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.

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.

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.

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.

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.

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.

Donor IL-4-treatment induces alternatively activated liver macrophages and IDO-expressing NK cells and promotes rat liver allograft acceptance.

Most approaches to transplant tolerance involve treatment of the recipient to prevent rejection. This study investigates donor treatment with IL-4 for its effect on subsequent rat liver allograft survival. Rat orthotopic liver transplants were performed in rejecting (PVG donor to Lewis recipient) or spontaneously tolerant (PVG to DA) strain combinations. Donors were untreated or injected intraperitoneally with IL-4 (30,000U/day) for 5days. Tissue infiltrates and gene expression were examined by immunohistochemistry and real-time quantitative PCR. IL-4 induced a marked leukocyte infiltrate in donor livers prior to transplant. Macrophages comprised the major population, although B cells, T cells and natural killer (NK) cells also increased. IL-4-induced liver macrophages had an alternatively activated phenotype with increased expression of mannose receptor but not inducible nitric oxide synthase (NOS2). IL-4 also induced IDO and IFN-gamma expression by NK cells. Donor IL-4-treatment converted rejection to acceptance in the majority of Lewis recipients (median survival time >96days) and did not prevent acceptance in DA recipients. Acceptance in Lewis recipients was associated with increased donor cell migration to recipient spleens and increased splenic IL-2, IFN-gamma and IDO expression 24h after transplantation. Donor IL-4-treatment increased leukocytes in the donor liver including potentially immunosuppressive populations of alternatively activated macrophages and IDO-expressing NK cells. Donor treatment led to long-term acceptance of most livers in association with early immune activation in recipient lymphoid tissues.

Overexpression of indoleamine dioxygenase in rat liver allografts using a high-efficiency adeno-associated virus vector does not prevent acute rejection.

The aim of this study was to evaluate the ability of local overexpression of indoleamine dioxygenase (IDO) to abrogate rat liver transplant rejection by the use of an adeno-associated virus vector [recombinant adeno-associated virus 2/8 (rAAV2/8)] to deliver the transgene to the allograft prior to transplantation. A green fluorescent protein (GFP)-expressing vector [recombinant adeno-associated virus 2/8-liver-specific promoter 1-enhanced green fluorescent protein (rAAV2/8-LSP1-eGFP)] was used to examine the kinetics of expression and optimal dosing for transduction of Piebald Virol Glaxo (PVG) rat livers. A vector encoding the rat IDO gene (rAAV2/8-LSP1-rIDO) was constructed and tested by its ability to induce tryptophan catabolism and kynurenine production in vitro and in vivo. PVG donor rats were injected, via the portal vein, with rAAV2/8-LSP1-rIDO 2 weeks before transplantation into PVG strain isograft or Lewis (LEW) strain allograft recipients. With the enhanced GFP vector, 29.5% and 47.4% of hepatocytes were found to express GFP at 3 and 6 weeks after injection, respectively. In untransplanted PVG animals, the rAAV2/8-LSP1-rIDO vector induced, 3 weeks after administration, a 1.8-fold increase (P = 0.0161) in liver IDO activity, which was associated with a fall in serum tryptophan to 0.5 times the baseline level (P < 0.001). PVG recipients of PVG liver isografts pretreated with the IDO-expressing vector had a 45% lower level of serum tryptophan than recipients of isografts pretreated with the GFP-expressing vector (P = 0.03). LEW recipients of PVG liver allografts pretreated with the rat IDO vector had a median survival time of 12 days, whereas recipients of allografts pretreated with rAAV2/8-LSP1-eGFP had a median survival time of 13 days (P = 0.38). Both groups displayed similar histological features of acute cellular rejection. In conclusion, rAAV2/8 vectors produce highly efficient, though delayed, hepatocyte transduction in vivo and provide a useful gene delivery tool for transplantation models. However, gene delivery using IDO was unsuccessful in prolonging rat liver allograft survival.

Arterial vascular conduits in adult orthotopic liver transplant recipients.

BACKGROUND: Vascular conduits may be required to gain arterial inflow to the donor hepatic artery in orthotopic liver transplantation. METHODS: From January 1986 to December 2003, arterial conduits were required in 31/582 (5.3%) adult liver transplant procedures. RESULTS: Indications for the conduit included recipient hepatic artery problems (20); hepatic artery thrombosis previous allograft (7) and other (4). The conduits used in 28/31 cases (90%) were deceased donor iliac arteries and the remainder prosthetic grafts. Patients requiring conduits were more likely to be already hospitalized (P = 0.038) or undergoing a retransplant procedure (P = 0.001) than patients not requiring conduits. Both sepsis and haemorrhage caused death in 8/31 (26%) patients requiring conduits versus 42/551 (7.6%) patients not requiring conduits. Death from thrombosis of the iliac artery conduit occurred in two cases and from bacterial infection of a prosthetic conduit in one case. For retransplant procedures, allograft loss was seen in 11/13 (84%) conduit cases versus 11/28 (39%) non-conduit cases (P = 0.016). Overall allograft survival was significantly lower in the conduit cases than in the non-conduit cases (P = 0.0001), with 12/31 (39%) allografts being lost within the first 3 months post-transplantation for the conduit cases. CONCLUSION: Arterial vascular conduits are more commonly required in adult liver transplant recipients who are hospitalized or undergoing retransplant procedures. Allograft survival is poorer in the conduit cases and is associated with complications, particularly sepsis and haemorrhage, following retransplantation procedures.

Posttransplant interleukin-4 treatment converts rat liver allograft tolerance to rejection.

BACKGROUND: Previous studies showed that liver transplant rejection in the Piebald Virol Glaxo (PVG)-to-Lewis combination was associated with more intragraft interleukin (IL)-4 mRNA expression than in spontaneously tolerant grafts in the PVG-to-Dark Agouti (DA) combination. There was also immunoglobulin (Ig) G1 antibody deposition, suggesting an IL-4-induced IgG class switch in rejection. The aim of this study was to investigate whether IL-4 treatment converts PVG-->DA liver transplant tolerance to rejection. METHODS: DA (RT1a) rats were recipients of orthotopic PVG (RT1c) liver transplants and DA liver transplants were syngeneic controls. Supernatant from IL-4-transfected Chinese hamster ovary cells (0.5 mL, 30,000 U) or from untransfected cells was injected intraperitoneally on days 3 through 7. Samples were taken for immunohistochemical staining of frozen tissue sections to analyze cellular infiltrate and antibody deposition. RESULTS: IL-4 treatment significantly reduced survival of liver allografts from greater than 100 days in untreated animals to 9 days (P=0.004). Pathologic analysis of IL-4-treated animals showed that death was caused by liver transplant rejection, with a heavy infiltrate of mononuclear cells, disruption of portal tract areas, and infarction. Immunohistochemistry revealed an extensive infiltrate of T cells, CD25-expressing cells, and B cells that was similar to the level in PVG--> Lewis liver allograft recipients that reject the liver. There was also a more extensive monocyte-macrophage infiltrate and more major histocompatibility complex class II expression in IL-4-treated animals compared with untreated animals. There was moderate increase of IgM, little IgG1, and no IgE or IgG2a antibody deposition. CONCLUSIONS: IL-4, a T-helper type 2 cytokine that has previously been shown to inhibit delayed-type hypersensitivity responses such as rejection, was found to promote rejection of liver allografts. There was only slight evidence of a graft-specific antibody response, showing that IL-4 induces liver allograft rejection in association with some, but not all, of the changes accompanying rejection in the PVG-->Lewis strain combination.