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It has been hypothesized that, in aggregate, rare variants in coding regions of genes explain a substantial fraction of the heritability of common diseases. We sequenced the exomes of 1,000 Danish cases with common forms of type 2 diabetes (including body mass index > 27.5 kg/m2 and hypertension) and 1,000 healthy controls to an average depth of 56×. Our simulations suggest that our study had the statistical power to detect at least one causal gene (a gene containing causal mutations) if the heritability of these common diseases was explained by rare variants in the coding regions of a limited number of genes. We applied a series of gene-based tests to detect such susceptibility genes. However, no gene showed a significant association with disease risk after we corrected for the number of genes analyzed. Thus, we could reject a model for the genetic architecture of type 2 diabetes where rare nonsynonymous variants clustered in a modest number of genes (fewer than 20) are responsible for the majority of disease risk. © 2013 The American Society of Human Genetics.

Abstract Background Pseudomonas syringae is a widespread bacterial plant pathogen, and strains of P. syringae may be assigned to different pathovars based on host specificity among different plant species. The genomes of P. syringae pv. syringae (Psy) B728a, pv. tomato (Pto) DC3000 and pv. phaseolicola (Pph) 1448A have been recently sequenced providing a major resource for comparative genomic analysis. A mechanism commonly found in bacteria for signal transduction is the two-component system (TCS), which typically consists of a sensor histidine kinase (HK) and a response regulator (RR). P. syringae requires a complex array of TCS proteins to cope with diverse plant hosts, host responses, and environmental conditions. Results Based on the genomic data, pattern searches with Hidden Markov Model (HMM) profiles have been used to identify putative HKs and RRs. The genomes of Psy B728a, Pto DC3000 and Pph 1448A were found to contain a large number of genes encoding TCS proteins, and a core of complete TCS proteins were shared between these genomes: 30 putative TCS clusters, 11 orphan HKs, 33 orphan RRs, and 16 hybrid HKs. A close analysis of the distribution of genes encoding TCS proteins revealed important differences in TCS proteins among the three P. syringae pathovars. Conclusion In this article we present a thorough analysis of the identification and distribution of TCS proteins among the sequenced genomes of P. syringae. We have identified differences in TCS proteins among the three P. syringae pathovars that may contribute to their diverse host ranges and association with plant hosts. The identification and analysis of the repertoire of TCS proteins in the genomes of P. syringae pathovars constitute a basis for future functional genomic studies of the signal transduction pathways in this important bacterial phytopathogen.

We present the case of a 58-year-old female patient who presented with an extramedullary B-ALL relapse after prior allogenic HSCT and blinatumomab therapy. The patient died from complications of a drug-induced acute liver failure after a salvage therapy combining inotuzumab ozogamicin (InO)-based induction followed by consolidation with high dose MTX and pegaspargase based on the GMALL protocol for older ALL patients. After a diagnosis of the extramedullary relapse in the form of a retro vesical chloroma, the patient received an individualized multi-agent chemotherapy based on induction chemotherapy for older patients in combination with InO. After four administrations of InO, in combination with vincristine, dexamethasone, cytarabine, and cyclophosphamide, CT-imaging showed a reduction in volume of the chloroma and response to therapy. Consolidation with high-dose methotrexate and pegaspargase was administered. The patient developed toxic liver damage manifested by hyperbilirubinemia and progressive hepatic encephalopathy. The diagnostic criteria for VOD were met, and therapy with defibrotide was initiated. Liver biopsy revealed no histological signs of VOD but instead steatohepatitis indicative of drug-induced toxicity. The patient ultimately died of hemorrhagic shock through postinterventional hemorrhage after liver biopsy. In conclusion, although InO shows promising results in the therapy of r/r ALL with and without additional chemotherapy, the combination with MTX and pegaspargase in an intensively pretreated patient with relapse after HCST may impart an increased risk for liver-related toxicity. Special caution is required when assessing fitness for further liver toxic regimens. A key takeaway is also the reminder that InO can cause liver damage not only in the form of VOD but also through direct hepatocellular toxicity., (© 2023. The Author(s).)

