Allografts In Rats Research Articles

Enhancing sciatic nerve regeneration with osteopontin-loaded acellular nerve allografts in rats: Effects on macrophage polarization

Osteopontin (OPN) is a multifunctional matrix glycoprotein with neuroprotective and immunomodulatory properties. This study explored the potential of OPN-loaded acellular nerve allografts (ANAs) to repair sciatic nerves in male Wistar rats. The research also delved into the impact of OPN on macrophage phenotypes. We reconstructed a 10 mm nerve gap with ANAs containing OPN at 2 nM and 4 nM. The sciatic functional index (SFI) and paw withdrawal reflex latency (WRL) showed the significant efficacy of ANA/OPN (2 nM) in enhancement of target organ reinnervation and subsequent sensorimotor recovery compared to other groups. Electrophysiological and histomorphometric analyses further supported the regenerative properties of ANA/OPN (2 nM). Additionally, ANA/OPN (2 nM) promoted macrophage polarization towards an M2 phenotype and reduced proinflammatory cytokines at the injury site. In conclusion, the study suggested that ANA loaded with 2 nM OPN effectively repaired transected sciatic nerves in rats, potentially through enhancing axonal sprouting and exerting anti-inflammatory effects.

Indoleamine 2, 3-dioxygenase-transfected bone marrow-derived mesenchymal stem cells promote corneal allograft survival by inhibiting T cell proliferation: A rat study

PurposeAllograft rejection is still the main cause of corneal transplantation failure. Therefore, we investigated the role of indoleamine 2,3-dioxygenase (IDO)-transfected bone marrow-derived mesenchymal stem cells (IDO-BMSCs) in corneal allograft rejection in rats. MethodsIDO-BMSCs were constructed and co-cultured with CD4+CD24− T cells to detect their effects on the proliferation of CD4+CD25−T cells in vitro. A corneal allograft rat model was used to confirm our in vitro and in vivo observations. Therefore, IDO-BMSCs were injected directly into the recipient's conjunctiva on the day of corneal transplantation and on day 5 after operation. Corneal graft rejection indices, including corneal neovascularization, opacity, and edema, were measured for up to 14 days after transplantation. The recipients' cervical lymph nodes and peripheral blood were collected to test the role of IDO-BMSCs in immune cells using flow cytometry. ResultsThe lentivirus-mediated IDO gene was successfully transfected into BMSCs, which stably secreted the IDO protein. The proliferation of CD4+CD25−T cells was significantly inhibited after their co-culture with IDO-BMSCs. Subconjunctival injection of IDO-BMSCs into corneal allografts of rats effectively reduced graft neovascularization, promoted allograft survival, and induced immune tolerance. Both CD4+ and CD8+ T cells in the local lymph nodes and peripheral blood, along with CD4+CD25−T cells in the local lymph nodes, were significantly reduced after transplantation. ConclusionOur results suggest that IDO-BMSC treatment enhances the direct immunomodulatory effect of corneal allograft transplants in rats, promoting corneal allograft survival by inhibiting the proliferation of CD4+, CD8+, and CD4+CD25−T cells. Therefore, modification of BMSCs by lentivirus-mediated IDO gene transfection may provide a novel strategy for controlling corneal allograft rejection.

24-hour supercooling preservation of vascularized composite allografts in rats: A proof-of-concept study

24-hour supercooling preservation of vascularized composite allografts in rats: A proof-of-concept study

Assessment of the anti-nociceptive effects of fetal ventral mesencephalic tissue allografts in a rat model of hemi-Parkinson's disease using fMRI.

