Early allogeneic immune modulation after establishment of donor hematopoietic cell-induced mixed chimerism in a nonhuman primate kidney transplant model

BackgroundMixed lymphohematopoietic chimerism is a proven strategy for achieving operational transplant tolerance, though the underlying immunologic mechanisms are incompletely understood.MethodsA post-transplant, non-myeloablative, tomotherapy-based total lymphoid (TLI) irradiation protocol combined with anti-thymocyte globulin and T cell co-stimulatory blockade (belatacept) induction was applied to a 3-5 MHC antigen mismatched rhesus macaque kidney and hematopoietic cell transplant model. Mechanistic investigations of early (60 days post-transplant) allogeneic immune modulation induced by mixed chimerism were conducted.ResultsChimeric animals demonstrated expansion of circulating and graft-infiltrating CD4+CD25+Foxp3+ regulatory T cells (Tregs), as well as increased differentiation of allo-protective CD8+ T cell phenotypes compared to naïve and non-chimeric animals. In vitro mixed lymphocyte reaction (MLR) responses and donor-specific antibody production were suppressed in animals with mixed chimerism. PD-1 upregulation was observed among CD8+ T effector memory (CD28-CD95+) subsets in chimeric hosts only. PD-1 blockade in donor-specific functional assays augmented MLR and cytotoxic responses and was associated with increased intracellular granzyme B and extracellular IFN-γ production.ConclusionsThese studies demonstrated that donor immune cell engraftment was associated with early immunomodulation via mechanisms of homeostatic expansion of Tregs and early PD-1 upregulation among CD8+ T effector memory cells. These responses may contribute to TLI-based mixed chimerism-induced allogenic tolerance

GPR30, but not estrogen receptor-α, is crucial in the treatment of experimental autoimmune encephalomyelitis by oral ethinyl estradiol

Abstract Background Remission of multiple sclerosis during periods of high ovarian hormone secretion (such as pregnancy) has led to a great deal of interest in the potential for estrogens to treat autoimmune disease. Previous work has established that 17β-estradiol can inhibit onset of experimental autoimmune encephalomyelitis (EAE), while ethinyl estradiol (EE) can reduce the severity of established disease. In the current study, the influence of estrogen receptor-α (ERα) and the G-protein coupled estrogen receptor (GPR30 or GPER) on EE's ability to treat EAE was explored. Results EE reduced disease severity in wild-type and ERα knockout (ERKO) mice, but did not alter disease in the GPR30KO group. Production of anti-inflammatory IL-10 increased in EE-ERKO mice (which showed reduced disease) but not in EE-GPR30KO mice (who did not have improved disease). Conclusions Differential production of IL-10 following EE treatment in ERKO and GPR30KO animals may be responsible for the distinctly different effects on disease severity. Increased IL-10 in ERKO-EE compared to ERKO-Controls is likely to be an important factor in reducing established disease. The inability of EE to reduce disease in GPR30KO mice indicates an important but still undefined role for GPR30 in regulating immune reactivity.</p

Complement inhibition attenuates acute kidney injury after ischemia-reperfusion and limits progression to renal fibrosis in mice.

The complement system is an essential component of innate immunity and plays a major role in the pathogenesis of ischemia-reperfusion injury (IRI). In this study, we investigated the impact of human C1-inhibitor (C1INH) on the early inflammatory response to IRI and the subsequent progression to fibrosis in mice. We evaluated structural damage, renal function, acute inflammatory response, progression to fibrosis and overall survival at 90-days post-injury. Animals receiving C1INH prior to reperfusion had a significant improvement in survival rate along with superior renal function when compared to vehicle (PBS) treated counterparts. Pre-treatment with C1INH also prevented acute IL-6, CXCL1 and MCP-1 up-regulation, C5a release, C3b deposition and infiltration by neutrophils and macrophages into renal tissue. This anti-inflammatory effect correlated with a significant reduction in the expression of markers of fibrosis alpha smooth muscle actin, desmin and picrosirius red at 30 and 90 days post-IRI and reduced renal levels of TGF-β1 when compared to untreated controls. Our findings indicate that intravenous delivery of C1INH prior to ischemic injury protects kidneys from inflammatory injury and subsequent progression to fibrosis. We conclude that early complement blockade in the context of IRI constitutes an effective strategy in the prevention of fibrosis after ischemic acute kidney injury

Guidelines for the management of a brain death donor in the rhesus macaque: A translational transplant model.

The development of a translatable brain death animal model has significant potential to advance not only transplant research, but also the understanding of the pathophysiologic changes that occur in brain death and severe traumatic brain injury. The aim of this paper is to describe a rhesus macaque model of brain death designed to simulate the average time and medical management described in the human literature.Following approval by the Institutional Animal Care and Use Committee, a brain death model was developed. Non-human primates were monitored and maintained for 20 hours after brain death induction. Vasoactive agents and fluid boluses were administered to maintain hemodynamic stability. Endocrine derangements, particularly diabetes insipidus, were aggressively managed.A total of 9 rhesus macaque animals were included in the study. The expected hemodynamic instability of brain death in a rostral to caudal fashion was documented in terms of blood pressure and heart rate changes. During the maintenance phase of brain death, the animal's temperature and hemodynamics were maintained with goals of mean arterial pressure greater than 60mmHg and heart rate within 20 beats per minute of baseline. Resuscitation protocols are described so that future investigators may reproduce this model.We have developed a reproducible large animal primate model of brain death which simulates clinical scenarios and treatment. Our model offers the opportunity for researchers to have translational model to test the efficacy of therapeutic strategies prior to human clinical trials

Optimal Time to Ship Human Islets Post Tissue Culture to Maximize Islet.

