Cohort Study Examining the Association of Immunosuppressant Drug Prescription With Major Adverse Cardiovascular and Limb Events in Patients With Peripheral Artery Disease.

AIM: Immune activation is strongly implicated in atherosclerotic plaque instability, however, the effect of immunosuppressant drugs on cardiovascular events in patients with peripheral artery disease (PAD) is not known. The aim of this study was to assess whether prescription of one or more immune suppressant drugs was associated with a lower risk of major adverse cardiovascular (MACE; i.e. myocardial infarction, stroke or cardiovascular events) or limb events (MALE; i.e. major amputation or requirement for peripheral revascularization) in patients with PAD. METHODS: A total of 1506 participants with intermittent claudication (n = 872) or chronic limb threatening ischemia (CLTI; n = 634) of whom 53 (3.5%) were prescribed one or more immunosuppressant drugs (prednisolone 41; methotrexate 17; leflunomide 5; hydroxychloroquine 3; azathioprine 2; tocilizumab 2; mycophenolate 1; sulfasalazine 1; adalimumab 1) were recruited from 3 Australian hospitals. Participants were followed for a median of 3.9 (inter-quartile range 1.2, 7.3) years. The association of immunosuppressant drug prescription with MACE or MALE was examined using Cox proportional hazard analyses. RESULTS: After adjusting for other risk factors, prescription of an immunosuppressant drug was associated with a significantly greater risk of MACE (Hazard ratio, HR, 1.83, 95% confidence intervals, CI, 1.11, 3.01; P = 0.017) but not MALE (HR 1.32, 95% CI 0.90, 1.92; P = 0.153). In a sub-analysis restricted to participants with CLTI findings were similar: MACE (HR 2.44, 95% CI 1.32, 4.51; P = 0.005); MALE (HR 1.38, 95% CI 0.87, 2.19; P = 0.175); major amputation (HR 1.37, 95% CI 0.49, 3.86; P = 0.547). CONCLUSIONS: This cohort study suggested that immunosuppressant drug therapy is associated with a greater risk of MACE amongst patients with PAD.

Intestinal Abnormalities in Patients With SARS-CoV-2 Infection: Histopathologic Changes Reflect Mechanisms of Disease.

Approximately 20% of patients with symptomatic syndrome-associated coronavirus-2 (SARS-CoV-2) infection have gastrointestinal bleeding and/or diarrhea. Most are managed without endoscopic evaluation because the risk of practitioner infection outweighs the value of biopsy analysis unless symptoms are life-threatening. As a result, much of what is known about the gastrointestinal manifestations of coronavirus disease-2019 (COVID-19) has been gleaned from surgical and autopsy cases that suffer from extensive ischemic injury and/or poor preservation. There are no detailed reports describing any other gastrointestinal effects of SARS-CoV-2 even though >3,000,000 people have died from COVID-19 worldwide. The purpose of this study is to report the intestinal findings related to SARS-CoV-2 infection by way of a small case series including one with evidence of direct viral cytopathic effect and 2 with secondary injury attributed to viral infection. Infection can be confirmed by immunohistochemical stains directed against SARS-CoV-2 spike protein, in situ hybridization for spike protein-encoding RNA, and ultrastructural visualization of viruses within the epithelium. It induces cytoplasmic blebs and tufted epithelial cells without inflammation and may not cause symptoms. In contrast, SARS-CoV-2 infection can cause gastrointestinal symptoms after the virus is no longer detected, reflecting systemic activation of cytokine and complement cascades rather than direct viral injury. Reversible mucosal ischemia features microvascular injury with hemorrhage, small vessel thrombosis, and platelet-rich thrombi. Systemic cytokine elaboration and dysbiosis likely explain epithelial cell injury that accompanies diarrheal symptoms. These observations are consistent with clinical and in vitro data and contribute to our understanding of the protean manifestations of COVID-19.

Mesenchymal Stem Cell Induced Foxp3(+) Tregs Suppress Effector T Cells and Protect against Retinal Ischemic Injury.

