DA Rats Research Articles

Transplantation of allogeneic hepatocyte‐like cells differentiated from adipose tissue mesenchymal stem cells (MSC) in rat models of liver regeneration and cirrhosis

It has been demonstrated that MSC may differentiate to hepatocyte‐like cells, which after syngeneic transplantation into a host liver persist in the parenchyma. Evidence emerges that MSC display tolerogenic attributes. Therefore, we investigated acceptance of allogeneic MSC transplants in rat models of liver regeneration and cirrhosis.MSC were isolated from adipose tissue of CD26 wildtype DA rats and cells either undifferentiated or after hepatocyte differentiation transplanted into the livers of CD26‐deficient Fischer rats via portal vein. After 10 weeks, small clusters of allogeneic donor cells were visible in the host parenchyma. Compared with undifferentiated cells, clusters were seemingly larger when differentiated cells were used but were by far smaller as compared to the transplantation of syngeneic MSC. When cells were applied systemically via tail vein injection, small clusters of allogeneic donor‐derived cells were visible in the host liver parenchyma after 5 days in animals challenged with partial hepatectomy and with CCl4‐induced liver cirrhosis. This demonstrates that allogeneic MSC home to the liver after acute and chronic liver injury. They integrate into the host liver indicating their acceptance by the host immune system at least in the short term range. This offers the opportunity to use allogeneic MSC for the therapy of liver diseases avoiding immune suppression.

Innate and adaptive immune responses in obliterative airway disease in rat tracheal allografts

We assessed cellular innate and adaptive immune responses in a rat heterotopic tracheal allograft model during the development of obliterative airway disease. Syngeneic tracheal grafts were transplanted heterotopically from DA to DA rats and fully MHC-mismatched allografts from DA to WF rats. The recipients received either no immunosuppression or two different doses of cyclosporine and were euthanized at 3, 10 and 30 days. Non-transplanted DA tracheas served as controls. Histologic, immunohistochemical and real-time RT-PCR analyses were performed. The syngrafts had normal epithelium at 10 days and no tracheal occlusion was seen at 30 days. In non-immunosuppressed allografts, almost total loss of epithelium was observed at 10 days, culminating in tracheal occlusion at 30 days. The activation of innate immune response was observed during the ischemic period at 3 days in both groups. Influx of the infiltrating inflammatory cells was more prominent in the allografts. In syngrafts, mRNA expression of pro-inflammatory, but also tolerogenic, cytokines was significantly upregulated, whereas Th1 and Th17 priming factors were significantly downregulated. In allografts, prominent mRNA expression of pro-inflammatory cytokines was seen and adaptive Th1 and Th17 alloresponses were increased. Cyclosporine treatment reduced tracheal occlusion and inhibited both tolerogenic and pro-inflammatory T-cell responses in allografts. Ischemia induced a self-limiting, alloantigen-independent innate immune response in syngrafts. In allografts, the predominant pro-inflammatory milieu and alloantigen-dependent Th1 and Th17 responses were linked to the development of obliterative airway disease and were inhibited by cyclosporine treatment.

Chiral analyses of dextromethorphan/levomethorphan and their metabolites in rat and human samples using LC-MS/MS

In order to develop an analytical method for the discrimination of dextromethorphan (an antitussive medicine) from its enantiomer, levomethorphan (a narcotic) in biological samples, chiral analyses of these drugs and their O-demethyl and/or N-demethyl metabolites in rat plasma, urine, and hair were carried out using LC-MS/MS. After the i.p. administration of dextromethorphan or levomethorphan to pigmented hairy male DA rats (5 mg/kg/day, 10 days), the parent compounds and their three metabolites in plasma, urine and hair were determined using LC-MS/MS. Complete chiral separation was achieved in 12 min on a Chiral CD-Ph column in 0.1% formic acid-acetonitrile by a linear gradient program. Most of the metabolites were detected as being the corresponding O-demethyl and N, O-didemethyl metabolites in the rat plasma and urine after the hydrolysis of O-glucuronides, although obvious differences in the amounts of these metabolites were found between the dextro and levo forms. No racemation was observed through O- and/or N-demethylation. In the rat hair samples collected 4 weeks after the first administration, those differences were more clearly detected and the concentrations of the parent compounds, their O-demethyl, N-demethyl, and N, O-didemethyl metabolites were 63.4, 2.7, 25.1, and 0.7 ng/mg for the dextro forms and 24.5, 24.6, 2.6, and 0.5 ng/mg for the levo forms, respectively. In order to fully investigate the differences of their metabolic properties between dextromethorphan and levomethorphan, DA rat and human liver microsomes were studied. The results suggested that there might be an enantioselective metabolism of levomethorphan, especially with regard to the O-demethylation, not only in DA rat but human liver microsomes as well. The proposed chiral analyses might be applied to human samples and could be useful for discriminating dextromethorphan use from levomethorphan use in the field of forensic toxicology, although further studies should be carried out using authentic human samples.

