Immunocompromised Mouse Model Research Articles

An Immunocompromised Mouse Model of Candida auris Systemic Infection.

With the recent emergence of multidrug-resistant Candida auris, there is an urgent need for new antifungal compounds with novel pharmacodynamic and pharmacokinetic properties that can treat systemic fungal infections caused by this emerging yeast. Historically, testing the efficacy of treatment for disseminated candidiasis was accomplished using a diverse array of in vivo animal models, including mice which offer an advantage both in their similarities to humans and their lower cost of maintenance. However, in order to create effective in vivo models for testing new antifungal compounds designed to treat systemic infections, it is important that these models also mimic several of the relevant predisposing conditions that can lead to disseminated candidiasis. Here, we describe an immunocompromised mouse model of hematogenously disseminated C. auris infection, which may have utility to test the efficacy of candidate antifungal compounds.

Deficiency of GPI Glycan Modification by Ethanolamine Phosphate Results in Increased Adhesion and Immune Resistance of Aspergillus fumigatus.

Glycosylphosphatidylinositol (GPI)-anchored proteins play important roles in maintaining the function of the cell wall and participating in pathogenic processes. The addition and removal of phosphoethanolamine (EtN-P) on the second mannose residue in the GPI anchor are vital for maturation and sorting of GPI-anchored proteins. Previously, we have shown that deletion of the gpi7, the gene that encodes an EtN-P transferase responsible for the addition of EtN-P to the second mannose residue of the GPI anchor, leads to the mislocalization of GPI-anchored proteins, abnormal polarity, reduced conidiation, and fast germination in Aspergillus fumigatus. In this report, the adherence and virulence of the A. fumigatus gpi7 deletion mutant were further investigated. The germinating conidia of the mutant exhibited an increased adhesion and a higher exposure of cell wall polysaccharides. Although the virulence was not affected, an increased adherence and a stronger inflammation response of the mutant were documented in an immunocompromised mouse model. An in vitro assay confirmed that the Δgpi7 mutant induced a stronger immune response and was more resistant to killing. Our findings, for the first time, demonstrate that in A. fumigatus, GPI anchoring is required for proper organization of the conidial cell wall. The lack of Gpi7 leads to fast germination, stronger immune response, and resistance to macrophage killing.

Abstract P145: The RNA helicase EIF4A is a therapeutic vulnerability in triple-negative breast cancer

Abstract Background: Translational control of oncoprotein expression has been implicated in the pathogenesis of multiple solid tumors. The eukaryotic translation initiation factor 4A (eIF4A) is an RNA helicase that catalyzes the unwinding of secondary structure in the 5′-untranslated region (5′-UTR) of mRNAs to facilitate translation initiation. eIF4A is an essential gene in many cancer cell lines and multiple oncogenes or anti-apoptotic proteins that contain structured 5’-UTRs have been shown to require enhanced eIF4A activity for translation. Zotatifin (eFT226) is a selective eIF4A inhibitor that has been shown previously to inhibit tumor growth in several immunocompromised mouse models and is currently in Phase I clinical trials as a single agent (NCT04092673). In this study we investigated the effects of Zotatifin both as a single agent and in combination with standard-of-care chemotherapy on tumor progression in several syngeneic triple-negative breast cancer (TNBC) mouse models. Methods: The activity of Zotatifin was assessed as single agent in eight different syngeneic p53-null mouse TNBC models across several intrinsic molecular subtypes. Synergism of Zotatifin with chemotherapy was evaluated in three responsive models. In addition, a Zotatifin-resistant model was developed following prolonged drug treatment. Drug response mechanisms and the effect on the tumor immune microenvironment were determined by tandem mass tag mass spectrometry, reverse phase protein array (RPPA) and mass cytometry. Results: Zotatifin treatment slowed tumor growth in six out of eight syngeneic TNBC models without obvious toxicity. Mass spectrometry of acutely treated tumors revealed that Zotatifin inhibited proliferative and stem cell signaling pathways including E2F targets, G2/M checkpoints, and NOTCH signaling, and induced proteins involved in Interferon-α and Interferon-γ responses. Mass cytometry analyses showed that Zotatifin suppressed the infiltration of M2 macrophages and neutrophils and promoted the infiltration of conventional dendritic cells to the tumor microenvironment. Interestingly, combination therapy of Zotatifin with chemotherapy significantly slowed tumor growth and improved survival. However, drug resistance emerged after prolonged treatment. The resistant tumors strikingly displayed a mesenchymal histology and an increased activity of the PI3K-AKT-mTOR pathway as determined by RPPA analysis. Combination treatment of Zotatifin with the mTOR inhibitor Everolimus prolonged survival. Conclusions: eIF4A is a therapeutic vulnerability in TNBC. Zotatifin inhibits tumor progression both through tumor-intrinsic mechanisms and by targeting the tumor immune microenvironment. Synergism between Zotatifin and chemotherapy supports the clinical potential of combination therapies. Citation Format: Na Zhao, Jeffrey Mark Rosen. The RNA helicase EIF4A is a therapeutic vulnerability in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P145.

