Microcirculation Inflammation Research Articles

New Insights into Hepatic and Intestinal Microcirculation and Pulmonary Inflammation in a Model of Septic Shock and Veno-Arterial Extracorporeal Membrane Oxygenation in the Rat.

Despite significant efforts toward improving therapy for septic shock, mortality remains high. Applying veno-arterial (V-A) extracorporeal membrane oxygenation (ECMO) in this context remains controversial. Since the cannulation of the femoral artery for V-A ECMO return leads to lower body hyperoxia, this study investigated the impact of V-A ECMO therapy on the intestinal and hepatic microcirculation during septic shock in a rodent model. Thirty male Lewis rats were randomly assigned to receive V-A ECMO therapy with low (60 mL/kg/min) or high (90 mL/kg/min) blood flow or a sham procedure. Hemodynamic data were collected through a pressure-volume catheter in the left ventricle and a catheter in the lateral tail artery. Septic shock was induced by intravenous administration of lipopolysaccharide (1 mg/kg). The rats received lung-protective ventilation during V-A ECMO therapy. The hepatic and intestinal microcirculation was measured by micro-lightguide spectrophotometry after median laparotomy for two hours. Systemic and pulmonary inflammation was detected via enzyme-linked immunosorbent assays (ELISA) of the plasma and bronchoalveolar lavage (BAL), respectively, measuring tumor necrosis factor-alpha (TNF-α), interleukins 6 (IL-6) and 10 (IL-10), and C-X-C motif ligands 2 (CXCL2) and 5 (CXCL5). Oxygen saturation and relative hemoglobin concentration were reduced in the hepatic and intestinal microcirculation during V-A ECMO therapy, independent of the blood flow rate. Further, rats treated with V-A ECMO therapy also presented elevated systolic, diastolic, and mean arterial blood pressure and increased stroke volume, cardiac output, and left ventricular end-diastolic volume. However, left ventricular end-diastolic pressure was only elevated during high-flow V-A ECMO therapy. Blood gas analysis revealed a dilutional anemia during V-A ECMO therapy. ELISA analysis showed an elevated plasma CXCL2 concentration only during high-flow V-A ECMO therapy and elevated BAL CXCL2 and CXCL5 concentrations only during low-flow V-A ECMO therapy. Rats undergoing V-A ECMO therapy exhibited impaired microcirculation of the intestine and liver during septic shock despite increased blood pressure and cardiac output. Increased pulmonary inflammation was detected only during low-flow V-A ECMO therapy in septic shock.

New Insights into Hepatic and Intestinal Microcirculation and Pulmonary Inflammation in a Model of Septic Shock and Venovenous Extracorporeal Membrane Oxygenation in the Rat.

Treatment of critically ill patients with venovenous (V-V) extracorporeal membrane oxygenation (ECMO) has gained wide acceptance in the last few decades. However, the use of V-V ECMO in septic shock remains controversial. The effect of ECMO-induced inflammation on the microcirculation of the intestine, liver, and critically damaged lungs is unknown. Therefore, the aim of this study was to measure the hepatic and intestinal microcirculation and pulmonary inflammatory response in a model of V-V ECMO and septic shock in the rat. Twenty male Lewis rats were randomly assigned to receive V-V ECMO therapy or a sham procedure. Hemodynamic data were measured by a pressure-volume catheter in the left ventricle and a catheter in the lateral tail artery. Septic shock was induced by the intravenous infusion of lipopolysaccharide (1 mg/kg). During V-V ECMO therapy, rats received lung-protective ventilation. The hepatic and intestinal microcirculation was assessed by micro-lightguide spectrophotometry after median laparotomy for 2 h. Systemic and pulmonary inflammation was measured by enzyme-linked immunosorbent assays of plasma and bronchoalveolar lavage (BAL), respectively, which included tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), IL-10, C-X-C motif ligand 2 (CXCL2), and CXCL5. Reduced oxygen saturation and relative hemoglobin concentration were measured in the hepatic and intestinal microcirculation during treatment with V-V ECMO. These animals also showed increased systolic, mean, and diastolic blood pressures. While no differences in left ventricular ejection fraction were observed, animals in the V-V ECMO group presented an increased heart rate, stroke volume, and cardiac output. Blood gas analysis showed dilutional anemia during V-V ECMO, whereas plasma analysis revealed a decreased concentration of IL-10 during V-V ECMO therapy, and BAL measurements showed increased concentrations of TNF-α, CXCL2, and CXCL5. Rats treated with V-V ECMO showed impaired microcirculation of the intestine and liver during septic shock despite increased blood pressure and cardiac output. Despite lung-protective ventilation, increased pulmonary inflammation was recognized during V-V ECMO therapy in septic shock.