Tissue engineering of bone an ectopic rat model

Tissue engineering is attempting to recreate the complexity of living tissues. In order to test a variety of scaffolds or cells that are constantly being developed, we describe here a model where tissue engineering of bone in a non-osseous environment at subcutaneous thoracic site of DA rats generates. In this model, cell - matix interactions can mimic the normal cascade of bone development into a well organized ossicle like structure including newly formed bone marrow, during 3-4 weeks. Histogenesis of cartilage, bone and bone marrow is closely related to changes in molecular expression of essential early transcriptional regulators of osteoblast differentiation. We tested different organic, anorganic and polymeric scaffolds and their interaction with mesenchymal stem cells present in fresh bone marrow. In another series of experiments we tested mesenchymal populations separated from cultures of calvaria and periosteum for their ability to form bone in the same rat model. It is concluded that this in vivo model is very potent in studying cell-scaffold interactions affecting the temporal and spatial tissue engineering of bone.

Prospective highlights of serum glycoproteins in spontaneous tolerance after orthotopic liver transplantation

Background We examined the effects and the mechanisms of serum glycoproteins on spontaneous tolerance after orthotopic liver transplantation (OLT) between DA (RT-1 a) and PVG (RT-1 c) rats. Methods A functional proteome analysis was introduced to investigate differently expressed proteins involved in overcoming major histocompatibility complex (MHC) barriers and lectin blotting was applied to survey the levels of fucosylated proteins. Results Two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry revealed statistically significant changes in the intensity of 19 proteins at 14 and 60 post-OLT days which respectively corresponded to rejection and tolerance. An interaction network analysis of the identified proteins indicated that the interleukin (IL)-6 signaling pathway might be correlated with the immune events in this model. The peak of IL-6 expression occurred during the recovery period might play a role in the remarkable shifts in the immune response towards spontaneous tolerance. Furthermore, increased levels of IL-6-modulated fucosylation of Igh-6 were also observed during the rejection response while fucosylated hemopexin and haptoglobin were obviously upregulated in the tolerogenic period. Conclusions These findings suggest that IL-6 and glycoproteins may play critical roles in this spontaneous tolerogenic DA/PVG OLT model and shed light on prolonging hepatic allograft survival in the future.

Influence of perineurial cells and Toll-like receptors 2 and 9 on Herpes simplex type 1 entry to the central nervous system in rat encephalitis.

Herpes simplex encephalitis (HSE) is a rare disease with high mortality and significant morbidity among survivors. We have previously shown that susceptibility to HSE was host-strain dependent, as severe, lethal HSE developed after injection of human Herpes simplex type 1 virus (HSV-1) into the whiskers area of DA rats, whereas PVG rats remained completely asymptomatic. In the present study we investigated the early immunokinetics in these strains to address the underlying molecular mechanisms for the observed difference. The virus distribution and the immunological responses were compared in the whiskers area, trigeminal ganglia and brain stem after 12 hours and the first four days following infection using immunohistochemistry and qRT-PCR. A conspicuous immunopathological finding was a strain-dependent difference in the spread of the HSV-1 virus to the trigeminal ganglia, only seen in DA rats already from 12 hpi. In the whiskers area infected perineurial cells were abundant in the susceptible DA strain after 2 dpi, whereas in the resistant PVG rats HSV-1 spread was confined only to the epineurium. In both strains activation of Iba1+/ED1+ phagocytic cells followed the distribution pattern of HSV-1 staining, which was visible already at 12 hours after infection. Notably, in PVG rats higher mRNA expression of Toll-like receptors (Tlr) -2 and -9, together with increased staining for Iba1/ED1 was detected in the whiskers area. In contrast, all other Tlr-pathway markers were expressed at higher levels in the susceptible DA rats. Our data demonstrate the novel observation that genetically encoded properties of the host nerve and perineurial cells, recruitment of phagocyting cells together with the low expression of Tlr2 and -9 in the periphery define the susceptibility to HSV-1 entry into the nervous system.

