MiR-499 enhances Porphyromonas gingivalis LPS-induced inflammatory response in macrophages by targeting NRIP1 via JAK/STAT pathway.

MicroRNA functions as an important part of the activity and development of immune cells. miR-499 has been demonstrated to play a significant role in the activity and development of immune cells. The precise mechanism by which miR-499 regulates the inflammatory response, however, remains unclear. This study was aimed to examine the role of microRNA miR-499 in the regulation of the inflammatory response in macrophages. RAW 264.7 macrophages were used as a cell model. The levels of miR-499 were measured in Porphyromonas gingivalis LPS-stimulated macrophages using qRT-PCR, and the levels of inflammatory cytokines (IL-6, IL-1ß, and TNF-α) were determined using both qRT-PCR and ELISA. StarBase was used to predict the binding sites between NRIP1 and miR-499, and the mRNA expression of NRIP1 was measured using qRT-PCR. The regulation of inflammatory factors controlled by miR-499 was also evaluated by using miR-499 inhibitor and sh-NRIP1. The activation of the JAK/STAT pathway was determined using western blotting to measure the levels of phosphorylated JAK2 and STAT1. Porphyromonas gingivalis LPS caused a high expression of miR-499, which promoted the inflammatory response in macrophages. miR-499 targeted the NRIP1 3' UTR and regulated the mRNA expression of inflammatory cytokines, including IL-6, IL-1ß, and TNF-α. The positive correlation between miR-499 and the expression of inflammatory factors and the negative correlation between NRIP1 and miR-499 suggests that the regulation of inflammatory factors controlled by miR-499 was associated with NRIP1. The phosphorylated proteins of the JAK/STAT pathway (p-JAK2 and p-STAT1) were activated by miR-499 through its regulation of NRIP1. These findings suggest that miR-499 regulates the P. gingivalis LPS-induced inflammatory response in macrophages and activates the JAK/STAT pathway through the regulation of NRIP1.

Long-term ex situ normothermic perfusion of human split livers for more than 1 week.

Current machine perfusion technology permits livers to be preserved ex situ for short periods to assess viability prior to transplant. Long-term normothermic perfusion of livers is an emerging field with tremendous potential for the assessment, recovery, and modification of organs. In this study, we aimed to develop a long-term model of ex situ perfusion including a surgical split and simultaneous perfusion of both partial organs. Human livers declined for transplantation were perfused using a red blood cell-based perfusate under normothermic conditions (36 °C) and then split and simultaneously perfused on separate machines. Ten human livers were split, resulting in 20 partial livers. The median ex situ viability was 125 h, and the median ex situ survival was 165 h. Long-term survival was demonstrated by lactate clearance, bile production, Factor-V production, and storage of adenosine triphosphate. Here, we report the long-term ex situ perfusion of human livers and demonstrate the ability to split and perfuse these organs using a standardised protocol.

Liver splitting during normothermic machine perfusion: a novel method to combine the advantages of both in-situ and ex-vivo techniques.

BACKGROUND: Split liver transplantation permits the transplant of two recipients using a single donor liver. Liver splitting can be performed using the ex-vivo technique (more convenient), or the in-situ technique (shorter cold ischaemic time). We aimed to develop a technique for liver splitting during normothermic machine perfusion which combines the advantages of both techniques and permits graft assessment prior to transplant. METHODS: Human livers declined for transplantation were perfused at 36 °C using a modified-commercial perfusion machine. We developed a six-step method to split whole livers into left lateral segment grafts and extended right grafts. Both partial livers were then perfused on separate machines for individual assessment. RESULTS: Using our technique, 10 whole livers were successfully split during normothermic perfusion resulting in 20 partial grafts. Apart from a single graft which failed due to a technical error, all grafts survived for 24-h after splitting. Survival was demonstrated by lactate clearance, bile production and synthesis of coagulation factors. CONCLUSIONS: Liver splitting during normothermic machine perfusion has the potential to revolutionise split liver transplantation. We describe a novel technique that reliably achieves two grafts from a single donor liver. This raises the possibility of semi-elective transplantation, and sophisticated graft assessment prior to implant.

Screening self-peptides for recognition by mouse alloreactive CD8+ T cells using direct ex vivo multimer staining.