Safe and Efficient Engraftment of CRISPR-Based ELANE Mono-Allelic Knocked out HSCs in Mice: Evidence for a Novel Treatment for ELANE Neutropenia

ELANE-related severe congenital neutropenia (SCN) is a disorder wherein numerous heterozygous mutations in the ELANE gene lead to misfolding and mislocalization of mutant neutrophil elastase, resulting in the death of myeloid cells and block in neutrophil differentiation.Currently, SCN is treated with daily injections of granulocyte colony-stimulating factor (G-CSF), but patients on this therapy are at risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The only other effective therapy is allogeneic hematopoietic stem cell transplantation that may involve graft versus host disease and risk of serious infections. Thus, there is great interest in safe and efficient therapeutic alternatives.Emendo Biotherapeutics has developed a novel CRISPR-based (OMNI A1) ex-vivo gene editing strategy that involves specific excision of disease-causing ELANE mutant allele. This treatment was tested on CD34 + HSCs from SCN patients and resulted in a significant improvement in the maturation of myeloid cells and their differentiation to normal functional neutrophils.To assess the feasibility and safety of this editing strategy, we conducted an in vivo study using human HSCs derived from two healthy donors that were engrafted into a NOD scid gamma (NSG) immunocompromised mice model. The study involved a single iv injection of HSCs that were either not-treated (NT), electroporated without delivery of CRISPR nuclease (mock) or edited with Emendo's novel nuclease, OMNI A1,(excision). Each group consisted of 6 NSG female mice and an additional group of 4 mice served as vehicle control (no HSCs). All animals were subjected to total body irradiation, 1 day prior to the treatment, at a dose of 250 cGy. Animals were subjected to retro-orbital blood collection, at 4 designated time points during the study (Figure A), for CD45 + cell counts using FACS. All animals were evaluated for systemic reactions, clinical signs and body weight changes during the study period. At the end of the study period, animals were subjected to peripheral blood (PB), bone marrow (BM) and spleen sampling. FACS analysis of CD45 + cells was performed on those samples to determine the engraftment efficiency. Furthermore, excision levels for ELANE were measured in the engrafted cells by droplet digital PCR (ddPCR). Finally, multilineage differentiation capacity of the engrafted cells was evaluated by FACS.Analysis of human CD45 + cells, at 20 weeks, showed comparable levels of engraftment of the not-treated, mock treated and excised HSCs in the mouse PB, BM and spleen (No statistically significant difference, Kruskal Wallis test) (Figure B). Excised cells were detected in the mouse BM and excision levels in some of the mice were comparable to those measured in the HSCs prior to engraftment (about 30-36%). Human-derived cells from the BM and spleen of mice engrafted with excised HSCs showed similar multilineage differentiation capacity as obtained in mice engrafted with either NT or mock-treated HSCs. Engrafted human CD45 + cells differentiated into neutrophils (CD66b +), myeloid cells (CD33 +), Lymphocytes: B-cells (CD19 +) and T-cells (CD3 +). In addition, a population of engrafted hematopoietic CD34 + cells was detected in the BM. Excised HSCs of both donors gave rise to all lineages tested, as efficient as the NT and the mock groups. Finally, engrafted mice showed no systemic reactions, abnormal clinical signs or loss of body weight.These data show that edited cells can be engrafted successfully, maintain their excision profile and populate the bone marrow. This study also supports the safety of the OMNI A1 therapeutic composition. [Display omitted] DisclosuresPovodovski: Emendo Biotherapeutic: Current Employment. Makaryan: Emendo Biotherapeutic: Research Funding. Sabo: Emendo Biotherapeutic: Research Funding. Dicken: Emendo Biotherapeutic: Ended employment in the past 24 months. Herman: Emendo Biotherapeutic: Current Employment. Emmanuel: Emendo Biotherapeutic: Current Employment. Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding.

The Effects of Bletilla striata Polysaccharides on Antioxidant and Immunomodulatory Activities of Mice In Vivo

Bletilla striata polysaccharide is a well-known antioxidant and immunomodulatory ingredient used in a variety of traditional Chinese medicine applications. A mouse aging model was constructed by intraperitoneal injection of D-galactose to explore the antioxidant and immunomodulatory effects of B. striata polysaccharides. B. striata polysaccharide was found to boost antioxidant capacity and retard aging in a subacutely aged mouse. In comparison to the cyclophosphamide alone group, the groups given 80 mg/kg and 160 mg/kg of B. striata polysaccharide showed considerably higher levels of total antioxidant capacity, superoxide dismutase, and catalase, with significantly lower malondialdehyde levels. To investigate the moderating effect of B. striata polysaccharide on immunological deficit, cyclophosphamide was injected into the abdominal cavity to create an immunocompromised mouse model. B. striata polysaccharide attenuated immunosuppressive effects of cyclophosphamide. The histological staining demonstrated that both liver and spleen cells in the control group were densely packed, with distinct nuclei and qualitative characteristics. The liver and spleen of the immunocompromised mice benefited significantly from 450 mg/kg B. striata polysaccharide. Taken together, B. striata polysaccharide exhibits an antioxidant and immunomodulatory effects in vivo.

