Passage In Mice Research Articles

On the contribution of the rodent model Plasmodium chabaudi for understanding the genetics of drug resistance in malaria.

Malaria is a devastating disease that still claims over half a million lives every year, mostly in sub-Saharan Africa. One of the main barriers to malaria control is the evolution and propagation of drug-resistant mutant parasites. Knowing the genes and respective mutations responsible for drug resistance facilitates the design of drugs with novel modes of action and allows predicting and monitoring drug resistance in natural parasite populations in real-time. The best way to identify these mutations is to experimentally evolve resistance to the drug in question and then comparing the genomes of the drug-resistant mutants to that of the sensitive progenitor parasites. This simple evolutive concept was the starting point for the development of a paradigm over the years, based on the use of the rodent malaria parasite Plasmodium chabaudi to unravel the genetics of drug resistance in malaria. It involves the use of a cloned parasite isolate (P. chabaudi AS) whose genome is well characterized, to artificially select resistance to given drugs through serial passages in mice under slowly increasing drug pressure. The end resulting parasites are cloned and the genetic mutations are then discovered through Linkage Group Selection, a technique conceived by Prof. Richard Carter and his group, and/or Whole Genome Sequencing. The precise role of these mutations can then be interrogated in malaria parasites of humans through allelic replacement experiments and/or genotype-phenotype association studies in natural parasite populations. Using this paradigm, all the mutations underlying resistance to the most important antimalarial drugs were identified, most of which were pioneering and later shown to also play a role in drug resistance in natural infections of human malaria parasites. This supports the use of P. chabaudi a fast-track predictive model to identify candidate genetic markers of resistance to present and future antimalarial drugs and improving our understanding of the biology of resistance.

Establishment of 6 pediatric rhabdomyosarcoma patient's derived xenograft models closely recapitulating patients' tumor characteristics.

The prognosis for patients with metastatic and recurrent pediatric rhabdomyosarcoma (RMS) remains poor. The availability of preclinical models is essential to identify promising treatments We established a series of pediatric RMS patient derived xenografts (PDXs), all faithfully mirroring primary tumor characteristics and representing a unique tool for clarifying the biological processes underlying RMS progression and relapse. Fresh tumor samples from 12 RMS patients were implanted subcutaneously in both flanks of immunocompromised mice. PDXs were considered as grafted after accomplishing three passages in mice. Characterization of tumor tissues and models was performed by comparing both morphology and immunoistochemical and fluorescence in situ hybridization (FISH) characteristics. Six PDXs were established, with a successful take rate of 50%. All models closely mirrored parental tumor characteristics. An increased grafting rate for tumors derived from patients with worse outcome (p = 0.006) was detected. For 50% PDXs grafting occurred when the corresponding patient was still alive. Our findings increase the number of available RMS PDX models and strengthen the role of PDXs as useful preclinical tools for patients with unmet medical needs and to develop personalized therapies.

Generation and characterization of patient-derived xenografts from patients with osteosarcoma

BackgroundStandard therapy of osteosarcoma (OS) rests on cytotoxic regimes, which dramatically limits the further improvement in the prognosis of patients with OS. Preclinical models of OS are extremely urgent to break this impasse. Materials and MethodsHere, we describe our experience of developing patient-derived tumor xenografts (PDXs) using surgical samples from patients with OS. All the xenografts growing subcutaneously in mice will be identified pathologically and genetically. Furthermore, we explored the consistency of the drug sensitivity between the PDX models and corresponding patients against specific regimens. ResultsWe generated nine OS PDXs from 21 surgical resections of primary OS tumors, with an engraftment rate of 42.9 %. Morphological and immunohistochemical (SATB2, MDM2, CDK4, and Ki67) studies and whole-exome sequencing showed a remarkable similarity between the patient’s tumor and corresponding PDX, which was maintained over several passages in mice. A therapeutic combination of Pirarubicin and Cisplatin was tested against two OS PDX models, in which the corresponding patient was insensitive and sensitive to the regimen, respectively. ConclusionThe panel of OS PDX models faithfully mirrored morphologic and genetic features of OS, representing reliable models to test therapeutic approaches.