Extensive studies showed increased subjective pain sensitivity in Parkinson's disease (PD), which appeared to be partially reversed by dopaminergic (DA) treatment. Although cell replacement represents an attractive therapeutic strategy, its potential for PD-related hyperalgesia remains unclear. We investigated re-establishment of DA function via allografting exogenic DA cells on pain hypersensitivity in a rat model of PD. We evaluated the anti-nociceptive effects of fetal ventral mesencephalic (rVM) tissue allografts in PD rats after unilateral 6-OHDA-induced toxicity in the medial forebrain bundle. The drug -induced rotation test was used to validate the severity of the nigrostriatal lesion; von Frey and thermal pain tests were employed to evaluate nociceptive function. Nociception-induced cerebral blood volume (CBV) response was measured using a 4.7-T MR system. Finally, the immunohistochemical (IHC) studies were performed and the results were compared with the imaging findings from functional magnetic resonance imaging (fMRI). The grafts significantly improved drug-induced rotation behavior and increased mechanical and thermal nociceptive thresholds in PD rats. The elevation of CBV signals significantly recovered on the grafted striatum, whereas this effect was inhibited by the D2R antagonist eticlopride in each striatum. Quantitative IHC analysis revealed the transplantation markedly increased the numbers of tyrosine hydroxylase immunoreactive cells. Therefore, we concluded transplantation of rVM tissue results in anti-nociceptive effects and improves motor function. Moreover, in vivo CBV response confirmed the key role of D2R-mediated pain modulation. Therefore, we demonstrate fMRI as a reliable imaging index in evaluating the anti-nociceptive therapeutic effects of fetal rVM transplantation in the rat model of PD.

A novel decellularized nerve graft for repairing peripheral nerve long gap injury in the rat

Decellularized nerve allografts are an alternative to autograft for repairing severe nerve injuries, since they have higher availability and do not induce rejection. In this study, we have assessed the regenerative potential of a novel decellularization protocol for human and rat nerves for repairing nerve resections, compared to the gold standard autograft. A 15-mm gap in the sciatic nerve was repaired with decellularized rat allograft (DC-RA), decellularized human xenograft (DC-HX), or fresh autograft (AG). Electrophysiology tests were performed monthly to evaluate muscle reinnervation, whereas histological and immunohistochemical analyses of the grafts were evaluated at 4 months. A short-term study was also performed to compare the differences between the two decellularized grafts (DC-RA and DC-HX) in early phases of regeneration. The decellularization process eliminated cellularity while preserving the ECM and endoneurial tubules of both rat and human nerves. Higher amount of reinnervation was observed in the AG group compared to the DC-RA group, while only half of the animals of the DC-HX showed distal muscle reinnervation. The density of myelinated axons was significantly higher in AG compared to both DC grafts, being this density significantly higher in DC-RA than in DC-HX. At short term, fibroblasts repopulated the DC-RA graft, supporting regenerated axons, whereas an important fibrotic reaction was observed around DC-HX grafts. In conclusion, the decellularized allograft sustained regeneration through a long gap in the rat although at a slower rate compared to the ideal autograft, whereas regeneration was limited or even failed when using a decellularized xenograft.

Effect of HO-1-modified BMMSCs on immune function in liver transplantation

We examined whether haem oxygenase-1 (HO-1) could enhance the immunosuppressive effects of bone marrow mesenchymal stem cells (BMMSCs) on the rejection of transplanted liver allografts in rats. The animals were divided into three groups: the normal saline (NS) group, BMMSC group and HO-1/BMMSCs group. In vitro, the extraction, culture and HO-1 transfection of BMMSCs were performed. Mixed lymphocyte response (MLR) analysis of HO-1/BMMSCs efficacy was performed. The rejection model of orthotopic liver transplantation in rats was established when BMMSCs and HO-1/BMMSCs were transfused via the portal vein. To reduce research bias, we established an isogenic Liver transplantation model of (LEW → LEW) and (BN → BN), which can achieve tolerance. Changes in histopathology and liver function in the transplanted liver and changes in regulatory T cell (Tregs), natural killer (NK) cells and cytokines after transplantation were observed in the different groups. The severe acute rejection after liver transplantation on postoperative Day 10 was observed in the NS group. The BMMSC group showed strong protective effects against rejection within the first 10 days after transplantation, while HO-1/BMMSCs showed stronger effects on rejection than BMMSCs alone. In addition, the activity of natural killer (NK) cells decreased significantly, the levels of regulatory T cells (Tregs), interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) increased significantly and the levels of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-23 (IL-23), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) decreased significantly in the HO-1/BMMSC group compared with the BMMSC group. HO-1/BMMSCs showed better immunosuppressive effects after liver transplantation than the other treatments. Our findings reveal that HO-1 can enhance the effects of BMMSCs on inhibiting acute rejection in orthotopic liver transplantation in rats.

