Assessment of biological organ age using molecular pathology in pre-transplant kidney biopsies.

Organ shortage is a major challenge in kidney transplantation but the use of older donors, often with co-morbidities, is hampered by inconsistent outcomes. Methods of accurately stratifying marginal donor organs by clinical and histological assessment are lacking. To better understand organ variability, we profiled the transcriptomes of 271 kidneys from deceased donors at retrieval. Following correction for biopsy composition, we assessed molecular pathways that associated with delayed, and sub-optimal one-year graft function. Analysis of cortical biopsies identified an adaptive immune gene-rich module that significantly associated with increasing age and worse outcomes. Cellular deconvolution using human kidney reference single cell transcriptomes confirmed an increase in kidney-specific B and T cell signatures, as well as kidney macrophage, myofibroblast and fibroblast gene sets in this module. Surprisingly, innate immune pathway and neutrophil gene signature enrichment was associated with better outcomes. Thus, our work uncovers cellular molecular features of pathological organ ageing, identifiable at kidney retrieval, with translational potential.

Early surveillance biopsy utilization and management of pediatric renal allograft acute T cell-mediated rejection in Canadian centers: Observations from the PROBE multicenter cohort study.

BACKGROUND: Early TCMR surveillance with protocol kidney biopsy is used differentially among pediatric kidney transplant centers. Little has been reported about actual center-based differences, and this variability may influence TCMR ascertainment, treatment, and monitoring more broadly. METHODS: Data from the PROBE multicenter study were used to identify patients from centers conducting ESB or LSIB. ESB was defined as >50% of patients having at least 1 surveillance biopsy in the first 9 months. Patients were compared for number of biopsies, rejection episodes, treatment, and follow-up monitoring. RESULTS: A total of 261 biopsies were performed on 97 patients over 1-2 years of follow-up. A total of 228 (87%) of biopsies were performed in ESB centers. Compared to LSIB centers, ESB centers had 7-fold more episodes of TCMR diagnosed on any biopsy [0.8 ± 1.2 vs 0.1 ± 0.4; P < .001] and a 3-fold higher rate from indication biopsies [0.3 ± 0.9 vs 0.1 ± 0.3; P = .04]. The proportion of rejection treatment varied based on severity: Banff borderline i1t1 (40%);>i1t1 and < Banff 1A (86%); and ≥ Banff 1A (100%). Biopsies for follow-up were performed after treatment in 80% of cases (n = 28) of rejection almost exclusively at ESB centers, with 17 (61%) showing persistence of TCMR (≥i1t1). CONCLUSIONS: Practice variation exists across Canadian pediatric renal transplant centers with ESB centers identifying more episodes of rejection. Additionally, treatment of Banff borderline is not universal and varies with severity regardless of center type. Lastly, follow-up biopsies are performed inconsistently and invariably show persistence of rejection.

Assessment of Organ Quality in Kidney Transplantation by Molecular Analysis and Why It May Not Have Been Achieved, Yet.

Donor organ shortage, growing waiting lists and substantial organ discard rates are key problems in transplantation. The critical importance of organ quality in determining long-term function is becoming increasingly clear. However, organ quality is difficult to predict. The lack of good measures of organ quality is a serious challenge in terms of acceptance and allocation of an organ. The underlying review summarizes currently available methods used to assess donor organ quality such as histopathology, clinical scores and machine perfusion characteristics with special focus on molecular analyses of kidney quality. The majority of studies testing molecular markers of organ quality focused on identifying organs at risk for delayed graft function, yet without prediction of long-term graft outcome. Recently, interest has emerged in looking for molecular markers associated with biological age to predict organ quality. However, molecular gene sets have not entered the clinical routine or impacted discard rates so far. The current review critically discusses the potential reasons why clinically applicable molecular quality assessment using early kidney biopsies might not have been achieved yet. Besides a critical analysis of the inherent limitations of surrogate markers used for organ quality, i.e., delayed graft function, the intrinsic methodological limitations of studies assessing organ quality will be discussed. These comprise the multitude of unpredictable hits as well as lack of markers of nephron mass, functional reserve and regenerative capacity.

Flow-controlled fluoroscopic angiography for the assessment of vascular integrity in bioengineered kidneys.

