Kaposi sarcoma-associated herpesvirus complement control protein (KCP) and glycoprotein K8.1 are not required for viral infection in vitro or in vivo.

Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causal agent of Kaposi sarcoma, a cancer that appears as tumors on the skin or mucosal surfaces, as well as primary effusion lymphoma and KSHV-associated multicentric Castleman disease, which are B-cell lymphoproliferative disorders. Effective prophylactic and therapeutic strategies against KSHV infection and its associated diseases are needed. To develop these strategies, it is crucial to identify and target viral glycoproteins involved in KSHV infection of host cells. Multiple KSHV glycoproteins expressed on the viral envelope are thought to play a pivotal role in viral infection, but the infection mechanisms involving these glycoproteins remain largely unknown. We investigated the role of two KSHV envelope glycoproteins, KSHV complement control protein (KCP) and K8.1, in viral infection in various cell types in vitro and in vivo. Using our newly generated anti-KCP antibodies, previously characterized anti-K8.1 antibodies, and recombinant mutant KSHV viruses lacking KCP, K8.1, or both, we demonstrated the presence of KCP and K8.1 on the surface of both virions and KSHV-infected cells. We showed that KSHV lacking KCP and/or K8.1 remained infectious in KSHV-susceptible cell lines, including epithelial, endothelial, and fibroblast, when compared to wild-type recombinant KSHV. We also provide the first evidence that KSHV lacking K8.1 or both KCP and K8.1 can infect human B cells in vivo in a humanized mouse model. Thus, these results suggest that neither KCP nor K8.1 is required for KSHV infection of various host cell types and that these glycoproteins do not determine KSHV cell tropism. IMPORTANCE: Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic human gamma-herpesvirus associated with the endothelial malignancy Kaposi sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman disease. Determining how KSHV glycoproteins such as complement control protein (KCP) and K8.1 contribute to the establishment, persistence, and transmission of viral infection will be key for developing effective anti-viral vaccines and therapies to prevent and treat KSHV infection and KSHV-associated diseases. Using newly generated anti-KCP antibodies, previously characterized anti-K8.1 antibodies, and recombinant mutant KSHV viruses lacking KCP and/or K8.1, we show that KCP and K8.1 can be found on the surface of both virions and KSHV-infected cells. Furthermore, we show that KSHV lacking KCP and/or K8.1 remains infectious to diverse cell types susceptible to KSHV in vitro and to human B cells in vivo in a humanized mouse model, thus providing evidence that these viral glycoproteins are not required for KSHV infection.

Neuron-specific gene NSG1 binds to and positively regulates sortilin ectodomain shedding via a metalloproteinase-dependent mechanism.

Increasing evidence suggests that aberrant regulation of sortilin ectodomain shedding can contribute to amyloid-ß pathology and frontotemporal dementia, although the mechanism by which this occurs has not been elucidated. Here, we probed for novel binding partners of sortilin using multiple and complementary approaches and identified two proteins of the neuron-specific gene (NSG) family, NSG1 and NSG2, that physically interact and colocalize with sortilin. We show both NSG1 and NSG2 induce subcellular redistribution of sortilin to NSG1- and NSG2-enriched compartments. However, using cell surface biotinylation, we found only NSG1 reduced sortilin cell surface expression, which caused significant reductions in uptake of progranulin, a molecular determinant for frontotemporal dementia. In contrast, we demonstrate NSG2 has no effect on sortilin cell surface abundance or progranulin uptake, suggesting specificity for NSG1 in the regulation of sortilin cell surface expression. Using metalloproteinase inhibitors and A disintegrin and metalloproteinase 10 KO cells, we further show that NSG1-dependent reduction of cell surface sortilin occurred via proteolytic processing by A disintegrin and metalloproteinase 10 with a concomitant increase in shedding of sortilin ectodomain to the extracellular space. This represents a novel regulatory mechanism for sortilin ectodomain shedding that is regulated in a neuron-specific manner. Furthermore, this finding has implications for the development of strategies for brain-specific regulation of sortilin and possibly sortilin-driven pathologies.

Phosphatidylinositol (3,5)-bisphosphate machinery regulates neurite thickness through neuron-specific endosomal protein NSG1/NEEP21.