Plasmacytoid dendritic cells mediate the tolerogenic effect of CD8+regulatory T cells in a rat tolerant liver transplantation model

BackgroundTolerance is more easily induced in liver transplant models than in other organs; CD8+CD45RClowregulatory T cells (Tregs) have been shown to induce tolerance in heart allografts. Whether CD8+CD45RClowTregs could induce tolerance in a liver transplant model and how dendritic cells (DCs) mediate the CD8+CD45RClowTregs effect remains to be investigated. MethodsA rat liver transplantation model was established and used to test tolerance and acute rejection compared to control groups. Liver function and histopathological changes of allograft were examined by enzyme-linked immunosorbent assay (ELISA) and haematoxylin and eosin (H&E) staining, respectively. The distribution and proportion of CD8+CD45RClowTregs and plasmacytoid dendritic cells (pDCs) in the allografts and spleen were determined using flow cytometry. Cytokine secretion levels were determined using ELISA and real-time quantitative PCR (qRT-PCR). ResultsThe rat liver transplantation model was well established, with a success rate of 93.3% (28/30). The mean survival time of the tolerant and acute-rejection rats were 156 and 14 days, respectively. The proportions of CD8+CD45RClowTegs were higher in the allografts of tolerant rats than in those of acute-rejection rats (33.1 ± 4.3 and 12.4 ± 4.6, respectively; P = 0.04). Significant accumulation of pDCs was observed in tolerant liver graft rats compared to that in acute-rejection rats (1.46 ± 0.23 and 0.80 ± 0.20, respectively; P = 0.02). Importantly, CD8+CD45RClowTregs were positively associated with the frequency of pDCs (P = 0.001, r2 = 0.775). The protein and mRNA expression of IL-10 and TGF-β in the allograft group were increased, possibly being responsible for tolerance induction. ConclusionCD8+CD45RClowT cells interact with pDCs through the induction of IL-10 and TGF-β expression and are responsible for inducing immune tolerance in rat liver transplantation.

Explorative study of the immobilizing effect of full-thickness skin subcutaneous grafting on allogeneic full-thickness skin graft in rats

Objective: To investigate the immobilizing effect of full-thickness skin subcutaneous grafting on allogeneic full-thickness skin graft in rats. Methods: The experimental research method was used. The inbred male Brown-Norway rats (n=10) and Lewis rats (n=10) were used as donors and recipients respectively. After subcutaneously full-thickness separation of a 2.2 cm×2.2 cm area on the nape of the recipient rat, a full-thickness skin of 2.0 cm×2.0 cm taken from the abdomen of the donor rat was subcutaneously grafted, and the donor site was pulled together and sutured. The autologous skin over the allograft in the recipient rat was excised 5-6 d after grafting, and the stitches were removed 7 d after excision. Within 2 months after grafting, the feeding, activity, and survival of the donor and recipient rats, behavior of tearing and scratching the wounds of the recipient rats, the wound condition after autologous skin excision in recipient rats, and the survival and hair growth of the grafted allogeneic skin were observed. Results: Within 2 months after grafting, the donor and recipient rats all ate normally and could move freely with no abnormal death. No tearing or scratching of the wounds occurred in recipient rats. There was a small amount of exudation and partial epidermal desquamation after autologous skin excision in recipient rats. All transplanted allografts survived, which were free of infection and necrosis, with new hairs growing out smoothly. Conclusions: The immobilizing method of full-thickness skin subcutaneous grafting of allogeneic full-thickness skin graft in rats is simple and time-saving without postoperative dressing change, with reliable pressure fixation and high survival rate of skin grafts, which can be promoted for animal skin grafting models.

Effects of gamma radiation and post-operative cisplatin injection on the incorporation of bone allografts in rats