Perfusion decellularization has been proposed as a promising method for generating nonimmunogenic organs from allogeneic or xenogeneic donors. Several imaging modalities have been used to assess vascular integrity in bioengineered organs with no consistency in the methodology used. Here, we studied the use of fluoroscopic angiography performed under controlled flow conditions for vascular integrity assessment in bioengineered kidneys. Porcine kidneys underwent ex vivo angiography before and after perfusion decellularization. Arterial and venous patencies were defined as visualization of contrast medium (CM) in distal capillaries and renal vein, respectively. Changes in vascular permeability were visualized and quantified. No differences in patency were detected in decellularized kidneys compared with native kidneys. However, focal parenchymal opacities and significant delay in CM clearance were detected in decellularized kidneys, indicating increased permeability. Biopsy-induced leakage was visualized in both groups, with digital subtraction angiography revealing minimal CM leakage earlier than nonsubtracted fluoroscopy. In summary, quantitative assessment of vascular permeability should be coupled with patency when studying the effect of perfusion decellularization on kidney vasculature. Flow-controlled angiography should be considered as the method of choice for vascular assessment in bioengineered kidneys. Adopting this methodology for organs premodified ex vivo under normothermic machine perfusion settings is also suggested.

Kidney biopsy-based management of maintenance immunosuppression is safe and may ameliorate flare rate in lupus nephritis.

The optimal duration of maintenance immunosuppressive therapy for patients with lupus nephritis who have achieved clinical remission has not been established. Furthermore, clinical and histologic remissions are often discordant. We postulated that continuing therapy for patients with persistent histologic activity on kidney biopsies done during maintenance and discontinuing therapy only for patients without histologic activity would minimize subsequent lupus nephritis flares. To test this, a cohort of 75 prospectively-followed patients with proliferative lupus nephritis was managed using kidney biopsies performed during maintenance therapy. These patients had been on immunosuppression for at least 42 months, had responded, and had maintained their clinical response for at least 12 months before the kidney biopsy was repeated. Maintenance therapy was withdrawn if the biopsy showed an activity index of zero, but was continued if the biopsy showed an activity index of one or more. A lupus nephritis flare developed in seven patients during the average 50 months from the third biopsy and the final clinic visit for a flare rate of 1.5/year; significantly less than reported flare rates. Baseline clinical parameters (serum creatinine, proteinuria) and serologic parameters (complement C3, C4 and anti-dsDNA) did not predict an activity index of zero on the third biopsy or who would have a lupus nephritis flare. No patients developed end-stage kidney disease. Four patients developed de novo chronic kidney disease. There were no serious adverse events related to biopsy. Thus, at an experienced center, biopsy-informed management of maintenance immunosuppression is safe and may improve the lupus nephritis flare rate compared to conventional clinical management.

Clinicopathologic Assessment of Monoclonal Immunoglobulin-associated Renal Disease in the Kidney Allograft: A Retrospective Study and Review of the Literature.

BACKGROUND: Monoclonal immunoglobulin (MIg)-associated renal disease (MIgARD) comprises a group of disorders caused by direct deposition of paraproteins in the kidney. Allograft MIgARD is infrequently encountered and poorly characterized. METHODS: First, we assessed our allograft biopsies diagnosed with MIgARD between 2007 and 2018. The cohort included the following 26 patients: proliferative glomerulonephritis with MIg deposits (PGNMID) (n = 13), AL amyloidosis (n = 5), light chain deposition disease (n = 5), light chain proximal tubulopathy (n = 2), and light chain cast nephropathy (n = 1). Second, we conducted a literature review to evaluate the rare non-PGNMID entities. We identified 20 studies describing 29 patients that were added to our cohort (total n = 42). RESULTS: Part 1: Patients' median age was 55 years; 31% were women, and 19% were blacks. Twelve patients (46%) lost their grafts at a median of 8 months after diagnosis. Compared to non-PGNMID, PGNMID patients had lower frequency of detectable paraproteins (31% versus 92%, P = 0.004) and hematologic neoplasms (23% versus 77%, P = 0.02). Within PGNMID group, 6 patients changed their apparent immunofluorescence phenotype between monotypic and polytypic, while all 3 patients with hematologic neoplasms had substructure on electron microscopy. Part 2: Whereas light chain cast nephropathy occurred the earliest and had the worst graft survival, AL amyloidosis occurred the latest and had the best graft survival. CONCLUSIONS: MIgARD in the kidney allograft is associated with poor prognosis. While posttransplant PGNMID can change its apparent clonality by immunofluorescence supporting oligoclonal immune responses, the presence of deposit substructure is an important indicator of underlying hematologic neoplasm. Non-PGNMID are often associated with hematologic neoplasms and varied prognosis.

