Transplantation of Donor-Recipient Chimeric Cells Restores Peripheral Blood Cell Populations and Increases Survival after Total Body Irradiation-Induced Injury in a Rat Experimental Model.

Current therapies for acute radiation syndrome (ARS) involve bone marrow transplantation (BMT), leading to graft-versus-host disease (GvHD). To address this challenge, we have developed a novel donor-recipient chimeric cell (DRCC) therapy to increase survival and prevent GvHD following total body irradiation (TBI)-induced hematopoietic injury without the need for immunosuppression. In this study, 20 Lewis rats were exposed to 7 Gy TBI to induce ARS, and we assessed the efficacy of various cellular therapies following systemic intraosseous administration. Twenty Lewis rats were randomly divided into four experimental groups (n = 5/group): saline control, allogeneic bone marrow transplantation (alloBMT), DRCC, and alloBMT + DRCC. DRCC were created by polyethylene glycol-mediated fusion of bone marrow cells from 24 ACI (RT1a) and 24 Lewis (RT11) rat donors. Fusion feasibility was confirmed by flow cytometry and confocal microscopy. The impact of different therapies on post-irradiation peripheral blood cell recovery was evaluated through complete blood count, while GvHD signs were monitored clinically and histopathologically. The chimeric state of DRCC was confirmed. Post-alloBMT mortality was 60%, whereas DRCC and alloBMT + DRCC therapies achieved 100% survival. DRCC therapy also led to the highest white blood cell counts and minimal GvHD changes in kidney and skin samples, in contrast to alloBMT treatment. In this study, transplantation of DRCC promoted the recovery of peripheral blood cell populations after TBI without the development of GVHD. This study introduces a novel and promising DRCC-based bridging therapy for treating ARS and extending survival without GvHD.

Subconjunctival injection of rapamycin-loaded polymeric microparticles for effective suppression of noninfectious uveitis in rats.

Noninfective uveitis is a major cause of vision impairment, and corticosteroid medication is a mainstay clinical strategy that causes severe side effects. Rapamycin (RAPA), a potent immunomodulator, is a promising treatment for noninfective uveitis. However, because high and frequent dosages are required, it is a great challenge to implement its clinical translation for noninfective uveitis therapy owing to its serious toxicity. In the present study, we engineered an injectable microparticulate drug delivery system based on biodegradable block polymers (i.e., polycaprolactone-poly (ethylene glycol)-polycaprolactone, PCEC) for efficient ocular delivery of RAPA via a subconjunctival injection route and investigated its therapeutic efficacy in an experimental autoimmune uveitis (EAU) rat model. RAPA-PCEC microparticles were fabricated using the emulsion-evaporation method and thoroughly characterized using scanning electron microscopy, fourier transform infrared spectroscopy, X-ray diffraction, and differential scanning calorimetry. The formed microparticles exhibited slow in vitro degradation over 28 days, and provided both in vitro and in vivo sustained release of RAPA over 4 weeks. Additionally, a single subconjunctival injection of PCEC microparticles resulted in high ocular tolerance. More importantly, subconjunctival injection of RAPA-PCEC microparticles significantly attenuated the clinical signs of EAU in a dose-dependent manner by reducing inflammatory cell infiltration (i.e., CD45+ cells and Th17 cells) and inhibiting microglial activation. Overall, this injectable microparticulate system may be promising vehicle for intraocular delivery of RAPA for the treatment of noninfective uveitis.

Role of the prefrontal cortical protease TACE/ADAM17 in neurobehavioral responses to chronic stress during adolescence.

