Well-established Murine Model Research Articles

Impacts of perR on oxygen sensitivity, gene expression, and murine infection in Clostridioides difficile 630∆erm.

Clostridioides difficile infection (CDI), characterized by colitis and diarrhea, afflicts approximately half a million people in the USA every year, burdening both individuals and the healthcare system. C. difficile 630Δerm is an erythromycin-sensitive variant of the clinical isolate C. difficile 630 and is commonly used in the C. difficile research community due to its genetic tractability. 630Δerm possesses a point mutation in perR, an autoregulated transcriptional repressor that regulates oxidative stress resistance genes. This point mutation results in a constitutively de-repressed PerR operon in 630Δerm. To address the impacts of perR on phenotypes relevant for oxygen tolerance and relevant to a murine model of CDI, we corrected the point mutant to restore PerR function in 630∆erm (herein, 630∆erm perRWT). We demonstrate that there is no difference in growth between 630Δerm and 630Δerm perRWT under anaerobic conditions or when exposed to concentrations of O2 that mimic those found near the surface of the colonic epithelium. However, 630∆erm perRWT is more sensitive to ambient oxygen than 630∆erm, which coincides with alterations in expression of a variety of perR-dependent and perR-independent genes. Finally, we show that 630∆erm and 630∆erm perRWT do not differ in their ability to infect and cause disease in a well-established murine model of CDI. Together, these data support the hypothesis that the perR mutation in 630∆erm arose as a result of exposure to ambient oxygen and that the perR mutation in 630∆erm is unlikely to impact CDI-relevant phenotypes in laboratory studies.IMPORTANCEClostridioides difficile is a diarrheal pathogen and a major public health concern. To improve humans' understanding of C. difficile, a variety of C. difficile isolates are used in research, including C. difficile 630Δerm. 630Δerm is a derivative of the clinical isolate 630 and is commonly studied because it is genetically manipulable. Previous work showed that a mutation in perR in 630Δerm results in a dysregulated oxidative stress response, but no work has been done to characterize perR-dependent effects on the transcriptome or to determine impacts of perR during infection. Here, we identify transcriptomic differences between 630∆erm and 630∆erm perRWT exposed to ambient oxygen and demonstrate that there is no strain-based difference in burdens in murine C. difficile infection.

Sciatic nerve analysis in thyroid hormone transporters Mct8 and Oatp1c1 knockout mice.

Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) cause Allan-Herndon-Dudley syndrome (AHDS), a severe form of psychomotor retardation with muscle hypoplasia and spastic paraplegia as key symptoms. These abnormalities have been attributed to impaired TH transport across brain barriers and into neural cells, thereby affecting brain development and function. Likewise, Mct8/Oatp1c1 (organic anion-transporting polypeptide 1c1) double knockout (M/Odko) mice, a well-established murine AHDS model, display a strongly reduced TH passage into the brain as well as locomotor abnormalities. To which extent the peripheral nervous system is affected by combined MCT8/OATP1C1 deficiency has not been addressed. Using the sciatic nerve as a model, we studied the spatiotemporal expression of TH transporters as well as the sciatic thyroidal state, sciatic nerve myelination and function in M/Odko mice by immunofluorescence, qPCR, Western blotting and electrophysiology. We detected MCT8 protein expression in sciatic nerve axons, whereas OATP1C1 expression was observed in a subset of endothelial cells early in postnatal development. The absence of MCT8 and OATP1C1 did not alter the thyroidal state of isolated nerves at P12. Moreover, electrophysiological studies did not disclose any significant alteration in sciatic nerve signal propagation parameters in adult M/Odko mice. Although Schwann cell numbers were similar, Western blot analysis showed a mild form of hypermyelination in adult M/Odko mice. Altogether, our data point to a largely unaffected sciatic nerve structure and function in the absence of MCT8 and OATP1C1.

Impacts of perR on oxygen sensitivity, gene expression, and murine infection in Clostridioides difficile 630Δerm.

Clostridioides difficile infection (CDI), characterized by colitis and diarrhea, afflicts approximately half a million people in the United States every year, burdening both individuals and the healthcare system. C. difficile 630Δerm is an erythromycin-sensitive variant of the clinical isolate C. difficile 630 and is commonly used in the C. difficile research community due to its genetic tractability. 630Δerm possesses a point mutation in perR, an autoregulated transcriptional repressor that regulates oxidative stress resistance genes. This point mutation results in a constitutively de-repressed PerR operon in 630Δerm. To address the impacts of perR on phenotypes relevant for oxygen tolerance and relevant to a murine model of CDI, we corrected the point mutant to restore PerR function in 630Δerm (herein, 630Δerm perR WT). We demonstrate that there is no difference in growth between 630Δerm and a 630Δerm perR WT under anaerobic conditions or when exposed to concentrations of O2 that mimic those found near the surface of the colonic epithelium. However, 630Δerm perR WT is more sensitive to ambient oxygen than 630Δerm, which coincides with alterations in expression of a variety of perR-dependent and perR-independent genes. Finally, we show that 630Δerm and 630Δerm perR WT do not differ in their ability to infect and cause disease in a well-established murine model of CDI. Together, these data support the hypothesis that the perR mutation in 630Δerm arose as a result of exposure to ambient oxygen and that the perR mutation in 630Δerm is unlikely to impact CDI-relevant phenotypes in laboratory studies.