Phosphatidylinositol (3,5)-bisphosphate [PtdIns(3,5)P2] is a critical signaling phospholipid involved in endolysosome homeostasis. It is synthesized by a protein complex composed of PIKfyve, Vac14, and Fig4. Defects in PtdIns(3,5)P2 synthesis underlie a number of human neurological disorders, including Charcot-Marie-Tooth disease, child onset progressive dystonia, and others. However, neuron-specific functions of PtdIns(3,5)P2 remain less understood. Here, we show that PtdIns(3,5)P2 pathway is required to maintain neurite thickness. Suppression of PIKfyve activities using either pharmacological inhibitors or RNA silencing resulted in decreased neurite thickness. We further find that the regulation of neurite thickness by PtdIns(3,5)P2 is mediated by NSG1/NEEP21, a neuron-specific endosomal protein. Knockdown of NSG1 expression also led to thinner neurites. mCherry-tagged NSG1 colocalized and interacted with proteins in the PtdIns(3,5)P2 machinery. Perturbation of PtdIns(3,5)P2 dynamics by overexpressing Fig4 or a PtdIns(3,5)P2-binding domain resulted in mislocalization of NSG1 to nonendosomal locations, and suppressing PtdIns(3,5)P2 synthesis resulted in an accumulation of NSG1 in EEA1-positive early endosomes. Importantly, overexpression of NSG1 rescued neurite thinning in PtdIns(3,5)P2-deficient CAD neurons and primary cortical neurons. Our study uncovered the role of PtdIns(3,5)P2 in the morphogenesis of neurons, which revealed a novel aspect of the pathogenesis of PtdIns(3,5)P2-related neuropathies. We also identified NSG1 as an important downstream protein of PtdIns(3,5)P2, which may provide a novel therapeutic target in neurological diseases.

Immunity mediates host specificity in the human hookworm Ancylostoma ceylanicum.

Hookworm infection affects millions globally, leading to chronic conditions like malnutrition and anaemia. Among the hookworm species, Ancylostoma ceylanicum stands out as a generalist, capable of infecting various hosts, including humans, cats, dogs and hamsters. Surprisingly, it cannot establish in mice, despite their close phylogenetic relationship to hamsters. The present study investigated the development of A. ceylanicum in immunodeficient NSG mice to determine the contribution of the immune system to host restriction. The infections became patent on day 19 post-infection (PI) and exhibited elevated egg production which lasted for at least 160 days PI. Infective A. ceylanicum larvae reared from eggs released by infected NSG mice were infectious to hamsters and capable of reproduction, indicating that the adults in the NSG mice were producing viable offspring. In contrast, A. ceylanicum showed limited development in outbred Swiss Webster mice. Furthermore, the closely related canine hookworm Ancylostoma caninum was unable to infect and develop in NSG mice, indicating that different mechanisms may determine host specificity even in closely related species. This is the first report of any hookworm species completing its life cycle in a mouse and implicate the immune system in determining host specificity in A. ceylanicum.

Humanization with CD34-positive hematopoietic stem cells in NOG-EXL mice results in improved long-term survival and less severe myeloid cell hyperactivation phenotype relative to NSG-SGM3 mice.

NSG-SGM3 and NOG-EXL mice combine severe immunodeficiency with transgenic expression of human myeloid stimulatory cytokines, resulting in marked expansion of myeloid populations upon humanization with CD34+ hematopoietic stem cells (HSCs). Humanized NSG-SGM3 mice typically develop a lethal macrophage activation syndrome and mast cell hyperplasia that limit their use in long-term studies (e.g., humanization followed by tumor xenotransplantation). It is currently unclear to what extent humanized NOG-EXL mice suffer from the same condition observed in humanized NSG-SGM3 mice. We compared the effects of human CD34+ HSC engraftment in these two strains in an orthotopic patient-derived glioblastoma model. NSG-SGM3 mice humanized in-house were compared to NOG-EXL mice humanized in-house and commercially available humanized NOG-EXL mice. Mice were euthanized at humane or study endpoints, and complete pathological assessments were performed. A semiquantitative multiparametric clinicopathological scoring system was developed to characterize chimeric myeloid cell hyperactivation (MCH) syndrome. NSG-SGM3 mice were euthanized at 16 weeks after humanization because of severe deterioration of clinical conditions. Humanized NOG-EXL mice survived to the study endpoint at 22 weeks after humanization and showed less-severe MCH phenotypes than NSG-SGM3 mice. Major differences included the lack of mast cell expansion and limited tissue/organ involvement in NOG-EXL mice compared to NSG-SGM3 mice. Engraftment of human lymphocytes, assessed by immunohistochemistry, was similar in the two strains. The longer survival and decreased MCH phenotype severity in NOG-EXL mice enabled their use in a tumor xenotransplantation study. The NOG-EXL model is better suited than the NSG-SGM3 model for immuno-oncology studies requiring long-term survival after humanization.