235.4: Analyzing the Impact of T-Cell Receptor Diversity in Acute Kidney Transplant Rejection

Purpose: Recent developments in T-cell receptor (TCR) immunosequencing allow for analysis of alloreactive human T-cell populations both before and after engraftment. In this study, we mapped T-cell repertoire from transplant recipients and assessed the role of chronological changes in T-cell clonality, immune repertoire turnover, T-cell Fraction (TCFr), and tissue pathological score to acute rejection (AR) risk stratification and management. Methods: The study included 339 blood samples, collected before and after transplantation, from 200 kidney transplant recipients; 100 with biopsy confirmed acute rejection (AR), 100 with biopsy confirmed stable (STA) allograft histology. Immunosequencing of the CDR3 regions of human TCRβ locus was performed using the immune SEQ Assay. Sequences were collapsed and filtered in order to identify and quantitate the absolute abundance of each unique TCRβ CDR3 region for further analysis. Results: More diverse T-cell Repertoire at baseline was associated with AR post-transplant. When compared to STA patients, AR patients had lower (TCFr) before transplantation (p=0.01). After transplantation, patients who developed rejection had greater repertoire turnover (p=0.0024) and increased TCFr. Significant increase in T-cell fraction at late rejection and in ABMR was observed which was significant in late rejection cases >6 mo post-transplantation (p<0.001)(Fig1A). There was an increase in T-cell fraction in ABMR compared to non-ABMR samples (p=0.05)(Fig1B). There was an increased peripheral T-cell fraction among AR subjects compared to STA among AR samples collected at 6 mo post-transplantation (p=0.003)(Fig1C). TCFr also correlated with microcirculation inflammation. Conclusions: In conclusion, our study demonstrated that T-cell repertoire changes with post-transplant rejection episodes and suggested that the T-cell repertoire changes may provide pre-transplant and post-transplant predictors of rejection risk which in turn can support immunosuppression management decisions at and after transplantation.

Xueshuantong injection alleviates cerebral microcirculation disorder in middle cerebral artery occlusion/reperfusion rats by suppressing inflammation via JNK mediated JAK2/STAT3 and NF-κB signaling pathways

Ethnopharmacological relevanceIn the long history of traditional Chinese medicine, Panax notoginseng has been used as a key herb for the treatment of blood diseases. Brain microvessels support adequate blood circulation to maintain normal physiological function, therefore, brain microcirculation disorder is an important therapeutic target for various brain diseases.However, the role of Xueshuantong (XST) injection composed of saponins from P. Notoginseng (PNS) in the amelioration of cerebral microcirculation disorder is unclear. Aims of the studyCerebral microcirculation disorder and inflammation play a vital role in stroke. Capillary endothelial cells and adjacent tight junctions are fundamental to the structure and function of cerebrovascule. XST injection has been used clinically in the treatment of stroke, but no studies have reported its indication in cerebral microcirculation disorder. This study is to explore the action and mechanism of XST injection in the alleviation of cerebral microcirculation disorder in middle cerebral artery occlusion/reperfusion (MCAO/R) rats. Materials and methodsMCAO/R rats and LPS-induced bEnd.3 cells were employed for the investigation of effect and mechanism of XST injection. Brain damages were evaluated by neurobehavioral assessment, 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, hematoxylin and eosin staining (H&E), and Nissl staining. Morphology and density changes of cerebral microvessels were monitored by immunohistochemistry. Cell permeability was detected by measurement of trans-endothelial electrical resistance (TEER) and sodium fluorescein (NaF) leakage. The mRNA and protein expressions of inflammatory cytokines, tight junction proteins, adhesion molecules, Janus kinase 2 (JAK2), signal transducer and activator of transcription-3 (STAT3), inhibitor of NF-κB (IκB), nuclear factor-κB (NF-κB) and c-jun N-terminal kinase (JNK) in brain microvessels and lipopolysaccharide (LPS)-induced bEnd.3 cells were measured by real-time PCR and Western blot, respectively. ResultsXST injection at 48 mg/kg significantly improved the neurological damage, inflammatory infiltration, and microvessel morphology, and increased microvessel density in brain of MCAO/R rats. The endothelial permeability was significantly mitigated by XST injection in LPS-induced bEnd.3 cells. Meanwhile, the tight junction proteins such as zona occludens 1 (ZO-1) and occludin were elevated remarkably in brain microvessel of MCAO/R rats and LPS-induced bEnd.3 cells. Moreover, the expression of inflammatory mediators including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), cycloocygenases 2 (COX-2), vascular cellular adhesion molecule-1 (VCAM-1), matrix metalloproteinase (MMP)-2, and MMP-9 were inhibited by XST injection. In addition, XST injection suppressed the phosphorylation of JAK2, STAT3, IκB, NF-κB and JNK, which could be abolished by anisomycin, the JNK agonist. ConclusionXST injection improved cerebral microvescular structure damage and dysfunction in MCAO/R rats through inhibiting inflammation activated by JNK mediated JAK2/STAT3 and NF-κB signaling pathways. The novel findings may provide theoretical basis for the clinical application in the treatment of cerebral microcirculation disorder.