Specific and Strain-Independent Effects of Dexamethasone in the Prevention and Treatment of Experimental Autoimmune Encephalomyelitis in Rodents

Experimental autoimmune encephalomyelitis in rodents (EAE) is a generally accepted in vivo model for immunopathogenic mechanisms underlying multiple sclerosis (MS). There are, however, different forms of rodent EAE, and therapeutic regimens may affect these forms differently. We have therefore tested the effects of dexamethasone (Dex) and found that both prophylactic and early therapeutic regimens were effective in suppressing the development of monophasic EAE in myelin basic protein-immunized Lewis rats, the relapsing-remitting forms of EAE induced in SJL mice by proteolipid protein and in DA rats by syngeneic spinal cord homogenate, and the progressive forms induced in C57BL/6 and DBA/1 mice by immunization with myelin oligodendrocyte glycoprotein. In addition, prophylactically administered Dex suppressed histological and immunological features of EAE such as spinal cord infiltration of inflammatory cells and the increased frequency of autoantigen-specific interferon-gamma-secreting lymph node mononuclear cells. The present data reproduced in rodent EAE models some of the beneficial effects observed with glucocorticoids in MS. This strengthens the validity of these five models as in vivo predictors of drug efficacy in at least some variants of human MS. Better understanding of the clinical and immunopharmacologic features of these models might prove useful when testing new drug candidates for MS treatment.

CD4+CD25+TREGS ACTIVATED IN VITRO WITH IL-2 AND IL-12P70 SUPPRESS REJECTION OF FULLY ALLOGENEIC GRAFTS IN ANIMALS WITH NO OTHER IMMUNOSUPPRESSION.

Hall, B. M.1; Verma, N. D.1; Boyd, R.1; Robinson, C. M.1; Tran, G. T.1; Wang, C.2; Bishop, G. A.3; Hodgkinson, S. J.1 Author Information

Effects of Gene Transfer CTLA4Ig and Anti-CD40L Monoclonal Antibody on Islet Xenograft Rejection in Mice

Blockade of a costimulatory pathway by adenovirus-mediated cytotoxic T lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) gene transfer and anti-CD40L mAb(MR1) have been reported to enhance graft survival in several experimental transplantation models. In this study, we investigated the effects of gene transfer of CTLA4Ig and MR1 on islet xenograft rejection in mice. Recombinant adenovirus AdCTLA4Ig was constructed to express CTLA4Ig. Islet grafts from adult male DA rats transferred with AdCTLA4Ig were transplanted to streptozocin-induced diabetic Balb/c mice. The diabetic mice were treated with MR1 after transplantation. We evaluated the islet xenograft mean survival time as well as changes in interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) levels in transplanted mice. The mean survival of islet xenografts in the MR1 treatment group was 34.9 ± 5.62 days, in the AdCTLA4Ig treatment group it was 56.5 ± 10.64 days, and in the AdCTLA4Ig plus MR1 treatment group it was 112.9 ± 19.26 days, all significantly prolonged compared with an untreated group (8.1 ± 0.83 days). Within 1 week after transplantation the levels of IL-2 and TNF-α showed sharp increases in the untreated group, being significantly higher than those observed prior to transplantation. In conclusion, using both AdCTLA4Ig and MR1 can improve the islet xenograft survival. The beneficial effects of the combined use of the 2 reagents were superior to either 1 alone, possibly related to down-regulated expression of Th1 cell–related cytokines.