INTRODUCTION: Chronic adolescent stress profoundly affects prefrontal cortical networks regulating top-down behavior control. However, the neurobiological pathways contributing to stress-induced alterations in the brain and behavior remain largely unknown. Chronic stress influences brain growth factors and immune responses, which may, in turn, disrupt the maturation and function of prefrontal cortical networks. The tumor necrosis factor alpha-converting enzyme/a disintegrin and metalloproteinase 17 (TACE/ADAM17) is a sheddase with essential functions in brain maturation, behavior, and inflammatory responses. This study aimed to determine the impact of stress on the prefrontal cortex and whether TACE/ADAM17 plays a role in these responses. METHODS: We used a Lewis rat model that incorporates critical elements of chronic psychosocial stress, such as uncontrollability, unpredictability, lack of social support, and re-experiencing of trauma. RESULTS: Chronic stress during adolescence reduced the acoustic startle reflex and social interactions while increasing extracellular free water content and TACE/ADAM17 mRNA levels in the medial prefrontal cortex. Chronic stress altered various ethological behavioral domains in the observation home cages (decreased ingestive behaviors and increased walking, grooming, and rearing behaviors). A group of rats was injected intracerebrally either with a novel Accell™ SMARTpool TACE/ADAM17 siRNA or a corresponding siRNA vehicle (control). The RNAscope Multiplex Fluorescent v2 Assay was used to visualize mRNA expression. Automated puncta quantification and analyses demonstrated that TACE/ADAM17 siRNA administration reduced TACE/ADAM17 mRNA levels in the medial prefrontal cortex (59% reduction relative to control). We found that the rats that received prefrontal cortical TACE/ADAM17 siRNA administration exhibited altered eating patterns (e.g., increased food intake and time in the feeding zone during the light cycle). CONCLUSION: This study supports that the prefrontal cortex is sensitive to adolescent chronic stress and suggests that TACE/ADAM17 may be involved in the brain responses to stress.

Antibiotics-Induced Intestinal Immunomodulation Attenuates Experimental Autoimmune Neuritis (EAN).

BACKGROUND: The composition of gut microbiota plays a pivotal role in priming the immune system and thus impacts autoimmune diseases. Data on the effects of gut bacteria eradication via systemic antibiotics on immune neuropathies are currently lacking. This study therefore assessed the effects of antibiotics-induced gut microbiota alterations on the severity of experimental autoimmune neuritis (EAN), a rat model of Guillain-Barré Syndrome (GBS). Myelin P0 peptide 180-199 (P0 180-199)-induced EAN severity was compared between adult Lewis rats (12 weeks old) that received drinking water with or without antibiotics (colistin, metronidazole, vancomycin) and healthy rats, beginning antibiotics treatment immediately after immunization (day 0), and continuing treatment for 14 consecutive days. Neuropathy severity was assessed via a modified clinical score, and then related to gut microbiota alterations observed after fecal 16S rRNA gene sequencing at baseline and after EAN induction. Effectors of gut mucosal and endoneurial immunity were assessed via immunostaining. EAN rats showed increased gut mucosal permeability alongside increased mucosal CD8+ T cells compared to healthy controls. Antibiotics treatment alleviated clinical EAN severity and reduced endoneurial T cell infiltration, decreased gut mucosal CD8+ T cells and increased gut bacteria that may be associated with anti-inflammatory mechanisms, like Lactobacillus or Parasutterella. Our findings point out a relation between gut mucosal immunity and the pathogenesis of EAN, and indicate that antibiotics-induced intestinal immunomodulation might be a therapeutic approach to alleviate autoimmunity in immune neuropathies. Further studies are warranted to evaluate the clinical transferability of these findings to patients with GBS.

Creation and characterization of novel rat model for recessive dystrophic epidermolysis bullosa: Frameshift mutation of the Col7a1 gene leads to severe blistered phenotype.