Association of HLA Mismatches and Histology Suggestive of Antibody-Mediated Injury in the Absence of Donor-Specific Anti-HLA Antibodies.

The histology of antibody-mediated rejection after kidney transplantation is observed frequently in the absence of detectable donor-specific anti-HLA antibodies. Although there is an active interest in the role of non-HLA antibodies in this phenotype, it remains unknown whether HLA mismatches play an antibody-independent role in this phenotype of microcirculation inflammation. To study this, we used the tools HLAMatchmaker, three-dimensional electrostatic mismatch score, HLA solvent accessible amino acid mismatches, and mismatched donor HLA-derived T cell epitope targets to determine the degree of HLA molecular mismatches in 893 kidney transplant recipients with available biopsy follow-up. Multivariable Cox proportional hazards models were applied to quantify the cause-specific hazard ratios of the different types of HLA mismatch scores for developing antibody-mediated rejection or histology of antibody-mediated rejection in the absence of donor-specific anti-HLA antibodies. In all survival analyses, the patients were censored at the time of the last biopsy. In total, 121 (14%) patients developed histology of antibody-mediated rejection in the absence of donor-specific anti-HLA antibodies, of which 44 (36%) patients had concomitant T cell-mediated rejection. In multivariable Cox analysis, all different calculations of the degree of HLA mismatch associated with developing histology of antibody-mediated rejection in the absence of donor-specific anti-HLA antibodies. This association was dependent neither on the presence of missing self (potentially related to natural killer cell activation) nor on the formation of de novo HLA antibodies. Also, glomerulitis and complement C4d deposition in peritubular capillaries associated with the degree of HLA mismatch in the absence of anti-HLA antibodies. The histology of antibody-mediated rejection and its defining lesions are also observed in patients without circulating anti-HLA antibodies and relate to the degree of HLA mismatch.

Notch3 expression in capillary pericytes predicts worse graft outcome in human renal grafts with antibody-mediated rejection.

Microvasculature consisting of endothelial cells and pericytes is the main site of injury during antibody‐mediated rejection (ABMR) of renal grafts. Little is known about the mechanisms of activation of pericytes in this pathology. We have found recently that activation of Notch3, a mediator of vascular smooth muscle cell proliferation and dedifferentiation, promotes renal inflammation and fibrosis and aggravates progression of renal disease. Therefore, we studied the pericyte expression of Notch3 in 49 non‐selected renal graft biopsies (32 for clinical cause, 17 for graft surveillance). We analysed its relationship with patients’ clinical and morphological data, and compared with the expression of partial endothelial mesenchymal transition (pEndMT) markers, known to reflect endothelial activation during ABMR. Notch3 was de novo expressed in pericytes of grafts with ABMR, and was significantly correlated with the microcirculation inflammation scores of peritubular capillaritis and glomerulitis and with the expression of pEndMT markers. Notch3 expression was also associated with graft dysfunction and proteinuria at the time of biopsy and in the long term. Multivariate analysis confirmed pericyte expression of Notch3 as an independent risk factor predicting graft loss. These data suggest that Notch3 is activated in the pericytes of renal grafts with ABMR and is associated with poor graft outcome.

Poor Long-Term Renal Allograft Survival in Patients with Chronic Antibody-Mediated Rejection, Irrespective of Treatment—A Single Center Retrospective Study

The Banff 2017 report permits the diagnosis of pure chronic antibody-mediated rejection (cAMR) in absence of microcirculation inflammation. We retrospectively investigated renal allograft function and long-term outcomes of 67 patients with cAMR, and compared patients who received antihumoral therapy (cAMR-AHT, n = 21) with patients without treatment (cAMRwo, n = 46). At baseline, the cAMR-AHT group had more concomitant T-cell-mediated rejection (9/46 (19.2%) vs. 10/21 (47.6%); p = 0.04), a higher g-lesion score (0.4 ± 0.5 versus 0.1 ± 0.3; p = 0.01) and a higher median eGFR decline in the six months prior to biopsy (6.6 vs. 3.0 mL/min; p = 0.04). The median eGFR decline six months after biopsy was comparable (2.6 vs. 4.9 mL/min, p = 0.61) between both groups, and three-year graft survival after biopsy was statistically lower in the cAMR-AHT group (35.0% vs. 61.0%, p = 0.03). Patients who received AHT had more infections (0.38 vs. 0.20 infections/patient; p = 0.04). Currently, antihumoral therapy is more often administered to patients with cAMR and rapidly deteriorating renal function or concomitant TCMR. However, long-term graft outcomes remain poor, despite treatment.