Immunocompromised Cas9 transgenic mice for rapid in vivo assessment of host factors involved in highly pathogenic virus infection

Targeting host factors for anti-viral development offers several potential advantages over traditional countermeasures that include broad-spectrum activity and prevention of resistance. Characterization of host factors in animal models provides strong evidence of their involvement in disease pathogenesis, but the feasibility of performing high-throughput in vivo analyses on lists of genes is problematic. To begin addressing the challenges of screening candidate host factors in vivo, we combined advances in CRISPR-Cas9 genome editing with an immunocompromised mouse model used to study highly pathogenic viruses. Transgenic mice harboring a constitutively expressed Cas9 allele (Cas9tg/tg) with or without knockout of type I interferon receptors served to optimize in vivo delivery of CRISPR single-guide RNA (sgRNA) using Invivofectamine 3.0, a simple and easy-to-use lipid nanoparticle reagent. Invivofectamine 3.0-mediated liver-specific editing to remove activity of the critical Ebola virus host factor Niemann-Pick disease type C1 in an average of 74% of liver cells protected immunocompromised Cas9tg/tg mice from lethal surrogate Ebola virus infection. We envision that immunocompromised Cas9tg/tg mice combined with straightforward sgRNA in vivo delivery will enable efficient host factor loss-of-function screening in the liver and other organs to rapidly study their effects on viral pathogenesis and help initiate development of broad-spectrum, host-directed therapies against emerging pathogens.

Successful Intratracheal Treatment of Phage and Antibiotic Combination Therapy of a Multi-Drug Resistant Pseudomonas aeruginosa Murine Model.

Background: Pseudomonas aeruginosa (PsA) is a common etiology of bacteria-mediated lower respiratory tract infections, including pneumonia, hospital acquired pneumonia (HAP), and ventilator-associated pneumonia (VAP). Given the paucity of novel antibiotics in our foreseeable pipeline, developing novel non-antibiotic antimicrobial therapies saliently targeting drug resistant PsA isolates remains a priority. Lytic bacteriophages (or phages) have come under scrutiny as a potential antimicrobial for refractory bacterial infections. We evaluated intratracheally and intraperitoneally (IP) administered phage therapy (with/without meropenem) in an acute immunocompromised mouse model of multi-drug resistant (MDR) PsA pulmonary infection. The MDR P. aeruginosa respiratory disease model used in these studies was developed to investigate novel therapies that might have efficacy as either monotherapies or as combination therapy with meropenem. Methods: We utilized eight-week-old, 18 g BALB/cJ female mice and an MDR strain of PsA (UNC-D). Mice were immunosuppressed with cyclophosphamide. We employed a three-phage cocktail targeting PsA (PaAH2ΦP (103), PaBAP5Φ2 (130), and PaΦ (134)), confirmed to exhibit in vitro suppression of the infecting isolate out to 45 h. Suppression was confirmed with phages acting in isolation and in combination with meropenem. Results: IP administration of phage did not protect mice from death. A one-time delivery of phage directly to the lungs via a single intubation-mediated, intratracheal (IMIT) instillation protected mice from lethal infection. Protection was observed despite delaying therapy out to 6 h. Finally, we observed that, by slowing the progression of infection by treatment with a sub-efficacious dose of meropenem, we could protect the mice from lethal infection via IP phage administration coupled to meropenem, observing partial additive effects of phage–antibiotic combination therapy. Conclusions: A personalized phage cocktail administered via IMIT exhibits high therapeutic efficacy, despite delayed treatment of 6 h in a lethal MDR PsA pneumonia model. IP phage alone did not forestall mortality, but exhibited efficacy when combined with meropenem and IMIT-administered phage. These additive effects of combined IP phage and meropenem confirm that phage may indeed reach the lung bed via the systemic circulation and protect mice if the infection is not too acute. Therefore, adjunctive phage therapy with concerted attention to identifying optimal phage targeting of the infecting isolate in vitro may exhibit transformative potential for combating the specter of MDR bacterial infections. Phage should serve as an integral component of a four-pronged approach coupled with antibiotics, source control, and immune optimization.

Optimization of the Administration Strategy for the Armed Oncolytic Adenovirus ZD55-IL-24 in Both Immunocompromised and Immunocompetent Mouse Models.