2013 Banff Criteria for Acute Antibody-Mediated Rejection Are Superior to 2007 Banff Criteria in the Diagnosis and Assessment of Renal Allograft Outcomes.

BACKGROUND: The 2013 Banff meeting updated the requirements for the diagnosis of acute/active antibody-mediated rejection (AAMR) in kidney allografts. There has been speculation that the changes lower the threshold for diagnosing AAMR, and may lead to possible unnecessary and expensive treatment. METHODS: We compared the 2013 Banff classification for AAMR to the previous 2007 Banff to determine if there was an increase in the number of patients receiving a diagnosis of AAMR and if the diagnosis affected allograft survival and post-biopsy 3-month and 6-month creatinine and eGFR values. RESULTS: A total of 212 renal allograft biopsies were compared to both 2007 and 2013 Banff classification requirements for AAMR. Ten patients (11 biopsies) met the 2007 criteria. An additional 15 patients (20 biopsies) met the 2013 criteria. These 2 groups showed no statistically significant demographic differences. By applying the 2013 Banff classification, we observed a 2.5-fold increase in the number of AAMR cases. One-year post-transplant allograft survival was higher in the 2013 group (.85 vs .55) and the 3-month and 6-month post-biopsy creatinine values were significantly lower for the 2013 group (1.6 ± .6 vs 3.3 ± 2.2, P value .01, and 1.7 ± .6 vs 3.4 ± 2.8, P value .03). The 3-month and 6-month eGFR values were higher in the 2013 group, although not statistically significant. CONCLUSIONS: These results suggest that use of Banff 2013 criteria in place of Banff 2007 may result in diagnosing milder and earlier cases of AAMR with the possibility of initiating earlier treatment and improving graft outcomes.

Benefits and risks of protocol biopsies in pediatric renal transplantation.

Protocol biopsies are defined as sampling of allograft tissue at predetermined times regardless of function. This procedure can be justified due to the lack of non-invasive methods to reliably diagnose rejection (acute or subclinical). Changes in creatinine are not seen with subclinical rejection or early acute rejection and do not always correlate with efficacy of treatment. Parents and providers are still hesitant to pursue protocol biopsy due to the potential complications and lack of definitive evidence of a benefit from doing this procedure. Importantly, the rate of transplant renal biopsy complications requiring additional intervention is low. It is unclear if detection and treatment of subclinical rejection detected on protocol biopsy will lead to improved graft survival. Our goal is to review the literature on this topic and share some of the experience in our center. Definition, indications, and complications of diagnostic transplant renal biopsies are not included in this review.

Assessment of the immunogenicity of residual host cell protein impurities of OsrHSA.

Human serum albumin (HSA) is the most abundant protein in human plasma and is widely used at high doses for treating various diseases. Recombinant HSA is an alternative approach to plasma-derived HSA, providing increased safety and an unlimited supply. However, the safety of the residual host cell proteins (HCPs) co-purified with Oryza sativa HSA (OsrHSA) remains to be determined. An animal system was used to assess the immunogenicity of OsrHSA and its residual HCPs. Low immunogenicity and immunotoxicity of the residual HCPs at a dose of 25 µg/kg, equivalent to 25 times the clinical dosage of HSA, were observed. An anti-drug-antibody (ADA) analysis revealed that anti-HSA, anti-OsrHSA or anti-HCP antibodies developed with a low frequency in pHSA and OsrHSA treatments, but the titers were as low as 1.0-2.0. Furthermore, the titer and the incidence of the specific antibodies were not significantly different between the pHSA and OsrHSA groups, indicating that OsrHSA presents similar immunogenicity to that of pHSA. More importantly, no cytokines were stimulated after the administration of OsrHSA and the residual HCPs, suggesting that there was no risk of a cytokine storm. These results demonstrated that the residual HCPs from OsrHSA have low immunogenicity, indicating that the rice endosperm is one of the best hosts for plant molecular pharming.