Human adipose-derived multipotent stromal cells enriched with IL-10 modRNA improve diabetic wound healing: Trigger the macrophage phenotype shift.

Diabetic wounds present a significant challenge in regenerative medicine due to impaired healing, characterized by prolonged inflammation and deficient tissue repair, primarily caused by a skewed pro-inflammatory macrophage phenotype. This study investigates the therapeutic potential of interleukin-10 (IL-10) chemically modified mRNA (modRNA)-enriched human adipose-derived multipotent stromal cells (hADSCs) in a well-established murine model of diabetic wounds. The modRNAs used in this study were chemically modified using N1-methylpseudouridine-5'-triphosphate (m1Ψ) by substituting uridine-5-triphosphate. In vitro experiments demonstrated that IL-10 modRNA-transfected hADSCs effectively modulated macrophage polarization towards an anti-inflammatory phenotype. In vivo experiments with a well-established murine model demonstrated that transplantation of hADSCsmodIL-10 on postoperative day 5 (POD5) significantly improved wound healing outcomes, including accelerated wound closure, enhanced re-epithelialization, promoted M2 polarization, improved collagen deposition, and increased neovascularization. This study concludes that IL-10 modRNA-enriched hADSCs offer a promising therapeutic approach for diabetic wound healing, with the timing of IL-10 administration playing a crucial role in its effectiveness. These cells modulate macrophage polarization and promote tissue repair, demonstrating their potential for improving the management of diabetic wounds.

Bactericidal and sterilizing activity of sudapyridine-clofazimine-TB47 combined with linezolid or pyrazinamide in a murine model of tuberculosis.

As an obligate aerobe, Mycobacterium tuberculosis relies on its branched electron transport chain (ETC) for energy production through oxidative phosphorylation. Regimens targeting ETC exhibit promising potential to enhance bactericidal activity against M. tuberculosis and hold the prospect of shortening treatment duration. Our previous research demonstrated that the bacteriostatic drug candidate TB47 (T) inhibited the growth of M. tuberculosis by targeting the cytochrome bc1 complex and exhibited synergistic activity with clofazimine (C). Here, we found synergistic activities between C and sudapyridine (S), a structural analog of bedaquiline (B). S has shown similar anti-tuberculosis efficacy and may share a mechanism of action with B, which inhibits ATP synthesis and the energy metabolism of bacteria. We evaluated the efficacy of SCT in combination with linezolid (L) or pyrazinamide (Z) using a well-established murine model of tuberculosis. Compared to the BPa(pretomanid)L regimen, SCT and SCTL demonstrated similar bactericidal and sterilizing activities. There was no significant difference in activity between SCT and SCTL. In contrast, SCZ and SCTZ showed much higher activities, with none of the 15 mice experiencing relapse after 2 months of treatment with either SCZ or SCTZ. However, T did not contribute to the activity of the SCZ. Our findings emphasize the efficacy and the potential clinical significance of combination therapy with ETC inhibitors. Additionally, cross-resistance exists not only between S and B but also between S/B and C. This is supported by our findings, as spontaneous S-resistant mutants exhibited mutations in Rv0678, which are associated with cross-resistance to B and C.

Cyclic guanosine monophosphate-adenosine monophosphate synthetase/stimulator of interferon genes signaling aggravated corneal allograft rejection through neutrophil extracellular traps

The activation of innate immunity following transplantation has been identified as a crucial factor in allograft inflammation and rejection. However, the role of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)/stimulator of interferon genes (STING) signaling-mediated innate immunity in the pathogenesis of allograft rejection remains unclear. Utilizing a well-established murine model of corneal transplantation, we demonstrated increased expression of cGAS and STING in rejected-corneal allografts compared with syngeneic (Syn) and normal (Nor) corneas, along with significant activation of the cGAS/STING pathway, as evidenced by the enhanced phosphorylation of TANK-binding kinase 1and interferon regulatory factor 3. Pharmacological and genetic inhibition of cGAS/STING signaling markedly delayed corneal transplantation rejection, resulting in prolonged survival time and reduced inflammatory infiltration. Furthermore, we observed an increase in the formation of neutrophil extracellular traps (NETs) in rejected allografts, and the inhibition of NET formation through targeting peptidylarginine deiminase 4 and DNase I treatment significantly alleviated immune rejection and reduced cGAS/STING signaling activity. Conversely, subconjunctival injection of NETs accelerated corneal transplantation rejection and enhanced the activation of the cGAS/STING pathway. Collectively, these findings demonstrate that NETs contribute to the exacerbation of allograft rejection via cGAS/STING signaling, highlighting the targeting of the NETs/cGAS/STING signaling pathway as a potential strategy for prolonging allograft survival.