ZD55-IL-24 is an armed oncolytic adenovirus similar but superior to ONYX-015. Virotherapeutic strategies using ZD55-IL-24 have been demonstrated to be effective against several cancer types. However, it is unclear whether the traditional administration strategy is able to exert the maximal antitumor efficacy of ZD55-IL-24. In this study, we sought to optimize the administration strategy of ZD55-IL-24 in both A375-bearing immunocompromised mouse model and B16-bearing immunocompetent mouse model. Although the underlying antitumor mechanisms are quite different, the obtained results are similar in these two mouse tumor models. We find that the antitumor efficacy of ZD55-IL-24 increases as injection times increase in both of these two models. However, no obvious increase of efficacy is observed as the dose of each injection increases. Our further investigation reveals that the administration strategy of sustained ZD55-IL-24 therapy can achieve a better therapeutic effect than the traditional administration strategy of short-term ZD55-IL-24 therapy. Furthermore, there is no need to inject every day; every 2 or 3 days of injection achieves an equivalent therapeutic efficacy. Finally, we find that the sustained rather than the traditional short-term ZD55-IL-24 therapy can synergize with anti-PD-1 therapy to reject tumors in B16-bearing immunocompetent mouse model. These findings suggest that the past administration strategy of ZD55-IL-24 is in fact suboptimal and the antitumor efficacy can be further enhanced through administration strategy optimization. This study might shed some light on the development of clinically applicable administration regimens for ZD55-IL-24 therapy.

Bone Marrow Stem Cells from Patients with Myelodysplastic Syndrome (MDS) Engraft and Migrate in-between the Humanized Niches in Immuno-Compromised Mouse Models

IntroductionMyelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized mainly by ineffective hematopoiesis. Xenograft models of MDS in immunodeficient mice have been instrumental in improving our understanding of clonal origin and evolution of this disease (Mian & Rouault-Pierre et al., Nat Commun. 2015; Rouault-Pierre & Mian et al Haematologica 2016; Leukemia, 2017); however, the low level of engraftment generally observed in these mouse models makes them impractical for testing therapeutic strategies. We used a 3D humanized scaffold in vivo system to test the MDS stem cell engraftment as well as study the cross talk between MDS-Initiating cells (MDS-ICs) and their niche.MethodologyIn this study bone marrow (BM) cells from 31 patients (27 MDS cases covering all WHO subtypes, 1 CMML, 1 MDS/MPD and 2 sAML) were studied. Initially, whole-exome sequencing was performed on all cases. Colony-forming assay was used to assess the colony forming ability of BM CD34+ cells from each patient. Mesenchymal stromal cells (MSCs) were isolated from the BM of the patient samples. We then used 3 immunodeficient mouse models (NSG, NSG-SGM3 and NBSGW) to study the engraftment kinetics and clonal evolution of the transplanted cells in gelatin-based 3D scaffolds coated with patient MSCs (RARS/RCMD, autologous; RAEB, autologous/allogenic).ResultsBM MSCs obtained from the MDS patients showed variable expansion levels, with the ability to differentiate into the adipogenic lineage whereas the osteogenic differentiation was highly aberrant.Next, 0.5x106 to 3x106 patient (n=31) mononuclear cells (MNCs T cell depleted) were seeded into 3D-scaffolds prior to implantation in NSG-SGM3 and/or NSG mice. We observed persistent long-term engraftment of hCD45+ cells ranging from 0.05% to 80% (up to 18 weeks). Importantly, 74% of the cases generated an engraftment level of >20% hCD45+ cells. Similar engraftment levels were observed in mice humanized scaffolds that were injected with BM cells from MDS/MPD, CMML and sAML (hCD45+ 10%, 22% and 86%, respectively). No difference in the hCD45+ cell engraftment between the patients with low-risk or high-risk MDS was observed.Remarkably, our 3D humanized scaffolds generated multi-lineage engraftment, with mice that received BM cells from RAEB cases yielding higher myeloid engraftment compared to RARS and RCMD (RARS hCD33+ 44%; RCMD hCD33+ 46.6%; RAEB hCD33+ 65%). Furthermore, transplantation of BM cells from 5 patients into NBSGW mice gave rise to engraftment levels of hCD45+ cells similar (hCD45+ cells, up to 40%) to the NSG and NSG-SGM3 mice. The MDS cells recovered from the mice maintained their genotypic characteristics, as demonstrated by the presence of the mutations that remained largely unchanged between the pre- and post-transplanted samples.Interestingly, Immuno-staining of the scaffolds revealed colonization by the host murine vasculature (mCD31+ endothelial cells). In line with this finding, mouse hematopoietic cells were also found to have migrated into the implanted humanized scaffolds. Surprisingly, we did not observe hCD45+ cell engraftment in the bone marrow of any of the xenotransplanted mouse strains (NSG or NSG-SGM3 mice with or without Irradiation and non-irradiated NBSGW mice). However, we demonstrated that MDS stem cells migrate by implanting a supplementary scaffold into NSG-SGM3 and NBSGW mice that was only coated with MDS-MSCs. Notably, these MDS-MSC seeded scaffolds were colonized by the circulating human MDS cells.Finally, we tested whether the cells recovered from the primary engrafted 3D humanized scaffolds would be able to generate secondary transplants. Our data shows that these cells were able to engraft in the secondary mice implanted with humanized 3D scaffolds.DiscussionOur model provides evidence that MDS BM cells are highly dependent on humanized MSC niche, confirming the importance of the cross-talk between MDS-ICs and MDS niche. Our data demonstrates that MDS stem cells do migrate out of the 3D Scaffold but require their “own niche” for colonization. Thus, our in vivo 3D model system, can be reliably used to better dissect the cross-talk between MDS stoma and MDS-ICs. This model requiring relatively low number of human MNCs, provides a robust preclinical assessment tool to study clonal evolution as well as to test therapeutic strategies on the MDS clonality prior to treatment of MDS patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Celastrol induces caspase-dependent apoptosis of hepatocellular carcinoma cells by suppression of mammalian target of rapamycin.