Mesenchymal stem/stromal cells (MSC) are well known for immunomodulation; however, the mechanisms involved in their benefits in the ischemic retina are unknown. This study tested the hypothesis that MSC induces upregulation of transcription factor forkhead box protein P3 (Foxp3) in T cells to elicit immune modulation, and thus, protect against retinal damage. Induced MSCs (iMSCs) were generated by differentiating the induced pluripotent stem cells (iPSC) derived from urinary epithelial cells through a noninsertional reprogramming approach. In in-vitro cultures, iMSC transferred mitochondria to immune cells via F-actin nanotubes significantly increased oxygen consumption rate (OCR) for basal respiration and ATP production, suppressed effector T cells, and promoted differentiation of CD4+CD25+ T regulatory cells (Tregs) in coculture with mouse splenocytes. In in-vivo studies, iMSCs transplanted in ischemia-reperfusion (I/R) injured eye significantly increased Foxp3+ Tregs in the retina compared to that of saline-injected I/R eyes. Furthermore, iMSC injected I/R eyes significantly decreased retinal inflammation as evidenced by reduced gene expression of IL1ß, VCAM1, LAMA5, and CCL2 and improved b-wave amplitudes compared to that of saline-injected I/R eyes. Our study demonstrates that iMSCs can transfer mitochondria to immune cells to suppress the effector T cell population. Additionally, our current data indicate that iMSC can enhance differentiation of T cells into Foxp3 Tregs in vitro and therapeutically improve the retina's immune function by upregulation of Tregs to decrease inflammation and reduce I/R injury-induced retinal degeneration in vivo.

Pre-operative Neutrophil to Lymphocyte Ratio is Associated With 30 Day Death or Amputation After Revascularisation for Acute Limb Ischaemia.

OBJECTIVE: Inflammation is an early feature of acute limb ischaemia (ALI), hence the potential prognostic significance of inflammatory biomarkers. This study aimed to assess the value of pre-operative inflammatory biomarkers, specifically the neutrophil to lymphocyte ratio (NLR) and the platelet to lymphocyte ratio (PLR), for predicting an adverse outcome after revascularisation for ALI. METHODS: All patients submitted to lower limb revascularisation for Rutherford IIa or IIb ALI at the authors' institution between 2009 and 2019 were screened retrospectively. Pre-operative NLR and PLR were analysed, along with other known prognostic factors. Primary outcome was the composite endpoint of 30 day death or amputation. RESULTS: A total of 345 patients were included, 84 of whom suffered the primary outcome (24.3%). The median follow up was 23.1 months (3.1 - 52.2). Higher age (OR 1.05 per year increase, 95% CI 1.01 - 1.09), diabetes (OR 2.63, 95% CI 1.14 - 6.06), Rutherford grade IIb vs. IIa (OR 5.51, 95% CI 2.11 - 14.42), higher NLR (OR 1.28 per unit increase, 95% CI 1.12 - 1.47), and fasciotomy need (OR 3.44, 95% CI 1.14 - 10.34) were independently associated with 30 day death or amputation, whereas pre-operative statin or anticoagulant medication were associated with a risk reduction (OR 0.23, 95% CI 0.53 - 0.96 and OR 0.20, 95% CI 0.05 - 0.84, respectively). PLR did not show an independent effect on this population. Pre-operative NLR presented a good discriminative ability (AUC 0.86, 95% CI 0.82 - 0.90). A cut off NLR level ≥ 5.4 demonstrated a 90.5% sensitivity and 73.6% specificity for 30 day death or amputation. Kaplan-Meier analysis showed that patients with pre-operative NLR ≥ 5.4 had significantly lower 30 day, six month and one year amputation free survival when compared with those with NLR < 5.4 (64.8 ± 4.0%, 44.1 ± 4.1%, and 37.5 ± 4.1% vs. 98.5 ± 0.9%, 91.9 ± 2.0%, and 85.9 ± 2.5%, log rank p < .001). CONCLUSION: In this study, higher pre-operative NLR was associated with 30 day death or amputation following intervention for Rutherford grade IIa or IIb ALI. NLR potentially stands as a simple, widely available and inexpensive biomarker that can refine decision making and possibly contribute to ALI morbidity and mortality reduction.