Background. The reconstruction of long bone defects that occur after resection of tumors is a problem that requires constant study. Bone allografts are often used in this scenario. Unfortunately, while they are prepared, allografts partially lose their strength and osteoinductive properties; their survivability in oncological patients is only 40% after 10 years. This is why the search for superior allograft treatment methods and the study of allograft remodeling and incorporation in oncological patients, whose state has been affected by radiation or chemotherapy, is an area of interest. Purpose – study the structure of bone tissue in the distal metaphysis of a rat’s femur after bone allograft implantation (sterilized using gamma radiation or antibiotic saturation) and post-operative intraperitoneal cisplatin injection. Materials and Methods. Experiments were performed on 20 male white rats aged 5–6 months at the beginning of the experiment and weighed 365.8 ± 6.4g. All rats underwent a surgery that created a defect in the distal metaphysis of the femur which was filled with a bone allograft treated with gamma radiation (Control-1 and Experimental-1 groups) or saturated with an antibiotic (Control-2 and Experimental-2 groups). 14 days after allograft implantation, animals from the control groups received an intraperitoneal injection of 2.0–2.4 ml of 0.9% NaCl, while animals from the experimental groups received 2.5mg/kg of cisplatin. Histological analysis and histomorphometry were completed 30 days after the surgery. Results. 30 days after the operation, the smallest relative area of bone tissue (11.79%) was observed in rats from the Experimental-1 group, with gamma radiation treated allografts and post-operative intraperitoneal cisplatin injections. A somewhat higher value was found in the Experimental-2 group (antibiotic saturation + cisplatin) – 31.64%. In the control groups, (intraperitoneal injection 0.9% NaCl), the relative area of bone tissue was 16.7% (Control-1, gamma radiation treatment) and 58.09% (Control-2, antibiotic saturation). The relative area of fibrous tissue was the largest in the Experimental-1 group – 31.55% and the smallest in the Control-2 group – 12.79%. Conclusions. Allograft remodeling occurs along with the formation of bone and fibrous tissue when allografts are used to fill defects in the distal femoral metaphysis of rats, However, the relative percentages of those tissues depend on the allograft sterilization method and the use of cytostatic agents. The largest relative percentage of bone tissue (58.09%) was obtained using an allograft saturated with antibiotics and without the administration of cisplatin. The smallest (11.79%), on the other hand, occurred in gamma radiation treated allografts with cisplatin injected intraperitoneally after the operation

ω-3 Polyunsaturated Fatty Acids Facilitate the Repair of Peripheral Nerve Defects with Chemically Extracted Acellular Allograft in Rats.

Acellular allograft (ACA) improves the repair and reconstruction of long peripheral nerve defects. ω-3 Polyunsaturated fatty acids (PUFAs) carry a neuroprotective potential, and their effects on ACA bridging were elucidated. Thirty rats with long gap sciatic nerve defects (15 mm long) were randomly divided into three groups (n = 10): ACA, ACA + PUFAs, and autograft (AU). Limb condition, wet weight of tibialis anterior muscle (TAM), nerve electrophysiology, S-100, horseradish peroxidase (HRP), and percentage of splenic CD4+ and CD8 + T-lymphocytes were evaluated for 12 weeks after the operation. Rats in the AU and ACA + PUFA groups showed superior condition in affected limbs compared to the ACA group. At 12 wk after surgery, the wet weight of TAM in the ACA + PUFA group was higher than that in the ACA group (0.4519 ± 0.1185 vs. 0.3049 ± 0.1272; P < 0.01) but lower than that in the AU group (0.4519 ± 0.1185, 0.5628 ± 0.0092; P < 0.05). In all the three groups, sole irritation elicited withdrawal reflex, and S-100 staining was detected in plantar skin. Moreover, horseradish peroxidase staining was overt in both the ventral horn and dorsal root ganglion of the spinal cord. Nerve conduction velocity (m/s), amplitude of action potential (mV), or somatosensory evoked potentials in ACA + PUFAs (28.81 ± 1.04, 2.20 ± 0.27, 6.98 ± 0.29) were significantly different from that in the AU (35.71 ± 1.28, 1.81 ± 0.19, 8.15 ± 0.52; P < 0.05) and ACA (20.03 ± 1.94, 2.95 ± 0.36, 5.22 ± 0.53; P < 0.01) groups. The percentages of splenic CD4+ and CD8+ cells were similar among the three groups. Omega-3 PUFAs improve the bridging effect of ACA on long gap peripheral nerve defects by promoting neuroprotection without arousing an immune response.