Functional Fc Gamma Receptor Gene Polymorphisms and Long-Term Kidney Allograft Survival

The functional Fc gamma receptor (FcγR) IIIA polymorphism FCGR3A-V/F158 was earlier suggested to determine the potential of donor-specific HLA antibodies to trigger microcirculation inflammation, a key lesion of antibody-mediated renal allograft rejection. Associations with long-term transplant outcomes, however, have not been evaluated to date. To clarify the impact of FCGR3A-V/F158 polymorphism on kidney transplant survival, we genotyped a cohort of 1,940 recipient/donor pairs. Analyzing 10-year death-censored allograft survival, we found no significant differences in relation to FCGR3A-V/F158. There was also no independent survival effect in a multivariable Cox model. Similarly, functional polymorphisms in two other activating FcγR, FCGR2A-H/R131 (FcγRIIA) and FCGR3B-NA1/NA2 (FcγRIIIB), were not associated with outcome. There were also no significant survival differences among patient subgroups at increased risk of rejection-related injury, such as pre-sensitized recipients (> 0% panel reactivity; n = 438) or recipients treated for rejection within the first year after transplantation (n = 229). Our study results suggest that the earlier shown association of FcγR polymorphism with microcirculation inflammation may not be strong enough to exert a meaningful effect on graft survival.

Impact of COVID-19 on the cerebrovascular system and the prevention of RBC lysis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) uses Angiotensin- converting enzyme 2 (ACE2) receptors to infect host cells which may lead to coronavirus disease (COVID-19). Given the presence of ACE2 receptors in the brain and the critical role of the renin-angiotensin system (RAS) in brain functions, special attention to brain microcirculation and neuronal inflammation is warranted during COVID-19 treatment. Neurological complications reported among COVID-19 patients range from mild dizziness, headache, hypogeusia, hyposmia to severe like encephalopathy, stroke, Guillain-Barre Syndrome (GBS), CNS demyelination, infarcts, microhemorrhages and nerve root enhancement. The pathophysiology of these complications is likely via direct viral infection of the CNS and PNS tissue or through indirect effects including post- viral autoimmune response, neurological consequences of sepsis, hyperpyrexia, hypoxia and hypercoagulability among critically ill COVID-19 patients. Further, decreased deformability of red blood cells (RBC) may be contributing to inflammatory conditions and hypoxia in COVID-19 patients. Haptoglobin, hemopexin, heme oxygenase-1 and acetaminophen may be used to maintain the integrity of the RBC membrane.

Trajectories of glomerular filtration rate and progression to end stage kidney disease after kidney transplantation

Although the gold standard of monitoring kidney transplant function relies on glomerular filtration rate (GFR), little is known about GFR trajectories after transplantation, their determinants, and their association with outcomes. To evaluate these parameters we examined kidney transplant recipients receiving care at 15 academic centers. Patients underwent prospective monitoring of estimated GFR (eGFR) measurements, with assessment of clinical, functional, histological and immunological parameters. Additional validation took place in seven randomized controlled trials that included a total of 14,132 patients with 403,497 eGFR measurements. After a median follow-up of 6.5 years, 1,688 patients developed end-stage kidney disease. Using unsupervised latent class mixed models, we identified eight distinct eGFR trajectories. Multinomial regression models identified seven significant determinants of eGFR trajectories including donor age, eGFR, proteinuria, and several significant histological features: graft scarring, graft interstitial inflammation and tubulitis, microcirculation inflammation, and circulating anti-HLA donor specific antibodies. The eGFR trajectories were associated with progression to end stage kidney disease. These trajectories, their determinants and respective associations with end stage kidney disease were similar across cohorts, as well as in diverse clinical scenarios, therapeutic eras and in the seven randomized control trials. Thus, our results provide the basis for a trajectory-based assessment of kidney transplant patients for risk stratification and monitoring.