Evolution of SIVmac239 following serial passaging in humanized mice.

Serial passage of SIVmac239 allows for greater understanding of the genetic changes necessary for cross‐species transmission of primate lentiviruses into humans. Using humanized mice, we show that adaptive mutations continue to accumulate in SIVmac239 during four serial passages, with persistent CD4+ T cell decline and increases in plasma viral loads.

Antiplasmodial effects of aqueous leaf extracts of Senna alata and Dennettia tripetalla in chloroquine-sensitive Plasmodium berghei berghei (NK65) infected mice

The antiplasmodial effects of Senna alata and Dennettia tripetalla on chloroquine-sensitive Plasmodium berghei berghei (NK65) were analysed. P. berghei was obtained from National Institute for Medical Research, Lagos, Nigeria, and maintained in the laboratory by serial passage in mice. Chemosuppressive, prophylactic, and curative procedures were used to evaluate the antiplasmodial potentials of the extracts against established infection. A total of 320 albino mice were used for this study. Twenty-five albino mice were divided into 5 groups and used for D. tripetalla acute toxicity study. Eighty-five albino mice were divided into 15 groups of five albino mice each and used for chemosuppressive tests while two hundred and ten mice were used for prophylactic and curative tests. The infected mice were orally treated daily with 5mg/kg chloroquine 500 mg/kg, 1000 mg/kg, 1500 mg/kg, and 2000 mg/kg body weight of S. alata, D. tripetalla, and S. alata + D. tripetalla respectively. The in vivo antiplasmodial effect of the extracts against P. berghei infection showed a significant (p?0.05) dose-dependent schizontocidal activity for the chemosuppressive, and prophylactic test. Results of the curative study showed that the combination of both S. alata and D. tripetalla exhibited a strong curative effect on malaria parasitaemia. There was a significant clearance (p?0.05) in parasiteamia level from day 1 to day 5 of treatment in a dose-dependent manner with a corresponding significant (p?0.05) percentage suppression. It was therefore concluded that the leaf extracts of S. alata and D. tripetalla possess antimalarial potencies which could be exploited for antimalarial therapy.

Expanding Mouse-Adapted Yamagata-like Influenza B Viruses in Eggs Enhances In Vivo Lethality in BALB/c Mice.

Despite the yearly global impact of influenza B viruses (IBVs), limited host range has been a hurdle to developing a readily accessible small animal disease model for vaccine studies. Mouse-adapting IBV can produce highly pathogenic viruses through serial lung passaging in mice. Previous studies have highlighted amino acid changes throughout the viral genome correlating with increased pathogenicity, but no consensus mutations have been determined. We aimed to show that growth system can play a role in mouse-adapted IBV lethality. Two Yamagata-lineage IBVs were serially passaged 10 times in mouse lungs before expansion in embryonated eggs or Madin–Darby canine kidney cells (London line) for use in challenge studies. We observed that virus grown in embryonated eggs was significantly more lethal in mice than the same virus grown in cell culture. Ten additional serial lung passages of one strain again showed virus grown in eggs was more lethal than virus grown in cells. Additionally, no mutations in the surface glycoprotein amino acid sequences correlated to differences in lethality. Our results suggest growth system can influence lethality of mouse-adapted IBVs after serial lung passaging. Further research can highlight improved mechanisms for developing animal disease models for IBV vaccine research.

Generation of a pdmH1N1 2018 Influenza A Reporter Virus Carrying a mCherry Fluorescent Protein in the PA Segment.