MTOR Regulates the Invasive Properties of Synovial Fibroblasts in Rheumatoid Arthritis

The invasive properties of fibroblast-like synoviocytes (FLS) correlate with radiographic and histologic damage in rheumatoid arthritis (RA) and pristane-induced arthritis (PIA). We previously determined that highly invasive FLS obtained from PIA-susceptible DA (blood type D, Agouti) rats have increased expression of genes associated with invasive cancers, including Villin-2/ezrin. Villin-2/ezrin mediates invasion via mTOR. In the present study we used the mTOR inhibitor rapamycin to assess the role of the ezrin-mTOR pathway on the invasive properties of FLS. FLS were isolated from synovial tissues from arthritic DA rats, and from RA patients. FLS were treated with rapamycin or dimethyl sulfoxide (DMSO) for 24 h and then studied in a Matrigel-invasion assay. Supernatants were assayed for matrix metalloproteinase (MMP) activity, and cell lysates were used for quantification of mTOR, p70S6K1, 4EBP1 and FAK, as well as their respective phosphorylated subsets. Actin filament and FAK localization were determined by immunofluorescence. Rapamycin decreased FLS invasion in DA and RA tissues by 93% and 82%, respectively. Rapamycin treatment reduced the phosphorylation of mTOR and its substrates, p70S6K1 and 4EBP1, confirming mTOR inhibition. In conclusion, rapamycin prevented actin reorganization in both DA and RA FLS, and inhibited the directional formation of lamellipodia. Phosphorylation of the lamellipodia marker FAK was also reduced by rapamycin. MMPs were not significantly affected by rapamycin. Rapamycin significantly reduced RA and DA rat FLS invasion via the suppression of the mTOR signaling pathway. This discovery suggests that rapamycin could have a role in RA therapy aimed at reducing the articular damage and erosive changes mediated by FLS.

Effect of Porphyromonas gingivalis‐induced inflammation on the development of rheumatoid arthritis

Periodontitis is an extra-synovial chronic inflammatory condition, which has been proposed to be inter-related with rheumatoid arthritis. We investigated the effect of an established extra-synovial chronic inflammatory lesion on the induction and severity of experimental arthritis. Chronic inflammatory lesions were induced by the implantation of polyurethane sponges impregnated with heat-killed Porphyromonas gingivalis into the backs of DA rats. Thirty-five days later, adjuvant arthritis (AA) was induced in the rats by injecting a mycobacterium cell wall in complete Freund's adjuvant. The development of arthritis was then monitored for 2 weeks. Histological assessment of the implanted sponges confirmed that a chronic inflammatory lesion had been established after 21 days. Following induction of adjuvant arthritis, the severity of disease was scored and paw swelling was measured. Severe arthritis developed more rapidly in animals with a pre-existing P. gingivalis-induced inflammatory lesion elsewhere. The results show that a pre-existing extra-synovial chronic inflammatory lesion induced by P. gingivalis promotes the development of arthritis in an animal model. These findings provide further evidence for a relationship between the presence of periodontal pathogen-associated inflammation and the development of rheumatoid arthritis.

Brain stem heme oxygenase 1 (HO‐1) mRNA expression in inbred rat strains after severe hemorrhage

Previously Klemcke et al. showed 8‐fold difference in survival time among inbred rat strains after severe hemorrhage (Shock 29:748, 2008). For Brown Norway (BN/Mcwi) rats we also found a 2.7‐fold increase in cardiac HO‐1 mRNA expression in catheterized plus hemorrhaged rats (Bled) compared to unmanipulated controls (CN; FASEB J. 23:801.2, 2009). To further examine HO‐1 as a possible intermediary of cellular responses to hemorrhage, in the same study we measured HO‐1 mRNA expression in brain stem of DA, Fawn Hooded Hypertensive (FHH), and BN/Mcwi strains. Using real time PCR, the relative quantity (RQ) of HO‐1 mRNA was measured with multiple housekeeping genes (Genome Biology 8:R19, 2007). Across all treatments (CN, catheterized only, and Bled) RQs were greater (P<0.05) in BN/Mcwi (0.47 ± 0.02; mean ± SEM) and FHH (0.48 ± 0.02) than in DA rats (0.41 ± 0.02). When considering all strains, HO‐1 mRNA RQ increased 12% (P<.05) in Bled rats compared with CN. Within strains and treatments, BN/Mcwi CN rats had greater (P<.05) RQs (0.46 ± 0.01) than did DA control rats (0.38 ± 0.03). The data imply that rat brain stem has minor strain‐dependent differences in HO‐1 mRNA expression, and that basal expression is modestly increased after the combined stressors of catheterization and hemorrhage.