SO030HISTOLOGICAL CHARACTERIZATION OF RENAL BIOPSIES FROM HCV-INFECTED DONORS: A VALUABLE SOURCE OF KIDNEY ALLOGRAFTS

Abstract Background and Aims The introduction of effective direct-acting antiviral agents (DAAs) has allowed safe transplantation of kidneys from HCV positive (HCV+) donors into HCV negative (HCV-) recipients. Some studies demonstrated an increased risk for rejection as well as polyomavirus infection in these situations (Molnar et al, Am J Transplant. 2019 Nov;19(11):3046-3057), possibly due to the development of an inflammatory milieu in the setting of HCV seroconversion and persistent viremia (Durand et al, Am J Transplant. 2019 Nov;19(11):2969-2970). With this concern for HCV-associated renal injury and increased risk for rejection, this retrospective study explores the histologic findings of allograft biopsies from HCV+ donors. Method Fifty renal allograft biopsies from 28 recipients of HCV+ donors evaluated at Vanderbilt University Medical Center between September 2018 and December 2019 were retrospectively reviewed with recording of clinical and histological features, and final diagnoses. All recipients were transplanted under a clinical protocol and were treated with DAA therapy. In the same period of time, 30 renal allograft biopsies from 24 recipients of HCV- donors were selected having the same post-transplant interval as the study cohort. Categorical variables are expressed in percentage and compared using Pearson’s chi-squared test. Continuous variables are expressed as mean±SD and compared used Student t-test. A p-value of &amp;lt;0.05 was considered as statistically significant. Results The complete results of the comparison between the two groups (HCV+ and HCV- donors) are demonstrated in Figure. The differences reaching statistical significance include higher frequency of males and tubulointerstitial fibrosis (IFTA) in the HCV+ group (p = 0.038 and 0.044, respectively) and the higher incidence of DSA positivity and acute tubular injury in the control group (p = 0.001 and 0.029). Notably, no significant differences were noted in terms of antibody-mediated events (microcirculation inflammation, transplant glomerulopathy, C4d positivity, or diagnostic ABMR), or T-cell mediated events including borderline or diagnostic T-cell mediated rejection (Banff 2013). The two groups also demonstrated the same incidence of polyomavirus nephropathy and de novo/recurrent glomerulonephritis. Interestingly, two of the HCV+ cases showed mesangial IgA deposits on immunofluorescence and electron microscopy on the first biopsy, of which one showed persistence of deposits, 18 and 44 days after transplant respectively. Once case demonstrated a de novo focal proliferative IgM-dominant glomerulonephritis on the 7th biopsy, 266 days after transplantation. Conclusion Kidneys from HCV+ donors represent a valuable resource in the era of DAA therapy. This reported experience demonstrates similar frequency of rejection and viral infection affecting allografts from HCV positive and negative donors. The presence of immune complex deposits and glomerulonephritis in some of these cases could represent either a donor-derived injury or an acquired post-seroconversion feature. In conclusion, HCV+ donors appear to be a safe resource for transplantation. Further studies with post-transplant intervals will provide additional characterization of their impact.

P1626LIQUID BIOPSY IN RENAL TRANSPLANT: THE ROLE OF DONOR-DERIVED CELL-FREE DNA TO DETECT REJECTION

Abstract Background and Aims Circulating donor-derived cell-free DNA (dd-cfDNA) has recently been proposed as an early non-invasive marker for renal allograft rejection (Bloom et al, J. Am. Soc. Nephrol. 2017;28, 2221–2232). Recent studies demonstrated its potential role in the detection of any kind of rejection with better performance in detecting antibody-mediated rejection (ABMR) (Huang et al, Am J Transplant. 2019;19:1663–1670). However, at the current cutoff value for a positive test, the test seems to be affected by low sensitivity for T-cell mediated rejection (TCMR) and false positive results in the setting of non-rejection related damage. In the present study, the performance of dd-cfDNA is assessed on a retrospective series of transplant renal biopsies. Method Sixty-three renal biopsies from 52 allograft recipients with available dd-cfDNA results, evaluated between November 2017 and December 2019 at Vanderbilt University Medical Center, Nashville, Tennessee, were reviewed. Clinical and histological features along with the final diagnosis were recorded. Renal allograft rejection was classified following Banff 2013 criteria. Pearson’s chi-squared test was used to compare cases positive and negative for dd-cfDNA. Receiving operator curve (ROC) analysis was used to assess the performance of the test in detecting any rejection and ABMR. A p-value of &amp;lt;0.05 was considered statistically significant. Results The patients were 53±12.5 years old, 43% were men, 51% and 49% were Caucasian and African-American, respectively. The median value of dd-cfDNA was 2.6% for ABMR and 2.4% for any kind of rejection, statistically different from the group without rejection (0.56%, p = 0.002 and 0.0003, respectively). TCMR failed to demonstrate a statistically significant difference in dd-cfDNA levels vs controls (1.8% vs 0.56%, p = 0.627). The comparison of groups with positive and negative dd-cfDNA test is reported in Figure (panel A). Positive patients demonstrated a higher rate of findings associated with ABMR such as DSA positivity, moderate microcirculation inflammation, and transplant glomerulopathy, in addition to diagnostic ABMR and chronic/active ABMR. Moreover, a significant difference was found between the two groups in the rate of any kind of rejection. ROC analysis for any ABMR (Figure, panel B) demonstrated an AUC of 0.76 (95% CI 0.61-0.91) with a sensitivity (Sn) of 92.3%, a specificity (Sp) of 60%, a positive predictive value (PPV) of 37.5% and a negative predictive value (NPP) of 96.8%. ROC analysis for any kind of rejection (Figure, panel C) demonstrated an AUC of 0.84 (95% CI 0.74-0.94) with a Sn of 94.1%, a Sp of 65.2%, a PPV of 50% and a NPP of 96.8%. Among the patients with positive dd-cfDNA and without rejection (n=16, 51%), allograft biopsy demonstrated borderline TCMR in 4, acute tubular injury in 8, chronic pyelonephritis in 1, mild interstitial fibrosis in 1, and 2 with minimal histological abnormalities. Notably, none of the cases with a positive test were diagnosed with polyomavirus nephropathy. Conclusion Similar to previous studies, dd-cfDNA represents a sensitive test for detection of ABMR, with high performance in any rejection setting with the Banff 2013 criteria. However, the high rate of false positive results and the presence of undetected T-cell mediated events (e.g. borderline cases) at the current recommended cut-off value for positivity limits its employment as a surrogate for the renal biopsy.

