Search

Mesenchymal stromal cells (MSC) have emerged as a promising therapy to minimize the immunosuppressive regimen or induce tolerance in solid organ transplantation. In this randomized open-label phase Ib/IIa clinical trial, 20 liver transplant patients were randomly allocated (1:1) to receive a single pre-transplant intravenous infusion of third-party bone marrow-derived MSC or standard of care alone. The primary end-point was the safety profile of MSC administration during the one-year follow-up. Nineteen patients completed the study, and none of those who received MSC experienced infusion-related complications. The incidence of serious and non-serious adverse events was similar in the two groups. Circulating Treg/memory Treg and tolerant NK subset of CD56bright NK cells increased slightly over baseline, albeit not to a statistically significant extent, in MSC-treated patients but not in the control group. Graft function and survival, as well as histologic parameters and intragraft expression of tolerance-associated transcripts in 1-year protocol biopsies were similar in the two groups. In conclusion, pre-transplant MSC infusion in liver transplant recipients was safe and induced mild positive changes in immunoregulatory T and NK cells in the peripheral blood. This study opens the way for a trial on possible tolerogenic efficacy of MSC in liver transplantation. ClinicalTrials.gov identifier: NCT02260375.

A 6-month-old boy presented with acute renal failure, thrombocytopenia, and severe non-immune hemolytic anemia. Infection by Shiga-like toxin-producing Escherichia coli and other causes of microangiopathic hemolysis were ruled out, leading to a diagnosis of atypical hemolytic uremic syndrome (aHUS). Neither pathogenic variants in HUS-associated genes nor anti-factor H antibodies were identified. Copy number variation analysis uncovered 4 copies of complement factor H related genes, CFHR1-CFHR4, conceivably leading to higher than normal levels of the corresponding proteins. However, this abnormality was also found in the healthy relatives, neither explaining the disease nor the excessive complement deposition on endothelial cells detected by an ex-vivo test. Whole-exome sequencing revealed a pathogenic homozygous variant in GRHPR encoding the glyoxylate and hydroxypyruvate reductase. Recessive GRHPR mutations cause primary hyperoxaluria type 2 (PH2). The presence of renal calculi in the patient and elevated oxalate levels in the urine were consistent with the genetic diagnosis of PH2. We hypothesize that, in this patient, hyperoxaluria caused by the GRHPR genetic defect triggered endothelial perturbation and complement activation, which was amplified by impaired factor H regulatory activity due to the increased ­CFHR1-CFHR4 copy numbers, resulting in aHUS.

We have previously shown that rat allogeneic DC, made immature by adenoviral gene transfer of the dominant negative form of IKK2, gave rise in-vitro to a unique population of CD4+CD25− regulatory T cells (dnIKK2-Treg). These cells inhibited Tcell response in-vitro, without needing cell-to-cell contact, and induced kidney allograft survival prolongation in-vivo. Deep insight into the mechanisms behind dnIKK2-Treg-induced suppression of Tcell proliferation remained elusive. Here we document that dnIKK2-Treg release extracellular vesicles (EV) riched in exosomes, fully accounting for the cell-contact independent immunosuppressive activity of parent cells. DnIKK2-Treg-EV contain a unique molecular cargo of specific miRNAs and iNOS, which, once delivered into target cells, blocked cell cycle progression and induced apoptosis. DnIKK2-Treg-EV-exposed T cells were in turn converted into regulatory cells. Notably, when administered in-vivo, dnIKK2-Treg-EV prolonged kidney allograft survival. DnIKK2-Treg-derived EV could be a tool for manipulating the immune system and for discovering novel potential immunosuppressive molecules in the context of allotransplantation.