Introduction: Endobronchial ultrasound-guided transbronchial needle aspiration remains the gold standard for the diagnosis of mediastinal pathologies. Its greatest limitation has been the low diagnostic yield in lymphoproliferative disorders as well as insufficient samples for molecular testing. Transesophageal examinations using an EBUS scope have helped increase the diagnostic yield by allowing for additional biopsies of paraesophageal and intra-abdominal lesions. Similarly, the novel approach of transbronchial mediastinal cryobiopsy has further increased the yield by providing larger and better-preserved biopsies. Both complimentary techniques have shown great individual feasibility and safety. However, the feasibility of transesophageal cryobiopsies in the evaluation of mediastinal pathologies remains a subject of debate., Aim: The aim of the study was to investigate the safety and feasibility of transesophageal EBUS-guided mediastinal cryobiopsies performed at our center., Methods: We conducted a retrospective review of 30 patients who underwent mediastinal cryobiopsy through the esophagus at our institution between October 2023 and March 2024. Data on patient demographics, diagnostic yield, and complications were collected and analyzed., Results: The mean patient age was 43 years, with a gender distribution of 60% male and 40% female. The primary indications included suspicion of lymphoproliferative disorders, suspected sarcoidosis, and malignancies with paraesophageal lesions. The overall diagnostic yield was 93%. No major complications were noted in any of the patients., Conclusion: Transesophageal mediastinal cryobiopsy appears to be a promising complimentary technique for mediastinal evaluation with a relatively high diagnostic yield and favorable safety profile. However, further studies with larger cohorts are warranted to validate the findings at our institution., (© 2024 S. Karger AG, Basel.)

Recurrent urinary tract infection (rUTI) remains a major problem for many women and therefore the pursuit for genomic and phenotypic traits which could define rUTI has been ongoing. The present study applied a genomic approach to investigate recurrent urinary tract infections by comparative analyses of recurrent and non-recurrent Escherichia coli isolates from general practice. From whole-genome sequencing data, phylogenetic clustering and genomic traits were studied on a collection of isolates which caused recurrent infection compared to non-recurrent isolates. In addition, genomic variation between the 1st and following infection was studied on a subset of the isolates. Evidence of limited adaptation between the recurrent infections based on single nucleotide polymorphism analyses with a range of 0-13 non-synonymous single nucleotide polymorphisms (SNPs) between the paired isolates. This included an overrepresentation of SNPs in metabolism genes. We identified several genes which were more common in rUTI isolates, including nine fimbrial genes, however, not significantly after false-discovery rate. Finally, the results show that recurrent isolates of the present dataset are not distinctive by variation in the core genome, and thus, did not cluster distinct from non-rUTI isolates in a SNP phylogeny.

Campylobacter jejuni is recognised as the leading cause of bacterial gastroenteritis in industrialised countries. Although the majority of Campylobacter infections are self-limiting, antimicrobial treatment is necessary in severe cases. Therefore, the development of antimicrobial resistance (AMR) in Campylobacter is a growing public health challenge and surveillance of AMR is important for bacterial disease control. The aim of this study was to predict antimicrobial resistance in C. jejuni from whole-genome sequencing data. A total of 516 clinical C. jejuni isolates collected between 2014 and 2017 were subjected to WGS. Resistance phenotypes were determined by standard broth dilution, categorising isolates as either susceptible or resistant based on epidemiological cutoffs for six antimicrobials: ciprofloxacin, nalidixic acid, erythromycin, gentamicin, streptomycin, and tetracycline. Resistance genotypes were identified using an in-house database containing reference genes with known point mutations and the presence of resistance genes was determined using the ResFinder database and four bioinformatical methods (modified KMA, ABRicate, ARIBA, and ResFinder Batch Upload). We identified seven resistance genes including tet(O), tet(O/32/O), ant(6)-Ia, aph(2″)-If, blaOXA, aph(3')-III, and cat as well as mutations in three genes: gyrA, 23S rRNA, and rpsL. There was a high correlation between phenotypic resistance and the presence of known resistance genes and/or point mutations. A correlation above 98% was seen for all antimicrobials except streptomycin with a correlation of 92%. In conclusion, we found that WGS can predict antimicrobial resistance with a high degree of accuracy and have the potential to be a powerful tool for AMR surveillance.