Abstract LB024: Permeation enhancer is necessary for the optimized PRV131 mediated cisplatin intratumoral delivery

Abstract PRV131 is an effective and safe polymeric nanoparticle suspension for intratumoral cisplatin delivery. Background: Systemic administration of platinum-based chemotherapy often leads to dose-limiting toxicity, affecting quality of life. While intratumoral delivery may reduce or eliminate the systemic toxicities and adverse side effects associated with intravenous cisplatin chemotherapy, this approach is not commonly adopted, in part due to the difficulty to obtain high drug retention in the tumor and control sustained drug release. PRV131 is a highly-concentrated cisplatin polymeric nanoparticle suspension carefully designed for intratumoral administration. The goal of this study was to assess the safety and efficacy of PRV131 as a monotherapy and in combination with a checkpoint inhibitor in a well-established preclinical murine colorectal adenocarcinoma model. Experimental Methods: MC38 cells were inoculated subcutaneously into the flanks of C57BL/6J female mice and allowed to grow to an average size of 100 mm3. Mice were then randomized into 6 groups of n=10: control (saline injection), 100 µg of cisplatin solution, 400 µg of cisplatin solution, 400 µg of PRV131, 400 µg of PRV131 + 200 µg systemic anti-PD1, and 200 µg systemic anti-PD1. A single injection of PRV131 or cisplatin solution was administered directly into the tumor on Day 1. Intraperitoneal anti-PD-1 was administered on Day 1 and Day 10. Mice were then monitored for efficacy (tumor measurements) and safety (overall health, weight loss and mortality). The durability of a single PRV131 injection was also studied. Efficacy and safety were adjudicated at 28 days post treatment and treatment durability assessment extended through 60 days post inoculation. Results: Our data demonstrates that PRV131 400 µg was significantly more effective than cisplatin solution at an equivalent dose. 80% of PRV131 treated mice survived to day 60 post inoculation, and no detectable tumor was found in 60% of PRV131 treated mice 38 days post administration. The safety profile was acceptable, with no mouse lost more than 20% body weight. Notably, efficacy profile of PRV131 monotherapy did not differ substantially from the addition of anti-PD-1 to the treatment, suggesting a high translational relevance of our drug delivery platform. Conclusion: A single intratumoral injection of PRV131 is well-tolerated in C57BL/6J female mice and elicits a durable antitumor response that lasts for over 30 days, laying the groundwork for its incorporation in clinical trial setting. Citation Format: Manijeh Goldberg, Aaron Manzi, Stefanie Cantin, Peter Conway, Jonathan Shikora, Adelaide McFarland, Matt Cayer, Lillyanna Dunn, Courtney Ball, Alexander T. Pearson, Ari J. Rosenberg, Nishant Agrawal, Evgeny Izumchenko. Permeation enhancer is necessary for the optimized PRV131 mediated cisplatin intratumoral delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB024.

Abstract LB440: PRV131 is an effective and safe polymeric nanoparticle suspension for intratumoral cisplatin delivery

Abstract Background: Systemic administration of platinum-based chemotherapy often leads to dose-limiting toxicity, affecting quality of life. While intratumoral delivery may reduce or eliminate the systemic toxicities and adverse side effects associated with intravenous cisplatin chemotherapy, this approach is not commonly adopted, in part due to the difficulty to obtain high drug retention in the tumor and control sustained drug release. PRV131 is a highly-concentrated cisplatin polymeric nanoparticle suspension carefully designed for intratumoral administration. The goal of this study was to assess the safety and efficacy of PRV131 as a monotherapy and in combination with a checkpoint inhibitor in a well-established preclinical murine colorectal adenocarcinoma model. Experimental Methods: MC38 cells were inoculated subcutaneously into the flanks of C57BL/6J female mice and allowed to grow to an average size of 100 mm3. Mice were then randomized into 6 groups of n=10: control (saline injection), 100 µg of cisplatin solution, 400 µg of cisplatin solution, 400 µg of PRV131, 400 µg of PRV131 + 200 µg systemic anti-PD1, and 200 µg systemic anti-PD1. A single injection of PRV131 or cisplatin solution was administered directly into the tumor on Day 1. Intraperitoneal anti-PD-1 was administered on Day 1 and Day 10. Mice were then monitored for efficacy (tumor measurements) and safety (overall health, weight loss and mortality). The durability of a single PRV131 injection was also studied. Efficacy and safety were adjudicated at 28 days post treatment and treatment durability assessment extended through 60 days post inoculation. Results: Our data demonstrates that PRV131 400 µg was significantly more effective than cisplatin solution at an equivalent dose. 80% of PRV131 treated mice survived to day 60 post inoculation, and no detectable tumor was found in 60% of PRV131 treated mice 38 days post administration. The safety profile was acceptable, with no mouse lost more than 20% body weight. Notably, efficacy profile of PRV131 monotherapy did not differ substantially from the addition of anti-PD-1 to the treatment, suggesting a high translational relevance of our drug delivery platform. Conclusion: A single intratumoral injection of PRV131 is well-tolerated in C57BL/6J female mice and elicits a durable antitumor response that lasts for over 30 days, laying the groundwork for its incorporation in clinical trial setting. Citation Format: Manijeh Goldberg, Aaron Manzi, Stefanie Cantin, Peter Conway, Jonathan Shikora, Adelaide McFarland, Matt Cayer, Lillyanna Dunn, Courtney Ball, Alexander T. Pearson, Ari J. Rosenberg, Nishant Agrawal, Evgeny Izumchenko. PRV131 is an effective and safe polymeric nanoparticle suspension for intratumoral cisplatin delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB440.