Access to functional high-quality pancreatic human islets is critical to advance diabetes research. The Integrated Islet Distribution Program (IIDP), a major source for human islet distribution for over 15 years, conducted a study to evaluate the most advantageous times to ship islets postisolation to maximize islet recovery. For the evaluation, three experienced IIDP Islet Isolation Centers each provided samples from five human islet isolations, shipping 10,000 islet equivalents (IEQ) at four different time periods postislet isolation (no 37°C culture and shipped within 0 to 18 hours; or held in 37°C culture for 18 to 42, 48 to 96, or 144 to 192 hours). A central evaluation center compared samples for islet quantity, quality, and viability for each experimental condition preshipment and postshipment, as well as post 37°C culture 18 to 24 hours after shipment receipt. Additional evaluations included measures of functional potency by static glucose-stimulated insulin release (GSIR), represented as a stimulation index. Comparing the results of the four preshipment holding periods, the greatest IEQ loss postshipment occurred with the shortest preshipment times. Similar patterns emerged when comparing preshipment to postculture losses. In vitro islet function (GSIR) was not adversely impacted by increased tissue culture time. These data indicate that allowing time for islet recovery postisolation, prior to shipping, yields less islet loss during shipment without decreasing islet function

Treatment with C1INH prevented the progression to fibrosis in kidneys subjected to ischemia-reperfusion injury.

<p>(A) Immunohistochemical staining for alpha smooth muscle actin (α-SMA), desmin and picrosirius red staining in renal tissue recovered at 30 and 90-days post-ischemic injury from sham + PBS (n = 6), sham + C1INH (n = 6), IRI + PBS (n = 6) and IRI + C1INH (n = 6) mice. Representative light microscopy images (200X magnification) from each group are depicted. Semi-automated quantification of (B) α-SMA(+), (C) Desmin(+) and (D) Picrosirius Red(+) area per HPF. Stained area is calculated and shown as a percentage relative to total tissue area per field. Data are mean ± SD. Statistical comparison was performed by one-way ANOVA followed by Bonferroni’s post-hoc correction. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Protective renal effect of complement blockade after ischemia-reperfusion injury.

<p>Mice were assigned to four groups: 1) Sham + PBS (n = 6), 2) Sham + C1INH (n = 6), 3) IRI + PBS (n = 9), IRI + C1INH (n = 15). Ischemia was induced by clamping of the renal hilum for 60 minutes and followed by contra-lateral nephrectomy. Sterile PBS or C1INH (750 U/kg) were given intravenously via tail vein injection 1 hour prior to surgery. Serum was collected at 24 and 72 hours after injury and survival was monitored for 90 days after reperfusion. (A) Pharmacological targeting of complement activation using C1INH significantly improves animal survival after IRI (Log-rank test, <i>P</i> = 0.0015). (B) Serum creatinine level at 24 and 72 hours after IRI. Groups: 1) Sham + PBS (n = 8), 2) Sham + C1INH (n = 8), 3) IRI + PBS (n = 10), IRI + C1INH (n = 10). (C) Histopathological analysis of kidneys at 24 hours after surgery. Representative light microscopy images of hematoxylin-eosin (H&E) staining of the cortex and medulla of kidneys (200X magnification). Arrows indicate necrotic tubules, and asterisks indicate tubular casts. (D) Renal tubular injury scores (0–4, arbitrary units). All data presented are mean ± SD. Survival data was analyzed by the Kaplan–Meier survival method and the log-rank test. Statistical comparison for creatinine values and tubular injury scoring was performed by one-way ANOVA followed by Bonferroni’s post-hoc correction. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Renal mRNA expression and circulating level of inflammatory cytokines and chemokines at 24 and 72-hours post-injury.

<p>RT-PCR analysis was performed on renal tissue recovered at the specified time points from mice in all groups (A) mRNA expression of monocyte chemoattractant protein-1 (MCP-1, n = 6), Interleukin-6 (IL-6, n = 6) and CXCL1 (KC, n = 4). Expression was normalized to baseline expression of native controls and GAPDH was used as the endogenous control. (B) Plasma KC and CCL2 levels post-injury. Cytokine levels determined by ELISA on plasma recovered at the specified time points from mice in all groups (n = 3 for each group). Data are mean ± SD. Statistical comparison was performed by Kruskal-Wallis and Dunn’s post-hoc correction. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

C1INH significantly decreased renal C5a cleavage and limited C3b deposition in kidneys subjected to IRI.

<p>(A) Immunoblotting analysis for C5a in renal tissue at 24 and 72 hours post-IRI. (B) Quantification of C5a band density from western blots normalized to β-tubulin. Results are expressed as mean ± SD fold changes (n = 3 for each group) compared with native controls. (C) Immunoblotting analysis of phosphorylated ERK1/2 normalized to total ERK1/2 (p44/42 MAPK) in renal tissue at 24 and 72 hours post-IRI in renal tissue. (D) Quantification of phosphorylated ERK band density from western blots normalized total ERK and β-tubulin. Results are expressed as mean ± SD fold changes (n = 3 for each group) compared with native controls. (E) Representative images of immunofluorescent microscopy performed on renal tissue at 72 hours post-injury targeting C3b deposition (200X). (F) mRNA expression of bradykinin 1 (BR1) and BR2 receptor in renal tissue. Expression was normalized to baseline expression of native controls and GAPDH was used as the endogenous control. Results are expressed as mean ± SD fold changes (n = 3 for each group).</p