Recessive dystrophic epidermolysis bullosa is a rare genodermatosis caused by a mutation of the Col7a1 gene. The Col7a1 gene codes for collagen type VII protein, a major component of anchoring fibrils. Mutations of the Col7a1 gene can cause aberrant collagen type VII formation, causing an associated lack or absence of anchoring fibrils. This presents clinically as chronic blistering, scarring, and fibrosis, often leading to the development of cutaneous squamous cell carcinoma. Patients also experience persistent pain and pruritus. Pain management and supportive bandaging remain the primary treatment options. The pathology of recessive dystrophic epidermolysis bullosa was first described in the 1980s, and there has since been a multitude of encouraging treatment options developed. However, in vivo research has been hindered by inadequate models of the disease. The various mouse models in existence possess longevity and surface area constraints, or do not adequately model a normal human disease state. In this paper, we describe a novel rat model of recessive dystrophic epidermolysis bullosa that offers an alternative to previous murine models. An 8-base pair deletion was induced in the Col7a1 gene of Lewis rats, which was subsequently found to cause a premature stop codon downstream. Homozygous mutants presented with a fragile and chronically blistered phenotype postnatally. Further histological analysis revealed subepidermal clefting and the absence of anchoring fibrils. The generation of this novel model offers researchers an easily maintained organism that possesses a larger surface area for experimental topical and transfused therapies to be tested, which may provide great utility in the future study of this debilitating disease.

Disulfiram, an Anti-alcoholic Drug, Targets Macrophages and Attenuates Acute Rejection in Rat Lung Allografts.

Macrophages contribute to post-transplant lung rejection. Disulfiram (DSF), an anti-alcoholic drug, has an anti-inflammatory effect and regulates macrophage chemotactic activity. Here, we investigated DSF efficacy in suppressing acute rejection post-lung transplantation. Male Lewis rats (280-300 g) received orthotopic left lung transplants from Fisher 344 rats (minor histocompatibility antigen-mismatched transplantation). DSF (0.75 mg/h) monotherapy or co-solvent only (50% hydroxypropyl-ß-cyclodextrin) as control was subcutaneously administered for 7 days (n = 10/group). No post-transplant immunosuppressant was administered. Grades of acute rejection, infiltration of immune cells positive for CD68, CD3, or CD79a, and gene expression of monocyte chemoattractant protein and pro-inflammatory cytokines in the grafts were assessed 7 days post-transplantation. The DSF-treated group had significantly milder lymphocytic bronchiolitis than the control group. The infiltration levels of CD68+ or CD3+ cells to the peribronchial area were significantly lower in the DSF than in the control groups. The normalized expression of chemokine ligand 2 and interleukin-6 mRNA in allografts was lower in the DSF than in the control groups. Validation assay revealed interleukin-6 expression to be significantly lower in the DSF than in the control groups. DSF can alleviate acute rejection post-lung transplantation by reducing macrophage accumulation around peripheral bronchi and suppressing pro-inflammatory cytokine expression.

Robust Rat and Mouse Models of Bilateral Renal Ischemia Reperfusion Injury.

BACKGROUND/AIM: Acute and chronic kidney diseases are a major contributor to morbidity and mortality worldwide, with no specific treatments currently available for these. To enable understanding the pathophysiology of and testing novel treatments for acute and chronic kidney disease, a suitable in vivo model of kidney disease is essential. In this article, we describe two reliable rodent models (rats and mice) of efficacious kidney injury displaying acute to chronic kidney injury progression, which is also reversible through novel therapeutic strategies such as ischemic preconditioning (IPC). MATERIALS AND METHODS: We utilized adult male Lewis rats and adult male wildtype (C57BL/6) mice, performed a midline laparotomy, and induced warm ischemia to both kidneys by bilateral clamping of both renal vascular pedicles for a set time, to mimic the hypoxic etiology of disease commonly found in kidney injury. RESULTS: Bilateral ischemia reperfusion injury caused marked structural and functional kidney injury as exemplified by histology damage scores, serum creatinine levels, and kidney injury biomarker levels in both rodents. Furthermore, this effect displayed a dose-dependent response in the mouse model. CONCLUSION: These rodent models of bilateral kidney IRI are reliable, reproducible, and enable detailed mechanistic study of the underlying pathophysiology of both acute and chronic kidney disease. They have been carefully optimised for single operator use with a strong track record of training both surgically trained and surgically naïve operators.