Eosinophils Are an Endogenous Source of Interleukin-4 during Filarial Infections and Contribute to the Development of an Optimal T Helper 2 Response

Plain Language SummaryFilarial nematodes can cause severe diseases like onchocerciasis and lymphatic filariasis, posing a significant public health challenge in tropical regions, putting over a billion people at risk. The WHO categorizes these infections as neglected tropical diseases and aims to eliminate onchocerciasis transmission and lymphatic filariasis as a public health issue by 2030. Filarial nematodes modulate the immune system of their host, and the induction of protective immune responses still requires a better understanding. Eosinophils have been identified as a key immune cell type in the well-established murine model for filarial infection, Litomosoides sigmodontis. However, their precise roles and interactions with other components of the type 2 immune response remain unclear. Our study reveals that eosinophils play a crucial role as a primary source of interleukin-4, the central cytokine in type 2 immunity. By using dblGATA mice, we found that the absence of eosinophils resulted in a reduced T helper 2 response but did not impact the alternative activation of macrophages or antibody production. In summary, our research uncovers an underappreciated function of eosinophils and their significant influence on type 2 immune responses.

Continuous topical application of microencapsulated recombinant human epidermal growth factor does not promote the progression of established melanoma in animals

Microencapsulated epidermal growth factor (EGF) device has been applied topically for the management of several types of wounds to accelerate wound healing and prevent scar formation. However, it remains unclear whether such EGF device induced neoplastic transformation in the skin. In this study, we exploited a well-established murine B16-F10 melanoma model, coupled with MTT viability and colony formation assays, to investigate the influence of microencapsulated recombinant EGF (Me-EGF; brand name NewEpi®) and its ingredients on the tumorigenicity of skin cancer cells in vitro and in vivo. The results indicated that Me-EGF did not stimulate the viability nor the anchorage-dependent growth of B16-F10 melanoma cells. Western blot analysis showed that Me-EGF treatment increased the total and phosphorylated EGFR expression without affecting the HER2 expression in B16-F10 melanoma cells. In mice bearing established B16-F10 melanoma, continuous application of Me-EGF for 14 days did not enhance the melanoma tumor burden compared with control groups. Immunohistochemical analysis also revealed the similar expression of proliferative index Ki-67 between Me-EGF-treated melanoma and other groups. Altogether, these results suggest that the application of Me-EGF device did not promote the oncogenic potential of B16-F10 melanoma in vitro and in vivo.

Line-field confocal optical coherence tomography for in vivo visualization of morphological characteristics of squamous cell carcinoma in a murine model.

Non-invasive imaging with line-field confocal optical coherence tomography (LC-OCT) can support the diagnosis of squamous cell carcinoma (SCC) through visualization of morphological characteristics specific to skin cancer. We aimed to visualize prominent morphological characteristics of SCC using LC-OCT in a well-established murine SCC model. Nine hairless mice were exposed to ultraviolet radiation three times weekly for 9 months to induce SCC development. Visible SCC tumors (n = 9) were imaged with LC-OCT and the presence of 10 well-described morphological characteristics of SCC were evaluated in the scans by two physicians with adjudication by a third. Overall, murine morphological characteristics resembled corresponding features previously reported in human SCCs. Interrupted dermal-epidermal junction occurred in 100% of tumors. In epidermis, the most frequently observed characteristics were severe epidermal dysplasia (100%) and tumor budding (89%). Common dermal characteristics included broad strands (100%) and collagen alterations (78%). LC-OCT imaging can be used to non-invasively visualize morphological characteristics specific to SCC in an in vivo preclinical model.