Reduction of diffusion barriers in isolated rat islets improves survival, but not insulin secretion or transplantation outcome

For people with type 1 diabetes and severe hypoglycemic unawareness, islet transplants offer hope for improving the quality of life. However, islet cell death occurs quickly during or after transplantation, requiring large quantities of islets per transplant. The purpose of this study was to determine whether poor function demonstrated in large islets was a result of diffusion barriers and if removing those barriers could improve function and transplantation outcomes. Islets were isolated from male DA rats and measured for cell viability, islet survival, glucose diffusion and insulin secretion. Modeling of diffusion barriers was completed using dynamic partial differential equations for a sphere. Core cell death occurred in 100% of the large islets (diameter > 150 μm), resulting in poor survival within 7 days after isolation. In contrast, small islets (diameter < 100 μm) exhibited good survival rates in culture (91%). Glucose diffusion into islets was tracked with 2-NBDG; 4.2 μm/min in small islets and 2.8 μm/min in large islets. 2-NBDG never permeated to the core cells of islets larger than 150μm diameter. Reducing the diffusion barrier in large islets improved their immediate and long-term viability in culture. However, reduction of the diffusion barrier in large islets failed to improve their inferior in vitro insulin secretion compared to small islets, and did not return glucose control to diabetic animals following transplantation. Thus, diffusion barriers lead to low viability and poor survival for large islets, but are not solely responsible for the inferior insulin secretion or poor transplantation outcomes of large versus small islets.

Survival time after hemorrhage (STaH) in inbred rats with known blood volumes

Eight‐fold differences in STaH were detected among 15 inbred rat strains (Klemcke et al., Shock 29:748, 2008) suggesting genetic effects on STaH. In that study, blood volumes (BV) for all strains were assumed to be 5.83 % of body weight and 55% of that BV was removed. We also showed that differences in STaH might reflect strain‐dependent differences in BV (Klemcke et al., FASEB J 23: 801.1, 2009). To address this hypothesis, each rat of 5 inbred rat strains was hemorrhaged based on its own BV measured using Evans Blue dye 24 hr prior to hemorrhage and its STaH determination. BV varied (P &lt;0.01) among Brown Norway (BN/Mcwi; n = 14; 7.43 ± .10 ml/100 g body weight, mean ± SEM), Salt Sensitive (S; n = 10; 7.08 ± .15), Fawn Hooded Hypertensive (FHH; n=10; 6.74 ± .08), Lewis (LEW; n=10; 6.74 ± .08), and DA (n=10; 6.74 ± .08) rat strains. With equal hemorrhages (47.1 ± .05 % of measured BV), STaH did not differ among rat strains (155 ± 12 min; P = 0.95). When considering both studies it is apparent that: 1) Strain‐related differences in BV contributed to differences in STaH in our 1st study; 2) In FHH rats 1.3% more of total BV was removed in the current study, yet they survived 1.5 hr longer; and 3) In LEW rats identical BV were removed in both studies, yet currently LEW rats survived 1 hr longer. Such observations suggest that Evans Blue dye and/or blood sampling for BV determinations might be preconditioning stimuli that improve STaH in some rat strains.