Influenza A virus (IAV) is a major human pathogen associated with significant morbidity and mortality worldwide. Through serial passage in mice, we generated a recombinant pdmH1N1 2009 IAV, A/Guangdong/GLW/2018 (GLW/18-MA), which encodes an mCherry gene fused to the C-terminal of a polymerase acidic (PA) segment and demonstrated comparable growth kinetics to the wild-type. Nine mutations were identified in the GLW/18-MA genome: PA (I61M, E351G, and G631S), NP (E292G), HA1 (T164I), HA2 (N117S and P160S), NA (W61R), and NEP (K44R). The recombinant IAV reporter expresses mCherry, a red fluorescent protein, at a high level and maintains its genetic integrity after five generations of serial passages in Madin-Darby Canine Kidney cells (MDCK) cells. Moreover, the imaging is noninvasive and permits the monitoring of infection in living mice. Treatment with oseltamivir or baicalin followed by infection with the reporter IAV led to a decrease in fluorescent protein signal in living mice. This result demonstrates that the IAV reporter virus is a powerful tool to study viral pathogenicity and transmission and to develop and evaluate novel anti-viral drugs, inhibitors, and vaccines in the future.

The Occurrence of Malignancy in Trypanosoma brucei brucei by Rapid Passage in Mice.

Pleomorphic Trypanosoma brucei are best known for their tightly controlled cell growth and developmental program, which ensures their transmissibility and host fitness between the mammalian host and insect vector. However, after long-term adaptation in the laboratory or by natural evolution, monomorphic parasites can be derived. The origin of these monomorphic forms is currently unclear. Here, we produced a series of monomorphic trypanosome stocks by artificially syringe-passage in mice, creating snapshots of the transition from pleomorphism to monomorphism. We then compared these artificial monomorphic trypanosomes, alongside several naturally monomorphic T. evansi and T. equiperdum strains, with the pleomorphic T. brucei. In addition to failing to generate stumpy forms in animal bloodstream, we found that monomorphic trypanosomes from laboratory and nature exhibited distinct differentiation patterns, which are reflected by their distinct differentiation potential and transcriptional changes. Lab-adapted monomorphic trypanosomes could still be induced to differentiate, and showed only minor transcriptional differences to that of the pleomorphic slender forms but some accumulated differences were observed as the passages progress. All naturally monomorphic strains completely fail to differentiate, corresponding to their impaired differentiation regulation. We propose that the natural phenomenon of trypanosomal monomorphism is actually a malignant manifestation of protozoal cells. From a disease epidemiological and evolutionary perspective, our results provide evidence for a new way of thinking about the origin of these naturally monomorphic strains, the malignant evolution of trypanosomes may raise some concerns. Additionally, these monomorphic trypanosomes may reflect the quantitative and qualitative changes in the malignant evolution of T. brucei, suggesting that single-celled protozoa may also provide the most primitive model of cellular malignancy, which could be a primitive and inherent biological phenomenon of eukaryotic organisms from protozoans to mammals.

In vitro selection of ketoconazole-pentamidine-resistant Leishmania (Viannia) braziliensis strains

The use of ketoconazole (KTZ) plus pentamidine (PMD) could be an interesting treatment option for New World cutaneous leishmaniasis. The aim of this work was to generate KTZ- and PMD-resistant strains and to determine some characteristics of the selection process and the resulting parasites. Resistance to one or two drugs was selected on promastigotes by progressively increasing drug concentrations for eleven months. The resistance levels (IC50) to one or two drugs (synergism assay) were determined using a colorimetric resazurin methodology. The stability of the resistance phenotype (without drug pressure or after mouse passage), cross resistance with paromomycin and miltefosine, and resistance transference to intracellular amastigotes were determined. In addition, some parasite attributes compared with WT, such as growth kinetics, amastigogenesis, THP-1 cells, and mouse infection, were determined. Promastigotes resistant to KTZ or PMD were obtained three times earlier than the combined KTZ + PMD-resistant strains. Resistant parasites (promastigotes and intracellular amastigotes) were three to twelve times less susceptible to KTZ and PMD than WT parasites. The resistance phenotype on parasites was unstable, and no cross resistance was observed. Similar parasite fitness related to our evaluated characteristics was observed except for in vivo infection, where a delay of the onset of cutaneous lesions was observed after KTZ + PMD-resistant parasite infection. ConclusionCombined treatment with KTZ and PMD delayed the onset of parasite resistance and was more effective in vitro than each drug separately for WT and all resistant strains. Parasites resistant to KTZ and PMD acquired similar in vitro behaviour to WT parasites, were less virulent to mice and maintained their resistance phenotype on intracellular amastigotes but not without drug pressure or after mouse infection.