Effects of VEGF on Prefabricated Vascularized Bone Allografts in Rats

Massive bone defects after wide resection of malignant bone tumors or a serious injury require treatment using vascularized bone grafts. Although cadaveric bone allografts combined with vascularized bone autografts are currently thought to be ideal in terms of size and durability, this treatment requires the scarification of healthy bone tissue. In a previous study, we attempted to improve this situation by prefabricating a vascularized bone allograft in recipient rats. In this study, we added vascular endothelial growth factor (VEGF)-containing hydroxyapatite/collagen composite (HAp/Col) to a prefabricated vascularized bone allograft to stimulate angiogenesis, which is known to be important for bone formation. Sprague Dawley rats (n = 50) were used as donors and Wistar rats (n = 50) as recipients. All rats were 9 weeks old. The recipient rats were divided into five groups according to the use of vascular bundles, HAp/Col, and an additive substance (VEGF). The bone allografts collected from the donors were transplanted into the thigh region of the recipients, and a saphenous vein and 10 μg HAp/Col with VEGF were inserted into the bone allografts through the slit. After 4 weeks, the transplanted bone allografts were harvested, and histologic and genetic evaluations were performed in relation to bone formation and resorption. The results showed that, compared with the control group, the implantation of the vascular bundles and VEGF-containing HAp/Col significantly stimulated angiogenesis and bone formation in the rats with the bone allografts. However, histological and genetic evaluations of bone resorption revealed that resorption was not observed in any group. These results suggest that VEGF-containing HAp/Col effectively stimulates angiogenesis and bone formation, but not bone resorption, in prefabricated vascularized bone allografts. This method could therefore become a useful tool for treating large bone defects.

Polyethylene glycol-fusion repair of sciatic allografts in female rats achieves immunotolerance via attenuated innate and adaptive responses.

Ablation/segmental loss peripheral nerve injuries (PNIs) exhibit poor functional recovery due to slow and inaccurate outgrowth of regenerating axons. Viable peripheral nerve allografts (PNAs) as growth-guide conduits are immunologically rejected and all anucleated donor/host axonal segments undergo Wallerian degeneration. In contrast, we report that ablation-type sciatic PNIs repaired by neurorrhaphy of viable sciatic PNAs and a polyethylene glycol (PEG)-fusion protocol using PEG immediately restored axonal continuity for many axons, reinnervated/maintained their neuromuscular junctions, and prevented much Wallerian degeneration. PEG-fused PNAs permanently restored many sciatic-mediated behaviors within 2-6weeks. PEG-fused PNAs were not rejected even though host/donors were neither immunosuppressed nor tissue-matched in outbred female Sprague Dawley rats. Innate and adaptive immune responses to PEG-fused sciatic PNAs were analyzed using electron microscopy, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction for morphological features, T cell and macrophage infiltration, major histocompatibility complex (MHC) expression, apoptosis, expression of cytokines, chemokines, and cytotoxic effectors. PEG-fused PNAs exhibited attenuated innate and adaptive immune responses by 14-21days postoperatively, as evidenced by (a) many axons and cells remaining viable, (b) significantly reduced infiltration of cytotoxic and total T cells and macrophages, (c) significantly reduced expression of inflammatory cytokines, chemokines, and MHC proteins, (d) consistently low apoptotic response. Morphologically and/or biochemically, PEG-fused sciatic PNAs often resembled sciatic autografts or intact sciatic nerves. In brief, PEG-fused PNAs are an unstudied, perhaps unique, example of immune tolerance of viable allograft tissue in a nonimmune-privileged environment and could greatly improve the clinical outcomes for PNIs relative to current protocols.

Transient antibody targeting of CD45RC inhibits the development of graft-versus-host disease

AbstractAllogeneic bone marrow transplantation (BMT) is a widely spread treatment of many hematological diseases, but its most important side effect is graft-versus-host disease (GVHD). Despite the development of new therapies, acute GVHD (aGVHD) occurs in 30% to 50% of allogeneic BMT and is characterized by the generation of effector T (Teff) cells with production of inflammatory cytokines. We previously demonstrated that a short anti-CD45RC monoclonal antibody (mAb) treatment in a heart allograft rat model transiently decreased CD45RChigh Teff cells and increased regulatory T cell (Treg) number and function allowing long-term donor-specific tolerance. Here, we demonstrated in rat and mouse allogeneic GVHD, as well as in xenogeneic GVHD mediated by human T cells in NSG mice, that both ex vivo depletion of CD45RChigh T cells and in vivo treatment with short-course anti-CD45RC mAbs inhibited aGVHD. In the rat model, we demonstrated that long surviving animals treated with anti-CD45RC mAbs were fully engrafted with donor cells and developed a donor-specific tolerance. Finally, we validated the rejection of a human tumor in NSG mice infused with human cells and treated with anti-CD45RC mAbs. The anti-human CD45RC mAbs showed a favorable safety profile because it did not abolish human memory antiviral immune responses, nor trigger cytokine release in in vitro assays. Altogether, our results show the potential of a prophylactic treatment with anti-human CD45RC mAbs in combination with rapamycin as a new therapy to treat aGVHD without abolishing the antitumor effect.