Here, we present a protocol to identify immunogenic self-peptide/allogeneic major histocompatibility complex (MHC) epitopes. We describe the generation of enriched alloreactive CD8+ T cells by priming mice with a skin graft expressing the allogeneic MHC class I molecule of interest, followed by boosting with a liver-specific AAV vector encoding the heavy chain of that donor MHC allomorph. We then use a peptide-exchange approach to assemble a range of peptide-MHC (pMHC) multimers for measuring recognition of the various epitopes by these alloreactive T cells. For complete details on the use and execution of this protocol, please refer to Son et al. (2021).1.

Targeting inflammatory monocytes by immune-modifying nanoparticles prevents acute kidney allograft rejection.

Inflammatory monocytes are a major component of the cellular infiltrate in acutely rejecting human kidney allografts. Since immune-modifying nanoparticles (IMPs) bind to circulating inflammatory monocytes via the specific scavenger receptor MARCO, causing diversion to the spleen and subsequent apoptosis, we investigated the therapeutic potential of negatively charged, 500-nm diameter polystyrene IMPs to prevent kidney allograft rejection. Kidney transplants were performed from BALB/c (H2d) to C57BL/6 (H2b) mice in two groups: controls (allo) and allo mice infused with IMPs. Groups were studied for 14 (acute rejection) or 100 (chronic rejection) days. Allo mice receiving IMPs exhibited superior survival and markedly less acute rejection, with better kidney function, less tubulitis, and diminished inflammatory cell density, cytokine and cytotoxic molecule expression in the allograft and lower titers of donor-specific IgG2c antibody in serum at day 14, as compared to allo mice. Cells isolated from kidneys from allo mice receiving IMPs showed reduced Ly6Chi monocytes, CD11b+ cells and NKT+ cells compared to allo mice. IMPs predominantly bound CD11b+ cells in the bloodstream and CD11b+ and CD11c-B220+ marginal zone B cells in the spleen. In the spleen, IMPs were found predominantly in red pulp, colocalized with MARCO and expression of cleaved caspase-3. At day 100, allo mice receiving IMPs exhibited reduced macrophage M1 responses but were not protected from chronic rejection. IMPs afforded significant protection from acute rejection, inhibiting both innate and adaptive alloimmunity. Thus, our current experimental findings, coupled with our earlier demonstration of IMP-induced protection in kidney ischemia-reperfusion injury, identify IMPs as a potential induction agent in kidney transplantation.

Heart rate reaction of basketball players under endurance training / Alteração da frequência cardíaca de jogadores de basquete em treino de resistência / Alteración de la frecuencia cardíaca de jugadores de básquet en entrenamiento de resistencia

ABSTRACT Introduction: In the current Chinese basketball team, many players have a high level of training. However, in official competitions, athletes tend to have heart rate problems. Therefore, it is importment to monitor and control the heart rate of basketball players to improve their performance. Objective: To explore the heart rate of basketball players in intermittent endurance training. Methods: The researchers selected 28 male basketball players from a university as the research objects. Athletes performed intermittent endurance training, and their heart rate variability, changes in frequency indicators, and changes in cardiac function were measured before and after training. Results: After training,(Total Power, TP), (High Frequency, HF), HFnorm, and (Low Frequency, LF) were significantly higher than before training. The effect sizes were medium for TP (0.7); moderate for HF (0.72); medium for HFnorm (0.59); and moderate for LFnorm (0.57). In the case of LF/HF and LF, the effect size was 0.48, close to the critical value of medium effect. Conclusions: Intermittent endurance training can improve the tension of the cardiovagal nerve of college basketball players and increase heart capacity and load, significantly improving heart function. Level of evidence II; Therapeutic studies - investigation of treatment results.

RESUMO Introdução: Muitos jogadores têm um alto nível de treinamento na atual equipe chinesa de basquetebol. Contudo, os atletas tendem a ter problemas relacionados à frequência cardíaca em competições oficiais. Portanto, é importante monitorar e controlar o batimento cardíaco visando obter a melhora de desempenho nos jogadores. Objetivo: Investigar o batimento cardíaco dos jogadores de basquetebol em treinamentos intermitentes de resistência. Métodos: Os pesquisadores selecionaram 28 jogadores de basquete em uma universidade como objeto de estudo. Esses atletas realizaram treinamento de resistência intermitente onde foram aferidas, antes e após do treino, a variabilidade de suas frequências cardíacas, as mudanças nos indicadores de frequência e as mudanças na função cardíaca. Resultados: Após o treinamento, a Potência Total (TP), a Alta Frequência (HF), a HFnorm e a Baixa Frequência (LF) foram significativamente mais altas que as aferidas previamente ao treino. O nível de alteração foi médio para TP (0,7), moderado para HF (0,72), médio para HFnorm (0,59) e moderado para LFnorm (0,57). No caso de LF/HF e LF, o tamanho da alteração foi de 0,48, próxima ao valor crítico do efeito médio. Conclusões: O treinamento de resistência intermitente pode melhorar a resistência do nervo cardiovagal nos jogadores universitários de basquete, aumentar a capacidade cardíaca e melhorar significativamente a função cardíaca. Nível de evidência II; Estudos terapêuticos - investigação de resultados de tratamento.