Raising the alarm: fosfomycin resistance associated with non-susceptible inner colonies imparts no fitness cost to the primary bacterial uropathogen.

While fosfomycin resistance is rare, the observation of non-susceptible subpopulations among clinical Escherichia coli isolates is a common phenomenon during antimicrobial susceptibility testing (AST) in American and European clinical labs. Previous evidence suggests that mutations eliciting this phenotype are of high biological cost to the pathogen during infection, leading to current recommendations of neglecting non-susceptible colonies during AST. Here, we report that the most common route to fosfomycin resistance, as well as novel routes described in this work, does not impair virulence in uropathogenic E. coli, the major cause of urinary tract infections, suggesting a re-evaluation of current susceptibility guidelines is warranted.

The Immune Checkpoint Siglec-15 in Promoting Immune Dysregulation in Non-Hodgkin's Lymphomas

Outcomes for patients with relapsed or refractory (r/r) hematologic malignancies remain dismal despite therapeutic advances over the last several years. This is particularly striking in patients with r/r aggressive mature B-cell lymphomas such as Diffuse Large B-cell Lymphoma (DLBCL) and Burkitt's Lymphoma (BL). With use of intensive multiagent chemo-immunotherapy, DLBCL, which accounts for approximately 10-20% of pediatric Non-Hodgkin's Lymphoma (NHL) cases and is the most common subtype in adults, is highly curable with 5-year event free survival >75%. However, approximately 30-40% of adults diagnosed with DLBCL will become refractory to or relapse after first-line treatment. Yet even with intensive salvage regimens, r/r DLBCL remains a clinical challenge. There remains an unmet need to identify specific defects in the immune response, among other pathways, that drive lymphomagenesis that will in turn inform the development of novel therapeutics to improve survival in this population of patients. Siglec-15 (Sig-15), a member of the sialic acid binding immunoglobulin-like lectin family of proteins, has recently been identified as a critical immune suppressor that is highly expressed in human cancers and intra-tumoral myeloid cells. Importantly, recent reports have demonstrated inhibition of Sig-15, has a restorative effect on local anti-tumor immune responses and abrogated tumor progression. While reported in solid malignancies and acute leukemias, a role for Siglec-15 in promoting disease progression in aggressive B-cell lymphomas has not yet been described. We have found higher expression of SIGLEC15 in DLBCL cell lines compared to normal B cells at the RNA level, through analyses of the publicly available ONCOMINE database. This was confirmed at the protein level by western blot demonstrating higher Sig-15 expression in various lymphoma cell lines (RAJI, SUDHL6, OCI-LY10, HBL-1) compared to healthy donor PBMCs. Further, immunohistochemistry evaluating Sig-15 expression was performed on a tumor microarray consisting of 139 cases of DLBCL from adult patients along with validation samples from 15 primary pediatric lymphoma cases (DLBCL, BL and Anaplastic Large Cell Lymphoma (ALCL)). Sig-15 was found to be highly expressed, with histologic scores ranging from weak (1+) to strong (3+) in 124 out of the 139 adult cases (Figure 1A) and in all pediatric lymphoma samples but with distinct staining patterns observed in the aggressive B-cell lymphomas. Specifically, Sig-15 appears to be highly expressed and associated with the cell membrane in most DLBCL and Burkitt's lymphoma. Expression is more variable in ALCL, primarily elevated in the cytoplasm at low levels and/or in macrophages (Figure 1A). There is heterogeneity in Sig-15 expression with preliminary analysis of RNA-seq data from a cohort of 693 DLBCL patient samples demonstrates higher levels in the germinal center B-cell (GCB) versus activated B-cell molecular (ABC) subtype of DLBCL when compared to healthy controls (Kruskal-Wallis, *p = 0.041). Finally, using a well-established murine lymphoma model, A20 cells were stably transduced with control, non-silencing shRNA (shNS) or shRNA against Sig-15 and injected into un-irradiated immune competent (wild type, WT) or immune deficient ( Rag1 -/-) BALB/C mice and monitored for signs of lymphoma development. Knockdown of Sig-15 in A20 cells abrogates disease progression in WT but not immune deficient mice (Log-rank, **p <0.0001, n=10 mice/group), consistent with a role for Sig-15 in immune evasion in lymphoma (Figure 1B). Together, these data implicate Sig-15 as an immune checkpoint that may be inhibited therapeutically to promote an immune response to lymphoma cells.

Pharmacokinetics and Biodistribution of 16,16 dimethyl Prostaglandin E2 in Non-Irradiated and Irradiated Mice and Non-Irradiated Non-Human Primates.