miR-107 Targets NSG1 to Regulate Hypopharyngeal Squamous Cell Carcinoma Progression through ERK Pathway.

Hypopharyngeal squamous cell carcinoma (HSCC) is a kind of malignant tumor with a poor prognosis and low quality of life in the otolaryngology department. It has been found that microRNA (miRNA) plays an important role in the occurrence and development of various tumors. This study found that the expression level of miRNA-107 (miR-107) in HSCC was significantly reduced. Subsequently, we screened out the downstream direct target gene Neuronal Vesicle Trafficking Associated 1 (NSG1) related to miR-107 through bioinformatics analysis and found that the expression of NSG1 was increased in HSCC tissues. Following the overexpression of miR-107 in HSCC cells, it was observed that miR-107 directly suppressed NSG1 expression, leading to increased apoptosis, decreased proliferation, and reduced invasion capabilities of HSCC cells. Subsequent experiments involving the overexpression and knockdown of NSG1 in HSCC cells demonstrated that elevated NSG1 levels enhanced cell proliferation, migration, and invasion, while the opposite effect was observed upon NSG1 knockdown. Further investigations revealed that changes in NSG1 levels in the HSCC cells were accompanied by alterations in ERK signaling pathway proteins, suggesting a potential regulatory role of NSG1 in HSCC cell proliferation, migration, and invasion through the ERK pathway. These findings highlight the significance of miR-107 and NSG1 in hypopharyngeal cancer metastasis, offering promising targets for therapeutic interventions and prognostic evaluations for HSCC.

A Double-Humanized Mouse Model for Studying Host Gut Microbiome-Immune Interactions in Gulf War Illness.

Unraveling the multisymptomatic Gulf War Illness (GWI) pathology and finding an effective cure have eluded researchers for decades. The chronic symptom persistence and limitations for studying the etiologies in mouse models that differ significantly from those in humans pose challenges for drug discovery and finding effective therapeutic regimens. The GWI exposome differs significantly in the study cohorts, and the above makes it difficult to recreate a model closely resembling the GWI symptom pathology. We have used a double engraftment strategy for reconstituting a human immune system coupled with human microbiome transfer to create a humanized-mouse model for GWI. Using whole-genome shotgun sequencing and blood immune cytokine enzyme linked immunosorbent assay (ELISA), we show that our double humanized mice treated with Gulf War (GW) chemicals show significantly altered gut microbiomes, similar to those reported in a Veteran cohort of GWI. The results also showed similar cytokine profiles, such as increased levels of IL-1ß, IL-6, and TNF R-1, in the double humanized model, as found previously in a human cohort. Further, a novel GWI Veteran fecal microbiota transfer was used to create a second alternative model that closely resembled the microbiome and immune-system-associated pathology of a GWI Veteran. A GWI Veteran microbiota transplant in humanized mice showed a human microbiome reconstitution and a systemic inflammatory pathology, as reflected by increases in interleukins 1ß, 6, 8 (IL-1ß, IL-6, IL-8), tumor necrosis factor receptor 1 (TNF R-1), and endotoxemia. In conclusion, though preliminary, we report a novel in vivo model with a human microbiome reconstitution and an engrafted human immune phenotype that may help to better understand gut-immune interactions in GWI.

Integrating Pharmacokinetic-Pharmacodynamic Modeling and Physiologically Based Pharmacokinetic Modeling to Optimize Human Dose Predictions for Plasmodium falciparum Malaria: a Chloroquine Case Study.