RESUMEN Introducción: Muchos jugadores tienen un alto nivel de entrenamiento en el actual equipo chino de baloncesto. Sin embargo, los atletas tienden a tener problemas relacionados a la frecuencia cardiaca en competiciones oficiales. Por lo tanto, es importante monitorear y controlar la frecuencia cardíaca con el fin de obtener mejoría de desempeño en los jugadores. Objetivo: Investigar la frecuencia cardíaca en los jugadores de baloncesto en entrenamientos intermitentes de resistencia. Métodos: Los investigadores seleccionaron 28 jugadores de baloncesto en una universidad como objeto de estudio. Estos atletas realizaron entrenamiento de resistencia intermitente donde fueron medidas, antes y después del entrenamiento, la variabilidad de sus frecuencias cardíacas, los cambios en los indicadores de frecuencia y los cambios en la función cardíaca. Resultados: Después del entrenamiento, la Potencia Total (TP), la Alta Frecuencia (HF), la HFnorm y la Baja Frecuencia (LF) fueron significativamente más altas que las mediciones previas al entrenamiento. El nivel de alteración fue medio para TP (0,7), moderado para HF (0,72), medio para HFnorm (0,59) y moderado para LFnorm (0,57). En el caso de LF/HF y LF, el tamaño de la alteración fue de 0,48, cercana al valor crítico del efecto medio. Conclusiones: El entrenamiento de resistencia intermitente puede mejorar la resistencia del nervio cardiovagal en los jugadores universitarios de baloncesto, aumentar la capacidad cardíaca y mejorar significativamente la función cardíaca.. Nivel de evidencia II; Estudios terapéuticos - investigación de resultados de tratamiento.

Interleukin-5 (IL-5) Therapy Prevents Allograft Rejection by Promoting CD4+CD25+ Ts2 Regulatory Cells That Are Antigen-Specific and Express IL-5 Receptor.

CD4+CD25+Foxp3+T cell population is heterogenous and contains three major sub-groups. First, thymus derived T regulatory cells (tTreg) that are naïve/resting. Second, activated/memory Treg that are produced by activation of tTreg by antigen and cytokines. Third, effector lineage CD4+CD25+T cells generated from CD4+CD25- T cells' activation by antigen to transiently express CD25 and Foxp3. We have shown that freshly isolated CD4+CD25+T cells are activated by specific alloantigen and IL-4, not IL-2, to Ts2 cells that express the IL-5 receptor alpha. Ts2 cells are more potent than naïve/resting tTreg in suppressing specific alloimmunity. Here, we showed rIL-5 promoted further activation of Ts2 cells to Th2-like Treg, that expressed foxp3, irf4, gata3 and il5. In vivo, we studied the effects of rIL-5 treatment on Lewis heart allograft survival in F344 rats. Host CD4+CD25+T cells were assessed by FACS, in mixed lymphocyte culture and by RT-PCR to examine mRNA of Ts2 or Th2-like Treg markers. rIL-5 treatment given 7 days after transplantation reduced the severity of rejection and all grafts survived ≥60d whereas sham treated rats fully rejected by day 31 (p<0.01). Treatment with anti-CD25 or anti-IL-4 monoclonal antibody abolished the benefits of treatment with rIL-5 and accelerated rejection. After 10d treatment with rIL-5, hosts' CD4+CD25+ cells expressed more Il5ra and responded to specific donor Lewis but not self. Enriched CD4+CD25+ cells from rIL-5 treated rats with allografts surviving >60 days proliferated to specific donor only when rIL-5 was present and did not proliferate to self or third party. These cells had more mRNA for molecules expressed by Th2-like Treg including Irf4, gata3 and Il5. These findings were consistent with IL-5 treatment preventing rejection by activation of Ts2 cells and Th2-like Treg.