Assessment of the T-SPOT.CMV interferon-γ release assay in renal transplant recipients: A single center cohort study.

BACKGROUND: The measurement of CMV specific cellular immunity in organ transplant recipients could contribute additional acuity to serology based, CMV infection risk stratification, facilitating optimisation of immunosuppression and anti-viral prophylaxis. METHODS: A pilot study of renal transplant recipient (RTR's) responses in the T-SPOT.CMV ELISPOT based assay. 108 RTR's were recruited 3 months post-transplantation, immediately prior to the cessation of stratified anti-viral prophylaxis, used in recipients from seropositive donors. RTR's were monitored for CMV viremia and disease. Cellular responses to peptides derived from CMV IE1 and pp65 were measured, using the T-SPOT.CMV assay. RESULTS: At recruitment, no CMV specific cellular immunity was detected by T-SPOT.CMV in CMV seronegative recipients (IE1 ≤ 1spot / 2.5x105 PBMC's; pp65 ≤ 3 spots / 2.5x105 PBMC's). At recruitment, CMV sero-positive recipients who made a robust response to both IE1 (>25 spots / 2.5x105 PBMC's) and pp65 (>50 spots / 2.5x105 PBMC's), were less likely to develop high level viremia than those who responded to one or neither antigen (0/28 vs 5/25; p<0.02). CONCLUSIONS: In CMV seronegative RTR's, CMV specific cellular immunity measured by T-SPOT.CMV was not detected prior to cessation of anti-viral prophylaxis. This differs from recent reports of CMV specific cellular immunity in a proportion of CMV seronegative RTR's, associated with protection from CMV infection. In seropositive RTR's, a dual response to IE1 and pp65 at recruitment, was associated with protection from subsequent viremia. This suggests that assessing the diversity of response to CMV antigens, may enhance risk stratification in this group.

Rice bran protein hydrolysates attenuate diabetic nephropathy in diabetic animal model.

INTRODUCTION: Diabetic nephropathy (DN) is an important microvascular complication of uncontrolled diabetes. The features of DN include albuminuria, extracellular matrix alterations, and progressive renal insufficiency. Rice bran protein hydrolysates (RBPs) have been reported to have antihyperglycemic, lipid-lowering, and anti-inflammatory effects in diabetic rats. Our study was to investigate the renoprotective effects of RBP in diabetic animals and mesangial cultured cells. METHODS: Eight-week-old male db/m and db/db mice were orally treated with tap water or RBP (100 or 500 mg/kg/day) for 8 weeks. At the end of the experiment, diabetic nephropathy in kidney tissues was investigated for histological, ultrastructural, and clinical chemistry changes, and biomarkers of angiogenesis, fibrosis, inflammation, and antioxidant in kidney were analyzed by Western blotting. Protection against proangiogenic proteins and induction of cytoprotection by RBP in cultured mesangial cells was evaluated. RESULTS: RBP treatment improved insulin sensitivity, decreased elevated fasting serum glucose levels, and improved serum lipid levels and urinary albumin/creatinine ratios in diabetic mice. RBP ameliorated the decreases in podocyte slit pore numbers, thickening of glomerular basement membranes, and mesangial matrix expansion and suppressed elevation of MCP-1, ICAM-1, HIF-1α, VEGF, TGF-ß, p-Smad2/3, and type IV collagen expression. Moreover, RBP restored suppressed antioxidant Nrf2 and HO-1 expression. In cultured mesangial cells, RBP inhibited high glucose-induced angiogenic protein expression and induced the expression of Nrf2 and HO-1. CONCLUSION: RBP attenuates the progression of diabetic nephropathy and restored renal function by suppressing the expression of proangiogenic and profibrotic proteins, inhibiting proinflammatory mediators, and restoring the antioxidant and cytoprotective system.

Development of CD3 cell quantitation algorithms for renal allograft biopsy rejection assessment utilizing open source image analysis software.