Pre-operative exercise therapy triggers anti-inflammatory trained immunity of Kupffer cells through metabolic reprogramming.

Pre-operative exercise therapy improves outcomes for many patients who undergo surgery. Despite the well-known effects on tolerance to systemic perturbation, the mechanisms by which pre-operative exercise protects the organ that is operated on from inflammatory injury are unclear. Here, we show that four-week aerobic pre-operative exercise significantly attenuates liver injury and inflammation from ischaemia and reperfusion in mice. Remarkably, these beneficial effects last for seven more days after completing pre-operative exercising. We find that exercise specifically drives Kupffer cells toward an anti-inflammatory phenotype with trained immunity via metabolic reprogramming. Mechanistically, exercise-induced HMGB1 release enhances itaconate metabolism in the tricarboxylic acid cycle that impacts Kupffer cells in an NRF2-dependent manner. Therefore, these metabolites and cellular/molecular targets can be investigated as potential exercise-mimicking pharmaceutical candidates to protect against liver injury during surgery.

Electroacupuncture alleviates intestinal inflammation and barrier dysfunction by activating dopamine in a rat model of intestinal ischaemia.

BACKGROUND: To investigate whether the mechanism underlying the anti-inflammatory effects of electroacupuncture (EA) at ST36 involves dopamine (DA) and its receptor and whether it is mediated by the vagus nerve in a rat model of intestinal ischaemia-reperfusion (I/R) injury. METHODS: Rats were subjected to gut ischaemia for 30 min and then received EA for 30 min with or without abdominal vagotomy or intraperitoneal administration of butaclamol (D1 receptor antagonist) or spiperone (D2 receptor antagonist). Plasma levels of DA and tumour necrosis factor (TNF)-α were assessed 1 or 4 h after reperfusion. Myeloperoxidase (MPO) activity and malondialdehyde (MDA) content in intestinal tissues were assessed using enzyme-linked immunosorbent assay (ELISA) kits. Intestinal tissue injury was assessed by observation of the pathological lesions and permeability to 4 kDa fluorescein isothiocyanate (FITC)-dextran. RESULTS: EA significantly increased levels of DA and lowered levels of TNF-α. EA also inhibited intestinal levels of MPO and MDA and intestinal tissue injury and decreased intestinal permeability to FITC-dextran. Abdominal vagotomy and intraperitoneal administration of butaclamol (but not spiperone) inhibited the effects of EA. CONCLUSION: These findings suggest that EA at ST36 could attenuate intestinal I/R-induced inflammatory injury and that the underlying mechanism may involve EA-induced increases in levels of DA, mediated by the vagus nerve and D1 receptors.

Flow-mediated outward arterial remodeling in aging.

The present review focuses on the effect of aging on flow-mediated outward remodeling (FMR) via alterations in estrogen metabolism, oxidative stress and inflammation. In ischemic disorders, the ability of the vasculature to adapt or remodel determines the quality of the recovery. FMR, which has a key role in revascularization, is a complex phenomenon that recruits endothelial and smooth muscle cells as well as the immune system. FMR becomes progressively less with age as a result of an increase in inflammation and oxidative stress, in part of mitochondrial origin. The alteration in FMR is greater in older individuals with risk factors and thus the therapy cannot merely amount to exercise with or without a mild vasodilating drug. Interestingly, the reduction in FMR occurs later in females. Estrogen and its alpha receptor (ERα) play a key role in FMR through the control of dilatory pathways including the angiotensin II type 2 receptor, thus providing possible tools to activate FMR in older subjects although only experimental data is available. Indeed, the main issue is the reversibility of the vascular damage induced over time, and to date promoting prevention and limiting exposure to the risk factors remain the best options in this regard.

Myeloid ALX/FPR2 regulates vascularization following tissue injury.