Scalable Synthesis, In Vitro cccDNA Reduction, and In Vivo Antihepatitis B Virus Activity of a Phosphonomethoxydeoxythreosyl Adenine Prodrug.

Standard literature procedures for the chemical synthesis of l-threose nucleosides generally employ l-ascorbic acid as starting material. Herein, we have explored two alternative routes that start from either l-arabitol or l-diethyl tartrate, both affording 2-O-methyl-l-threofuranose as a key building block for nucleobase incorporation. The access to multigram quantities of this glycosyl donor in a reproducible fashion allows for the preparation of 2'-deoxy-α-l-threofuranosyl phosphonate nucleosides on a large scale. This methodology was applied to the gram scale synthesis of an aryloxy amidate prodrug of phosphonomethoxydeoxythreosyl adenine. This prodrug exerted potent activity against an entecavir-resistant hepatitis B virus (HBV) strain, while leading to a significant reduction in the levels of HBV covalently closed circular DNA in a cellular assay. Furthermore, its remarkable anti-HBV efficacy was also confirmed in vivo using a hydrodynamic injection-based HBV mouse model, without relevant toxicity and systemic exposure occurring.

Gut Microbial Metabolites Induce Donor-Specific Tolerance of Kidney Allografts through Induction of T Regulatory Cells by Short-Chain Fatty Acids.

BACKGROUND: Short-chain fatty acids derived from gut microbial fermentation of dietary fiber have been shown to suppress autoimmunity through mechanisms that include enhanced regulation by T regulatory cells (Tregs). METHODS: Using a murine kidney transplantation model, we examined the effects on alloimmunity of a high-fiber diet or supplementation with the short-chain fatty acid acetate. Kidney transplants were performed from BALB/c(H2d) to B6(H2b) mice as allografts in wild-type and recipient mice lacking the G protein-coupled receptor GPR43 (the metabolite-sensing receptor of acetate). Allograft mice received normal chow, a high-fiber diet, or normal chow supplemented with sodium acetate. We assessed rejection at days 14 (acute) and 100 (chronic), and used 16S rRNA sequencing to determine gut microbiota composition pretransplantation and post-transplantation. RESULTS: Wild-type mice fed normal chow exhibited dysbiosis after receiving a kidney allograft but not an isograft, despite the avoidance of antibiotics and immunosuppression for the latter. A high-fiber diet prevented dysbiosis in allograft recipients, who demonstrated prolonged survival and reduced evidence of rejection compared with mice fed normal chow. Allograft mice receiving supplemental sodium acetate exhibited similar protection from rejection, and subsequently demonstrated donor-specific tolerance. Depletion of CD25+ Tregs or absence of the short-chain fatty acid receptor GPR43 abolished this survival advantage. CONCLUSIONS: Manipulation of the microbiome by a high-fiber diet or supplementation with sodium acetate modified alloimmunity in a kidney transplant model, generating tolerance dependent on Tregs and GPR43. Diet-based therapy to induce changes in the gut microbiome can alter systemic alloimmunity in mice, in part through the production of short-chain fatty acids leading to Treg cell development, and merits study as a potential clinical strategy to facilitate transplant acceptance.

Early Prognostic Utility of Gp210 Antibody-Positive Rate in Primary Biliary Cholangitis: A Meta-Analysis.

BACKGROUND: The prevalence of primary biliary cholangitis (PBC), which is an autoimmune liver disease, has increased over time. PBC often leads to severe consequences, such as liver failure and death. Stratification tools using biochemical liver tests are needed to assess and predict the progression of this disease at the time of PBC diagnosis. METHODS: We searched PubMed, Cochrane Library, Web of Science, and Embase for studies focused on the relationship between positive rates of Gp210 antibodies and poor prognosis of PBC. The primary end point was the number of PBC patients with poor outcome in the Gp210 antibody (+) and Gp210 antibody (-) groups. The secondary end point was the basic serum level of alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (TBIL), and IgM in the two groups. The age and number of female patients were also measured. RESULTS: A total of 5 studies, comprising 737 patients, were included in this analysis. A positive rate of Gp210 antibodies was positively correlated with poor outcomes and with many types of progression in PBC, especially liver failure. Mortality was also higher in the Gp210 antibody (+) group. Furthermore, the serum levels of ALP and IgM were associated with the positive rate of Gp210 antibodies, while the serum levels of ALT and TBIL were not. The age and number of female patients were also not associated with the positive rate of Gp210 antibodies. CONCLUSION: PBC-specific Gp120 antibodies are optimal predictors of PBC prognosis at the time of diagnosis. Some other liver function indicators, such as ALP and IgM, can be used as predictors to complement Gp210 antibodies to establish a stratification tool to predict the prognosis of PBC at the time of diagnosis.

