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.

Lentiviral vector ALS20 yields high hemoglobin levels with low genomic integrations for treatment of beta-globinopathies.

Ongoing clinical trials for treatment of beta-globinopathies by gene therapy involve the transfer of the beta-globin gene, which requires integration of three to four copies per genome in most target cells. This high proviral load may increase genome toxicity, potentially limiting the safety of this therapy and relegating its use to total body myeloablation. We hypothesized that introducing an additional hypersensitive site from the locus control region, the complete sequence of the second intron of the beta-globin gene, and the ankyrin insulator may enhance beta-globin expression. We identified a construct, ALS20, that synthesized significantly higher adult hemoglobin levels than those of other constructs currently used in clinical trials. These findings were confirmed in erythroblastic cell lines and in primary cells isolated from sickle cell disease patients. Bone marrow transplantation studies in beta-thalassemia mice revealed that ALS20 was curative at less than one copy per genome. Injection of human CD34+ cells transduced with ALS20 led to safe, long-term, and high polyclonal engraftment in xenograft experiments. Successful treatment of beta-globinopathies with ALS20 could potentially be achieved at less than two copies per genome, minimizing the risk of cytotoxic events and lowering the intensity of myeloablation.

Pathology of macrophage activation syndrome in humanized NSGS mice.

"Humanized" immunodeficient mice generated via the transplantation of CD34+ human hematopoietic stem cells (hHSC) are an important preclinical model system. The triple transgenic NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mouse line is increasingly used as recipient for CD34+ hHSC engraftment. NSGS mice combine the features of the highly immunodeficient NSG mice with transgenic expression of the human myeloid stimulatory cytokines GM-CSF, IL-3, and Kit ligand. While generating humanized NSGS (huNSGS) mice from two independent cohorts, we encountered a fatal macrophage activation syndrome (MAS)-like phenotype resulting from the transplantation of CD34+ hHSC. huNSGS mice exhibiting this phenotype declined clinically starting at approximately 10 weeks following CD34+ hHSC engraftment, with all mice requiring euthanasia by 16 weeks. Gross changes comprised small, irregular liver, splenomegaly, cardiomegaly, and generalized pallor. Hematological abnormalities included severe thrombocytopenia and anemia. Pathologically, huNSGS spontaneously developed a disseminated histiocytosis with infiltrates of activated macrophages and hemophagocytosis predominantly affecting the liver, spleen, bone marrow, and pancreas. The infiltrates were chimeric with a mixture of human and mouse macrophages. Immunohistochemistry suggested activation of the inflammasome in both human and murine macrophages. Active Epstein-Barr virus infection was not a feature. Although the affected mice exhibited robust chimerism of the spleen and bone marrow, the phenotype often developed in the face of low chimerism of the peripheral blood. Given the high penetrance and early lethality associated with the MAS-like phenotype here described, we urge caution when considering the use of huNSGS mice for the development of long-term studies.

Metastatic Mixed Germ Cell Tumour with Embryonal Carcinoma and Choriocarcinoma in a Female Eurasian Harvest Mouse (Micromys minutus).

Mixed germ cell tumours occur rarely in veterinary species. This report describes a case of metastatic mixed germ cell tumour in a female Eurasian harvest mouse (Micromys minutus). The tumour was extensive in one ovary and the uterus, and was characterized by two distinct tumour cell populations with features typical of embryonal carcinoma (EC) and choriocarcinoma (CC). Metastases of CC to the lungs and liver were observed. The exact origin of the CC was unclear, but the possibility of a non-gestational CC is favoured, given the context of a mixed germ cell tumour and lack of p53 expression. EC diagnosis was confirmed by immunohistochemical labelling of CD30 and lack of immunoreactivity for c-Kit. In addition, membranous ß-catenin expression was present in the EC component, indicating an inactive Wnt/ß-catenin pathway, which is required for the maintenance of pluripotency.

Spontaneous Incidental Brain Lesions in C57BL/6J Mice.

Genetically engineered mouse lines on a C57BL/6J background are widely employed as preclinical models to study neurodegenerative human disorders and brain tumors. However, because of the lack of comprehensive data on the spontaneous background neuropathology of the C57BL/6J strain, discriminating between naturally occurring changes and lesions caused by experimental mutations can be challenging. In this context, this study aims at defining the spectrum and frequency of spontaneous brain changes in a large cohort of C57BL/6J mice and their association with specific biological variables, including age and sex. Brains from 203 experimentally naive and clinically unremarkable C57BL/6J mice were collected and analyzed by means of histopathology and immunohistochemistry. Mice ranged in age from 3 to 110 weeks with 89 females, 111 males, and 3 unknowns. Sixteen different spontaneous lesion categories were described in this cohort. Age-related neurodegenerative and/or neuroinflammatory findings represented the most common pathologic changes and included (1) Hirano-like inclusions in the thalamic neurons, (2) neuroaxonal dystrophy in the medulla oblongata, (3) periodic acid-Schiff-positive granular deposits in the neuropil of the hippocampus, and (4) progressive neuroinflammation characterized by microgliosis and astrogliosis. Neoplastic conditions, developmental abnormalities, and circulatory disorders were rarely observed incidental findings. In conclusion, this study describes spontaneous age-related brain lesions of the C57BL/6J mouse and provides a reference for evaluating and interpreting the neuropathological phenotype in genetically engineered mouse models developed and maintained on this congenic background.

Co-occurrence of Nasal Polyps and Neoplasms of the Canine Nasal Cavity.

Nasal polyps in dogs are space-occupying soft-tissue masses that have been encountered concurrently with intranasal neoplasia in surgical biopsy specimens. The proportion of nasal polyp co-occurrence with primary nasal tumors was examined, and follow-up biopsies on dogs initially diagnosed with nasal polyp were reviewed. Histologic sections from 321 cases of intranasal neoplasia and 50 cases of nasal polyp from 2004 to 2017 were reviewed. Of the 321 cases of intranasal neoplasia, 51 (16%) had concurrent nasal polyps, and most of these (47/51) had intranasal carcinoma. Twenty-five of the 50 dogs with a primary diagnosis of nasal polyp were rebiopsied, and the diagnoses in these subsequent biopsies were nasal polyp in 15, malignant neoplasm in 9, and intranasal nematode in 1. Nasal polyps occurred frequently in conjunction with nasal carcinoma. In dogs with a diagnosis of nasal polyp, repeat biopsy to reveal possible neoplasia is warranted.