Natural Killer Cells: Critical Effectors During Antibody-mediated Rejection of Solid Organ Allografts.

Antibody-mediated rejection (AMR) is an important cause of graft loss and continues to present a formidable obstacle to successful transplantation. Unresolved problems continue to be the absence of effective strategies to ablate the donor-specific antibody (DSA) response as well as to attenuate the antibody-mediated graft tissue injury. While the properties of DSA that cause greater graft tissue injury and the characteristic microvascular pathology of the graft injury are well documented, the mechanisms underlying the injury mediated by the antibodies remains unclear. Recent transcriptome interrogation of kidney and heart biopsies procured during ongoing AMR has indicated the expression of genes associated with natural killer (NK) cell activation that is absent during T cell-mediated rejection. The expression of NK cell transcripts during AMR correlates with the presence of CD56+ cells in the microcirculation inflammation observed during AMR. Several mouse models have recently demonstrated the role of NK cells in antibody-mediated chronic vasculopathy in heart allografts and the requirement for NK cell activation during acute AMR of kidney allografts. In the latter model, NK cell activation within kidney allografts is regulated by the activation of myeloid cells producing myeloperoxidase. Overall, the studies to date indicate that AMR constitutes a complex series of DSA-induced interactions with components of the innate immune response. The innate immune participants and their expressed effector functions resulting in the rejection are beginning to be identified. The identification of these components should uncover novel targets that can be used to attenuate acute graft tissue injury in the presence of DSA.

Non-invasive Chemokine Detection: Improved Prediction of Antibody-Mediated Rejection in Donor-Specific Antibody-Positive Renal Allograft Recipients.

Background: Screening for donor-specific antibodies (DSA) has limited diagnostic value in patients with late antibody-mediated rejection (ABMR). Here, we evaluated whether biomarkers reflecting microcirculation inflammation or tissue injury—as an adjunct to DSA detection—are able to improve non-invasive ABMR monitoring.Methods: Upon prospective cross-sectional antibody screening of 741 long-term kidney transplant recipients with a silent clinical course, 86 DSA-positive patients were identified and biopsied. Serum and urine levels of E-selectin/CD62E, vascular cell adhesion molecule 1 (VCAM-1), granzyme B, hepatocyte growth factor (HGF), C-C motif chemokine ligand (CCL)3, CCL4, C-X-C motif chemokine ligand (CXCL)9, CXCL10, and CXCL11 in DSA-positive recipients were investigated applying multiplexed bead-based immunoassays.Results: Diagnosis of ABMR (50 patients) was associated with significantly higher levels of CXCL9 and CXCL10 in blood and urine and of HGF in blood. Overall, urinary CXCL9 had the highest diagnostic accuracy for ABMR (area under the receiver operating characteristic curve: 0.77; accuracy: 80%) and its combined evaluation with the mean fluorescence intensity of the immunodominant DSA (DSAmax MFI) revealed a net reclassification improvement of 73% compared to DSAmax MFI alone.Conclusions: Our results suggest urinary CXCL9 testing, combined with DSA analysis, as a valuable non-invasive tool to uncover clinically silent ABMR late after transplantation.