Low-molecular-weight fucoidan increases telomere length and immunostimulatory effects on NK-92 cells following inhaled anesthetic injury.

Inhaled anesthetics, such as isoflurane, may cause side effects, including short-term immunosuppression and DNA damage. In contrast, low molecular weight fucoidan (LMF), derived from brown seaweed, exhibits promising immunomodulatory effects. In this study, we determined the effect of isoflurane on telomeres and examined the potential of LMF to ameliorate the harmful effects of isoflurane. Male Lewis rats, the mouse lymphoma cell line YAC-1, and the human nature killer cell line NK-92 MI were exposed to isoflurane. The relative telomere length (T/S) ratio and mRNA expression were determined by quantitative PCR. The viability assay was used to assess cell viability. In vivo, 2% isoflurane exposure, which is a clinically relevant concentration, reduced telomere length, and correlated with exposure frequency and duration. Isoflurane concentrations above 2% shortened YAC-1 telomeres, with minimal impact on cell viability. LMF pre-treatment enhanced NK-92 MI cell survival resulting from isoflurane exposure and exerted superior telomere protection compared with LMF post-treatment. Furthermore, adding LMF during isoflurane exposure resulted in a significant increase in IFN-γ, TNF-α, and IL-10 mRNA compared with the untreated group. LMF protected against isoflurane-induced telomere shortening, enhanced NK cell viability, and modulated cytokine expression, thus mitigating postoperative immune suppression and risk of tumor metastasis.

Osteogenic human MSC-derived extracellular vesicles regulate MSC activity and osteogenic differentiation and promote bone regeneration in a rat calvarial defect model.

BACKGROUND: There is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects. METHODS: EVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10 µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50 µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry. RESULTS: Although Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM. CONCLUSIONS: This study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.

Metformin alleviates lung ischemia-reperfusion injury in a rat lung transplantation model.

Background: Lung ischemia-reperfusion injury (LIRI) is among the complications observed after lung transplantation and is associated with morbidity and mortality. Preconditioning of the donor lung before organ retrieval may improve organ quality after transplantation. We investigated whether preconditioning with metformin (Met) ameliorates LIRI after lung transplantation. Methods: Twenty Lewis rats were randomly divided into the sham, LIRI, and Met groups. The rats in the LIRI and Met groups received saline and Met, respectively, via oral gavage. Subsequently, a donor lung was harvested and kept in cold storage for 8 h. The LIRI and Met groups then underwent left lung transplantation. After 2 h of reperfusion, serum and transplanted lung tissues were examined. Results: The partial pressure of oxygen (PaO2) was greater in the Met group than in the LIRI group. In the Met group, wet-to-dry (W/D) weight ratios, inflammatory factor levels, oxidative stress levels and apoptosis levels were notably decreased. Conclusions: Met protects against ischemia-reperfusion injury after lung transplantation in rats, and its therapeutic effect is associated with its anti-inflammatory, antioxidative, and antiapoptotic properties.

Topical Tacrolimus and Mycophenolic Acid Therapy Synergizes with Low Dose Systemic Immunosuppression to Sustain Vascularized Composite Allograft Survival.