Complement activation has been increasingly implicated in the pathogenesis of type 2 diabetes and its chronic complications. It is unknown whether complement factor H (CFH) genetic variants, which have been previously associated with complement-mediated organ damage likely due to inefficient complement modulation, influence the risk of renal and cardiovascular events and response to therapy with angiotensin-converting enzyme inhibitors (ACEi) in type 2 diabetic patients. Here, we have analyzed the c.2808G> T, (p.Glu936Asp) CFH polymorphism, which tags the H3 CFH haplotype associated to low plasma factor H levels and predisposing to atypical hemolytic uremic syndrome, in 1,158 type 2 diabetics prospectively followed in the Bergamo nephrologic complications of type 2 diabetes randomized, controlled clinical trial (BENEDICT) that evaluated the effect of the ACEi trandolapril on new onset microalbuminuria. At multivariable Cox analysis, the p.Glu936Asp polymorphism (Asp/Asp homozygotes, recessive model) was associated with increased risk of microalbuminuria [adjusted hazard ratio (HR) 3.25 (95% CI 1.46-7.24), P = 0.0038] and cardiovascular events [adjusted HR 2.68 (95% CI 1.23-5.87), P = 0.013]. The p.Glu936Asp genotype significantly interacted with ACEi therapy in predicting microalbuminuria. ACEi therapy was not nephroprotective in Asp/Asp homozygotes [adjusted HR 1.54 (0.18-13.07), P = 0.691 vs. non-ACEi-treated Asp/Asp patients], whereas it significantly reduced microalbuminuria events in Glu/Asp or Glu/Glu patients [adjusted HR 0.38 (0.24-0.60), P < 0.0001 vs. non-ACEi-treated Glu/Asp or Glu/Glu patients]. Among ACEi-treated patients, the risk of developing cardiovascular events was higher in Asp/Asp homozygotes than in Glu/Asp or Glu/Glu patients [adjusted HR 3.26 (1.29-8.28), P = 0.013]. Our results indicate that type 2 diabetic patients Asp/Asp homozygotes in the p.Glu936Asp CFH polymorphism are at increased risk of microalbuminuria and cardiovascular complications and may be less likely to benefit from ACEi therapy. Further studies are required to confirm our findings.

Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and renal failure. It is caused by genetic or acquired defects of the complement alternative pathway. Factor H autoantibodies (anti-FHs) have been reported in 10% of aHUS patients and are associated with the deficiency of factor H-related 1 (FHR1). However, FHR1 deficiency is not enough to cause aHUS, since it is also present in about 5% of Caucasian healthy subjects. In this study we evaluated the prevalence of genetic variants in CFH, CD46, CFI, CFB, C3, and THBD in aHUS patients with anti-FHs, using healthy subjects with FHR1 deficiency, here defined “supercontrols,” as a reference group. “Supercontrols” are more informative than general population because they share at least one risk factor (FHR1 deficiency) with aHUS patients. We analyzed anti-FHs in 305 patients and 30 were positive. The large majority were children (median age: 7.7 [IQR, 6.6–9.9] years) and 83% lacked FHR1 (n = 25, cases) due to the homozygous CFHR3-CFHR1 deletion (n = 20), or the compound heterozygous CFHR3-CFHR1 and CFHR1-CFHR4 deletions (n = 4), or the heterozygous CFHR3-CFHR1 deletion combined with a frameshift mutation in CFHR1 that generates a premature stop codon (n = 1). Of the 960 healthy adult subjects 48 had the FHR1 deficiency (“supercontrols”). Rare likely pathogenetic variants in CFH, THBD, and C3 were found in 24% of cases (n = 6) compared to 2.1% of the “supercontrols” (P-value = 0.005). We also found that the CFH H3 and the CD46GGAAC haplotypes are not associated with anti-FHs aHUS, whereas these haplotypes are enriched in aHUS patients without anti-FHs, which highlights the differences in the genetic basis of the two forms of the disease. Finally, we confirm that common infections are environmental factors that contribute to the development of anti-FHs aHUS in genetically predisposed individuals, which fits with the sharp peak of incidence during scholar-age. Further studies are needed to fully elucidate the complex genetic and environmental factors underlying anti-FHs aHUS and to establish whether the combination of anti-FHs with likely pathogenetic variants or other risk factors influences disease outcome and response to therapies.