Ischemic injury initiates a sterile inflammatory response that ultimately participates in the repair and recovery of tissue perfusion. Macrophages are required for perfusion recovery during ischemia, in part because they produce growth factors that aid in vascular remodeling. The input signals governing this pro-revascularization phenotype remain of interest. Here we found that hindlimb ischemia increases levels of resolvin D1 (RvD1), an inflammation-resolving lipid mediator that targets macrophages via its receptor, ALX/FPR2. Exogenous RvD1 enhances perfusion recovery during ischemia, and mice deficient in Alx/Fpr2 have an endogenous defect in this process. Mechanistically, RNA sequencing revealed that RvD1 induces a transcriptional program in macrophages characteristic of a pro-revascularization phenotype. Vascularization of ischemic skeletal muscle, as well as cutaneous wounds, is impaired in mice with myeloid-specific deficiency of Alx/Fpr2, and this is associated with altered expression of pro-revascularization genes in skeletal muscle and macrophages isolated from skeletal muscle. Collectively, these results uncover a role of ALX/FPR2 in revascularization that may be amenable to therapeutic targeting in diseases associated with altered tissue perfusion and repair.

IL10 Alters Peri-Collateral Macrophage Polarization and Hind-Limb Reperfusion in Mice after Femoral Artery Ligation.

Arteriogenesis is a process by which a pre-existing arterioarterial anastomosis develops into a functional collateral network following an arterial occlusion. Alternatively activated macrophages polarized by IL10 have been described to promote collateral growth. This study investigates the effect of different levels of IL10 on hind-limb reperfusion and the distribution of perivascular macrophage activation types in mice after femoral artery ligation (FAL). IL10 and anti-IL10 were administered before FAL and the arteriogenic response was measured by Laser-Doppler-Imaging perioperatively, after 3, 7, and 14 d. Reperfusion recovery was accelerated when treated with IL10 and impaired with anti-IL10. Furthermore, symptoms of ischemia on ligated hind-limbs had the highest incidence after application of anti-IL10. Perivascular macrophages were immunohistologically phenotyped using CD163 and CD68 in adductor muscle segments. The proportion of alternatively activated macrophages (CD163+/CD68+) in relation to classically activated macrophages (CD163-/CD68+) observed was the highest when treated with IL10 and suppressed with anti-IL10. This study underlines the proarteriogenic response with increased levels of IL10 and demonstrates an in-vivo alteration of macrophage activation types in the perivascular bed of growing collaterals.

CD4+ T Cell-Derived NGAL Modifies the Outcome of Ischemic Acute Kidney Injury.

CD4+ T cells mediate the pathogenesis of ischemic and nephrotoxic acute kidney injury (AKI). However, the underlying mechanisms of CD4+ T cell-mediated pathogenesis are largely unknown. We therefore conducted unbiased RNA-sequencing to discover novel mechanistic pathways of kidney CD4+ T cells after ischemia compared with normal mouse kidney. Unexpectedly, the lipocalin-2 (Lcn2) gene, which encodes neutrophil gelatinase-associated lipocalin (NGAL) had the highest fold increase (∼60). The NGAL increase in CD4+ T cells during AKI was confirmed at the mRNA level with quantitative real-time PCR and at the protein level with ELISA. NGAL is a potential biomarker for the early detection of AKI and has multiple potential biological functions. However, the role of NGAL produced by CD4+ T cells is not known. We found that ischemic AKI in NGAL knockout (KO) mice had worse renal outcomes compared with wild-type (WT) mice. Adoptive transfer of NGAL-deficient CD4+ T cells from NGAL KO mice into CD4 KO or WT mice led to worse renal function than transfer of WT CD4+ T cells. In vitro-simulated ischemia/reperfusion showed that NGAL-deficient CD4+ T cells express higher levels of IFN-γ mRNA compared with WT CD4+ T cells. In vitro differentiation of naive CD4+ T cells to Th17, Th1, and Th2 cells led to significant increase in Lcn2 expression. Human kidney CD4+ T cell NGAL also increased significantly after ischemia. These results demonstrate an important role for CD4+ T cell NGAL as a mechanism by which CD4+ T cells mediate AKI and extend the importance of NGAL in AKI beyond diagnostics.