Incidence of infectious complications is associated with a high mortality in patients with hepatitis B virus-related acute-on-chronic liver failure.

BACKGROUND: In China, hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) is the most common liver failure characterized by serious clinical syndromes of liver decompensation with a very high mortality. Bacterial and/or fungal infections are the most common complications that are associated with high short-term mortality. Bacterial translocation from the intestine, impaired hepatic clearance, and immune paralysis of circulating immune cells are thought to contribute to infectious complications in liver failure. The control of bacterial and fungal infections is the key to improving HBV-ACLF outcomes. Active prevention, early diagnosis, and timely treatment of bacterial and fungal infections are essential for treating HBV-ACLF. AIM: To investigate the frequency and role of bacterial and fungal infections in patients with HBV-ACLF. METHODS: Patients with HBV-ACLF hospitalized at Taihe Hospital, Hubei University of Medicine from January 2014 to December 2017 were retrospectively enrolled. Patient-related information was retrieved from the hospital case database, including general information, blood biochemistry, complications, etc. According to the occurrence of secondary infection or not, the patients were divided into an infection group and a non-infection group. The sites, types, and incidences of bacterial and fungal infections and the influence of infections on the prognosis of HBV-ACLF were statistically analyzed. The risk factors for infections were assessed by unconditional logistic regression. RESULTS: There were 174 cases of HBV-ACLF that met the enrollment criteria, of which 114 (65.52%) were diagnosed with infectious complications. Infections occurred in the abdominal cavity (87 cases), respiratory tract (51 cases), urinary tract (18 cases), and biliary tract (10 cases). Patients with infectious complications had a significantly higher 28-d mortality (70.18%, 80/114) than those without (40.00%, 24/60) (70.18% vs 40.00%, P < 0.05). And patients with infectious complications had a much higher incidence of non-infectious complications (54.39%, 62/114) (54.39% vs 15.00%, P < 0.05), leading to an extremely high 28-d mortality of 88.71% (55/62) (P < 0.05). The grade of liver failure, period of hospital stay ≥ 30 d, age ≥ 45 years, and percentage of neutrophils > 70% were identified as risk factors for infectious complications. CONCLUSION: The high incidence of infectious complications in patients with HBV-ACLF is associated with severity and deterioration of the disease and may contribute to the extremely high mortality of these patients.

Direct recognition of hepatocyte-expressed MHC class I alloantigens is required for tolerance induction.

Adeno-associated viral vector-mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). The relative contributions of direct and indirect allorecognition to tolerance induction in this setting are unknown. Using hepatocyte-specific AAV vectors encoding WT allogeneic MHC class I molecules, or class I molecules containing a point mutation (D227K) that impedes direct recognition of intact allogeneic MHC class I by CD8+ T cells without hampering the presentation of processed peptides derived from allogeneic MHC class I, we show here that tolerance induction depends upon recognition of intact MHC class I. Indirect recognition alone yielded a modest prolongation of subsequent skin graft survival, attributable to the generation of CD4+ Tregs, but it was not sufficient to induce tolerance.

A modified method for heterotopic mouse heart transplantion.

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37 ° C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

Interleukin-12 (IL-12p70) Promotes Induction of Highly Potent Th1-Like CD4(+)CD25(+) T Regulatory Cells That Inhibit Allograft Rejection in Unmodified Recipients.