Rationale & Objective Although primary atypical hemolytic uremic syndrome (aHUS) is associated with abnormalities in complement genes and antibodies to complement factor H, the role of complement in secondary aHUS remains debatable. We evaluated the usefulness of an ex vivo test to: (1) detect complement activation within the endothelium in primary and secondary aHUS, (2) differentiate active disease from remission, (3) monitor the effectiveness of eculizumab therapy, and (4) identify relapses during eculizumab dosage tapering and after discontinuation of treatment. Study Design Case series. Setting & Participants 121 patients with primary aHUS and 28 with secondary aHUS. Serum samples were collected during acute episodes, following remission, and during eculizumab treatment and were assessed using a serum-induced ex vivo C5b-9 endothelial deposition test. Results Serum-induced C5b-9 deposition on cultured microvascular endothelium was quantified by calculating the endothelial area covered by C5b-9 staining; values were expressed as percentage of C5b-9 deposits induced by a serum pool from healthy controls. Testing with adenosine diphosphate–activated endothelium demonstrated elevated C5b-9 deposits for all untreated patients with aHUS independent of disease activity, while testing with unstimulated endothelium demonstrated deposits only in active disease. Similar findings were observed in secondary aHUS. Serum-induced C5b-9 deposits on activated and unstimulated endothelium normalized during eculizumab treatment. 96% (22/23) of patients receiving eculizumab at extended 3- or 4-week dosing intervals demonstrated normal C5b-9 deposits on activated endothelium, despite most patients having CH50Eq (serum complement activity) > 20 UEq/mL, indicating that adequate complement control was achieved even with incomplete blockade of circulating C5. During eculizumab dosage tapering or after treatment discontinuation, all patients experiencing relapses versus only 6% (1/17) of those in stable remission had elevated C5b-9 deposits on unstimulated endothelium. Limitations The C5b-9 endothelial deposition test can be performed in only specialized laboratories. Findings on eculizumab dosage tapering need to be confirmed with longitudinal monitoring of C5b-9 deposition. Conclusions The C5b-9 endothelial deposition assay may represent an advance in our ability to monitor aHUS activity and individualize therapy.

Background and objectives Genetic and acquired abnormalities causing dysregulation of the complement alternative pathway contribute to atypical hemolytic uremic syndrome (aHUS), a rare disorder characterized by thrombocytopenia, nonimmune microangiopathic hemolytic anemia, and acute kidney failure. However, in a substantial proportion of patients the disease-associated alterations are still unknown. Design, setting, participants, & measurements Whole-exome and whole-genome sequencing were performed in two unrelated families with infantile recessive aHUS. Sequencing of cDNA from affected individuals was used to test for the presence of aberrant mRNA species. Expression of mutant diacylglycerol kinase epsilon (DGKE) protein was evaluated with western blotting. Results Whole-exome sequencing analysis with conventional variant filtering parameters did not reveal any obvious candidate mutation in the first family. The report of aHUS-associated mutations in DGKE , encoding DGKE, led to re-examination of the noncoding DGKE variants obtained from next-generation sequencing, allowing identification of a novel intronic DGKE mutation (c.888+40A>G) that segregated with disease. Sequencing of cDNA from affected individuals revealed aberrant forms of DGKE mRNA predicted to cause profound abnormalities in the protein catalytic site. By whole-genome sequencing, the same mutation was found in compound heterozygosity with a second nonsense DGKE mutation in all affected siblings of another unrelated family. Homozygous and compound heterozygous patients presented similar clinical features, including aHUS presentation in the first year of life, multiple relapsing episodes, and proteinuria, which are prototypical of DGKE -associated aHUS. Conclusions This is the first report of a mutation located beyond the exon-intron boundaries in aHUS. Intronic mutations such as these are underreported because conventional filtering parameters used to process next-generation sequencing data routinely exclude these regions from downstream analyses in both research and clinical settings. The results suggest that analysis of noncoding regions of aHUS-associated genes coupled with mRNA sequencing might provide a tool to explain genetically unsolved aHUS cases.

Camera traps have proven very useful in ecological, conservation and behavioral research. Camera traps non-invasively record presence and behavior of animals in their natural environment. Since the introduction of digital cameras, large amounts of data can be stored. Unfortunately, processing protocols did not evolve as fast as the technical capabilities of the cameras. We used camera traps to record videos of Eurasian beavers (Castor fiber). However, a large number of recordings did not contain the target species, but instead empty recordings or other species (together non-target recordings), making the removal of these recordings unacceptably time consuming. In this paper we propose a method to partially eliminate non-target recordings without having to watch the recordings, in order to reduce workload. Discrimination between recordings of target species and non-target recordings was based on detecting variation (changes in pixel values from frame to frame) in the recordings. Because of the size of the target species, we supposed that recordings with the target species contain on average much more movements than non-target recordings. Two different filter methods were tested and compared. We show that a partial discrimination can be made between target and non-target recordings based on variation in pixel values and that environmental conditions and filter methods influence the amount of non-target recordings that can be identified and discarded. By allowing a loss of 5% to 20% of recordings containing the target species, in ideal circumstances, 53% to 76% of non-target recordings can be identified and discarded. We conclude that adding an extra processing step in the camera trap protocol can result in large time savings. Since we are convinced that the use of camera traps will become increasingly important in the future, this filter method can benefit many researchers, using it in different contexts across the globe, on both videos and photographs.