To investigate the efficacy of celastrol treatment of hepatocellular carcinoma (HCC) cells in vitro and in vivo and to propose a mechanism of action. A human HepG2 liver cancer cell line and a xenograft tumor model were used to investigate the effects of celastrol on HCC in vitro and in vivo. A CCK-8 kit was used to detect cell viability. Flow cytometry and terminal-deoxynucleoitidyl transferase mediated nick end labeling staining were used to detect apoptosis. Western blotting and immunohistochemistry were used to detect the expression of cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9, cleaved-PARP, mammalian target of rapamycin (mTOR), and p-mTOR. Hematoxylin-eosin staining was used to observe the tissue morphology. Celastrol decreased the viability of HepG2 cells and induced apoptosis. Western blot assays indicated that celastrol up-regulated cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9, and cleaved-PARP by inhibiting the phosphorylation of mTOR in HepG2 cells. Moreover, celastrol inhibited the tumor growth in a xenograft model. Celastrol also induced caspase-dependent apoptosis (up-regulation of cleaved-caspase- 3, -8, -9, and cleaved-PARP) and inhibited the activation of mTOR in vivo. Celastrol induces caspase-dependent apoptosis in HCC cells by inhibiting the activation of mTOR.

A novel phosphoramide compound, DCZ0805, shows potent anti-myeloma activity via the NF-\u03baB pathway

BackgroundMultiple myeloma (MM) is a highly aggressive and incurable clonal plasma cell disease with a high rate of recurrence. Thus, the development of new therapies is urgently needed. DCZ0805, a novel compound synthesized from osalmide and pterostilbene, has few observed side effects. In the current study, we intend to investigate the therapeutic effects of DCZ0805 in MM cells and elucidate the molecular mechanism underlying its anti-myeloma activity.MethodsWe used the Cell Counting Kit-8 assay, immunofluorescence staining, cell cycle assessment, apoptosis assay, western blot analysis, dual-luciferase reporter assay and a tumor xenograft mouse model to investigate the effect of DCZ0805 treatment both in vivo and in vitro.ResultsThe results showed that DCZ0805 treatment arrested the cell at the G0/G1 phase and suppressed MM cells survival by inducing apoptosis via extrinsic and intrinsic pathways. DCZ0805 suppressed the NF-κB signaling pathway activation, which may have contributed to the inhibition of cell proliferation. DCZ0805 treatment remarkably reduced the tumor burden in the immunocompromised xenograft mouse model, with no obvious toxicity observed.ConclusionThe findings of this study indicate that DCZ0805 can serve as a novel therapeutic agent for the treatment of MM.

In vitro and in vivo interaction of caspofungin with isavuconazole against Candida auris planktonic cells and biofilms.

The in vitro and in vivo efficacy of caspofungin was determined in combination with isavuconazole against Candida auris. Drug-drug interactions were assessed utilizing the fractional inhibitory concentration indices (FICIs), the Bliss independence model and an immunocompromised mouse model. Median planktonic minimum inhibitory concentrations (pMICs) of 23 C. auris isolates were between 0.5 and 2mg/l and between 0.015 and 4mg/l for caspofungin and isavuconazole, respectively. Median pMICs for caspofungin and isavuconazole in combination showed 2-128-fold and 2-256-fold decreases, respectively. Caspofungin and isavuconazole showed synergism in 14 out of 23 planktonic isolates (FICI range 0.03-0.5; Bliss cumulative synergy volume range 0-4.83). Median sessile MICs (sMIC) of 14 biofilm-forming isolates were between 32 and >32mg/l and between 0.5 and >2mg/l for caspofungin and isavuconazole, respectively. Median sMICs for caspofungin and isavuconazole in combination showed 0-128-fold and 0-512-fold decreases, respectively. Caspofungin and isavuconazole showed synergistic interaction in 12 out of 14 sessile isolates (FICI range 0.023-0.5; Bliss cumulative synergy volume range 0.13-234.32). In line with the in vitro findings, synergistic interactions were confirmed by in vivo experiments. The fungal kidney burden decreases were more than three log volumes in mice treated with combination of 1mg/kg caspofungin and 20mg/kg isavuconazole daily; this difference was statistically significant compared with control mice (P<0.001). Despite the favorable effect of isavuconazole in combination with caspofungin, further studies are needed to confirm the therapeutic advantage of this combination when treating an infection caused by C. auris.