There is an increasing concern about the role of animals as reservoirs of Clostridioides difficile. In this study, we investigated prevalence, antimicrobial resistance and zoonotic potential of C. difficile in dogs. Two-hundred and twenty-five dog faecal deposits were collected from trashcans in nine public gardens. C. difficile was isolated using selective plating and enrichment culture, identified by MALDI-TOF, tested for susceptibility to seven antibiotics by E-test, and sequenced on an Illumina NextSeq platform. Genome sequences were analysed to determine multilocus sequence types and resistance and toxin gene profiles. Zoonotic potential was assessed by measuring genetic variations of core genome (cg)MLST types between canine isolates and 216 temporally and spatially related human clinical isolates from a national database. C. difficile was isolated from 11 samples (4.9%). Seven isolates were toxigenic (tcdA+, tcdB+, cdtA/B-) and belonged to the sequence types ST2, ST6, ST10 and ST42. The four non-toxigenic isolates were assigned to ST15, ST26 and one novel ST. ST2, corresponding to PCR ribotype RT014/020, was the dominating lineage (n = 4) and, together with ST26 and ST42 isolates, showed close resemblance to human isolates, i.e. 2-5 allelic differences among the 1999 genes analysed by cgMLST. Three non-toxigenic isolates displayed resistance to clindamycin, erythromycin and tetracycline mediated by erm(B) and tet(M). Resistance to metronidazole, moxifloxacine, rifampicin or vancomycin was not detected. In conclusion, a small proportion of faecal deposits contained toxigenic C. difficile such as ST2 (RT014/020), which is a major cause of community-acquired infections. Our finding suggests that pathogenic strains can be exchanged between dogs and humans., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest related to this study., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Despite phages' ubiquitous presence and great importance in shaping microbial communities, little is known about the diversity of specific phages in different ecological niches. Here, we isolated, sequenced, and characterized 38 Escherichia coli-infecting phages (coliphages) from poultry faeces to gain a better understanding of the coliphage diversity in the poultry intestine. All phages belonged to either the Siphoviridae or Myoviridae family and their genomes ranged between 44,324 and 173,384 bp, with a G+C content between 35.5 and 46.4%. Phylogenetic analysis was performed based on single "marker" genes; the terminase large subunit, portal protein, and exonucleases, as well as the full draft genomes. Single gene analysis resulted in six distinct clusters. Only minor differences were observed between the different phylogenetic analyses, including branch lengths and additional duplicate or triplicate subclustering. Cluster formation was according to genome size, G+C content and phage subfamily. Phylogenetic analysis based on the full genomes supported these clusters. Moreover, several of our Siphoviridae phages might represent a novel unclassified phage genus. This study allowed for identification of several novel coliphages and provides new insights to the coliphage diversity in the intestine of poultry. Great diversity was observed amongst the phages, while they were isolated from an otherwise similar ecosystem.

In industrialized countries, the leading cause of bacterial gastroenteritis is Campylobacter jejuni. However, outbreaks are rarely reported, which may reflect limitations of surveillance, for which molecular typing is not routinely performed. To determine the frequency of genetic clusters among patients and to find links to concurrent isolates from poultry meat, broiler chickens, cattle, pigs, and dogs, we performed whole-genome sequencing on 1,509 C. jejuni isolates from 774 patients and 735 food or animal sources in Denmark during 2015-2017. We found numerous clusters; 366/774 (47.3%) clinical isolates formed 104 clusters of >2 isolates. A total of 41 patient clusters representing 199/366 (54%) patients matched a potential source, primarily domestic chickens/broilers. This study revealed serial outbreaks and numerous matches to concurrent food and animal isolates and highlighted the potential of whole-genome sequencing for improving routine surveillance of C. jejuni by enhancing outbreak detection, source tracing, and potentially prevention of human infections.