Induction of Lymphocyte Apoptosis in Rat Liver Allograft by Adenoviral Gene Transfection of Human Interleukin-10

Background/Aims: Gene therapy can provide a possible avenue in organ transplantation to treat acute allograft rejection. This study was designed to investigate the effect of adenovirus-mediated human IL-10 (hIL-10) gene transfer on the apoptosis of infiltrating lymphocytes and examine the efficacy of hIL-10 gene transfer in combination with subtherapeutic doses of cyclosporine A (CsA) in a rat liver transplantation model. Methods: Inbred male DA and LEW rats were used for liver donors and recipients, respectively. The rats were divided into saline, Ad-lacZ, CsA, Ad-hIL-10 and Ad-hIL-10 + CsA groups. Graft survival, histopathological, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction and flow cytometry were performed in liver specimens obtained from different time points after transplantation in the 5 groups. Results: Ad-hIL-10 pretreatment inhibited allograft rejection, prolonged the survival of hepatic allografts, and downregulated the expression of IFN-γ and IL-2 mRNA, with simultaneous upregulation of IL-4 mRNA. In addition, Ad-hIL-10 pretreatment upregulated the expression of Fas mRNA in the isolated graft-infiltrating lymphocytes and induced graft-infiltrating lymphocyte apoptosis. A single subtherapeutic dose of CsA acted synergistically with it. Conclusion: hIL-10 gene therapy induced alloreactive lymphocyte apoptosis via Fas/FasL pathway. hIL-10 gene transfection in combination with subtherapeutic doses of CsA facilitates the long-term survival of liver grafts.

Toll-like receptor 3 upregulation in macrophages participates in the initiation and maintenance of pristane-induced arthritis in rats

IntroductionToll-like receptors (TLRs) are involved in both innate and adaptive immune responses and are likely to play a complex role in the pathogenesis of human rheumatoid arthritis (RA) and experimental arthritis. The objective of this study was to identify the key TLR in pristane-induced arthritis (PIA), a rat model for RA, and to clarify its roles in the initiation and maintenance of arthritis.MethodsArthritis in DA rats was induced by pristane and the severity was evaluated by macroscopic and microscopic score systems. Spleen TLR and cytokine expression was detected at different time points by real-time polymerase chain reaction (PCR) and flow cytometry. Polyinosine-polycytidylic acid (polyI:C, a ligand of TLR3) or TLR3 specific short-hairpin RNA plasmid for RNA interference was administrated to PIA rats in vivo. Serum nitrogen oxide concentration was determined by Griess method, and tumor necrosis factor alpha (TNF-α) was determined by L929 biotest. In splenic macrophages, TLR3 expression was measured by flow cytometry. A rat macrophage cell line (NR8383) was stimulated by pristane, and anti-TLR3 antibody were used to block TLR3 pathway. TLR3 and cytokine expression in NR8383 were detected by real-time PCR.ResultsBy screening the TLR expression profile in spleen of DA rats after pristane injection, we found that TLR3 was the most early and prominently upregulated TLR. Both TLR3 mRNA and protein expression of spleen were upregulated at 6 and 26 days after pristane injection. Furthermore, administration of polyI:C exacerbated, whereas RNA interference targeting TLR3 ameliorated, the arthritis. Particularly, TLR3 expression was induced in splenic macrophages of PIA rats, and also in the NR8383 cell line after pristane stimulation in a dose- and time- dependent manner. Upregulation of interferon beta (IFN-β) and TNF-α by pristane stimulation was blocked by anti-TLR3 antibody in NR8383.ConclusionsTLR3 plays a pivotal role in the initiation and development of PIA which may dependent on macrophage. These findings are useful to understand the pathogenesis of RA and may provide an intriguing therapeutic opportunity for RA.

Influence of bone‐derived matrices on generation of bone in an ectopic rat model

Most bone regeneration experimental models that test bone-derived matrices take place in conjunction with the native bone. Here, we compared the relative effectiveness of bone matrix components on bone-marrow-directed osteogenesis in an ectopic model. Cortical bone cylinders consisted of diaphysis of DA rat femurs. They were either demineralized (DBM), deproteinized (HABM), or nontreated (MBM). Fresh bone marrow was placed into cylinders and implanted at subcutaneous thoracic sites of 2-month-old DA rats. At designated times the cylinders were surgically removed from the animals. Microradiographs of DBM and histology of DBM and MBM cylinders demonstrated progressive increase in mineralized bone volume and its trabecular configuration. Bone filled the inner volume of DBM and MBM cylinders within 4 weeks, while in HABM cylinders mostly granulation tissue developed. In the DBM cylinders cartilage deposited within 10 days, while in the MBM cylinders bone was directly deposited. As early as day 3 after marrow transplantation, marrow cells interacting with DBM increased significantly the genes that express the cartilage and the bone phenotype. In conclusion, organic components of bone are needed for marrow-directed osteogenesis.