Subunit vaccine protects against a clinical isolate of Mycobacterium avium in wild type and immunocompromised mouse models

The nontuberculous mycobacteria (NTM) Mycobacterium avium is a clinically significant pathogen that can cause a wide range of maladies, including tuberculosis-like pulmonary disease. An immunocompromised host status, either genetically or acutely acquired, presents a large risk for progressive NTM infections. Due to this quietly emerging health threat, we evaluated the ability of a recombinant fusion protein ID91 combined with GLA-SE [glucopyranosyl lipid adjuvant, a toll like receptor 4 agonist formulated in an oil-in-water stable nano-emulsion] to confer protection in both C57BL/6 (wild type) and Beige (immunocompromised) mouse models. We optimized an aerosol challenge model using a clinical NTM isolate: M. avium 2-151 smt, observed bacterial growth kinetics, colony morphology, drug sensitivity and histopathology, characterized the influx of pulmonary immune cells, and confirmed the immunogenicity of ID91 in both mouse models. To determine prophylactic vaccine efficacy against this M. avium isolate, mice were immunized with either ID91 + GLA-SE or bacillus Calmette–Guérin (BCG). Immunocompromised Beige mice displayed a delayed influx of innate and adaptive immune cells resulting in a sustained and increased bacterial burden in the lungs and spleen compared to C57BL/6 mice. Importantly, both ID91 + GLA-SE and BCG vaccines significantly reduced pulmonary bacterial burden in both mouse strains. This work is a proof-of-concept study of subunit vaccine-induced protection against NTM.

Transition state analogue of MTAP extends lifespan of APCMin/+ mice

A mouse model of human Familial Adenomatous Polyposis responds favorably to pharmacological inhibition of 5′-methylthioadenosine phosphorylase (MTAP). Methylthio-DADMe-Immucillin-A (MTDIA) is an orally available, transition state analogue inhibitor of MTAP. 5′-Methylthioadenosine (MTA), the substrate for MTAP, is formed in polyamine synthesis and is recycled by MTAP to S-adenosyl-l-methionine (SAM) via salvage pathways. MTDIA treatment causes accumulation of MTA, which inhibits growth of human head and neck (FaDu) and lung (H359, A549) cancers in immunocompromised mouse models. We investigated the efficacy of oral MTDIA as an anti-cancer therapeutic for intestinal adenomas in immunocompetent APCMin/+ mice, a murine model of human Familial Adenomatous Polyposis. Tumors in APCMin/+ mice were decreased in size by MTDIA treatment, resulting in markedly improved anemia and doubling of mouse lifespan. Metabolomic analysis of treated mice showed no changes in polyamine, methionine, SAM or ATP levels when compared with control mice but indicated an increase in MTA, the MTAP substrate. Generation of an MTDIA-resistant cell line in culture showed a four-fold amplification of the methionine adenosyl transferase (MAT2A) locus and expression of this enzyme. MAT2A is downstream of MTAP action and catalyzes synthesis of the SAM necessary for methylation reactions. Immunohistochemical analysis of treated mouse intestinal tissue demonstrated a decrease in symmetric dimethylarginine, a PRMT5-catalyzed modification. The anti-cancer effects of MTDIA indicate that increased cellular MTA inhibits PRMT5-mediated methylations resulting in attenuated tumor growth. Oral dosing of MTDIA as monotherapy has potential for delaying the onset and progression of colorectal cancers in Familial Adenomatous Polyposis (FAP) as well as residual duodenal tumors in FAP patients following colectomy. MTDIA causes a physiologic inactivation of MTAP and may also have efficacy in combination with inhibitors of MAT2A or PRMT5, known synthetic-lethal interactions in MTAP−/− cancer cell lines.

Immune-Enhancing Effects of Lactobacillus plantarum 200655 Isolated from Korean Kimchi in a Cyclophosphamide-Induced Immunocompromised Mouse Model.