Cilostazol Mediates Immune Responses and Affects Angiogenesis During the Acute Phase of Hind Limb Ischemia in a Mouse Model.

BACKGROUND: Cilostazol is a drug of choice for the treatment of intermittent claudication that also affects innate and adaptive immune cells. The purpose of our study was the evaluation of cilostazol's impact on the immune and angiogenic response in murine models of hind limb ischemia. METHODS: We used 108 immunodeficient NOD.CB17-Prkdcscid/J mice and 108 wild-type CB17 mice. At day 0 (D0), all animals underwent hind limb ischemia. Half of them in both groups received daily cilostazol starting at D0 and for the next 7 postoperative days, while the rest of them served as controls, receiving vehicle. Interleukin (IL) 2, IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ) serum concentrations were measured by flow cytometry on postsurgery days D1, D3, D5, and D7. On D7, both groups underwent positron emission tomography scan with 68Ga-RGD. Mice were euthanatized and gastrocnemius muscles were obtained for histological evaluation. RESULTS: There was a statistically significant augmentation (P < .05) in IL-4, IL-10, IL-6, and IFN-γ concentrations in treated CB17 animals, while IL-2 was significantly suppressed. Significant difference was detected between the CiBisch and Bisch groups on D1 and D7 (P < .05) in CD31 staining. In treated NOD.CB17 animals, TNF-α, IL-6, and IFN-γ presented significant augmentation, while 68Ga-NODAGA-RGDfK uptake and CD31 expression were found significantly lower for both legs in comparison to the control. CONCLUSION: Cilostazol seems to significantly increase angiogenesis in wild-type animals during the first postoperational week. It also influences immune cells, altering the type of immune response by promoting anti-inflammatory cytokine production in wild-type animals, while it helps toward inflammation regression in immunodeficient animals.

Capillary-resident EphA7+ pericytes are multipotent cells with anti-ischemic effects through capillary formation.

The presence of pericytes (PCs) with multipotency and broad distribution along capillary suggests that microvasculature plays a role not only as a duct for blood fluid transport but also as a stem cell niche that contributes to tissue maintenance and regeneration. The lack of an appropriate marker for multipotent PCs still limits our understanding of their pathophysiological roles. We identified the novel marker EphA7 to detect multipotent PCs using microarray analysis of an immortalized PC library. PCs were isolated from microvessels of mouse subcutaneous adipose tissues, then EphA7+ PCs called capillary stem cells (CapSCs) were separated from EphA7- control PCs (ctPCs) using fluorescence-activated cell sorting system. CapSCs had highly multipotency that enabled them to differentiate into mesenchymal and neuronal lineages compared with ctPCs. CapSCs also differentiated into endothelial cells and PCs to form capillary-like structures by themselves. Transplantation of CapSCs into ischemic tissues significantly improved blood flow recovery in hind limb ischemia mouse model due to vascular formation compared with that of ctPCs and adipose stromal cells. These data demonstrate that EphA7 identifies a subpopulation of multipotent PCs that have high angiogenesis and regenerative potency and are an attractive target for regenerative therapies.

Batroxobin accelerated tissue repair via neutrophil extracellular trap regulation and defibrinogenation in a murine ischemic hindlimb model.