Exposure to high-dose ionizing radiation can lead to life-threatening injuries and mortality. Bone marrow is the most sensitive organ to radiation damage, resulting in the hematopoietic acute radiation syndrome (H-ARS) with the potential sequelae of infection, hemorrhage, anemia, and death if untreated. The development of medical countermeasures (MCMs) to protect or mitigate radiation injury is a medical necessity. In our well-established murine model of H-ARS we have demonstrated that the prostaglandin E2 (PGE2) analog 16,16 dimethyl-PGE2 (dmPGE2) has survival efficacy as both a radioprotectant and radiomitigator. The purpose of this study was to investigate the pharmacokinetics (PK) and biodistribution of dmPGE2 when used as a radioprotector in irradiated and non-irradiated inbred C57BL/6J mice, PK in irradiated and non-irradiated Jackson Diversity Outbred (JDO) mice, and the PK profile of dmPGE2 in non-irradiated non-human primates (NHPs). The C57BL/6J and JDO mice each received a single subcutaneous (SC) dose of 35 ug of dmPGE2 and were randomized to either receive radiation 30 min later or remain non-irradiated. Plasma and tissue PK profiles were established. The NHP were dosed with 0.1 mg/kg by SC administration and the PK profile in plasma was established. The concentration time profiles were analyzed by standard non-compartmental analysis and the metrics of AUC0-Inf, AUC60-480 (AUC from 60-480 min), Cmax, and t1/2 were evaluated. AUC60-480 represents the postirradiation time frame and was used to assess radiation effect. Overall, AUC0-Inf, Cmax, and t1/2 were numerically similar between strains (C57BL/6J and JDO) when combined, regardless of exposure status (AUC0-Inf: 112.50 ng·h/ml and 114.48 ng·h/ml, Cmax: 44.53 ng/ml and 63.96 ng/ml; t1/2: 1.8 h and 1.1 h, respectively). PK metrics were numerically lower in irradiated C57BL/6J mice than in non-irradiated mice [irradiation ratio: irradiated values/non-irradiated values = 0.71 for AUC60-480 (i.e., 29% lower), and 0.6 for t1/2]. In JDO mice, the radiation ratio was 0.53 for AUC60-480 (i.e., 47% lower), and 1.7 h for t1/2. The AUC0-Inf, Cmax, and t1/2 of the NHPs were 29.20 ng·h/ml, 7.68 ng/ml, and 3.26 h, respectively. Despite the numerical differences seen between irradiated and non-irradiated groups in PK parameters, the effect of radiation on PK can be considered minimal based on current data. The biodistribution in C57BL/6J mice showed that dmPGE2 per gram of tissue was highest in the lungs, regardless of exposure status. The radiation ratio for the different tissue AUC60-480 in C57BL/6J mice ranged between 0.5-1.1 (50% lower to 10% higher). Spleen, liver and bone marrow showed close to twice lower exposures after irradiation, whereas heart had a 10% higher exposure. Based on the clearance values from mice and NHP, the estimated allometric scaling coefficient was 0.81 (95% CI: 0.75, 0.86). While slightly higher than the current literature estimates of 0.75, this scaling coefficient can be considered a reasonable estimate and can be used to scale dmPGE2 dosing from animals to humans for future trials.

A randomized, blinded study of photobiomodulation in a mouse model of Alzheimer’s disease showed no preventive effect

Photobiomodulation (PBM), the process of exposing tissue to red or near-infrared light, has become a topic of great interest as a therapy for diverse pathologies, including neurodegenerative disorders. Here, we aimed to evaluate the potential beneficial effect of PBM on Alzheimer’s disease (AD) using behavioral and histological readouts from a well-established transgenic murine AD model (5xFAD mice) in a randomized and fully blinded long-term in-vivo study following GLP (Good Laboratory Practices) guidelines. The heads of the mice were illuminated with no (sham), low or high power 810 nm light, three times a week for 5 months from the first to the sixth month of life corresponding to the prodromal phase of the pathology. The results showed that there were no significant differences between the groups in behavioral tests, including the Morris water maze, novel object recognition, and Y-maze. Similarly, histological analyses showed no differences in amyloid load, neuronal loss or microglial response. In conclusion, under the conditions of our experiment, we were unable to demonstrate any therapeutic effect of PBM for AD. This study calls for further evidence and caution when considering PBM as an effective treatment for AD.

Abstract 14198: Therapy for Myocardial Ischemia-Reperfusion Injury Using Novel Allogenic Mitochondria and Associated Organelle Complex