The translation of a preclinical antimalarial drug development candidate to the clinical phases should be supported by rational human dose selection. A model-informed strategy based on preclinical data, which incorporates pharmacokinetic-pharmacodynamic (PK-PD) properties with physiologically based pharmacokinetic (PBPK) modeling, is proposed to optimally predict an efficacious human dose and dosage regimen for the treatment of Plasmodium falciparum malaria. The viability of this approach was explored using chloroquine, which has an extensive clinical history for malaria treatment. First, the PK-PD parameters and the PK-PD driver of efficacy for chloroquine were determined through a dose fractionation study in the P. falciparum-infected humanized mouse model. A PBPK model for chloroquine was then developed for predicting the drug's PK profiles in a human population, from which the human PK parameters were determined. Lastly, the PK-PD parameters estimated in the P. falciparum-infected mouse model and the human PK parameters derived from the PBPK model were integrated to simulate the human dose-response relationships against P. falciparum, which subsequently allowed the determination of an optimized treatment. The predicted efficacious human dose and dosage regimen for chloroquine were comparable to those recommended clinically for the treatment of uncomplicated, drug-sensitive malaria, which provided supportive evidence for the proposed model-based approach to antimalarial human dose predictions.

Humanized mice for investigating SARS-CoV-2 lung infection and associated human immune responses.

There is an urgent need for animal models of coronavirus disease 2019 to study immunopathogenesis and test therapeutic intervenes. In this study, we showed that NOD/SCID IL2rg-/- (NSG) mice engrafted with human lung (HL) tissue (NSG-L mice) could be infected efficiently by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and that live virus capable of infecting Vero cells was found in the HL grafts and multiple organs from infected NSG-L mice. RNA-Sequencing identified a series of differentially expressed genes, which are enriched in viral defense responses, chemotaxis, IFN stimulation and pulmonary fibrosis, between HL grafts from infected and control NSG-L mice. Furthermore, when infected with SARS-CoV-2, humanized mice with both human immune system (HIS) and autologous HL grafts (HISL mice) had bodyweight loss and hemorrhage and immune cell infiltration in HL grafts, which were not observed in immunodeficient NSG-L mice, indicating the development of anti-viral immune responses in these mice. In support of this possibility, the infected HISL mice showed bodyweight recovery and lack of detectable live virus at the later time. These results demonstrate that NSG-L and HISL mice are susceptible to SARS-CoV-2 infection, offering a useful in vivo model for studying SARS-CoV-2 infection and the associated immune response and immunopathology, and testing anti-SARS-CoV-2 therapies.

Senescence-associated secretory proteins induced in lung adenocarcinoma by extended treatment with dexamethasone enhance migration and activation of lymphocytes.

There is a need to improve response rates of immunotherapies in lung adenocarcinoma (AC). Extended (7-14 days) treatment of high glucocorticoid receptor (GR) expressing lung AC cells with dexamethasone (Dex) induces an irreversible senescence phenotype through chronic induction of p27. As the senescence-associated secretory phenotype (SASP) may have either tumor supporting or antitumor immunomodulatory effects, it was interest to examine the effects of Dex-induced senescence of lung AC cells on immune cells. Dex-induced senescence resulted in sustained production of CCL2, CCL4, CXCL1 and CXCL2, both in vitro and in vivo. After Dex withdrawal, secretion of these chemokines by the senescent cells attracted peripheral blood monocytes, T-cells, and NK cells. Following treatment with Dex-induced SASP protein(s), the peripheral blood lymphocytes exhibited higher cell count and tumor cytolytic activity along with enhanced Ki67 and perforin expression in T and NK cells. This cytolytic activity was partially attributed to NKG2D, which was upregulated in NK cells by SASP while its ligand MICA/B was upregulated in the senescent cells. Enhanced infiltrations of T and NK cells were observed in human lung AC xenografts in humanized NSG mice, following treatment with Dex. The findings substantiate the idea that induction of irreversible senescence in high-GR expressing subpopulations of lung AC tumors using Dex pretreatment enhances tumor immune infiltration and may subsequently improve the clinical outcome of current immunotherapies.