AIM: The goal of this study was to evaluate whether topical administration of tacrolimus (TAC) and mycophenolic acid (MPA) at the transplant site enables vascularized composite allograft (VCA) survival with significant minimization of the dose and adverse effects of systemic TAC (STAC) immunosuppression. MATERIALS AND METHODS: Lewis (Lew) rats received orthotopic hind limb allotransplants from fully mismatched Brown Norway (BN) donors. Group 1 (Controls) received no treatment. Other groups were treated with STAC at a dose of 1 mg/kg/day for 7 days. On post-operative day (POD) 8, the STAC dose was dropped to 0.1 mg/kg/day for Group 2 and maintained at 1 mg/kg for Group 3. Group 4 received topical application of TAC and MPA on the transplanted (Tx) limb starting POD 8 without STAC. Group 5 received topical TAC and MPA on the contralateral non-Tx limb and Group 6 received topical TAC and MPA on the Tx limb starting POD 8 along with low dose STAC (0.1 mg/kg/day). Treatment was continued until the study end point was reached, defined as either grade 3 rejection or allograft survival exceeding 100 days. .We conducted sequential LC-MS/MS measurements to assess TAC and MPA concentrations in both blood/plasma and allograft tissues. Additionally, we evaluated markers indicative of organ toxicity associated with STAC immunosuppression. RESULTS: Compared to controls, topical therapy with TAC+MPA significantly prolonged allograft survival beyond 100 daysat very low dose STAC (0.1 mg/kg/day) (Group 6). The histopathological assessment of the grafts was consistent with the clinical outcomes. .Drug levels in blood/plasma remained low or undetectable, while allograft tissues showed higher drug concentrations compared to contralateral limb tissues (P<0.05). . Urinary creatinine clearance remained within the normal range at 2.5 mL/min. CONCLUSION: Combination therapy with topical TAC and MPA synergizes with a very low dose, corticosteroid- free-STAC regimen and facilitates rejection-free, prolonged VCA survival without morbidity.

Dietary polyamines promote intestinal adaptation in an experimental model of short bowel syndrome.

Intestinal adaptation does not necessarily recover absorptive capacity in short bowel syndrome (SBS), sometimes resulting in intestinal failure-associated liver disease (IFALD). Additionally, its therapeutic options remain limited. Polyamines (spermidine and spermine) are known as one of the autophagy inducers and play important roles in promoting the weaning process; however, their impact on intestinal adaptation is unknown. The aim of this study was to investigate the impact of polyamines ingestion on adaptation and hepatic lipid metabolism in SBS. We performed resection of two-thirds of the small intestine in male Lewis rats as an SBS model. They were allocated into three groups and fed different polyamine content diets (0%, 0.01%, 0.1%) for 30 days. Polyamines were confirmed to distribute to remnant intestine, whole blood, and liver. Villous height and number of Ki-67-positive cells in the crypt area increased with the high polyamine diet. Polyamines increased secretory IgA and mucin content in feces, and enhanced tissue Claudin-3 expression. In contrast, polyamines augmented albumin synthesis, mitochondrial DNA copy number, and ATP storage in the liver. Moreover, polyamines promoted autophagy flux and activated AMP-activated protein kinase with suppression of lipogenic gene expression. Polyamines ingestion may provide a new therapeutic option for SBS with IFALD.

A Model to Study Wound Healing Over Exposed Avascular Structures in Rodents With a 3D-Printed Wound Frame.

BACKGROUND: Soft tissue defects with exposed avascular structures require reconstruction with well-vascularized tissues. Extensive research is ongoing to explore tissue engineered products that provide durable coverage. However, there is a lack of controlled and affordable testbeds in the preclinical setting to reflect this challenging clinical scenario. We aimed to address this gap in the literature and develop a feasible and easily reproducible model in rodents that reflects an avascular structure in the wound bed. METHODS: We created 20 × 20 mm full thickness wounds on the dorsal skin of Lewis rats and secured 0.5-mm-thick silicone sheets of varying sizes to the wound bed. A 3D-printed wound frame was designed to isolate the wound environment. Skin graft and free flap survival along with exposure of the underlying silicone was assessed. Rats were followed for 4 weeks with weekly dressing changes and photography. Samples were retrieved at the endpoint for tissue viability and histologic analysis. RESULTS: The total wound surface area was constant throughout the duration of the experiment in all groups and the wound frames were well tolerated. The portion of the skin graft without underlying silicone demonstrated integration with the underlying fascia and a histologically intact epidermis. Gradual necrosis of the portion of the skin graft overlying the silicone sheet was observed with varying sizes of the silicone sheet. When the size of the silicone sheet was reduced from 50% of the wound surface area, the portion surviving over the silicone sheet increased at the 4-week timepoint. The free flap provided complete coverage over the silicone sheet. CONCLUSION: We developed a novel model of rodent wound healing to maintain the same wound size and isolate the wound environment for up to 4 weeks. This model is clinically relevant to a complex wound with an avascular structure in the wound bed. Skin grafts failed to completely cover increasing sizes of the avascular structure, whereas the free flap was able to provide viable coverage. This cost-effective model will establish an easily reproducible platform to evaluate more complex bioengineered wound coverage solutions.