Different Chronic Allograft Pathology Lesions in Two Orthotopic Lung Transplant Rat Strain Combinations

Chronic lung allograft dysfunction (CLAD) remains the major obstacle to long-term survival after lung transplantation. Two different phenotypes have recently been described. About 80% of patients have primarily obstructive lung disease associated with obliterative bronchiolitis, airway fibrosis with intraluminal narrowing (OB-CLAD) while a smaller subset has lung restriction with hypoxemia and parenchymal fibrosis (R-CLAD). Preclinical modeling is essential to better analyze the pathophysiological processes leading to CLAD development. Lung allografts from a large cohort of two different rat strain combinations orthotopically transplanted in the last decade were re-evaluated. From 2007 to 2017 127 rats were transplanted in our centre: 55 Lewis (Lew; RT-1l) into Fisher 344 (F344; RT-1v) (Group 1, G1) and 72 Brown-Norway (BN; RT1n) into Lew (Group 2, G2). Chronic rejection was detected in 39 cases, 18 G1 and 21 G2 rats, respectively, at a mean time of 61 and 17 days, respectively. Several pathological lesions (airway, pleural and vascular fibrosis, airway obliteration, endothelialitis, lymphoid aggregates, epithelial hyperplasia and flattening) were graded following a recent proposed grading system for animal model CLAD (Martinu et al, 2019, AJT). An end stage lung remodelling was detected in 33% of G1 and 48% of G2 models. A vanishing obliterative bronchiolitis (end stage OB-CLAD) was observed in 33% and 20% of G1 and G2 rats, respectively, while signs of fibrosis/fibroelastosis (end stage R-CLAD) were detected in 67% and 80% of G1 and G2 rats, respectively. Acute fibrinous and organizing pneumonia (AFOP) was present in 37.5% of G2 strains and never observed in G1 animals. Interestingly, G2 rats showed higher endothelialitis, vascular fibrosis and lower lymphoid aggregates than G1 strain (p=0.005, p=0.01, p=0.03, respectively). A combination score of highly suggestive R-CLAD lesions (parenchymal, pleural, vascular fibrosis and endothelialitis) was more frequently detected in G2 rats (p=0.03). CLAD associated lesions in lung rat allografts mimic those observed in humans. The earlier R-CLAD developed in G2 may be related to a higher degree of mismatch in this strain combination. These animal models could allow future mechanistic studies to better understand CLAD pathogenesis.

Influence of the new standardized clinical cryopreservation/slow thawing protocol on immunogenicity of arterial allografts in rats.

At the present time there are two waiting list for patients with vascular prosthetic infection indicated for arterial transplantation in the Czech Republic. The inclusion of each patient for cold-stored or cryopreserved arterial transplantation is the preference of indicating surgeon. In this experimental work we studied the immunogenicity of rat aortal allografts treated by our new clinical cryopreservation/slow thawing protocol. Brown-Norway (BN) (N = 6, 203-217 g) or Lewis (LEW) (N = 6, 248-254 g) abdominal aortal grafts treated in accordance with our new clinical cryopreservation/slow thawing protocol were orthotopically transplanted to Lewis recipients (N = 12, 191-245 g). Aortal wall histology and infiltration by recipient immune cells, as well as donor specific anti MHC class I and II antibodies in recipient serum were studied in both isografts and allografts on day 30 postransplant. Core data of cryopreserved allografts were compared to our previous data of cold-stored aortal allografts treated in accordance with our clinical cold-storage protocol. Cryopreserved allografts showed regular morphology of aortal wall with clear differentiation of all three basic anatomical layers on day 30 postransplant. Intimal layer showed no hyperplasia, luminal surface was covered by endothelial cells. No statistical difference was observed in tunica media thickness between isografts and allografts. The medial layer showed no necrosis, shrinkage or immunoglobuline G deposition in any experimental group. The adventitial infiltration by immune cells was significantly higher (P<0.05) in allografts. Cryopreserved allografts showed significant lower activation of both cell- and antibody mediated immunity compared to historical data of cold-stored allografts. Aortal wall histology of rat allografts treated by our new standardized clinical cryopreservation/slow thawing protocol was comparable to that of the cryopreserved isografts on day 30 posttranspant. The immunogenicity of cryopreserved aortal allografts was significantly lower compared to that of cold-stored aortal allografts.