In this study, we evaluated the immune-enhancing activity of kimchi-derived Lactobacillus plantarum 200655 on immune suppression by cyclophosphamide (CP) in ICR mice. Animals were fed distilled water or 1×109 colony-forming unit/kg B.W. 200655 or Lactobacillus rhamnosus GG as a positive control for 14 days. An in vivo model of immunosuppression was induced using CP 150 and 100 mg/kg B.W. at 7 and 10 days, respectively. Body weight, spleen index, spleen weight, and gene expression were measured to estimate the immune-enhancing effects. The dead 200655 (D-200655) group showed an increased spleen weight compared to the sham control (SC) group. Similarly, the spleen index was significantly higher than that in the CP-treated group. The live 200655 (L-200655) group showed an increased mRNA expression of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 in splenocytes. Also, the iNOS and COX-2 mRNA expression was upregulated in the L-200655 group compared to the CP-only (SC) group. The phosphorylation of ERK and MAPK was also upmodulated in the L-200655 group. These results indicate that L. plantarum 200655 ameliorated CP-induced immune suppression, suggesting that L. plantarum 200655 may have the potential to enhance the immune system.

Mesenchymal stem cell carriers enhance antitumor efficacy induced by oncolytic reovirus in acute myeloid leukemia

Chemotherapy is the main treatment for acute myeloid leukemia (AML), but the therapeutic efficacy is modest, and most commonly manifests as relapse from remission. Thus, improving long-term AML survival is a crucial clinical challenge. In recent years, oncolytic virotherapy has provided an alternative approach for AML treatment. The use of oncolytic reoviruses has been explored in more than 30 clinical trials for safety and feasibility issues. However, like other oncolytic viruses, neutralizing antibodies (NAbs) reduce therapeutic efficacy. To tackle this problem, human umbilical cord mesenchymal stem cells (hUC-MSCs) were used to deliver reovirus using in vitro and in vivo models. Human UC-MSCs were successfully loaded with reovirus, without impairing biological function.We also observed in vitro protective effects of hUC-MSCs on reovirus in the presence of NAbs. In the immunocompromised AML mouse model, hUC-MSCs effectively carried reoviruses to tumor lesions and significantly prolonged the survival of AML xenografts in mice in the presence of a high titer anti-reovirus antibody (p = 0.001). However, reovirus-induced activation of AKT, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and NF-κB signaling led to the maintenance of intrinsic migratory properties and secretion of pro-inflammatory cytokines from hUC-MSCs, particularly CXCL10. In immuno-competent AML mice, MSCs carrying reovirus triggered immune responses, and eventually inhibited tumor growth. Therefore, these results suggest that MSCs as carriers of oncolytic reoviruses can enhance the antitumor efficacy of virotherapy.

MicroRNA-3613-3p functions as a tumor suppressor and represents a novel therapeutic target in breast cancer

BackgroundMicroRNAs have been reported to participate in tumorigenesis, treatment resistance, and tumor metastasis. Novel microRNAs need to be identified and investigated to guide the clinical prognosis or therapy for breast cancer.MethodThe copy number variations (CNVs) of MIR3613 from Cancer Genome Atlas (TCGA) or Cancer Cell Line Encyclopedia (CCLE) were analyzed, and its correlation with breast cancer subtypes or prognosis was investigated. The expression level of miR-3613-3p in tumor tissues or serum of breast cancer patients was detected using in situ hybridization and qPCR. Gain-of-function studies were performed to determine the regulatory role of miR-3613-3p on proliferation, apoptosis, and tumor sphere formation of human breast cancer cells MDA-MB-231 or MCF-7. The effects of miR-3613-3p on tumor growth or metastasis in an immunocompromised mouse model of MDA-MB-231-luciferase were explored by intratumor injection of miR-3613-3p analogue. The target genes, interactive lncRNAs, and related signaling pathways of miR-3613-3p were identified by bioinformatic prediction and 3′-UTR assays.ResultsWe found that MIR3613 was frequently deleted in breast cancer genome and its deletion was correlated with the molecular typing, and an unfavorable prognosis in estrogen receptor-positive patients. MiR-3613-3p level was also dramatically lower in tumor tissues or serum of breast cancer patients. Gain-of-function studies revealed that miR-3613-3p could suppress proliferation and sphere formation and promote apoptosis in vitro and impeded tumor growth and metastasis in vivo. Additionally, miR-3613-3p might regulate cell cycle by targeting SMS, PAFAH1B2, or PDK3 to restrain tumor progression.ConclusionOur findings indicate a suppressive role of miR-3613-3p in breast cancer progression, which may provide an innovative marker or treatment for breast cancer patients.