Renal allograft rejection diagnosis depends on assessment of parameters such as interstitial inflammation; however, studies have shown interobserver variability regarding interstitial inflammation assessment. Since automated image analysis quantitation can be reproducible, we devised customized analysis methods for CD3+ T-cell staining density as a measure of rejection severity and compared them with established commercial methods along with visual assessment. Renal biopsy CD3 immunohistochemistry slides (n = 45), including renal allografts with various degrees of acute cellular rejection (ACR) were scanned for whole slide images (WSIs). Inflammation was quantitated in the WSIs using pathologist visual assessment, commercial algorithms (Aperio nuclear algorithm for CD3+ cells/mm2 and Aperio positive pixel count algorithm), and customized open source algorithms developed in ImageJ with thresholding/positive pixel counting (custom CD3+%) and identification of pixels fulfilling "maxima" criteria for CD3 expression (custom CD3+ cells/mm2). Based on visual inspections of "markup" images, CD3 quantitation algorithms produced adequate accuracy. Additionally, CD3 quantitation algorithms correlated between each other and also with visual assessment in a statistically significant manner (r = 0.44 to 0.94, p = 0.003 to < 0.0001). Methods for assessing inflammation suggested a progression through the tubulointerstitial ACR grades, with statistically different results in borderline versus other ACR types, in all but the custom methods. Assessment of CD3-stained slides using various open source image analysis algorithms presents salient correlations with established methods of CD3 quantitation. These analysis techniques are promising and highly customizable, providing a form of on-slide "flow cytometry" that can facilitate additional diagnostic accuracy in tissue-based assessments.

Mycophenolate mofetil or cyclophosphamide in indian patients with lupus nephritis: Which is better? A single-center experience.

Mycophenolate mofetil (MMF) is used extensively for the induction therapy of lupus nephritis (LN) and has even outpaced intravenous (i.v.) cyclophosphamide (CyP) as the initial choice of therapy. There are no studies comparing the response of MMF with standard dose i.v. CyP in Indian patients with LN. We conducted a 24-week prospective, randomized, open-label trial comparing oral MMF with monthly i.v. CyP as induction therapy for active biopsy proven Class III and IV LN. The primary end-point was response to treatment at 24 weeks, and the secondary end-points were complete remission, Systemic Lupus Erythematosus Disease Activity Index scores (SLEDAI) and adverse reactions. Of the 40 patients, 17 were randomized to the MMF group and 23 to the i.v. CyP group. Complete remission was seen in nine (52.94%) patients in the MMF group and 11 (47.82%) in the i.v. CyP group. Partial remission was seen in six (35.30%) in the MMF group and nine (39.13%) in the i.v. CyP group. At six months, the cumulative probability of response was not statistically significant between the two groups (P = 1.000). MMF is comparable to i.v. CyP in the management of LN in Indian patients having an equal safety profile. The dose of MMF required was lower than the conventional doses used in other studies suggesting genetic or environmental factors in the Indian population influencing the metabolism of MMF, which requires further evaluation. The cost of MMF is a limiting factor in its use. The use of i.v. CyP is favorable as the monthly doses ensure compliance and is also cost-effective.

Systematic assessment of the influence of complement gene polymorphisms on kidney transplant outcome.

The importance of the innate immune system, including complement, in causing transplant injury and augmenting adaptive immune responses is increasingly recognized. Therefore variability in graft outcome may in part be due to genetic polymorphism in genes encoding proteins of the immune system. This study assessed the relationship between single nucleotide polymorphisms (SNPs) in complement genes and outcome after transplantation. Analysis was performed on two patient cohorts of 650 and 520 transplant recipients. 505 tagged SNPs in 47 genes were typed in both donor and recipient. The relationships between SNPs and graft survival, serum creatinine, delayed graft function and acute rejection were analyzed. One recipient SNP in the gene encoding mannose binding lectin was associated with graft outcome after correction for analysis of multiple SNPs (p=6.41 × 10(-5)). When further correction was applied to account for analysis of the effect of SNPs in both donor and recipient this lost significance. Despite association p values of <0.001 no SNP was significantly associated with clinical phenotypes after Bonferroni correction. In conclusion, the variability seen in transplant outcome in this patient cohort cannot be explained by variation in complement genes. If causal genetic effects exist in these genes, they are too small to be detected by this study.