Batroxobin, isolated from Bothrops moojeni, is a defibrinogenating agent used as a thrombin-like serine protease against fibrinogen for improving microcirculation. Here, we investigated whether, and if so, how batroxobin restores ischemic tissue injury in terms of anti-inflammatory effects. In an in vitro flow cytometry assay for human neutrophil extracellular traps (NETs), batroxobin (DF-521; Defibrase) inhibited human NETs induced by tumor necrosis factor-α (TNF-α) in the presence of human fibrinogen. Next, the effect of batroxobin was investigated by immunohistochemistry of the anterior tibial muscle (ATM) in an ischemic hindlimb model using C57BL/6J mice intraperitoneally injected with DF-521 versus the saline control. NETs and fibrinogen deposition in the ischemic ATM decreased in DF-521-treated mice on day 2 after ischemia. Meanwhile, reverse transcription-quantitative PCR assay of the ischemic ATM unveiled continuous downregulation in the expression of the genes; Tnf-α and nitric oxide synthase2 (Nos2) with hypoxia-inducible factor-1α (Hif-1α) and vascular endothelial growth factor-a (Vegf-a) from day 3 to day 7, but the upregulation of arginase-1 (Arg-1) and placental growth factor (Plgf) with myogenin (Myog) on day 7. Daily intraperitoneal DF-521 injection for the initial 7 days into mice with ischemic hindlimbs promoted angiogenesis and arteriogenesis on day 14. Moreover, DF-521 injection accelerated myofiber maturation after day 14. Laser doppler imaging analysis revealed that blood perfusion in DF-521-injected mice significantly improved on day 14 versus the saline control. Thus, DF-521 improves microcirculation by protecting NETs with tissue defibrinogenation, thereby protecting against severe ischemic tissue injury and accelerating vascular and skeletal muscular regeneration. To our knowledge, batroxobin might be the first clinically applicable NET inhibitor against ischemic diseases.

Interactions between the complement and endothelin systems in normal pregnancy and following placental ischemia.

Preeclampsia is characterized by new onset hypertension and fetal growth restriction and is associated with aberrant activation of the innate immune complement system and stressed or ischemic placenta. Previous studies have suggested a role for both endothelin and complement system activation products in new onset hypertension in pregnancy, but inter-relationships of the pathways are unclear. We hypothesized that complement activation following placental ischemia stimulates the endothelin pathway to cause hypertension and impair fetal growth. The Reduced Uterine Perfusion Pressure (RUPP) model results in hypertension and fetal growth restriction in a pregnant rat due to placental ischemia caused by mechanical obstruction of blood flow to uterus and placenta. The effect of inhibitor of complement activation soluble Complement Receptor 1 (sCR1) and endothelin A receptor (ETA) antagonist atrasentan on hypertension, fetal weight, complement activation (systemic circulating C3a and local C3 placental deposition) and endothelin [circulating endothelin and message for preproendothelin (PPE), ETA and endothelin B receptor (ETB) in placenta] in the RUPP rat model were determined. Following placental ischemia, sCR1 attenuated hypertension but increased message for PPE and ETA in placenta, suggesting complement activation causes hypertension via an endothelin independent pathway. With ETA antagonism the placental ischemia-induced increase in circulating C3a was unaffected despite inhibition of hypertension, indicating systemic C3a alone is not sufficient. In normal pregnancy, inhibiting complement activation increased plasma endothelin but not placental PPE message. Atrasentan treatment increased fetal weight, circulating endothelin and placental ETA message, and unexpectedly increased local complement activation in placenta (C3 deposition) but not C3a in circulation, suggesting endothelin controls local placental complement activation in normal pregnancy. Atrasentan also significantly decreased message for endogenous complement regulators Crry and CD55 in placenta and kidney in normal pregnancy. Results of our study indicate that complement/endothelin interactions differ in pregnancies complicated with placental ischemia vs normal pregnancy, as well as locally vs systemically. These data clearly illustrate the complex interplay between complement and endothelin indicating that perturbations of either pathway may affect pregnancy outcomes.

Natural killer cells contribute to mitochondrial dysfunction in response to placental ischemia in reduced uterine perfusion pressure rats.