Evaluation of bacteriophage cocktail on septicaemia caused by colistin-resistant Acinetobacter baumannii in immunocompromised mice model

Background & objectives:Since the bacterium, Acinetobacter baumannii (AB) has acquired resistance to almost all commercially available antibiotics, the search for alternative treatment options continues to be need of the hour. Bacteriophage therapy seems to be the most promising amongst various proposed alternatives (e.g. antimicrobial peptides, bacteriocin, probiotics, etc.). The present study, therefore, aimed to evaluate the effect of different dosages of specific phages in immunocompromised rodents in a septicaemia model caused by AB mimicking real clinical situations.Methods:The three most active and unique phages (ɸAb4, ɸAb7 and ɸAb14) were selected for this study. A constant dose (100 µl of 108 pfu/ml) of AB was given in all the experiments. Five different sets of experiments were designed: prophylactic administration of phage cocktail in the volume of 100 µl (109 pfu/ml) before and simultaneous with the bacterial challenge; and therapeutic i.e. administration of phage cocktail six, 12 and 24 h after bacterial challenge. Since there were deaths in mice when phage was given 24 h after bacterial challenge, the reduced dosage i.e. 100 µl of 107, 106, 105 pfu/ml of phage cocktail was also evaluated.Results:The administration of 100 µl (109 pfu/ml) of phage cocktail after six, 12 and 24 h of the bacterial challenge resulted in the mortality ranging between 20 to 60 per cent. However, no mortality could be observed with simultaneous or prophylactic administration of phages with the bacterial challenge. No mortality was observed with reduced doses of the cocktail (106 and105 pfu/ml).Interpretation & conclusions:As per the results of this study, it may be concluded that even if patients with acute infections report late to the hospital, a relatively low dose of the phage cocktail may be therapeutically beneficial.

Exosomal lncRNA-H19 promotes osteogenesis and angiogenesis through mediating Angpt1/Tie2-NO signaling in CBS-heterozygous mice.

Rationale: Emerging evidence indicates that the growth of blood vessels and osteogenesis is tightly coordinated during bone development. However, the molecular regulators of intercellular communication in the bone microenvironment are not well studied. Therefore, we aim to investigate whether BMMSC-Exo promotes osteogenesis and angiogenesis via transporting lnc-H19 in the CBS- heterozygous mouse model.Methods: Using RT2 lncRNA PCR array screening, we identify a bone-specific, long noncoding RNA-H19 (lncRNA-H19/lnc-H19) in exosomes derived from bone marrow mesenchymal stem cells (BMMSC-Exo) during osteogenesis. Using bioinformatics analysis, we further discovered the seed sequence of miR-106a that could bind to lnc-H19. A luciferase reporter assay was performed to demonstrate the direct binding of miR-106a to the target gene angiopoietin 1 (Angpt1). We employed an immunocompromised Nude mouse model, to evaluate the effects of BMMSC-Exo on angiogenesis in vivo. Using a micro-CT scan, we monitored microstructural changes of bone in the experimental mice.Results: BMMSC-Exo possessed exosomal characteristics including exosome size, and typical markers including CD63, CD9, and TSD101. In vitro, BMMSC-Exo significantly promoted endothelial angiogenesis and osteogenesis. Mechanistic studies have shown that exosomal lnc-H19 acts as “sponges” to absorb miR-106 and regulate the expression of angiogenic factor, Angpt1 that activates lnc-H19/Tie2-NO signaling in mesenchymal and endothelial cells. Both of these effects on osteogenesis and angiogenesis are inhibited by antagonizing Tie2 signaling. Treatment of BMMSC-Exo also restored the bone formation and mechanical quality in vivo.Conclusion: These findings provide a novel insight into how the extracellular role of exosomal lnc-H19 affects osteogenesis and angiogenesis through competing endogenous RNA networks.

Gain-of-function hot spot mutant p53R248Q regulation of integrin/FAK/ERK signaling in esophageal squamous cell carcinoma

PurposeTP53, encoding the protein p53, is among the most frequently mutated genes in all cancers. A high frequency of 60 – 90% mutations is seen in esophageal squamous cell carcinoma (ESCC) patients. Certain p53 mutants show gain-of-function (GoF) oncogenic features unrelated to its wild type functions. MethodsThis study functionally characterized a panel of p53 mutants in individual ESCC cell lines and assayed for GoF oncogenic properties. ResultsThe ESCC cell line with endogenous p53R248Q expression showed suppressed tumor growth in an immunocompromised mouse model and suppressed colony growth in in vitro three-dimensional culture, when depleted of the endogenous p53 protein expression. This suppression is accompanied by suppressed cell cycle progression, along with reduced integrin expression and decreased focal adhesion kinase and extracellular-regulated protein kinase signaling and can be compensated by expression of a constitutively active mitogen-activated protein. P53R248Q enhances cell proliferation upon glutamine deprivation, as compared to other non-GoF mutants. ConclusionsIn summary, study of the functional contributions of endogenous p53 mutants identified a novel GoF mechanism through which a specific p53 mutant exerts oncogenic features and contributes to ESCC tumorigenesis.