Background: Salmonella Infantis (S. Infantis) is one of the most frequent Salmonella serovars isolated from human cases of salmonellosis and the most detected serovar from animal and food sources in Europe. The serovar is commonly associated with poultry and there is increasing concern over multidrug resistant clones spreading worldwide, as the dominating clones are characterized by presence of large plasmids carrying multiple resistance genes. Increasing the knowledge of the S. Infantis population and evolution is important for understanding and preventing further spread. In this study, we analysed a collection of strains representing different decades, sources and geographic locations. We analysed the population structure and the accessory genome, in particular we identified prophages with a view to understand the role of prophages in relation to the evolution of this serovar., Results: We sequenced a global collection of 100 S. Infantis strains. A core-genome SNP analysis separated five strains in e-Burst Group (eBG) 297 with a long branch. The remaining strains, all in eBG31, were divided into three lineages that were estimated to have separated approximately 150 years ago. One lineage contained the vast majority of strains. In five of six clusters, no obvious correlation with source or geographical locations was seen. However, one cluster contained mostly strains from human and avian sources, indicating a clone with preference for these sources. The majority of strains within this cluster harboured a pESI-like plasmid with multiple resistance genes. Another lineage contained three genetic clusters with more rarely isolated strains of mainly animal origin, possibly less sampled or less infectious clones. Conserved prophages were identified in all strains, likely representing bacteriophages which integrated into the chromosome of a common ancestor to S. Infantis. We also saw that some prophages were specific to clusters and were probably introduced when the clusters were formed., Conclusions: This study analysed a global S. Infantis population and described its genetic structure. We hypothesize that the population has evolved in three separate lineages, with one more successfully emerging lineage. We furthermore detected conserved prophages present in the entire population and cluster specific prophages, which probably shaped the population structure.

We present the LiSEQ (Listeria SEQuencing) project, funded by the European Food Safety Agency (EFSA) to compare Listeria monocytogenes isolates collected in the European Union from ready-to-eat foods, compartments along the food chain (e.g. food-producing animals, food-processing environments) and humans. In this article, we report the molecular characterization of a selection of this data set employing whole-genome sequencing analysis. We present an overview of the strain diversity observed in different sampled sources, and characterize the isolates based on their virulence and resistance profile. We integrate into our analysis the global L. monocytogenes genome collection described by Moura and colleagues in 2016 to assess the representativeness of the LiSEQ collection in the context of known L. monocytogenes strain diversity.

Avian-pathogenic Escherichia coli (APEC) is a subgroup of extraintestinal pathogenic E. coli (ExPEC) presumed to be zoonotic and to represent an external reservoir for extraintestinal infections in humans, including uropathogenic E. coli (UPEC) causing urinary tract infections. Comparative genomics has previously been applied to investigate whether APEC and human ExPEC are distinct entities. Even so, whole-genome-based studies are limited, and large-scale comparisons focused on single sequence types (STs) are not available yet. In this study, comparative genomic analysis was performed on 323 APEC and human ExPEC genomes belonging to sequence type 95 (ST95) to investigate whether APEC and human ExPEC are distinct entities. Our study showed that APEC of ST95 did not constitute a unique ExPEC branch and was genetically diverse. A large genetic overlap between APEC and certain human ExPEC was observed, with APEC located on multiple branches together with closely related human ExPEC, including nearly identical APEC and human ExPEC. These results illustrate that certain ExPEC clones may indeed have the potential to cause infection in both poultry and humans. Previously described ExPEC-associated genes were found to be encoded on ColV plasmids. These virulence-associated plasmids seem to be crucial for ExPEC strains to cause avian colibacillosis and are strongly associated with strains of the mixed APEC/human ExPEC clusters. The phylogenetic analysis revealed two distinct branches consisting of exclusively closely related human ExPEC which did not carry the virulence-associated plasmids, emphasizing a lower avian virulence potential of human ExPEC in relation to an avian host. IMPORTANCE APEC causes a range of infections in poultry, collectively called colibacillosis, and is the leading cause of mortality and is associated with major economic significance in the poultry industry. A growing number of studies have suggested APEC as an external reservoir of human ExPEC, including UPEC, which is a reservoir. ExPEC belonging to ST95 is considered one of the most important pathogens in both poultry and humans. This study is the first in-depth whole-genome-based comparison of ST95 E. coli which investigates both the core genomes as well as the accessory genomes of avian and human ExPEC. We demonstrated that multiple lineages of ExPEC belonging to ST95 exist, of which the majority may cause infection in humans, while only part of the ST95 cluster seem to be avian pathogenic. These findings further support the idea that urinary tract infections may be a zoonotic infection., (Copyright © 2019 Jørgensen et al.)