Background: Ischemia and reperfusion (I/R) injury induces intracellular calcium overload and generation of reactive oxygen species, which trigger intracellular mitochondrial damage, resulting in cardiomyocyte apoptosis. Freshly prepared autologous mitochondria improved damaged cardiac function, however, little is known whether allogenic mitochondrial organelle complex -Q (MRC-Q) can be replenished in the heart to achieve cardioprotective effects against I/R injury. Purpose: The aim of this study was to investigate whether the exogenous administration of allergenically isolated mitochondria leads to a protective effect on the myocardium after I/R injury using a well-established murine model. Methods and Results: The MRC-Q used in the current experiments were isolated by a proprietary technology ( WO2021132735A2) . All MRC-Q was frozen and stored at -80 °C before use. First, purified GFP-labeled HUVEC-derived MRC-Q were added to a rat cardiomyocyte cell line, and observed to be uptaken in cardiomyocytes in a time-dependent manner, which was also confirmed by quantitative polymerase chain reaction detecting human mitochondria DNA in rat cardiomyocytes. In addition, we demonstrated that cell viability was improved in the MRC-Q-treated group 96 hours after hypoxia/reoxygenation (H/R). Further examinations suggested the one of mechanism on cardioprotection by incorporating MRC-Q into the cardiomyocyte to be mediated by the secretion of mitochondria-derived peptides (MDP), which could inhibit apoptosis-promoting signals in addition to anti-ROS activity of MRC-Q. Next, we examined potential cardioprotective effect of MRC-Q administration on I/R injury in a murine model. Simultaneously at reperfusion, MRC-Q were administered intramyocardially in the damaged myocardium area. Compared to the vehicle group, MRC-Q-treated group showed a reduction in infarct size with fewer apoptotic cells, a decrease in circulating cardiac enzymes, and an improvement in cardiac function 24 hours after reperfusion. Finally, a preclinical pig model also confirmed the cardioprotective effects of MRC-Q. Conclusion: Our data demonstrated that MRC-Q have a protective effect on the heart by modulating cardiac apoptosis after ischemia/reperfusion injury.

Progressive Gut Microbiome Dysbiosis Is Associated with Chronic Lymphocytic Leukemia Pathogenesis and Development in the Eµ-TCL1 Mouse Model

Introduction: The gut microbiome is home to a community of microorganisms such as bacteria, fungi, viruses, archaea, and protozoa. Maintaining a balance between “favorable” and “unfavorable” bacteria within the gut microbiome is essential for homeostatic physiological processes including proper immune system development, regulation of metabolic pathways, protection against pathogens, and gut epithelium integrity. Chronic lymphocytic leukemia (CLL) is a mature B-cell malignancy characterized by the accumulation of CD5 + B-cells in the blood, bone marrow, and secondary lymphoid tissues. The pathogenesis of CLL is related to the activation of critical survival pathways such as B-cell and toll-like receptor signaling pathways, where microbial antigens are known to be key stimulators driving CLL B-cell proliferation. In this study, we investigated how the gut microbiota composition contributes and correlates to CLL disease progression. Methods: Using a well-established murine model representative of CLL development in humans, we profiled the gut microbiota of transgenic Eµ-TCL1 mice and age-matched wild-type B6 (WT B6) mice over the course of 12 months (n = 7-10/group). Fecal pellets were collected aseptically from study mice at four time points (4, 7, 10, and 12 months) for DNA extraction using the QIAamp® PowerFecal® Pro DNA Kit. To characterize the gut microflora, we utilized 16S rRNA sequencing using the Illumina 16S metagenomics protocol. Bacterial taxonomy was assigned and analyzed using the QIIME2™ platform. Linear Discriminant Analysis (LDA) Effect Size (LEfSe) analysis was employed to identify differentially expressed bacterial taxa between cohorts. Beta diversity indices were used to evaluate intersample differences in the fecal microbiota. Mice were monitored for peripheral expansion of leukemia cells (%CD45 +/CD19 +/CD5 + cells) via flow cytometry. Next, we evaluated the microbiome in an aggressive model of CLL. Utilizing the Eµ-TCL1 adoptive transfer murine model, similar methods were adopted to profile the gut microbiome over the course of 7 weeks. Lastly, to evaluate the contribution of the gut microbiota to CLL development, we employed an antibiotic-mediated gut microflora ablation to the adoptive transfer Eµ-TCL1 model, with leukemia expansion monitored post-engraftment on a weekly basis. Results: A unique microbiome signature was identified in both the indolent and aggressive model of CLL. Evaluation of beta diversity, using Bray-Curtis distance metrics, confirmed distinct differences in the gut microbial communities between Eµ-TCL1 and WT B6 cohorts. At the phylum level, the gut microbiota of the Eµ-TCL1 and WT B6 cohorts were similarly dominated by Firmicutes and Bacteroidota. However, Proteobacteria Deferribacterota and Desulfobacterota were more abundant in the Eµ-TCL1 mice over 12 months as compared to age-matched WT B6. Abundance of Actinobacteriota increased specifically at 12 months in the Eµ-TCL1 mice. Additionally, LEfSe analysis showed enrichment of certain phyla in the Eµ-TCL1 mice at 4 months, 10 months, and 12 months. Presence of these bacteria paralleled with expansion of disease. In the aggressive model of CLL, beta diversity confirmed distinct differences in gut microbial communities as CLL disease progressed. Moreover, certain phyla were enriched in leukemic mice at 7 weeks post-engraftment including Proteobacteria and Actinobacteriota. Lastly, in the antibiotic-mediated gut flora ablation model, a delay of disease onset was reflected in mice with ablated gut microbiomes compared to mice with intact gut microbiomes, resulting in prolonged overall survival. Conclusion: We provide novel results associating the gut microbiome with the initiation and progression of CLL disease. In both the indolent and aggressive CLL disease models, we showed that there is a distinct gut microbiome as evident by beta diversity. A dysbiotic signature represented by increasing Proteobacteria abundance was similarly identified in both models. Proteobacteria are known to be evident in diseases characterized by inflammation. These findings support future studies to determine a potential causal mechanism between gut microbiota changes and CLL pathogenesis.