In rat models, CD4(+)CD25(+) T regulatory cells (Treg) play a key role in the induction and maintenance of antigen-specific transplant tolerance, especially in DA rats with PVG cardiac allografts (1, 2). We have previously described generation of alloantigen-specific Treg (Ts1), by culture of naïve natural CD4(+)CD25(+) Treg (nTreg) with specific alloantigen and IL-2 for 4 days. These cells express mRNA for IFN-γ receptor (ifngr) and suppress donor but not third party cardiac allograft rejection mediated by alloreactive CD4(+) T cells at ratios of <1:10. Here, we show that Ts1 also expressed the IL-12p70 specific receptor (il-12rß2) and that rIL-12p70 can induce their proliferation. Ts1 cells re-cultured with rIL-12p70 alone or rIL-12p70 and recombinant interleukin-2 (rIL-2), suppressed proliferation of CD4(+) T cells in mixed lymphocyte culture at <1:1024, whereas Ts1 cells re-cultured with rIL-2 and alloantigen only suppressed at 1:32-64. The rIL-12p70 alloactivated Ts1 cells markedly delayed PVG, but not third party Lewis, cardiac allograft rejection in normal DA recipients. Ts1 cells re-cultured for 4 days with rIL-12p70 alone, but not those re-cultured with rIL-12p70 and rIL-2, expressed more il-12rß2, t-bet, and ifn-γ, and continued to express the markers of Ts1 cells, foxp3, ifngr, and il-5 indicating Th1-like Treg were induced. Ts1 cells re-cultured with rIL-2 and alloantigen remained of the Ts1 phenotype and did not suppress cardiac graft rejection in normal DA rats. We induced highly suppressive Th1-like Treg from naïve nTreg in 7 days by culture with alloantigen, first with rIL-2 then with rIL-12p70. These Th1-like Treg delayed specific donor allograft rejection demonstrating therapeutic potential.

Optimized method for ureteric reconstruction in a mouse kidney transplant model.

BACKGROUND: Murine kidney transplantation is an important model for studies of transplantation immunobiology. The most challenging aspect of the difficult surgical procedure is the ureteric anastomosis. METHODS: Two different approaches to ureteric reconstruction are compared here. Method 1, Patch: this involves anastomosis of the donor ureter together with a patch of donor bladder to recipient bladder. Method 2, Implant: this utilizes a 5-0 suture to pull the ureter through the bladder wall. The ureter's peripheral tissue is then fixed to the bladder wall at the implant site with 10-0 micro-sutures. RESULTS: In animals transplanted with the patch method, the initial success rate, defined as survival up to the third post-operative day, was 79% (n = 62), whereas the initial success rate for the implant method was 86.1% (n = 101; P = 0.28). The death rate from unknown and/or unspecified causes in the initial period was 16.1% (10/62) for the patch method, and 8.9% (9/101) for the implant method (P = 0.21). The average donor/recipient operation time with the implant method was 14.8 ± 2.2/61.4 ± 4.7 min (76 min per transplant), whereas operation time with the patch method was 28.3 ± 2.4/77.8 ± 5.5 min (106 min per transplant; P < 0.001). The ureteric implant method resulted in a lower rate of urinary leak compared with the patch method (1.1% versus 10.2%; P = 0.02). CONCLUSIONS: The ureteric implant method for mouse kidney transplantation is a reliable approach with at least as high a success rate as the bladder patch method and with a shorter operation time.

Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs.

The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.

Gene therapy for tolerance: high-level expression of donor major histocompatibility complex in the liver overcomes naive and memory alloresponses to skin grafts.

BACKGROUND: The liver has long been recognized as having tolerogenic properties. We investigated whether recombinant adenoassociated virus (rAAV)-mediated expression of donor major histocompatibility complex in recipient livers could induce tolerance to donor-strain grafts. METHODS: Naive B10.BR (H-2) or B10.BR recipients primed with a H-2K-expressing (K) skin graft were injected with rAAV-expressing H-2K (rAAV-K) to induce K expression on hepatocytes 7 days before challenge with a K skin graft. K-specific responses were measured by interferon (IFN)-γ ELISpot and flow cytometric assessment of directly H-2K reactive cells. Fully allogeneic grafts from C57BL/6 (H-2) donors were transplanted onto longstanding B10.BR recipients of K skin to test for linked epitope suppression. RESULTS: rAAV-K-treated B10.BR mice accepted K skin grafts with increased median survival time (MST) more than 169 days compared to uninoculated (MST=18.5 days) and rAAV-K-treated controls (MST=19 days). rAAV-K-treated B10.BR animals primed with K skin grafts also accepted secondary K skin grafts in the long term (MST>100 days) compared to accelerated rejection in primed, uninoculated mice (MST=12 days). Treatments did not induce liver pathology, assessed by serum alanine aminotransferase levels and histology. IFN-γ ELISpot analysis of splenocytes from rAAV-K-treated mice indicated reduced responses to donor K antigen, but protection was not extended to fully allogeneic C57BL/6 skin or heart grafts, even in recipients that had accepted K skin grafts in the long term. CONCLUSIONS: High-level expression of donor major histocompatibility complex in recipient livers promotes tolerance to skin allografts, even in animals primed to produce a memory response. This provides proof of concept for an approach using liver-targeted gene delivery for tolerance induction to donor antigen.