Generation and characterization of an immunodeficient mouse model of mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (Hunter syndrome, MPS II) is an inherited X-linked recessive disease caused by deficiency of iduronate-2-sulfatase (IDS), resulting in the accumulation of the glycosaminoglycans (GAG) heparan and dermatan sulfates. Mouse models of MPS II have been used in several reports to study disease pathology and to conduct preclinical studies for current and next generation therapies. Here, we report the generation and characterization of an immunodeficient mouse model of MPS II, where CRISPR/Cas9 was employed to knock out a portion of the murine IDS gene on the NOD/SCID/Il2rγ (NSG) immunodeficient background. IDS-/- NSG mice lacked detectable IDS activity in plasma and all analyzed tissues and exhibited elevated levels of GAGs in those same tissues and in the urine. Histopathology revealed vacuolized cells in both the periphery and CNS of NSG-MPS II mice. This model recapitulates skeletal disease manifestations, such as increased zygomatic arch diameter and decreased femur length. Neurocognitive deficits in spatial memory and learning were also observed in the NSG-MPS II model. We anticipate that this new immunodeficient model will be appropriate for preclinical studies involving xenotransplantation of human cell products intended for the treatment of MPS II.

Enhanced development of functional human NK cells in NOD-scid-IL2rgnull mice expressing human IL15.

Human innate immunity plays a critical role in tumor surveillance and in immunoregulation within the tumor microenvironment. Natural killer (NK) cells are innate lymphoid cells that have opposing roles in the tumor microenvironment, including NK cell subsets that mediate tumor cell cytotoxicity and subsets with regulatory function that contribute to the tumor immune suppressive environment. The balance between effector and regulatory NK cell subsets has been studied extensively in murine models of cancer, but there is a paucity of models to study human NK cell function in tumorigenesis. Humanized mice are a powerful alternative to syngeneic mouse tumor models for the study of human immuno-oncology and have proven effective tools to test immunotherapies targeting T cells. However, human NK cell development and survival in humanized NOD-scid-IL2rgnull (NSG) mice are severely limited. To enhance NK cell development, we have developed NSG mice that constitutively expresses human Interleukin 15 (IL15), NSG-Tg(Hu-IL15). Following hematopoietic stem cell engraftment of NSG-Tg(Hu-IL15) mice, significantly higher levels of functional human CD56+ NK cells are detectable in blood and spleen, as compared to NSG mice. Hematopoietic stem cell (HSC)-engrafted NSG-Tg(Hu-IL15) mice also supported the development of human CD3+ T cells, CD20+ B cells, and CD33+ myeloid cells. Moreover, the growth kinetics of a patient-derived xenograft (PDX) melanoma were significantly delayed in HSC-engrafted NSG-Tg(Hu-IL15) mice as compared to HSC-engrafted NSG mice demonstrating that human NK cells have a key role in limiting the tumor growth. Together, these data demonstrate that HSC-engrafted NSG-Tg(Hu-IL15) mice support enhanced development of functional human NK cells, which limit the growth of PDX tumors.

Spontaneous early-onset neurodegeneration in the brainstem and spinal cord of NSG, NOG, and NXG mice.

The spectrum of background, incidental, and experimentally induced lesions affecting NSG and NOG mice has been the subject of intense investigation. However, comprehensive studies focusing on the spontaneous neuropathological changes of these immunocompromised strains are lacking. This work describes the development of spontaneous early-onset neurodegeneration affecting both juvenile and adult NSG, NOG, and NXG mice. The study cohort consisted of 367 NSG mice of both sexes (including 33 NSG-SGM3), 61 NOG females (including 31 NOG-EXL), and 4 NXG females. These animals were primarily used for preclinical CAR T-cell testing, generation of humanized immune system chimeras, and/or tumor xenograft transplantation. Histopathology of brain and spinal cord and immunohistochemistry (IHC) for AIF-1, GFAP, CD34, and CD45 were performed. Neurodegenerative changes were observed in 57.6% of the examined mice (affected mice age range was 6-36 weeks). The lesions were characterized by foci of vacuolation with neuronal degeneration/death and gliosis distributed throughout the brainstem and spinal cord. IHC confirmed the development of gliosis, overexpression of CD34, and a neuroinflammatory component comprised of CD45-positive monocyte-derived macrophages. Lesions were significantly more frequent and severe in NOG mice. NSG males were considerably more affected than NSG females. Increased lesion frequency and severity in older animals were also identified. These findings suggest that NSG, NOG, and NXG mice are predisposed to the early development of identical neurodegenerative changes. While the cause of these lesions is currently unclear, potential associations with the genetic mutations shared by NSG, NOG, and NXG mice as well as unidentified viral infections are considered.