Autotaxin Inhibitor Protects from Chronic Allograft Injury in Rat Kidney Allotransplantation

Background: Tissue fibrosis is the final common phase of chronic allograft injury, the leading cause of late graft loss in kidney transplantation. Preclinical evidence points to the involvement of lysophosphatidic acid (LPA), a bioactive phospholipid, in the development of renal fibrosis. Objectives: We assessed whether treatment with an orally available inhibitor of autotaxin (ATXi), the main LPA-producing enzyme, could slow the progression of chronic allograft injury in a fully major histocompatibility complex-mismatched rat kidney transplant model and compared its effects with those of the angiotensin-converting enzyme inhibitor lisinopril. Methods: Kidney allograft recipients were given ciclosporin for the first 15 postoperative days to prevent early acute rejection. Thereafter, they received either no treatment or ATXi or lisinopril and were followed for 180 days after transplantation. Results: Renal LPA levels were increased in allograft rats, providing the rationale for using ATXi in this model. Chronic treatment with ATXi or lisinopril limited progressive proteinuria and ameliorated tubulointerstitial fibrosis compared with allograft rats, although the effects were more robust under ATX inhibition. The administration of ATXi, but not lisinopril, attenuated systemic hypertension, reduced intragraft T cell infiltration, and eventually improved renal graft survival. Conclusions: In summary, ATXi had protective effects on indices of chronic allograft injury and could be of therapeutic add-on value in the kidney transplant setting. Notably, an ATX inhibitor is currently being investigated in 2 large phase 3 studies in idiopathic pulmonary fibrosis, underscoring the clinical relevance of our findings.

Proteasome Expression in Ovarian Heterotopic Allografts of Wistar and August Rats under Induction of Donor-Specific Tolerance

The aim of this work is to investigate the dynamics for the ovarian tissue engraftment of inbred August rats transplanted to outbred Wistar rats and vice versa on the background of the induction of donor-specific tolerance and to identify the features of proteasome pools in the surviving grafts and tissue replacing the rejected grafts in animals of both groups. By day 14 after transplantation, a slight difference in graft engraftment was revealed in studied recipients: 87% in Wistar rats and 80% in August rats. At the same time, ovarian tissue allografts with well-preserved structure and significant vascularization represented 76% in Wistar rats and only 29% in August rats on day 37. The difference revealed in the remote period is apparently connected with the special condition of the central nervous system of August rats caused by the increased content of monoamines and heat-shock protein 70. The total proteasome level on day 37 was the same both in intact donor tissue and in the surviving grafts, as well as in the tissue replacing the rejected grafts, and did not depend on the donor–recipient differences. However, the increased expression of immune proteasomes was found in the surviving tissue and tissue replacing the rejected grafts compared to intact donor tissue. The surviving grafts were enriched with proteasomes with immune subunit LMP2, and the tissue replacing the rejected grafts was enriched with proteasomes with immune subunit LMP7 equally in August and Wistar rats. In addition, the surviving grafts in both groups of animals were characterized by equally high expression of proteasome activator РА28αβ compared to the replacement and intact donor tissues. Thus, transplant proteasomes containing immune subunits LMP2, and probably associated with the activator RA28αβ, serve as one of the key factors for engraftment regardless of the genetically determined differences of the recipients.

