Diverse molecular causes of unsolved autosomal dominant tubulointerstitial kidney diseases

Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD) is caused by mutations in one of at least five genes and leads to kidney failure usually in mid adulthood. Throughout the literature, variable numbers of families have been reported, where no mutation can be found and therefore termed ADTKD-not otherwise specified. Here, we aim to clarify the genetic cause of their diseases in our ADTKD registry. Sequencing for all known ADTKD genes was performed, followed by SNaPshot minisequencing for the dupC (an additional cytosine within a stretch of seven cytosines) mutation of MUC1. A virtual panel containing 560 genes reported in the context of kidney disease (nephrome) and exome sequencing were then analyzed sequentially. Variants were validated and tested for segregation. In 29 of the 45 registry families, mutations in known ADTKD genes were found, mostly in MUC1. Sixteen families could then be termed ADTKD-not otherwise specified, of which nine showed diagnostic variants in the nephrome (four in COL4A5, two in INF2 and one each in COL4A4, PAX2, SALL1 and PKD2). In the other seven families, exome sequencing analysis yielded potential disease associated variants in novel candidate genes for ADTKD; evaluated by database analyses and genome-wide association studies. For the great majority of our ADTKD registry we were able to reach a molecular genetic diagnosis. However, a small number of families are indeed affected by diseases classically described as a glomerular entity. Thus, incomplete clinical phenotyping and atypical clinical presentation may have led to the classification of ADTKD. The identified novel candidate genes by exome sequencing will require further functional validation

Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro

Background: Bacterial invasion through the blood-cerebrospinal fluid barrier (BCSFB) during bacterial meningitis causes secretion of proinflammatory cytokines/chemokines followed by the recruitment of leukocytes into the CNS. In this study, we analyzed the cellular and molecular mechanisms of polymorphonuclear neutrophil (PMN) and monocyte transepithelial transmigration (TM) across the BCSFB after bacterial infection. Methods: Using an inverted transwell filter system of human choroid plexus papilloma cells (HIBCPP), we studied leukocyte TM rates, the migration route by immunofluorescence, transmission electron microscopy and focused ion beam/scanning electron microscopy, the secretion of cytokines/chemokines by cytokine bead array and posttranslational modification of the signal regulatory protein (SIRP) α via western blot. Results: PMNs showed a significantly increased TM across HIBCPP after infection with wild-type Neisseria meningitidis (MC58). In contrast, a significantly decreased monocyte transmigration rate after bacterial infection of HIBCPP could be observed. Interestingly, in co-culture experiments with PMNs and monocytes, TM of monocytes was significantly enhanced. Analysis of paracellular permeability and transepithelial electrical resistance confirmed an intact barrier function during leukocyte TM. With the help of the different imaging techniques we could provide evidence for para- as well as for transcellular migrating leukocytes. Further analysis of secreted cytokines/chemokines showed a distinct pattern after stimulation and transmigration of PMNs and monocytes. Moreover, the transmembrane glycoprotein SIRPα was deglycosylated in monocytes, but not in PMNs, after bacterial infection. Conclusions: Our findings demonstrate that PMNs and monoctyes differentially migrate in a human BCSFB model after bacterial infection. Cytokines and chemokines as well as transmembrane proteins such as SIRPα may be involved in this process

Associations Between Deceased-Donor Urine MCP-1 and Kidney Transplant Outcomes

Existing methods to predict recipient allograft function during deceased-donor kidney procurement are imprecise. Understanding the potential renal reparative role for monocyte chemoattractant protein-1 (MCP-1), a cytokine involved in macrophage recruitment after injury, might help to predict allograft outcomes. Methods: We conducted a substudy of the multicenter prospective Deceased Donor Study cohort that evaluated deceased kidney donors from 5 organ procurement organizations from May 2010 to December 2013. We measured urine MCP-1 (uMCP-1) concentrations from donor samples collected at nephrectomy to determine associations with donor acute kidney injury (AKI), recipient delayed graft function (DGF), 6-month estimated glomerular filtration rate (eGFR), and graft failure. We also assessed perfusate MCP-1 concentrations from pumped kidneys for associations with DGF and 6-month eGFR. Results: AKI occurred in 111 donors (9%). The median (interquartile range) uMCP-1 concentration was higher in donors with AKI compared with donors without AKI (1.35 [0.41–3.93] ng/ml vs. 0.32 [0.11–0.80] ng/ml, P < 0.001). DGF occurred in 756 recipients (31%), but uMCP-1 was not independently associated with DGF. Higher donor uMCP-1 concentrations were independently associated with a higher 6-month eGFR in those without DGF (0.77 [0.10–1.45] ml/min per 1.73 m2 per doubling of uMCP1). However, there were no independent associations between uMCP-1 and graft failure over a median follow-up of ∼2 years. Lastly, perfusate MCP-1 concentrations significantly increased during pump perfusion but were not associated with DGF or 6-month eGFR. Discussion: Donor uMCP-1 concentrations were modestly associated with higher recipient 6-month eGFR in those without DGF. However, the results suggest that donor uMCP-1 has minimal clinical utility given no associations with graft failure