The Vascular Side of Chronic Bed Rest: When a Therapeutic Approach Becomes Deleterious

The interplay between chronic constraint and advanced aging on blood flow, shear-rate, vascular function, nitric oxide (NO)-bioavailability, microcirculation, and vascular inflammation factors is still a matter of debate. Ninety-eight individuals (Young, n = 28, 23 ± 3 yrs; Old, n = 36, 85 ± 7 yrs; Bedridden, n = 34, 88 ± 6 yrs) were included in the study. The bedridden group included old individuals chronically confined to bed (3.8 ± 2.3 yrs). A blood sample was collected and analyzed for plasma nitrate, and vascular inflammatory markers. Hyperemic response (∆peak) during the single passive leg movement (sPLM) test was used to measure vascular function. Skeletal muscle total hemoglobin was measured at the vastus lateralis during the sPLM test, by means of near infrared spectroscopy (NIRS). Bedridden subjects revealed a depletion of plasma nitrates compared with Old (−23.8%) and Young (−31.1%). Blood flow was lower in the Bedridden in comparison to Old (−20.1%) and Young (−31.7%). Bedridden presented lower sPLM ∆peak compared Old (−72.5%) and the Young (−83.3%). ∆peak of NIRS total hemoglobin was lower in the Bedridden compared to that in the Young (−133%). All vascular inflammatory markers except IL-6 were significantly worse in the Bedridden compared to Old and Young. No differences were found between the Old and Young in inflammatory markers. Results of this study confirm that chronic physical constraint induces an exacerbation of vascular disfunction and differential regulation of vascular-related inflammatory markers. The mechanisms involved in these negative adaptations seems to be associated with endothelial dysfunction and consequent diminished NO-bioavailability likely caused by the reduced shear-rate consequential to long-term reduction of physical activity.

Immunoadsorption Combined with Membrane Filtration to Counteract Early Treatment-Refractory Antibody-Mediated Rejection

Introduction: Immunoadsorption (IA) represents a therapeutic option for acute antibody-mediated rejection (ABMR) after kidney transplantation. The addition of membrane filtration (MF) to enhance elimination of macromolecular components that potentially contribute to rejection, such as key complement component C1q and alloreactive IgM, may be an effective strategy to further improve its therapeutic efficiency. Results: Here we present 4 consecutive patients with episodes of HLA donor-specific antibody-positive ABMR nonresponsive to cycles of 6–16 sessions of IA treatment. Rejection episodes were characterized by severe microvascular injury (high-grade microcirculation inflammation and/or signs of thrombotic microangiopathy) and evidence of intense complement activation in peritubular capillaries (diffuse C4d-positivity). IA combined with MF led to substantial morphologic improvement (follow-up biopsies: g + ptc and C4d scores ≤1) and stabilization of allograft function. Conclusions: Our findings provide evidence for an effect of combination of IA + MF in refractory early acute/active ABMR in kidney transplant recipients.

High-activity Classical and Alternative Complement Pathway Genotypes-Association With Donor-specific Antibody-triggered Injury and Renal Allograft Survival.

Complement may contribute to donor-specific antibody (DSA)-triggered transplant injury. Here, we investigated whether the intrinsic strength of classical pathway and alternative pathway (AP) relates to the pathogenicity of DSA. Classical pathway and AP high-activity genotypes were defined according to C4 gene copy number and the presence of functional polymorphisms in C3 (C3102G), factor B (fB32R), and factor H (fH62V) genes. Associations of these genotypes with blood complement profiles and morphologic/molecular rejection features were evaluated in a cohort of 83 DSA-positive patients (antibody-mediated rejection [AMR], n = 47) identified upon cross-sectional screening of 741 kidney allograft recipients ≥180 days posttransplantation. Associations with long-term graft survival were evaluated in a larger kidney transplant cohort (n = 660) not enriched for a specific type of rejection. In the cohort of DSA-positive subjects, the number of C4 gene copies was related to C4 protein levels in serum and capillary C4d staining, but not AMR activity. Patients with a high-activity AP complotype, which was associated with complement consumption in serum, showed enhanced microcirculation inflammation (median glomerulitis plus peritubular capillaritis score, 2 [interquartile range, 0-4 versus 1 0-2]; P = 0.037). In the larger transplant cohort, this complotype was associated with a slightly increased risk of graft loss (hazard ratio, 1.52; 95% confidence interval, 1.02-2.25; P = 0.038 and multivariable Cox model, 1.55; 1.04-2.32; P = 0.031). Our study suggests a contribution of complement genetics to the phenotypic presentation of AMR. Future studies will have to clarify whether a possible association of AP strength with graft survival relates to enhanced antibody-triggered injury.

Early central blood pressure elevation in adult patients with 21-hydroxylase deficiency.