Patient-Derived Xenografts of High-Grade Serous Ovarian Cancer Subtype as a Powerful Tool in Pre-Clinical Research.

(1) Background. PDX models have become the preferred tool in research laboratories seeking to improve development and pre-clinical testing of new drugs. PDXs have been shown to capture the cellular and molecular characteristics of human tumors better than simpler cell line-based models. More recently, however, hints that PDXs may change their characteristics over time have begun to emerge, emphasizing the need for comprehensive analysis of PDX evolution. (2) Methods. We established a panel of high-grade serous ovarian carcinoma (HGSOC) PDXs and developed and validated a 300-SNP signature that can be successfully utilized to assess genetic drift across PDX passages and detect PDX contamination with lymphoproliferative tissues. In addition, we performed a detailed histological characterization and functional assessment of multiple PDX passages. (3) Results. Our data show that the PDXs remain largely stable throughout propagation, with marginal genetic drift at the time of PDX initiation and adaptation to mouse host. Importantly, our PDX lines retained the major histological characteristics of the original patients' tumors even after multiple passages in mice, demonstrating a strong concordance with the clinical responses of their corresponding patients. (4) Conclusions. Our data underline the value of defined HGSOC PDXs as a pre-clinical tumor model.

Intercellular interactions between mast cells and stromal fibroblasts obtained from canine cutaneous mast cell tumours

Mast cell tumours (MCTs) are the most frequent malignant skin neoplasm in dogs. Due to the difficulty in purifying large numbers of canine neoplastic mast cells, relatively little is known about their properties. A reproducible in vitro model is needed to increase the understanding about the phenotype and functional properties of neoplastic mast cells. In the present study, we describe the establishment of primary cocultures of neoplastic mast cells from canine cutaneous MCTs and cancer-associated fibroblasts. We confirmed the inability of canine neoplastic mast cells to remain viable for long periods in vitro without the addition of growth factors or in vivo passages in mice. Using a transwell system, we observed that mast cell viability was significantly higher when there is cell-to-cell contact in comparison to non-physical contact conditions and that mast cell viability was significantly higher in high-grade than in low-grade derived primary cultures. Moreover, the use of conditioned medium from co-cultured cells led to a significantly higher tumoral mast cell viability when in monoculture. Signalling mechanisms involved in these interactions might be attractive therapeutic targets to block canine MCT progression and deserve more in-depth investigations.

Development and characterization of an animal model of Japanese encephalitis virus infection in adolescent C57BL/6 mouse.

ABSTRACTA mouse-adapted isolate of Japanese encephalitis virus (JEV), designated as JEV-S3, was generated by serially passaging the P20778 strain of the virus in 3- to 4-week-old C57BL/6 mice. Blood-brain barrier leakage was evident in JEV-S3-infected mice, in which viral antigens and RNA were consistently demonstrated in the brain, along with infiltration of activated immune cells, as evidenced by an increased CD45+CD11b+ cell population. Histopathology studies showed the presence of perivascular cuffing, haemorrhage and necrotic foci in the virus-infected brain, conforming to the pathological changes seen in the brain of JEV-infected patients. Mass spectrometry studies characterized the molecular events leading to brain inflammation in the infected mice. Notably, a significant induction of inflammatory cytokines, such as IFNγ, IL6, TNFα and TGFβ, was observed. Further, genome sequencing of the JEV-S3 isolate identified the mutations selected during the mouse passage of the virus. Overall, we present an in-depth characterization of a robust and reproducible mouse model of JEV infection. The JEV-S3 isolate will be a useful tool to screen antivirals and study virus pathogenesis in the adolescent mouse model.

Neuroinvasiveness of the MR766 strain of Zika virus in IFNAR-/-mice maps to prM residues conserved amongst African genotype viruses.