Model Characterization: Total Body Irradiation or Busulfan for Conditioning in Human Cell Therapy Toxicology and Tumorigenicity Studies using NOD/SCID/IL2Rγnull (NSG) Mice.

The NOD/SCID/IL2Rγnull (NSG) mouse is a relevant model for toxicology and tumorigenicity studies evaluating human cell therapies. Data was compiled from toxicology study control NSG mice exposed to gamma irradiation (0 or 200 cGy) or busulfan. Retrospective data evaluation included mortality, clinical observations, body weights, hematology, and external and internal macroscopic observations. There was no mortality in any of the 129 toxicology control (irradiated and non-irradiated) mice up to the 20-week observation period. Mortalities occurred between Days 1 and 25 among animals given busulfan ≥25 mg/kg/day at 1 or 2 doses via intraperitoneal (i.p.) injection. There were 4/10, 6/10 and 4/10 deaths at 25, 30 and 35 mg/kg/day busulfan, respectively. Busulfan-treated mice presented with dose-dependent clinical signs including signs of anemia in some individuals. Hematology, including white blood cell (WBC) and neutrophil (NEUT) counts, from irradiated mice at Weeks 12 and 20 revealed comparable values to non-irradiated animals. In contrast, irradiated mice treated with a positive control (HL-60) were euthanized prior to Week 12. There were no irradiation-related differences in macroscopic observations with lymphoid atrophy identified comparably in irradiated and non-irradiated groups. These results suggest that irradiation was suitable for conditioning to enable cell engraftment in NSG mice in the context of regulatory toxicology and tumorigenicity studies. Busulfan administered at 20 mg/kg/day for 2 days, i.p. was also well-tolerated, and it could be considered for toxicology studies of genetically modified human cells.

Humanized NSG Mouse Models as a Preclinical Tool for Translational Research in Inflammatory Bowel Diseases.

The development of animal models reflecting the pathologies of ulcerative colitis (UC) and Crohn's disease (CD) remains a major challenge. The NOD/SCID/IL2rγnull (NSG) mouse strain, which is immune-compromised, tolerates the engraftment of human peripheral blood mononuclear cells (PBMC) derived from patients with UC (NSG-UC) or CD (NSG-CD). This offers the opportunity to examine the impact of individual immunological background on the development of pathophysiological manifestations. When challenged with ethanol, NSG-UC mice exhibited a strong pro-inflammatory response, including the development of edemas, influx of human T cells, B cells and monocytes into the mucosa and submucosa, and elevated expression of the inflammatory markers CRP and CCL-7. Fibrotic alterations were characterized by an influx of fibroblasts and a thickening of the muscularis mucosae. In contrast, the development of pathological manifestations in NSG-CD mice developed without challenge and was signified by extensive collagen deposition between the muscularis propria and muscularis mucosae, as observed in the areas of strictures in CD patients. Vimentin-expressing fibroblasts supplanting colonic crypts and elevated expression of HGF and TGFß corroborated the remodeling phenotype. In summary, the NSG-UC and NSG-CD models partially reflect these human diseases and are powerful tools to examine the mechanism underlying the inflammatory processes in UC and CD.

Identifying serum miRNA biomarkers for radiation exposure in hematopoietic humanized NSG-SGM3 mice.