Osteopontin mediates necroptosis in lung injury after transplantation of ischaemic renal allografts in rats

BackgroundRespiratory complications after surgery are associated with morbidity and mortality. Acute lung injury can result from the systemic inflammatory response after acute kidney injury. The mechanisms behind this remote injury are not fully understood. In this study, a renal transplantation model was used to investigate remote lung injury and the underlying molecular mechanisms, especially the role of osteopontin (OPN). MethodsIn vitro, human lung epithelial cell line (A549) and monocyte/macrophage cell line (U937) were challenged with tumour necrosis factor-alpha (TNF-α) in combination with OPN. In vivo, the Fischer rat renal grafts were extracted and stored in 4°C University of Wisconsin preserving solution for up to 16 h, and transplanted into Lewis rat recipients. Lungs were harvested on Day 1 after grafting for further analysis. ResultsRenal engraftment was associated with pathological changes and an increase in TNF-α and interleukin-1 beta in the lung of the recipient. OPN, endoplasmic reticulum (ER) stress, and necroptosis were increased in both the recipient lung and A549 cells challenged with TNF-α. Exogenous OPN exacerbated lung injury and necroptosis. Suppression of OPN through siRNA reduced remote lung injury by mitigation of ER stress, necroptosis, and the inflammatory response. ConclusionsRenal allograft transplant triggers recipient remote lung injury, which is, in part, mediated by OPN signalling. This study may provide a molecular basis for strategies to be developed to treat such perioperative complications.

Lanthanum carbonate, a phosphate binder, inhibits calcification of implanted aortic allografts in a rat model.

Calcification is one of the major postoperative problems after aortic allograft implantation. We hypothesized that phosphate binders, lanthanum carbonate and calcium carbonate inhibit calcification of implanted aortic allografts and verified this hypothesis using a rat model. Aortas were harvested from 4-week-old BrownNorway rats and implanted into the subdermal space of 4-week-old Lewis rats. Twenty-seven recipient Lewis rats were divided into Group N, Group L, and Group C (9 rats per group), which were fed a normal diet, a normal diet containing 3% lanthanum carbonate, and a normal diet containing 3% calcium carbonate, respectively. Implanted aortic allografts were explanted 2weeks later. Calcification of aortic allografts was evaluated using von Kossa staining and calcium content assay. Calcification score was defined in von Kossa staining as 0 (none), 1 (mild), 2 (moderate), and 3 (severe). Serum calcium and phosphorus levels at euthanasia were measured. Calcification scores were 2.6, 1.2, and 0.8, and calcium content was 48.9, 15.8, and 8.9mg/dry·g, in Groups N, L, and C, respectively. Calcification was significantly reduced in Groups L and C. Serum calcium level was 11.5, 12.2, and 13.5mg/dl, and serum phosphorus level was 15.4, 12.5, and 11.7mg/dl, in Groups N, L, and C, respectively. Serum calcium level in Group C was significantly higher than in the other two groups. Lanthanum carbonate and calcium carbonate significantly reduced calcification of implanted aortic allografts in young rats. Although calcium carbonate induced hypercalcemia, lanthanum carbonate has significant potential to inhibit calcification of implanted aortic allografts.

Expression of Allograft Inflammatory Factor-1 (AIF-1) in Hepatocellular Carcinoma.

BackgroundAllograft inflammatory factor-1 (AIF-1) is a cytoplasmic protein cloned from activated macrophages in human and rat allografts. AIF-1 has been identified as a modulator of inflammatory response, and recently published studies have shown its increased expression in carcinogenesis. However, there are still limited data on the potential functional role of AIF-1 in hepatocellular carcinoma (HCC).Material/MethodsWe evaluated the expression of AIF-1 in 104 cases of paired HCC and adjacent non-cancerous liver tissues using immunohistochemistry, Western blotting, and qPCR analysis, and sought to determine whether its expression was correlated with clinicopathological features. In vitro assays, including cell proliferation and migration assays, were used to study the effects of AIF-1 knockdown in L02 human hepatocyte, and Huh7 and SMMC7721 liver cancer cell lines.ResultsExpression of AIF-1 was increased in HCC compared to adjacent normal liver tissues and was positively correlated with median tumor size (p=0.046), number of tumor deposits (p=0.009), the Barcelona Clinic Liver Cancer (BCLC) stage (p=0.004), and portal vein tumor thrombus (PVTT) (p<0.001). Huh7 and SMMC7721 human HCC cells demonstrated upregulated AIF-1 expression compared to normal hepatocytes. Small interfering RNA (siRNA)-mediated silencing of AIF-1 expression resulted in a reduction in cell proliferation and migration in human HCC cells.ConclusionsThese findings suggest AIF-1 may have roles as a diagnostic or prognostic biomarker and a promising therapeutic target in HCC.