Preeclampsia (PE) is characterized by new-onset hypertension during pregnancy and is associated with immune activation and placental oxidative stress. Mitochondrial dysfunction is a major source of oxidative stress and may play a role in the pathology of PE. We (Vaka VR, et al. Hypertension 72: 703-711, 2018. doi: 10.1161/HYPERTENSIONAHA.118.11290 .) have previously shown that placental ischemia is associated with mitochondrial oxidative stress in the reduced uterine perfusion pressure (RUPP) model of PE. Furthermore, we have also shown that placental ischemia induces natural killer (NK) cell activation in RUPP. Thus, we hypothesize that NK cell depletion could improve mitochondrial function associated with hypertension in the RUPP rat model of PE. Pregnant Sprague-Dawley rats were divided into three groups: normal pregnant (NP), RUPP, and RUPP+NK cell depletion rats (RUPP+NKD). On gestational day (GD)14, RUPP surgery was performed, and NK cells were depleted by administering anti-asialo GM1 antibodies (3.5 µg/100 µl ip) on GD15 and GD17. On GD19, mean arterial pressure (MAP) was measured, and placental mitochondria were isolated and used for mitochondrial assays. MAP was elevated in RUPP versus NP rats (119 ± 1 vs.104 ± 2 mmHg, P = 0.0004) and was normalized in RUPP+NKD rats (107 ± 2 mmHg, P = 0.002). Reduced complex IV activity and state 3 respiration rate were improved in RUPP+NKD rats. Human umbilical vein endothelial cells treated with RUPP+NKD serum restored respiration with reduced mitochondrial reactive oxygen species (ROS). The restored placental or endothelial mitochondrial function along with attenuated endothelial cell mitochondrial ROS with NK cell depletion indicate an important role of NK cells in mediating mitochondrial oxidative stress in the pathology of PE.

A Flow Dynamic Rationale for Accelerated Vascularized Composite Allotransplant Rejection.

BACKGROUND: From 1996 to 2000, Diefenbeck et al. carried out six knee vascularized composite allotransplants. The allotransplants were composed of bone, soft tissue, and femoral vascular pedicle (25 to 40 cm). All rejected between 14 and 56 months. Failures were attributed to chronic rejection. In 2008, the Louisville team lost their fourth patient's hand transplant at 8 months. During the rejection workup, intraoperative findings noted a thickened arterial pedicle attributed to intimal hyperplasia with significant fibrotic perivascular tissue and a near "no-flow phenomenon." No cutaneous rejection was appreciated and failure was attributed to chronic rejection. METHODS: Data were collected from two teams, one in Germany and the other in Louisville, Kentucky. The population under study consisted of the six knee and one hand transplants. The factor of interest was the long donor arterial pedicle. The outcome measurements were transplant survival time and histopathologic results. RESULTS: There are only seven published vascularized composite allotransplant cases where a donor artery longer than 25 cm was used. This cohort represents a 100 percent accelerated failure rate. The cause of these losses remains unexplained. The donor arteries suffered from T-cell-mediated rejection and ischemia-induced media/adventitial necrosis. CONCLUSIONS: We hypothesize that the donor artery rejected at an accelerated rate because of ischemia caused by disruption of the external vasa vasorum in conjunction with intimal hyperplasia induced by T-cell-mediated rejection that led to disruption of the Windkessel effect. Loss of this effect presented as intimal hyperplasia accelerated by ischemia causing an expedited transplant failure. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Emphysematous pyelonephritis in renal allograft related to antibody-mediated rejection: A case report and literature review.

Emphysematous pyelonephritis (EPN) is a rare condition which can rapidly progress to sepsis and multiple organ failure with high mortality. We experienced a rare case of EPN in a renal allograft related to antibody-mediated rejection (AMR). The patient received a deceased donor kidney transplant due to end-stage renal disease secondary to diabetes mellitus. Cross-match test was negative but she had remote history of anti-HLA-A2 antibody corresponding with the donor HLA. Surgery concluded without any major events. Anti-thymoglobulin was given perioperatively for induction. She was compliant with her immunosuppressive medications making urine of 2 L/d with serum creatinine of 1.9 mg/dL at discharge on post-operative day (POD) 6. She did well until POD 14 when she presented to the clinic with features of sepsis, pain over the transplanted kidney area and decline in urine volume with elevated serum creatinine. CT revealed extensive gas throughout the transplanted kidney. Renal scan revealed non-functional transplant kidney with no arterial flow. Based on these findings, a decision to perform transplant nephrectomy was made. At laparotomy, the kidney was completely necrotic. Pathology showed non-viable kidney parenchyma with the tubules lacking neutrophilic casts suggestive of ischemic necrosis. Donor-specific antibody (DSA) returned positive with high intensity anti-HLA-A2 antibody. This is the first case of early EPN in allograft considered to have occurred as a result of thrombotic ischemia secondary to AMR. This case suggests consideration of perioperative anti-B-cell and/or anti-plasma cell therapies for historical DSA and strict post-operative follow-up in immunologically high-risk recipients to detect early signs of rejection and avoid deleterious outcomes.