BACKGROUND: Humans are commonly exposed to ionizing radiation. The conventional approach for estimating radiation exposure is to integrate physical and clinical measurements for optimizing the dose calculation. However, these methods have several limitations. The present study attempted to identify candidate microRNA (miRNA) biomarkers for radiation exposure in a hematopoietic humanized NSGS (hu-NSGS) mouse model. METHODS: We grafted human CD34+ hematopoietic stem cells into NSG-SGM3 (NSGS) mice. The hu-NSGS mice underwent total body irradiation at doses of 2, 3, and 4 Gy. Tissues from the spleen, thymus, and lymph nodes of hu-NSGS mice were prepared to analyze levels of CD45+ and CD3+ T cells and CD 20+ B cells using flow cytometry and immunohistochemistry. Serum miRNAs were profiled using a digital multiplexed NanoString n-Counter. RESULTS: The expression of 45 miRNAs was upregulated/downregulated hu-NSGS mice. The miRNAs hsa-mir-188-5p, hsa-let-7a-5p, hsa-mir-612, hsa-mir-671-5p, and hsa-mir-675-5p were highly radiation-responsive in irradiated hu-NSGS mice. When compared with control mice, radiation-exposed mice exhibited significant upregulated of hsa-let-7a-5p expression and significant downregulation of hsa-mir-188-5p expression. CONCLUSIONS: Single miRNAs or combinations of hsa-mir-188-5p, hsa-let-7a-5p, hsa-mir-675-5p, hsa-mir-612, and hsa-mir-671-5p can be used as biomarkers for predicting the impact of radiation exposure. The current findings suggest the usefulness of hu-NSGS models for investigating radiation biomarkers.

Lenvatinib inhibits the growth of gastric cancer patient-derived xenografts generated from a heterogeneous population.

BACKGROUND: Lenvatinib is a multitargeted tyrosine kinase inhibitor that is being tested in combination with immune checkpoint inhibitors to treat advanced gastric cancer; however, little data exists regarding the efficacy of lenvatinib monotherapy. Patient-derived xenografts (PDX) are established by engrafting human tumors into immunodeficient mice. The generation of PDXs may be hampered by growth of lymphomas. In this study, we compared the use of mice with different degrees of immunodeficiency to establish PDXs from a diverse cohort of Western gastric cancer patients. We then tested the efficacy of lenvatinib in this system. METHODS: PDXs were established by implanting gastric cancer tissue into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) or Foxn1nu (nude) mice. Tumors from multiple passages from each PDX line were compared histologically and transcriptomically. PDX-bearing mice were randomized to receive the drug delivery vehicle or lenvatinib. After 21 days, the percent tumor volume change (%Δvtumor) was calculated. RESULTS: 23 PDX models were established from Black, non-Hispanic White, Hispanic, and Asian gastric cancer patients. The engraftment rate was 17% (23/139). Tumors implanted into NSG (16%; 18/115) and nude (21%; 5/24) mice had a similar engraftment rate. The rate of lymphoma formation in nude mice (0%; 0/24) was lower than in NSG mice (20%; 23/115; p < 0.05). PDXs derived using both strains maintained histologic and gene expression profiles across passages. Lenvatinib treatment (mean %Δvtumor: -33%) significantly reduced tumor growth as compared to vehicle treatment (mean %Δvtumor: 190%; p < 0.0001). CONCLUSIONS: Nude mice are a superior platform than NSG mice for generating PDXs from gastric cancer patients. Lenvatinib showed promising antitumor activity in PDXs established from a diverse Western patient population and warrants further investigation in gastric cancer.

Contribution of the innate and adaptive immune systems to aortic dilation in murine mucopolysaccharidosis type I.