Effect of metallo-beta-lactamase production and multidrug resistance on clinical outcomes in patients with Pseudomonas aeruginosa bloodstream infection: a retrospective cohort study

BACKGROUND: Blood stream infections (BSI) with Pseudomonas aeruginosa lead to poor clinical outcomes. The worldwide emergence and spread of metallo-β-lactamase (MBL) producing, often multidrug-resistant organisms may further aggravate this problem. Our study aimed to investigate the effect of MBL-producing P. aeruginosa (MBL-PA) and various other resistance phenotypes on clinical outcomes. METHODS: A retrospective cohort study was conducted in three German hospitals. Medical files from 2006 until 2012 were studied, and a number of 113 patients with P. aeruginosa BSI were included. The presence of VIM, IMP and NDM genes was detected using molecular techniques. Genetic relatedness was assessed through multilocus sequence typing (MLST). The effect of resistance patterns or MBL production on clinical outcomes was investigated by using multivariate Cox regression models. RESULTS: In-hospital mortality was significantly higher in patients with MBL-PA and multidrug-resistant P. aeruginosa. However, neither BSI with MBL-PA nor BSI with various resistance phenotypes of P. aeruginosa were independently associated with mortality or length of hospital stay. In multivariate models, the SAPS II score (HR 1.046), appropriate definitive treatment (HR range 0.25-0.26), and cardiovascular disease (HR range 0.44-0.46) were independent predictors of mortality. Concomitant infections were associated with an excess length of stay (HR < 1). CONCLUSIONS: Medication with appropriate antimicrobial agents at any time during the course of infection remains the key for improving clinical outcomes in patients with P. aeruginosa BSI and should be combined with a strict implementation of routine infection control measures

The relationship between microbial biomass and disease in the Arabidopsis thaliana phyllosphere

A central goal in microbiome research is to learn what distinguishes a healthy from a dysbiotic microbial community. Shifts in diversity and taxonomic composition are important indicators of dysbiosis, but a full understanding also requires knowledge of absolute microbial biomass. Simultaneous information on both microbiome composition and the quantity of its components can provide insight into microbiome function and disease state. Here we use shotgun metagenomics to simultaneously assess microbiome composition and microbial load in the phyllosphere of wild populations of the plant Arabidopsis thaliana. We find that wild plants vary substantially in the load of colonizing microbes, and that high loads are typically associated with the proliferation of single taxa, with only a few putatively pathogenic taxa achieving high abundances in the field. Our results suggest (i) that the inside of a plant leaf is on average sparsely colonized with an estimated two bacterial genomes per plant genome and an order of magnitude fewer eukaryotic microbial genomes, and (ii) that higher levels of microbial biomass often indicate successful colonization by pathogens. Lastly, our results show that load is a significant explanatory variable for loss of estimated Shannon diversity in phyllosphere microbiomes, implying that reduced diversity may be a significant predictor of microbial dysbiosis in a plant leaf

Analysis of a long-term outbreak of XDR Pseudomonas aeruginosa: a molecular epidemiological study

OBJECTIVES: Here we report on a long-term outbreak from 2009 to 2012 with an XDR Pseudomonas aeruginosa on two wards at a university hospital in southern Germany. METHODS: Whole-genome sequencing was performed on the outbreak isolates and a core genome was constructed for molecular epidemiological analysis. We applied a time-place-sequence algorithm to improve estimation of transmission probabilities. RESULTS: By using conventional infection control methods we identified 49 P. aeruginosa strains, including eight environmental isolates that belonged to ST308 (by MLST) and carried the metallo-β-lactamase IMP-8. Phylogenetic analysis on the basis of a non-recombinant core genome that contained 22 outbreak-specific SNPs revealed a pattern of four dominant clades with a strong phylogeographic structure and allowed us to determine the potential temporal origin of the outbreak to July 2008, 1 year before the index case was diagnosed. Superspreaders at the root of clades exhibited a high number of probable and predicted transmissions, indicating their exceptional position in the outbreak. CONCLUSIONS: Our results suggest that the initial expansion of dominant sublineages was driven by a few superspreaders, while environmental contamination seemed to sustain the outbreak for a long period despite regular environmental control measures.This work was supported by a grant from ‘la Caixa’ to L.Z