Caloric Restriction: A Novel Conditioning Strategy to Improve the Survival of Ischemically Challenged Musculocutaneous Random Pattern Flaps.

Caloric restriction (CR) is a cost-effective and easy-to-perform approach to counteracting surgical stress. The present study therefore evaluates the tissue-protective effects of a 30% CR in musculocutaneous flaps undergoing ischemia. For this purpose, a well-established murine dorsal skinfold chamber model, in combination with random pattern musculocutaneous flaps, was used. C57BL/6N mice were divided at random into a CR group (n = 8) and a control group with unrestricted access to standard chow (n = 8). The CR animals were subjected to a 30% reduction in caloric intake for 10 days before flap elevation. Intravital fluorescence microscopy was carried out on days 1, 3, 5, 7 and 10 after flap elevation to assess the nutritive blood perfusion, angiogenesis and flap necrosis. Subsequently, the flap tissue was harvested for additional histological and immunohistochemical analyses. The CR-treated animals exhibited a significantly higher functional capillary density and more newly formed microvessels within the flap tissue when compared to the controls; this was associated with a significantly higher flap survival rate. Immunohistochemical analyses showed a decreased invasion of myeloperoxidase-positive neutrophilic granulocytes into the flap tissue of the CR-treated mice. Moreover, the detection of cleaved caspase-3 revealed fewer cells undergoing apoptosis in the transition zone between the vital and necrotic tissue in the flaps of the CR-treated mice. These results demonstrate that a CR of 30% effectively prevents flap necrosis by maintaining microperfusion on a capillary level and inhibiting inflammation under ischemic stress. Hence, CR represents a promising novel conditioning strategy for improving the survival of musculocutaneous flaps with random pattern perfusion.

LB1753 OR101, next generation selective JAK3, shows significant effectiveness in well-established murine models of inflammatory skin disorders

LB1753 OR101, next generation selective JAK3, shows significant effectiveness in well-established murine models of inflammatory skin disorders

Cigarette smoke attenuates mesenchymal stem cell-based suppression of immune cell-driven acute liver failure

Detrimental effects of smoking on mesenchymal stem cell (MSC)-dependent immunosuppression and hepatoprotection are unknown. Herewith, by using α-galactosylceramide (α-GalCer)-induced liver injury, a well-established murine model of fulminant hepatitis, we examined molecular mechanisms which were responsible for negative effects of cigarette smoke on MSC-dependent immunomodulation. MSC which were grown in cigarette smoke-exposed medium (MSCWS-CM) obtained pro-inflammatory phenotype, were not able to optimally produce hepatoprotective and immunosuppressive cytokines (TGF-β, HGF, IL-10, NO, KYN), and secreted significantly higher amounts of inflammatory cytokines (IFN-γ, TNF-α, IL-17, IL-6) than MSC that were cultured in standard medium never exposed to cigarette smoke (MSCCM). In contrast to MSCCM, which efficiently attenuated α-GalCer-induced hepatitis, MSCWS-CM were not able to prevent hepatocyte injury and liver inflammation. MSCWS-CM had reduced capacity for the suppression of liver-infiltrated inflammatory macrophages, dendritic cells (DCs) and lymphocytes. Although significantly lower number of IL-12-producing macrophages and DCs, TNF-α, IFN-γ or IL-17-producing CD4 + and CD8 +T lymphocytes, NK and NKT cells were noticed in the livers of α-GalCer+MSCCM-treated mice compared to α-GalCer+saline-treated animals, this phenomenon was not observed in α-GalCer-injured mice that received MSCWS-CM. MSCWS-CM could not induce expansion of anti-inflammatory IL-10-producing FoxP3 +CD4 + and CD8 + T regulatory cells and were not able to create immunosuppressive microenvironment in the liver as MSCCM. Similarly as it was observed in mice, MSCWS-CM were not able to optimally inhibit production of inflammatory and hepatototoxic cytokines in activated human Th1/Th17 and NKT1/NKT17 cells, confirming the hypothesis that cigarette smoke significantly attenuates therapeutic potential of MSC in cell-based immunotherapy of inflammatory liver diseases.