The CD40-ATP-P2X 7 Receptor Pathway: Cell to Cell Cross-Talk to Promote Inflammation and Programmed Cell Death of Endothelial Cells.

Extracellular adenosine 5'-triphosphate (ATP) functions not only as a neurotransmitter but is also released by non-excitable cells and mediates cell-cell communication involving glia. In pathological conditions, extracellular ATP released by astrocytes may act as a "danger" signal that activates microglia and promotes neuroinflammation. This review summarizes in vitro and in vivo studies that identified CD40 as a novel trigger of ATP release and purinergic-induced inflammation. The use of transgenic mice with expression of CD40 restricted to retinal Müller glia and a model of diabetic retinopathy (a disease where the CD40 pathway is activated) established that CD40 induces release of ATP in Müller glia and triggers in microglia/macrophages purinergic receptor-dependent inflammatory responses that drive the development of retinopathy. The CD40-ATP-P2X7 pathway not only amplifies inflammation but also induces death of retinal endothelial cells, an event key to the development of capillary degeneration and retinal ischemia. Taken together, CD40 expressed in non-hematopoietic cells is sufficient to mediate inflammation and tissue pathology as well as cause death of retinal endothelial cells. This process likely contributes to development of degenerate capillaries, a hallmark of diabetic and ischemic retinopathies. Blockade of signaling pathways downstream of CD40 operative in non-hematopoietic cells may offer a novel means of treating diabetic and ischemic retinopathies.

Functional muscle recovery with nanoparticle-directed M2 macrophage polarization in mice.

Persistence of inflammation, and associated limits in tissue regeneration, are believed to be due in part to the imbalance of M1 over M2 macrophages. Here, we hypothesized that providing a sustained source of an antiinflammatory polarizing cytokine would shift the balance of macrophages at a site of tissue damage to improve functional regeneration. Specifically, IL-4-conjugated gold nanoparticles (PA4) were injected into injured murine skeletal muscle, resulting in improved histology and an ∼40% increase in muscle force compared with mice treated with vehicle only. Macrophages were the predominant infiltrating immune cell, and treatment with PA4 resulted in an approximately twofold increase in the percentage of macrophages expressing the M2a phenotype and an approximately twofold decrease in M1 macrophages, compared with mice treated with vehicle only. Intramuscular injection of soluble IL-4 did not shift macrophage polarization or result in functional muscle improvements. Depletion of monocytes/macrophages eliminated the therapeutic effects of PA4, suggesting that improvement in muscle function was the result of M2-shifted macrophage polarization. The ability of PA4 to direct macrophage polarization in vivo may be beneficial in the treatment of many injuries and inflammatory diseases.

Collectin-11 (CL-11) Is a Major Sentinel at Epithelial Surfaces and Key Pattern Recognition Molecule in Complement-Mediated Ischaemic Injury.

The complement system is a dynamic subset of the innate immune system, playing roles in host defense, clearance of immune complexes and cell debris, and priming the adaptive immune response. Over the last 40 years our understanding of the complement system has evolved from identifying its presence and recognizing its role in the blood to now focusing on understanding the role of local complement synthesis in health and disease. In particular, the local synthesis of complement was found to have an involvement in mediating ischaemic injury, including following transplantation. Recent work on elucidating the triggers of local complement synthesis and activation in renal tissue have led to the finding that Collectin-11 (CL-11) engages with L-fucose at the site of ischaemic stress, namely at the surface of the proximal tubular epithelial cells. What remains unknown is the precise structure of the damage-associated ligand that participates in CL-11 binding and subsequent complement activation. In this article, we will discuss our hypothesis regarding the role of CL-11 as an integral tissue-based pattern recognition molecule which we postulate has a significant contributory role in complement-mediated ischaemic injury.