Zika virus (ZIKV) strains are classified into the African and Asian genotypes. The higher virulence of the African MR766 strain, which has been used extensively in ZIKV research, in adult IFNα/β receptor knockout (IFNAR-/-) mice is widely viewed as an artifact associated with mouse adaptation due to at least 146 passages in wild-type suckling mouse brains. To gain insights into the molecular determinants of MR766’s virulence, a series of genes from MR766 were swapped with those from the Asian genotype PRVABC59 isolate, which is less virulent in IFNAR-/- mice. MR766 causes 100% lethal infection in IFNAR-/- mice, but when the prM gene of MR766 was replaced with that of PRVABC59, the chimera MR/PR(prM) showed 0% lethal infection. The reduced virulence was associated with reduced neuroinvasiveness, with MR766 brain titers ≈3 logs higher than those of MR/PR(prM) after subcutaneous infection, but was not significantly different in brain titers of MR766 and MR/PR(prM) after intracranial inoculation. MR/PR(prM) also showed reduced transcytosis when compared with MR766 in vitro. The high neuroinvasiveness of MR766 in IFNAR-/- mice could be linked to the 10 amino acids that differ between the prM proteins of MR766 and PRVABC59, with 5 of these changes affecting positive charge and hydrophobicity on the exposed surface of the prM protein. These 10 amino acids are highly conserved amongst African ZIKV isolates, irrespective of suckling mouse passage, arguing that the high virulence of MR766 in adult IFNAR-/- mice is not the result of mouse adaptation.

The Ablation of Envelope Protein Glycosylation Enhances the Neurovirulence of ZIKV and Cell Apoptosis in Newborn Mice.

Zika virus (ZIKV) has attracted the wide global attention due to its causal link to microcephaly. In this study, two amino acid (aa) mutation (E143K and R3394K) were identified at the fourth generation (named ZKC2P4) during the serial passage of ZIKV-Asian lineage ZKC2/2016 strain in the newborn mouse brain, while another seven aa deletions in envelope (E) protein were detected in ZKC2P6. ZKC2P6 is a novel nonglycosylated E protein Asian ZIKV we first identified and provides the first direct supporting evidence that glycosylation motif could be lost during the passage in neonatal mice. To study the impact of E protein glycosylation ablation, we compared the pathogenicity of ZKC2P6 with that of ZKC2P4. The results showed that the loss of E protein glycosylation accelerated the disease progression, as evidenced by an earlier weight loss and death, a thinner cerebral cortex, and more serious tissue lesions and inflammation/necrosis. Furthermore, ZKC2P6 exhibited a greater ability to replicate and caused severer cell apoptosis than that of ZKC2P4. Therefore, the ablation of E glycosylation generally enhances the neurovirulence of ZIKV and cell apoptosis in newborn mice.

Abstract 1075: AXL is a potential druggable target in trastuzumab resistance in HER2+ breast cancer patients