Immuno-histological assessment of sub-clinical acute and borderline rejection in renal allograft recipients: Data from a transplant center in India.

This single-center study was carried out on living related and unrelated renal transplant recipients (RTRs) to evaluate the usefulness of surveillance biopsies in monitoring stable renal allografts using immuno-histological markers for immune-activation. This is a prospective, longitudinal study. Protocol biopsies of 60 RTRs with stable graft function were evaluated at three, six and 12 months post-transplant. Immuno-histological evaluation was carried out using immune-activation markers (perforins, granzyme and interleukin-2R), phenotypic markers (CD-3 and CD-20), viral markers and C4d. The demographic and clinical profile was recorded for each patient. All cases of acute sub-clinical rejection (SCR) were treated and borderline SCR cases were followed-up without treatment. SCR at three and six months post-transplant was evident in 16.7% and 3.7% of RTRs, respectively. Positive statistical association of SCR was seen with HLA-DR mismatches, whereas patients receiving induction therapy and tacrolimus-based immunosuppression exhibited a lower incidence of SCR. T cell phenotype with persistent expression of immune-activation markers exhibited positive statistical association with interstitial fibrosis and tubular atrophy at 12-month follow-up biopsy. The mean creatinine levels were significantly lower in the protocol biopsy group than the non-protocol biopsy group. No significant difference was found between the mean creatinine levels of the SCR group after treatment and the non-SCR cases within the protocol biopsy group. Early treatment of sub-clinical acute rejection leads to better functional outcomes. However, persistent immune-activation is associated with chronicity and may have implications on long-term graft survival.

Functional assessment of mouse complement pathway activities and quantification of C3b/C3c/iC3b in an experimental model of mouse renal ischaemia/reperfusion injury.

The complement system is an essential component of our innate immunity, both for the protection against infections and for proper handling of dying cells. However, the complement system can also contribute to tissue injury and inflammatory responses. In view of novel therapeutic possibilities, there is an increasing interest in measurement of the complement system activation in the systemic compartment, both in the clinical setting as well as in experimental models. Here we describe in parallel a sensitive and specific sandwich ELISA detecting mouse C3 activation fragments C3b/C3c/iC3b, as well as functional complement ELISAs detecting specific activities of the three complement pathways at the level of C3 and at the level of C9 activation. In a murine model of renal ischaemia/reperfusion injury (IRI) we found transient complement activation as shown by generation of C3b/C3c/iC3b fragments at 24 h following reperfusion, which returned to base-line at 3 and 7 days post reperfusion. When the pathway specific complement activities were measured at the level of C3 activation, we found no significant reduction in any of the pathways. However, the functional complement activity of all three pathways was significantly reduced when measured at the level of C9, with the strongest reduction being observed in the alternative pathway. For all three pathways there was a strong correlation between the amount of C3 fragments and the reduction in functional complement activity. Moreover, at 24 h both C3 fragments and the functional complement activities showed a correlation with the rise in serum creatinine. Together our results show that determination of the systemic pathway specific complement activity is feasible in experimental mouse models and that they are useful in understanding complement activation and inhibition in vivo.

A Banff Component Scoring-based Histologic Assessment of Bortezomib-based Antibody-mediated Rejection Therapy.

BACKGROUND: Histology remains a cornerstone for antibody-mediated rejection (AMR) diagnosis. Little data exist supporting histology for assessing therapeutic responses. This study evaluates histologic components in assessing AMR therapeutic responses. METHODS: Antibody-mediated rejection was diagnosed using Antibody Working Group criteria and Banff component scoring, and C4d staining data were analyzed. Statistics included independent and paired samples t test, χ(2), Fisher exact, or the Wilcoxon-signed rank test. Fifty-five AMR patients were analyzed. Early AMR was defined as occurring within 6 months after transplantation and treated with a single rituximab dose and 4 bortezomib doses preceded by plasmapheresis. Allograft biopsies were performed within 48 hours of treatment; repeat biopsy was performed 14 to 21 days later. RESULTS: Early AMR demonstrated histologic improvement in mean scores for acute Banff components glomerulitis (g), C4d, g+ peritubular capillaritis (ptc) and acute composite score, but showed deterioration in chronic Banff components tubular atrophy and interstitial fibrosis. Late AMR showed improved mean scores for acute Banff components tubulitis, interstitial inflammation, g, ptc, g + ptc, C4d, and acute composite score, but chronic scores did not change. Significant changes in distribution of Banff scores after treatment were observed for g, C4d, tubular atrophy, and interstitial fibrosis scores in early AMR patients and tubulitis, interstitial inflammation, g, ptc, and C4d in late AMR. CONCLUSIONS: These results show that: (1) Banff component scoring provides insights into histologic responses to AMR therapy and may provide a potential endpoint for clinical AMR trials. (2) Early and late AMR demonstrate differences in acute and chronic Banff components at the time of the AMR diagnostic biopsy, as well as differential responses to AMR therapy.