Definition of arthritis candidate risk genes by combining rat linkage-mapping results with human case-control association data

Objective:To define genomic regions that link to rat arthritis and to determine the potential association with rheumatoid arthritis (RA) of the corresponding human genomic regions.Methods:Advanced intercross lines (AIL) between arthritis...

Total body irradiation of the donor in a spontaneous tolerance rat liver transplantation model and the effects on CD4(+)CD25(+) regulatory T cells content

To study the effect of total body irradiation of the donor in a spontaneous tolerance rat liver transplantation model and the role of CD4(+)CD25(+) regulatory T cells on induction of immunotolerance in the recipient. Liver transplantation was performed using male Lewis rats as donors and male DA rats as recipients. These rats were randomly allocated into the following groups:Control group, Homogeneity Liver Transplantation group, Idio-immunotolerance group and Acute Rejection group. After transplantation, survival time rate of each group were observed. Serum ALT, TB level, Foxp3(+)CD4(+)CD25(+) regulatory T cells, expression of GITR on T cell subgroup, histopathology of the hepatic graft on day 14, spleen CTL lytic activity on day 14 were measured. In the Idio-immunotolerance group, the recipients suffered from transient rejection after surgery but acquired immunotolerance and survived long. In the Acute Rejection group, the donors were preconditioned with total body irradiation before liver transplantation. All recipients died between day 17 to 21. Serum ALT and TB increased significantly and the ratio of Foxp3(+)CD4(+)CD25(+) regulatory T cells decreased significantly compared with the Idio-immunotolerance group, the Homogeneity Liver Transplantation group and the Control group. The expression of GITR on CD3(+)CD4(+)T cells in the peripheral blood decreased, the expression of GITR on CD3(+)CD8(+) T cells and CTL lytic activity of the recipients increased by preconditioning of the donors with total body irradiation. Preconditioning of the donors with total body irradiation eliminated the passenger lymphocytes of the liver graft, decreased the expression of Foxp3(+)CD4(+)CD25(+) regulatory T cells in peripheral blood, and increased the expression of GITR on CD3(+)CD8(+) T cells, thus affected the course of tolerance and induced acute rejection after liver transplantation.

Role of the liver in determining alloimmune response in vitro following donor-specific spleen cell injection

The aim of our study was to investigate the allogeneic influence inside and outside the liver in vitro following donor-specific cell injection (DSI). DA rats (RT1a) were used as donors and WS rats (RT1k) as recipients. WS were sensitized with DA spleen cells, followed 24 h later by total hepatectomy. The liver was transplanted into another WS (sensitized liver-grafted; SL-Grafted). The hepatectomized WS underwent liver transplantation from a naive WS (sensitized liver-removed; SL-Removed). Alloantigens accumulated in the liver in SL-Grafted and in the extrahepatic tissue/organ(s) in SL-Removed. DA hearts were transplanted 10 days after antigen administration. To analyze the immune responses, we measured Th1/Th2 cytokine profiles, and perforin mRNA in various organs, allogeneic mixed lymphocyte reaction (MLR), and donor-specific immunoglobulin. Th1 cytokine levels in the liver of SL-Grafted and in spleen of SL-Removed were highly and rapidly upregulated but decreased thereafter. IFN-γ and perforin mRNAs were significantly higher in SL-Grafted and lower in SL-Removed. MLR was significantly higher in SL-Grafted than SL-Removed and controls. There was no significant difference in the donor-specific immunoglobulin level. Our findings suggest that liver and other organs may behave differently to alloantigen, suggesting the importance of an early Th1 reaction in the liver and spleen.