Abstract Introduction HER2+ correlates with aggressive phenotype and poor prognosis. The use of several antiHER2 drugs has dramatically improved prognosis and survival of HER2+ breast cancer (BC) patients. However, ≈20% of these patients will present resistance to antiHER2 therapies and relapse. The purpose of this study is to explore the role of AXL in antiHER2 treatment resistance and its potential as a druggable biomarker in HER2+ BC. Methods We generated 2 trastuzumab (T) resistant models from a HER2+ BC patient. The first one was a PDX derived resistant cell line established by chronic treatment for 6 months with T in vitro (TR1 and TR2). The second resistant model (TR4) was established by chronic treatment with T for 4 months and two serial passages in mice. The prognostic value of AXL was evaluated in primary tumor cohort of HER2+ BC patients treated by standard guidelines (n=51). AXL was also explored in a cohort from PAMELA trial, a neoadjuvant phase II study. Patients were given lapatinib and T for 18 weeks (Luminal-HER2+ patients were additionally given hormonotherapy). Tumor samples were collected at baseline, day 14 and surgery (n= 96). Results PDX resistant cell lines (TR1 and TR2) and resistant tumors (TR4) exhibit upregulation of AXL in comparison to the parental cells (PDX118). In vitro, the combination of an AXL inhibitor (TP-0903) + T resensitized resistant cells to T. In 3D organoids models from TR4, the combination decreased cell number in organoids more than both single agents. In TR4 in vivo model, TP-0903 + T completely abrogate tumor growth for more than 2 months.To validate our preclinical findings, we analyzed the level of AXL in our retrospectively cohort of patients. AXL level was significantly upregulated in patients who relapsed (p<0.0001). Patients with high levels of AXL had significantly shorter disease-free survival (DFS) and overall survival (OS) (log-rank p<0.0001; HR=15.78; 95% CI 3.89-19.80 for DFS; log-rank p=0.013; HR= 5.43; 95% CI 1.35-13.07 for OS). The median DFS and OS were 36 and 77 months in patients with high AXL level, whereas patients with low AXL level presented 92.5 and 92.5 months.To identify early molecular changes induced by dual HER2 blockade in patients with HER2+ disease, AXL was analyzed in PAMELA trial cohort. A significant increase of AXL was detected at day 14 compared to baseline (p=5.8e-20). This effect was observed in both luminal and non-luminal HER2+ BC patients and across all PAM50 subtypes. This rapid response suggests the implication of AXL as a mechanism of antiHER2 resistance. Levels of AXL decreased as long as treatment was on hold at surgery compared to day 14 (p=6.04e-14). Conclusion AXL level was correlated with poor outcome of HER2+ BC patients. On our preclinical models, we showed that combination of TP-0903 and T abrogate tumor growth in acquired resistant models to T in PDXs. We postulate AXL as a promising new therapeutic strategy to overcome resistance to antiHER2 therapies Citation Format: Anna Adam-Artigues, Raimundo Cervera, Enrique Javier Arenas, Fara Brasó-Maristany, Alex Martinez-Sabadell, Eduardo Tormo, Cristina Hernando, Maria Teresa Martinez, Soraya Simon, Jesús Poveda, Octavio Burgués, Ana Rovira, Federico Rojo, Joan Albanell, Begoña Bermejo, Ana Lluch, Pilar Eroles, Aleix Prat, Joaquin Arribas, Juan Miguel Cejalvo. AXL is a potential druggable target in trastuzumab resistance in HER2+ breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1075.