Th-1 biased immunomodulation and synergistic antileishmanial activity of stable cationic lipid-polymer hybrid nanoparticle: biodistribution and toxicity assessment of encapsulated amphotericin B.

To address issues related to Amphotericin B (AmpB) clinical applications, we developed macrophage targeted cationic stearylamine lipid-polymer hybrid nanoparticles (LPNPs) with complementary characteristics of both polymeric nanoparticles and liposomes, for enhancement of therapeutic efficacy and diminishing toxic effect of encapsulated AmpB. The LPNPs (size 198.3 ± 3.52 nm, PDI 0.135 ± 0.03, zeta potential +31.6 ± 1.91 mV) provide core-shell type structure which has the ability to encapsulate amphiphilic AmpB in higher amount (Encapsulation efficiency 96.1 ± 2.01%), sustain drug release and stabilize formulation tremendously. Attenuated erythrocytes and J774A.1 toxicity of LPNPs demonstrated safe applicability for parenteral administration. Elevated macrophage uptake of LPNPs, rapid plasma clearance and higher drug allocation in macrophage abundant liver and spleen illustrated admirable antileishmanial efficacy of AmpB-LPNPs in vitro (IC50, 0.16 ± 0.04 µg AmpB/ml) and in vivo (89.41 ± 3.58% parasite inhibition) against visceral leishmaniasis models. Augmentation in antileishmanial activity due to Th-1 biased immune-alteration mediated by drug-free LPNPs which elevated microbicidal mediators of macrophages. Moreover, minimal distribution to kidney tissues and low level of nephrotoxicity markers (creatinine and BUN) demonstrated the safety profile of AmpB-LPNPs. Conclusively, reliable safety and macrophage directed therapeutic performance of AmpB-LPNPs suggest it as promising alternative to commercial AmpB-formulations for the eradication of intra-macrophage diseases.

Assessment of an anti-alpha-toxin monoclonal antibody for prevention and treatment of Staphylococcus aureus-induced pneumonia.

Alpha-toxin (AT) is a major virulence factor in the disease pathogenesis of Staphylococcus aureus. We previously identified a monoclonal antibody (MAb) against AT that reduced disease severity in a mouse dermonecrosis model. Here, we evaluate the activity of an affinity-optimized variant, LC10, in a mouse model of S. aureus pneumonia. Passive immunization with LC10 increased survival and reduced bacterial numbers in the lungs and kidneys of infected mice and showed protection against diverse S. aureus clinical isolates. The lungs of S. aureus-infected mice exhibited bacterial pneumonia, including widespread inflammation, whereas the lungs of mice that received LC10 exhibited minimal inflammation and retained healthy architecture. Consistent with reduced immune cell infiltration, LC10-treated animals had significantly lower (P < 0.05) proinflammatory cytokine and chemokine levels in the bronchoalveolar lavage fluid than did those of the control animals. This reduction in inflammation and damage to the LC10-treated animals resulted in reduced vascular protein leakage and CO2 levels in the blood. LC10 was also assessed for its therapeutic activity in combination with vancomycin or linezolid. Treatment with a combination of LC10 and vancomycin or linezolid resulted in a significant increase (P < 0.05) in survival relative to the monotherapies and was deemed additive to synergistic by isobologram analysis. Consistent with improved survival, the lungs of animals treated with antibiotic plus LC10 exhibited less inflammatory tissue damage than those that received monotherapy. These data provide insight into the mechanisms of protection provided by AT inhibition and support AT as a promising target for immunoprophylaxis or adjunctive therapy against S. aureus pneumonia.