Expression of common gamma chain signalling cytokines and their receptors distinguishes rejection from tolerance in a rat organ transplant model.

BACKGROUND: Signalling through the cytokine common γ chain (γc) is crucial for survival of activated T cells. In its absence, severe combined immunodeficiency ensues and transplanted tissues are not rejected. METHODS: To determine whether differences in the availability of γc signalling cytokines correlate with rejection or acceptance, we examined expression of all γc signalling components in organs transplanted between PVG donors and DA recipients. In this combination hearts or kidneys are rejected in <10 days while livers survive >100 days. Expression of the γc cytokines IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 and their receptors γc, IL-2Rα, IL-2Rß/IL-15Rß, IL-4Rα, IL-7Rα, IL-9Rα, IL-15Rα and IL-21Rα was determined by real-time PCR pre-transplant and on days 3, 5 and 7 after transplantation. RESULTS: Most increased after transplantation, although there were significantly lower levels of IL-2, IL-2Rα, IL-4 and IL-15Rα in tolerant livers compared to rejecting hearts or kidneys. IL-9 was only expressed in normal kidneys and decreased during rejection. IL-15 was constitutively expressed and did not change after transplantation. IL-21 and IL-21R increased in all transplanted organs to a similar extent. IL-7Rα in liver was considerably increased compared with heart or kidney, consistent with its known inverse relationship to global levels of γc signalling. CONCLUSIONS: In transplanted livers, acceptance is associated with low levels of all γc cytokines or receptors except IL-21. This is consistent with "dilution" of γc cytokines from a finite clone size of alloreactive T cells in livers, which are ten times larger than kidneys or hearts.

Reliable and reproducible murine models for commonly used abdominal plastic surgical flaps.

Animal models have been used for many years in surgical research to develop different surgical techniques, improve understanding of anatomy and physiology and hone surgical skills. The benefit of such models has been particularly important in developing relatively young specialties like plastic surgery and many plastic surgical techniques are designed and studied in animals long before they are used in humans. We describe techniques for raising several reliable and reproducible abdominal flaps in rodents, including transverse rectus abdominis myocutaneous flaps in rats and mice, superficial inferior epigastric artery flaps in rats and perforator flaps in rats. The intention of this paper is to act as a point of reference for any microvascular or plastic surgeon who is planning to perform abdominal plastic surgical flap research or further microvascular skills.

Spontaneous acceptance of mouse kidney allografts is associated with increased Foxp3 expression and differences in the B and T cell compartments.

Spontaneous acceptance of organ allografts can identify novel mechanisms of drug-free transplantation tolerance. Spontaneous acceptance occurs in both mouse kidney transplants and rat liver transplants however the early immune processes of mouse kidney acceptance have not been studied. Acceptance of C57BL/6 strain kidney allografts in fully MHC-incompatible B10.BR recipients was compared with rejection (REJ) of heart allografts in the same strain combination. Graft infiltrate and antibody deposition were examined by immunohistochemical staining. Expression of mRNA was measured by quantitative real-time PCR. Apoptosis was examined by TUNEL staining. The majority of kidney allografts were accepted long-term and induced tolerance (TOL) of donor-strain skin grafts, showing that acceptance was not due to immune ignorance. There was an extensive infiltrate of T cells in the TOL kidney that exceeded the level in REJ hearts but subsequently declined. The main differences were deposition of IgG2a antibody in REJ that was absent in TOL, more B cells infiltrating TOL kidneys and a progressive increase in the ratio of CD8:CD4 cells during rejection. There was also significantly greater Foxp3 mRNA expression in TOL. Kidneys from RAG-/- donors were accepted, showing that donor lymphocytes were not necessary for acceptance. Neutralising antibodies to TGF-ß administered from day 0 to day 6 did not prevent TOL. On the basis of cytokine expression and apoptosis there was no evidence for immune deviation or deletion as mechanisms of acceptance. In accord with the findings of spontaneous acceptance of liver allografts in rats, the main difference between mouse kidney TOL and heart REJ was in the B cell compartment. The major difference to rat liver allograft acceptance was that apoptosis of infiltrate did not appear to play a role. Instead, increased Foxp3 expression in TOL kidneys implies that regulatory T cells might be important.