Establishing Mouse Models of B-Cell Non-Hodgkin's Lymphoma

There are numerous types of B-cell lymphoma including Burkitt lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma/mucosa-associated lymphoid tissue lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma and other less frequent subtypes. However, the molecular pathogenesis of B-cell lymphoma remain largely unknown. Here we chose three different B-cell lymphoma subtype cell lines to establish mouse models, which could be useful to determine the efficacy and mechanisms of novel therapies.Firstly, we injected subcutaneously lymphoma cells into the right axillary region of BALB/c (nu/nu) nude mice. DOHH2 F0, Ly8 F0, Raji F0 appeared 7, 7, 21 days later. When tumors growed to 1.5cm at length, successful passage of mouse models was established by injecting cell suspension obtained from mincing and grinding tumors through a 70-μm cell strainer. The cell suspension was cultured at 37°C in a humidified atmosphere containing 5% CO2. The cells were cryopreserved in cryopreservation-medium (90% FBS, 10% DMSO), stored in liquid nitrogen, thawed again (with a viability of more than 70%) and successfully reconstituted. The above cell lines mouse models have been passaged to the 16th, 18th, 13th generation.For histological examination, an autopsy was performed on each passage of nude mice, and immunohistochmistry was use to determine the expression of surface markers. The tumor tissues from DOHH2 F0, DOHH2 F6, DOHH2 F12 were positive for CD20, cytoplasmic CD3, CD10, CD5, CD21, Ki67, CyclinD1, Bcl-2, Bcl-6. EBV was also detected in situ hybridization. Grade 3B Follicular lymphoma was identified in this B-cell lymphoma and was consistent with the primary origination of DOHH2. Immunohistochemical staining of Ly8 F0, Ly8 F6, Ly8 F12 showed the large atypical cells were positive for CD20, CD3, CD10, Bcl-2, mum-1, no reactivity to antibodies for bcl-6 and negative to EBER. Ki-67 values were graded as 30%. The pathologic expression in Raji F0, Raji F6, Raji F12 is like the following: CD20+/CD3+-/Ki67+-/CD10+/Bcl2+-/ Bcl6- /Mum1+/EBER++.Flow cytometry analysis was performed on cell suspensions cultured from each passage tumor tissues. DOHH2 F0, DOHH2 F6,DOHH2 F12 cells were CD19+/CD20-/CD10+/CD5-/CD38+/HLA-DR+/ CD22+ /CD23+/Ki-67+CD71+, and the kappa light chain was expressed monoclonally. Similar to DOHH2 cells, the serial Ly8 cells were CD19+ /CD20-/CD10+/CD5-/CD38+/HLA-DR+/CD22+/CD23+Ki-67+CD71+/EBER-, and the kappa light chain was expressed monoclonally. As Burkitt lymphoma, Raji and its serial cell clones were CD19+/CD20+/CD10+/CD5-/CD38+/HLA-DR+/CD22+/Ki-67+/CD71+/EBER++.In conclusions, we firstly and systematically established mouse models of B-cell lymphoma and take into passage successfully. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Risk of Environmental Exposure to H7N9 Influenza Virus via Airborne and Surface Routes in a Live Poultry Market in Hebei, China

Environmental transmission of viruses to humans has become an early warning for potential epidemic outbreaks, such as SARS-CoV-2 and influenza virus outbreaks. Recently, an H7N9 virus, A/environment/Hebei/621/2019 (H7N9), was isolated by environmental swabs from a live poultry market in Hebei, China. We found that this isolate could be transmitted by direct contact and aerosol in mammals. More importantly, after 5 passages in mice, the virus acquired two adaptive mutations, PB1-H115Q and B2-E627K, exhibiting increased virulence and aerosol transmissibility. These results suggest that this H7N9 virus might potentially be transmitted between humans through environmental or airborne routes.

SARS-CoV-2 infection, neuropathogenesis and transmission among deer mice: Implications for spillback to New World rodents.

Coronavirus disease-19 (COVID-19) emerged in late 2019 in China and rapidly became pandemic. As with other coronaviruses, a preponderance of evidence suggests the virus originated in horseshoe bats (Rhinolophus spp.) and may have infected an intermediate host prior to spillover into humans. A significant concern is that SARS-CoV-2 could become established in secondary reservoir hosts outside of Asia. To assess this potential, we challenged deer mice (Peromyscus maniculatus) with SARS-CoV-2 and found robust virus replication in the upper respiratory tract, lungs and intestines, with detectable viral RNA for up to 21 days in oral swabs and 6 days in lungs. Virus entry into the brain also occurred, likely via gustatory-olfactory-trigeminal pathway with eventual compromise to the blood-brain barrier. Despite this, no conspicuous signs of disease were observed, and no deer mice succumbed to infection. Expression of several innate immune response genes were elevated in the lungs, including IFNα, IFNβ, Cxcl10, Oas2, Tbk1 and Pycard. Elevated CD4 and CD8β expression in the lungs was concomitant with Tbx21, IFNγ and IL-21 expression, suggesting a type I inflammatory immune response. Contact transmission occurred from infected to naive deer mice through two passages, showing sustained natural transmission and localization into the olfactory bulb, recapitulating human neuropathology. In the second deer mouse passage, an insertion of 4 amino acids occurred to fixation in the N-terminal domain of the spike protein that is predicted to form a solvent-accessible loop. Subsequent examination of the source virus from BEI Resources determined the mutation was present at very low levels, demonstrating potent purifying selection for the insert during in vivo passage. Collectively, this work has determined that deer mice are a suitable animal model for the study of SARS-CoV-2 respiratory disease and neuropathogenesis, and that they have the potential to serve as secondary reservoir hosts in North America.