Controversial data exist on cardiovascular damages in patients with congenital adrenal hyperplasia (CAH). To assess blood pressure and early cardiovascular damages on a large cohort of adult CAH patients and control individuals. Case-control study. Referral Center for Rare Disease, Pitié Salpêtrière Hospital, Paris, France. Fifty-eight women and 26 men with CAH diagnosed in childhood and 85 controls matched-paired for sex, age and smoking status were prospectively included. Measurement of large arteries and microcirculatory anatomical and functional indices as well as hormonal status and cardiovascular risk factors evaluation. The primary objective was to compare carotid intima-media thickness (cIMT) in CAH patients and controls. The secondary objectives were to compare blood pressure (BP), radial augmentation index (rAI), central BP, carotid-femoral pulse wave velocity (PWV), skin microcirculation indices and inflammation parameters in CAH patients and controls. Although PWV and cIMT were identical in patients and controls, higher rAI (64.6 ± 1.7 vs. 59.9 ± 1.6%, P = 0.02) and higher central SBP (101.8 ± 1.5 vs. 95.1 ± 1.5 mmHg, P < 0.001) were observed in CAH patients. Regarding microcirculatory indices, there was a higher functional resting capacity and a lower anatomical capillary density in CAH patients. In multivariate analysis, we found an independant association between CAH and central SBP elevation. We found an early rise in central SBP in CAH patients whereas sublinical arterial damages markers were normal. Our study suggest that vascular damages and increased cardiovascular risk could be mainly BP-driven.

Pretransplant angiotensin II type 1-receptor antibodies point to an increase in renal graft sub-intimal fibrosis in living- donor kidney transplant recipients

The association between anti-AT1Rabs and microvascular injury observed in antibody-mediated rejection has been described in kidney graft Biopsies (KGBx). MethodsWe herein describe the histopathologic findings of KGBx performed during the first year of transplantation (Tx) in 134 patients tested for pre-Tx anti-AT1Rabs in cryopreserved sera (04/2009 to 09/2013). Protocol KGBx before implantation (time-zero), 1 year after Tx and for cause KGBx were included. 21/134 Tx patients were anti-AT1Rab positive (≥17 U/mL); 7/21 experienced acute rejection. For comparison a control group with anti-AT1Rabs <17 U/mL, with (n = 16) and without (n = 31) acute rejection was included. ResultsPreimplantation KGBx showed no differences in inflammatory and chronic findings, nor in subintimal fibrosis (25 vs 12.8%, p = .42) between patients with anti-AT1Rabs ≥17 U/mL and those with <17 U/mL. Follow-up KGBx revealed a significantly greater proportion of arterial sub-intimal fibrosis (52.3 vs. 27.6%, p = .049) and extension (15.7 vs. 5.3, p = .015) in anti-AT1Rabs ≥17 U/mL compared to anti-AT1Rabs <17 U/mL KGBx. No differences were observed in microcirculation inflammation, nor in interstitial fibrosis or tubular atrophy between groups. Also, anti-AT1Rabs ≥17 U/mL (β 10.1, 2.3 to 17.8, p = .012) and more importantly anti-AT1Rabs ≥ 30 U/mL (β12.1, 3.1 to 20.9, p < .01), were independent risk factors associated with vascular occlusion resulting from sub-intimal fibrosis. ConclusionOur study findings have shown that anti-AT1Rab values ≥17 U/mL are significantly associated to sub-intimal fibrosis and a greater percentage of vessel occlusion in kidney graft biopsies obtained during the first year posttransplant, particularly in coexistence with inflammation and de novo DSA.

Histological picture of antibody-mediated rejection without donor-specific anti-HLA antibodies: Clinical presentation and implications for outcome.

In this cohort study (n=935 transplantations), we investigated the phenotype and risk of graft failure in patients with histological criteria for antibody-mediated rejection (ABMR) in the absence of circulating donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA), and compared this to patients with definite ABMR and HLA-DSA-positivity. The histological picture did not differ between HLA-DSA-positive (n=85) and HLA-DSA-negative (n=123) cases of ABMR histology, apart from increased complement split product 4d (C4d) deposition in the peritubular capillaries in HLA-DSA-positive cases. Histology of ABMR without HLA-DSA was more transient than DSA-positive ABMR, and patients with ABMR histology without HLA-DSA had graft survival superior to that of HLA-DSA-positive patients, independent of concomitant T cell-mediated rejection (38.2%) or borderline changes (17.9%). Multivariate analysis showed that the risk of graft failure was not higher in patients with histological picture of ABMR (ABMRh ) in the absence of HLA-DSA, compared to patients without ABMRh . Despite an association between C4d deposition and HLA-DSA-positivity, using C4d deposition as alternative for the DSA criterion in the diagnosis of ABMR, as proposed in Banff 2017, did not contribute to the prognosis of graft function and graft failure. We concluded that biopsies with ABMRh but without detectable HLA-DSA represent a distinct, often transient phenotype with superior allograft survival.