Significantly Improved Cold Preservation of Rat Hind Limb Vascularized Composite Allografts Using the New PrC-210 Free Radical Scavenger.

Vascularized composite allotransplantation (VCA) represents a promising reconstructive solution primarily conducted to improve quality of life. However, tissue damage caused by cold-ischemia (CI) storage prior to transplant represents a major factor limiting widespread application. This study investigates the addition of the novel free radical scavenger PrC-210 to UW Organ Preservation Solution (UW Solution) to suppress CI-induced skeletal muscle injury in a rat hind limb amputation model. Lewis rats received systemic perfusion of UW solution +/- PrC-210 (0 mM control, 10 mM, 20 mM, 30 mM, or 40 mM), followed by bilateral transfemoral amputation. Limbs were stored in 40 mL of the same perfusate at 4 °C for 48 h. Muscle punch biopsies were taken at set times over the 48 h cold-storage period and analyzed for caspase-3,7 activity, cytochrome C levels, and qualitative histology. A single 15 s perfusion of PrC-210-containing UW Solution conferred a dose-dependent reduction in CI-induced muscle cell death over 48 h. In the presence of PrC-210, muscle cell mitochondrial cytochrome C release was equivalent to 0 h controls, with profound reductions in the caspase-3,7 apoptotic marker that correlated with limb histology. PrC-210 conferred complete prevention of ROS-induced mitochondrial lysis in vitro, as measured by cytochrome C release. We conclude that the addition of 30 mM PrC210 to UW Solution conferred the most consistent reduction in CI limb damage, and it warrants further investigation for clinical application in the VCA setting.

Sintered fluorapatite scaffolds as an autograft-like engineered bone graft.

Hydroxyapatite (HA)-based materials are widely used as bone substitutes due to their inherent biocompatibility, osteoconductivity, and bio-absorption properties. However, HA scaffolds lack compressive strength when compared to autograft bone. It has been shown that the fluoridated form of HA, fluorapatite (FA), can be sintered to obtain this desired strength as well as slower degradation properties. Also, FA surfaces have been previously shown to promote stem cell differentiation toward an osteogenic lineage. Thus, it was hypothesized that FA, with and without stromal vascular fraction (SVF), would guide bone healing to an equal or better extent than the clinical gold standard. The regenerative potentials of these scaffolds were tested in 32 Lewis rats in a femoral condylar defect model with untreated (negative), isograft (positive), and commercial HA as controls. Animals were survived for 12 weeks post-implantation. A semi-quantitative micro-CT analysis was developed to quantify the percent new bone formation within the defects. Our model showed significantly higher (p < .05) new bone depositions in all apatite groups compared to the autograft group. Overall, the FA group had the most significant new bone deposition, while the differences between HA, FA, and FA + SVF were insignificant (p > .05). Histological observations supported the micro-CT findings and highlighted the presence of healthy bone tissues without interposing capsules or intense immune responses for FA groups. Most importantly, the regenerating bone tissue within the FA + SVF scaffolds resembled the architecture of the surrounding trabecular bone, showing intertrabecular spaces, while the FA group presented a denser cortical bone-like architecture. Also, a lower density of cells was observed near FA granules compared to HA surfaces, suggesting a reduced immune response. This first in vivo rat study supported the tested hypothesis, illustrating the utility of FA as a bone scaffold material.

Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model." However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasite's replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

Evaluating the Effects of Kidney Preservation at 10 °C with Hemopure and Sodium Thiosulfate in a Rat Model of Syngeneic Orthotopic Kidney Transplantation.

Kidney transplantation is preferred for end-stage renal disease. The current gold standard for kidney preservation is static cold storage (SCS) at 4 °C. However, SCS contributes to renal graft damage through ischemia-reperfusion injury (IRI). We previously reported renal graft protection after SCS with a hydrogen sulfide donor, sodium thiosulfate (STS), at 4 °C. Therefore, this study aims to investigate whether SCS at 10 °C with STS and Hemopure (blood substitute), will provide similar protection. Using in vitro model of IRI, we subjected rat renal proximal tubular epithelial cells to hypoxia-reoxygenation for 24 h at 10 °C with or without STS and measured cell viability. In vivo, we preserved 36 donor kidneys of Lewis rats for 24 h in a preservation solution at 10 °C supplemented with STS, Hemopure, or both followed by transplantation. Tissue damage and recipient graft function parameters, including serum creatinine, blood urea nitrogen, urine osmolality, and glomerular filtration rate (GFR), were evaluated. STS-treated proximal tubular epithelial cells exhibited enhanced viability at 10 °C compared with untreated control cells (p < 0.05). Also, STS and Hemopure improved renal graft function compared with control grafts (p < 0.05) in the early time period after the transplant, but long-term function did not reach significance. Overall, renal graft preservation at 10 °C with STS and Hemopure supplementation has the potential to enhance graft function and reduce kidney damage, suggesting a novel approach to reducing IRI and post-transplant complications.

Ivabradine Alleviates Experimental Autoimmune Myocarditis-Mediated Myocardial Injury.

Ivabradine (IVA) reduces heart rate by inhibiting hyperpolarization-activated cyclic nucleotide-gated channels (HCNs), which play a role in the promotion of pacemaker activity in cardiac sinoatrial node cells. HCNs are highly expressed in neural and myocardial tissues and are involved in the modulation of inflammatory neuropathic pain. However, whether IVA exerts any effect on myocardial inflammation in the pathogenesis of heart failure is unclear. We employed single-cell RNA sequencing (scRNA-seq) in porcine cardiac myosin-induced experimental autoimmune myocarditis rat model to determine the effects and mechanisms of IVA. Lewis rats (n = 32) were randomly divided into the normal, control, high-dose-IVA, and low-dose-IVA groups. Heart rate and blood pressure were measured on days 0 and 21, respectively. Echocardiography was performed on day 22, and inflammation of the myocardium was evaluated via histopathological examination. Western blot was employed to detect the expression of HCN1-4, MinK-related protein 1 (MiRP1), matrix metalloproteinase 2 (MMP-2), MMP-9, and transforming growth factor-ß (TGF-ß). Furthermore, enzyme-linked immunosorbent assay was performed to measure serum IL-1, IL-6, and TNF-α. The relative mRNA levels of collagen I, collagen III, and α-smooth muscle actin (α-SMA) were determined via qRT-PCR. We found that IVA reduced the total number of cells infiltrated into the myocardium, particularly in the subset of fibroblasts, endocardia, and monocytes. IVA administration ameliorated cardiac inflammation and reduced collagen production. Results of the echocardiography indicated that left ventricular internal diameter at end-systole LVIDs increased whereas left ventricular ejection fraction and left ventricular fractional shortening decreased in the control group. IVA improved cardiac performance. The expression of HCN4 and MiRP1 protein and the level of serum IL-1, IL-6, and TNF-α were decreased by IVA treatment. In conclusion, HCNs and the helper proteins were increased in the profile of myocardial inflammation. HCNs may be involved in the regulation of myocardial inflammation by inhibiting immune cell infiltration. Our findings can contribute to the development of IVA-based combination therapies for the future treatment of cardiac inflammation and heart failure.