A novel pseudovirus\u2010based mouse model of SARS-CoV-2 infection to test COVID-19 interventions

BackgroundThe spread of SARS-CoV-2, the virus that causes Coronavirus Disease 2019 (COVID-19), has been characterized as a worldwide pandemic. Currently, there are few preclinical animal models that suitably represent infection, as the main point of entry to human cells is via human angiotensin-converting enzyme 2 (ACE2) which is not present in typical preclinical mouse strains. Additionally, SARS-CoV-2 is highly virulent and unsafe for use in many research facilities. Here we describe the development of a preclinical animal model using intranasal administration of ACE2 followed by non-infectious SARS-CoV-2 pseudovirus (PsV) challenge.MethodsTo specifically generate our SARS-CoV-2 PsV, we used a lentivirus system. Following co-transfection with a packaging plasmid containing HIV Gag and Pol, luciferase-expressing lentiviruses, and a plasmid carrying the SARS-CoV-2 spike protein, SARS-CoV-2 PsVs can be isolated and purified. To better understand and maximize the infectivity of SARS-CoV-2 PsV, we generated PsV carrying spike protein variants known to have varying human ACE2 binding properties, including 19 deletion (19del) and 19del + D614G.ResultsOur system demonstrated the ability of PsVs to infect the respiratory passage of mice following intranasal hACE2 transduction. Additionally, we demonstrate in vitro and in vivo manipulability of our system using recombinant receptor-binding domain protein to prevent PsV infection.ConclusionsOur PsV system is able to model SARS-CoV-2 infections in a preclinical mouse model and can be used to test interventions or preventative treatments. We believe that this method can be extended to work in various mouse strains or to model infection with different coronaviruses. A simple in vivo system such as our model is crucial for rapidly and effectively responding to the current COVID-19 pandemic in addition to preparing for future potential coronavirus outbreaks.

RGB-Marking to Identify Patterns of Selection and Neutral Evolution in Human Osteosarcoma Models.

Simple SummaryNowadays, we are assisting a re-discovered interest in the field of cancer heterogeneity and in defining the clonal dynamics governing tumor growth, progression, and therapy resistance. Sequencing data suggest different models of cancers development and a relationship with patients’ prognosis and therapeutic response. Only a few studies have attempted to reconstruct osteosarcoma evolution, providing evidence of linear and branched pattern of clonal development. In this study, we employed a single-cell marking strategy to study the clonal dynamics of human, canine, and murine osteosarcoma models. With our collection of primary samples and cell lines, we demonstrate that different clones can evolve in parallel and generate sub-clones with similar tumorigenic potential, in a sort of extremely branched development of neutrally coexisting clones.Osteosarcoma (OS) is a highly aggressive tumor characterized by malignant cells producing pathologic bone; the disease presents a natural tendency to metastasize. Genetic studies indicate that the OS genome is extremely complex, presenting signs of macro-evolution, and linear and branched patterns of clonal development. However, those studies were based on the phylogenetic reconstruction of next-generation sequencing (NGS) data, which present important limitations. Thus, testing clonal evolution in experimental models could be useful for validating this hypothesis. In the present study, lentiviral LeGO-vectors were employed to generate colorimetric red, green, blue (RGB)-marking in murine, canine, and human OS. With this strategy, we studied tumor heterogeneity and the clonal dynamics occurring in vivo in immunodeficient NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ (NSG) mice. Based on colorimetric label, tumor clonal composition was analyzed by confocal microscopy, flow cytometry, and different types of supervised and unsupervised clonal analyses. With this approach, we observed a consistent reduction in the clonal composition of RGB-marked tumors and identified evident clonal selection at the first passage in immunodeficient mice. Furthermore, we also demonstrated that OS could follow a neutral model of growth, where the disease is defined by the coexistence of different tumor sub-clones. Our study demonstrates the importance of rigorous testing of the selective forces in commonly used experimental models.