless of host, pathogenic mycobacteria of the Mycobacterium tuberculosiscomplex such as Mycobacterium bovis, induce a characteristic lesion known as agranuloma, tubercle or tuberculoid granuloma. Granulomas represent a distinct host response to chronic antigenic stimuli, such as foreign bodies, certain bacterial components, or persistent pathogens such as M. bovis. Granulomas are composed of specific cell types including epithelioid macrophages, lymphocytes and a morphologically distinctive cell type, the multinucleated giant cell. Multinucleated giant cells are formed by the fusion of multiple macrophages; however, their function remains unclear. In humans, giant cells in tuberculous granulomas have been shown to express various cytokines, chemokines and enzymes important to the formation and maintenance of the granuloma. The objective of this study was to quantitatively assess multinucleated giant cell cytokine expression in bovine tuberculoid granulomas; focusing on cytokines of suspected relevance to bovine tuberculosis. Using calves experimentally infected with M. bovis, in situ cytokine expression was quantitatively assessed using RNAScope^® for the following cytokines TNF-α, IFN-γ, TGF-β, IL-17A and IL-10. Multinucleated giant cells in bovine tuberculoid granulomas expressed all examined cytokines to varying degrees, with differential expression of TGF-β, IL-17A and IL-10 in giant cells from early versus late stage granulomas. There was a modest, positive correlation between the level of cytokine expression and cell size or number of nuclei. These results suggest that multinucleated giant cells are active participants within bovine tuberculoid granulomas, contributing to the cytokine milieu necessary to form and maintain granulomas.

We previously developed optimized DNA vaccines against both Lassa fever and Ebola hemorrhagic fever viruses and demonstrated that they were protective individually in guinea pig and nonhuman primate models. In this study, we vaccinated groups of strain 13 guinea pigs two times, four weeks apart with 50 µg of each DNA vaccine or a mock vaccine at discrete sites by intradermal electroporation. Five weeks following the second vaccinations, guinea pigs were exposed to lethal doses of Lassa virus, Ebola virus, or a combination of both viruses simultaneously. None of the vaccinated guinea pigs, regardless of challenge virus and including the coinfected group, displayed weight loss, fever or other disease signs, and all survived to the study endpoint. All of the mock-vaccinated guinea pigs that were infected with Lassa virus, and all but one of the EBOV-infected mock-vaccinated guinea pigs succumbed. In order to determine if the dual-agent vaccination strategy could protect against both viruses if exposures were temporally separated, we held the surviving vaccinates in BSL-4 for approximately 120 days to perform a cross-challenge experiment in which guinea pigs originally infected with Lassa virus received a lethal dose of Ebola virus and those originally infected with Ebola virus were infected with a lethal dose of Lassa virus. All guinea pigs remained healthy and survived to the study endpoint. This study clearly demonstrates that DNA vaccines against Lassa and Ebola viruses can elicit protective immunity against both individual virus exposures as well as in a mixed-infection environment.

Survivors of Ebola virus infection may become subclinically infected, but whether animal models recapitulate this complication is unclear. Using histology in combination with immunohistochemistry and in situ hybridization in a retrospective review of a guinea pig confirmation-of-virulence study, we demonstrate for the first time Ebola virus infection in hepatic oval cells, the endocardium and stroma of the atrioventricular valves and chordae tendinae, satellite cells of peripheral ganglia, neurofibroblasts and Schwann cells of peripheral nerves and ganglia, smooth muscle cells of the uterine myometrium and vaginal wall, acini of the parotid salivary glands, thyroid follicular cells, adrenal medullary cells, pancreatic islet cells, endometrial glandular and surface epithelium, and the epithelium of the vagina, penis and, prepuce. These findings indicate that standard animal models for Ebola virus disease are not as well-described as previously thought and may serve as a stepping stone for future identification of potential sites of virus persistence.

Following the largest Ebola virus disease outbreak from 2013 to 2016, viral RNA has been detected in survivors from semen and breast milk long after disease recovery. However, as there have been few cases of sexual transmission, it is unclear whether every RNA positive fluid sample contains infectious virus. Virus isolation, typically using cell culture or animal models, can serve as a tool to determine the infectivity of patient samples. However, the sensitivity of these methods has not been assessed for the Ebola virus isolate, Makona. Described here is an efficiency comparison of Ebola virus Makona isolation using Vero E6, Huh-7, monocyte-derived macrophage cells, and suckling laboratory mice. Isolation sensitivity was similar in all methods tested. Laboratory mice and Huh-7 cells were less affected by toxicity from breast milk than Vero E6 and MDM cells. However, the advantages associated with isolation in Huh-7 cells over laboratory mice, including cost effectiveness, sample volume preservation, and a reduction in animal use, make Huh-7 cells the preferred substrate tested for Ebola virus Makona isolation.