The association between activation of the ERK1/2-NF-κB signaling pathway by TIMP2 expression and chronic renal allograft dysfunction in the CRAD rat model.

PURPOSE: The tissue inhibitor of metalloproteinase 2 (TIMP2), a natural inhibitor of matrix metalloproteinase (MMP), regulates inflammation, fibrosis, and cell proliferation. Chronic renal allograft dysfunction (CRAD) is a primary factor affecting the long-term survival of renal allografts. We assessed whether up-regulation of TIMP2 expression may affect the ERK1/2-NF-κB signaling pathway and CRAD development. METHODS: Lewis rats received orthotopic F344 kidney allografts to establish the classical CRAD model. The treatment group was injected with a lentivirus encoding a TIMP2-targeting small hairpin (sh)RNA (LTS) at 5 × 108 TU/ml monthly after kidney transplantation. A second CRAD group was injected with a lentivirus TIMP2-control vector (LTC). After 12 weeks, blood, urine, and kidney tissue were harvested to evaluate renal function and pathological examinations. Hematoxylin and eosin staining, Masson staining, and Periodic acid-Schiff staining were performed for renal histopathological evaluation according to the Banff criteria. TIMP2, phospho (p)-ERK1/2, p-p65 (NF-κB) expression levels were measured via immunohistochemical and Western blot analyses. RESULTS: Compared to the F344 and Lewis control groups, the expression of TIMP2, p-ERK1/2, and p-p65 were significantly higher in the CRAD and CRAD+LTC renal tissues (p < 0.05). There were also increased levels of serum creatinine, nitrogen, and 24 h urinary protein in these two groups (p < 0.05). Typical histopathological changes of CRAD were observed in the CRAD and CRAD+LTC groups. Administration of LTS effectively decreased the expression of TIMP2, p-ERK1/2, and p-P65, and reduced interstitial fibrosis and macrophage infiltration in the treatment group (p < 0.05). Additionally, MCP1 and ICAM-1, which are downstream cytokines of the NF-κB pathway, were also inhibited in the renal rat kidney from the LTS group (p < 0.05). Furthermore, renal function was well preserved in the LTS group compared to the CRAD group and CRAD+LTC group. CONCLUSION: A decrease of TIMP2 can alleviate the progression of inflammation in CRAD via inhibition of the ERK1/2-NF-κB signaling pathway.

Immunosuppressive enzyme-responsive nanoparticles for enhanced accumulation in liver allograft to overcome acute rejection.

Acute rejection is a life-threatening complication after liver transplantation. Immunosuppressants such as tacrolimus are used to inhibit acute rejection of liver grafts in clinic. However, inefficient intragraft accumulation may reduce the therapeutic outcomes of tacrolimus. Here, an enzyme-responsive nanoparticle is developed to selectively enhance the accumulation of tacrolimus in liver allograft through enzyme-induced aggregation to refine immunotherapeutic efficacy of tacrolimus. The nanoparticles are composed of amphiphilic tacrolimus prodrugs synthesized by covalently conjugating tacrolimus and matrix metalloproteinase 9 (MMP9)-cleavable peptide-containing methoxy poly (ethylene glycol) to poly (l-glutamic acid). Upon exposure to MMP9, which is overexpressed in rejected liver allografts, the nanoparticles undergo a morphological transition from spherical micellar nanoparticles to microscale aggregate-like scaffolds. Intravenous administration of MMP9-responsive nanoparticles into a rat model of acute liver graft rejection results in enhanced nanoparticle accumulation in allograft as compared to nonresponsive nanoparticles. Consequently, the MMP9-responsive nanoparticles significantly inhibit intragraft inflammatory cell infiltration and proliferation, maintain intragraft immunosuppressive environment, alleviate graft damage, improve liver allograft function, abate weight loss and prolong recipient survival. This work proves that morphology-switchable enzyme-responsive nanoparticles represent an innovative strategy for selectively enhancing intragraft accumulation of immunosuppressive agents to improve treatment of liver allograft rejection.