BACKGROUND: Adult immunocompetent male C57Bl/6 mucopolysaccharidosis, type I (MPSI) mice develop aortic insufficiency (AI), dilated ascending aortas and decreased cardiac function, findings not observed in immune incompetent adult male NSG MPSI mice. We sought to determine why. METHODS: Cardiac ultrasound measurements of ascending aorta and left ventricular dimensions and Doppler interrogation for AI were performed in 6-month-old male B6 MPSI (N = 12), WT (N = 6), NSG MPSI (N = 8), NSG (N = 6) mice. Urinary glycosaminoglycans, RNA sequencing with quantitative PCR were performed and aortic pathology assessed by routine and immunohistochemical staining on subsets of murine aortas. RESULTS: Ascending aortic diameters were significantly greater, left ventricular function significantly decreased, and AI significantly more frequent in B6 MPSI mice compared to NSG MPSI mice (p < 0.0001, p = 0.008 and p = 0.02, respectively); NSG and B6 WT mice showed no changes. Urinary glycosaminoglycans were significantly greater in B6 and NSG MPSI mice and both were significantly elevated compared to WT controls (p = 0.003 and p < 0.0001, respectively). By RNA sequencing, all 11 components of the inflammasome pathway were upregulated in B6 MUT, but only Aim2 and Ctsb in NSG MUT mice and none in WT controls. Both B6 and NSG MUT mice demonstrated variably-severe intramural inflammation, vacuolated cells, elastin fragmentation and disarray, and intense glycosaminoglycans on histological staining. B6 MPSI mice demonstrated numerous medial MAC2+ macrophages and adventitial CD3+ T-cells while MAC2+ macrophages were sparse and CD3+ T-cells absent in NSG MPSI mice. CONCLUSIONS: Aortic dilation, AI and decreased cardiac function occur in immunocompetent B6 MPSI male mice but not in immune incompetent NSG MPSI mice, unrelated to GAG excretion, upregulation of Ctsb, or routine histologic appearance. Upregulation of all components of the inflammasome pathway in B6 MUT, but not NSG MUT mice, and abundant medial MAC2 and adventitial CD3 infiltrates in B6, but not NSG, MPSI aortas differentiated the two strains. These results suggest that the innate and adaptive immune systems play a role in these cardiac findings which may be relevant to human MPSI.

Degenerative Myelopathy and Neuropathy in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) Mice Caused by Lactate Dehydrogenase-Elevating Virus (LDV).

Following implantation of patient-derived xenograft (PDX) breast carcinomas from three separate individuals, 33/51 female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice presented with progressive, unilateral to bilateral, ascending hindlimb paresis to paralysis. Mice were mildly dehydrated, in thin to poor body condition, with reduced to absent hindlimb withdrawal reflex and deep pain sensation. Microscopically, there was variable axonal swelling, vacuolation, and dilation of myelin sheaths within the ventral spinal cord and spinal nerve roots of the thoracolumbar and sacral spinal cord, as well as within corresponding sciatic nerves. Results of PCR screening of PDX samples obtained at necropsy and pooled environmental swabs from the racks housing affected animals were positive for lactate dehydrogenase-elevating virus (LDV). LDV is transmitted through animal-animal contact or commonly as a contaminant of biologic materials of mouse origin. Infection is associated with progressive degenerative myelopathy and neuropathy in strains of mice harboring endogenous retrovirus (AKR, C58), or in immunosuppressed strains (NOD-SCID, Foxn1nu), and can interfere with normal immune responses and alter engraftment and growth of xenograft tumors in immunosuppressed mice. This is the first reported series of LDV-induced poliomyelitis in NSG mice and should be recognized as a potentially significant confounder to biomedical studies utilizing immunodeficient xenograft models.

Investigating the response of paediatric leukaemia-propagating cells to BCL-2 inhibitors.

Relapse of paediatric acute lymphoblastic leukaemia (ALL) may occur due to persistence of resistant cells with leukaemia-propagating ability (LPC). In leukaemia, the balance of B-cell lymphoma-2 (BCL-2) family proteins is disrupted, promoting survival of malignant cells and possibly LPC. A direct comparison of BCL-2 inhibitors, navitoclax and venetoclax, was undertaken on LPC subpopulations from B-cell precursor (BCP) and T-cell ALL (T-ALL) cases in vitro and in vivo. Responses were compared to BCL-2 levels detected by microarray analyses and Western blotting. In vitro, both drugs were effective against most BCP-ALL LPC, except CD34- /CD19- cells. In contrast, only navitoclax was effective in T-ALL and CD34- /CD7- LPC were resistant to both drugs. In vivo, navitoclax was more effective than venetoclax, significantly improving survival of mice engrafted with BCP- and T-ALL samples. Venetoclax was not particularly effective against T-ALL cases in vivo. The proportions of CD34+ /CD19- , CD34- /CD19- BCP-ALL cells and CD34- /CD7- T-ALL cells increased significantly following in vivo treatment. Expression of pro-apoptotic BCL-2 genes was lower in these subpopulations, which may explain the lack of sensitivity. These data demonstrate that some LPC were resistant to BCL-2 inhibitors and sustained remission will require their use in combination with other therapeutics.