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is a subtype of Hodgkin lymphoma that frequently shows a nodal growth pattern with abundant reactive B cells in the microenvironment. Early NLPHL cases can be particularly difficult to differentiate from progressively transformed germinal centers (PTGC). Since PTGC have been described to be IgG4 associated in a relatively high proportion of cases, the aim of the present study was to determine if IgG4 immunostaining can be helpful in the differential diagnosis between NLPHL and PTGC. We furthermore aimed to learn if LP cells can express IgG4. For this purpose, 58 cases of PTGC and 56 cases of NLPHL were assessed using IgG4 immunostaining. We could confirm that a significant number of PTGC cases showed high numbers of IgG4-positive plasma cells (22/58, 38%), whereas hot spot areas of IgG4-positive plasma cells were not found in any of the NLPHL cases. In lymph node areas with the differential diagnosis of NLPHL and PTGC, IgG4 immunostaining can therefore provide a helpful diagnostic tool to rule out NLPHL when a high number of IgG4-positive plasma cells are encountered. We also assessed 13 cases with a combination of NLPHL and PTGC in the same lymph node. Five of these cases presented hot spot areas of IgG4-positive plasma cells in the PTGC regions, while no significant numbers of IgG4-positive plasma cells were observed in the NLPHL part of the lymph node. LP cells were never IgG4 positive. Furthermore, immunoglobulin heavy chain rearrangements of single IgG4-positive plasma cells were analyzed, revealing a polyclonal plasma cell population. In summary, our data suggest that IgG4 immunostaining can provide additional information in the diagnostic workup of cases with the differential diagnosis of NLPHL and PTGC. IgG4's inefficiency in clearing antigens may explain why lymph nodes with PTGC are usually strongly enlarged and develop a high number of hyperplastic germinal centers. Polyclonal immunoglobulin heavy chain rearrangements in IgG4-positive plasma cells further support the hypothesis that PTGC represent a misled immune reaction.

The faecal flora is a common reservoir for urinary tract infection (UTI), and Escherichia coli (E. coli) is frequently found in this reservoir without causing extraintestinal infection. We investigated these E. coli reservoirs by whole-genome sequencing a large collection of E. coli from healthy controls (faecal), who had never previously had UTI, and from UTI patients (faecal and urinary) sampled from the same geographical area. We compared MLST types, phylogenetic relationship, accessory genome content and FimH type between patient and control faecal isolates as well as between UTI and faecal-only isolates, respectively. Comparison of the accessory genome of UTI isolates to faecal isolates revealed 35 gene families which were significantly more prevalent in the UTI isolates compared to the faecal isolates, although none of these were unique to one of the two groups. Of these 35, 22 belonged to a genomic island and three putatively belonged to a type VI secretion system (T6SS). MLST types and SNP phylogeny indicated no clustering of the UTI or faecal E. coli from patients distinct from the control faecal isolates, although there was an overrepresentation of UTI isolates belonging to clonal lineages CC73 and CC12. One combination of mutations in FimH, N70S/S78N, was significantly associated to UTI, while phylogenetic analysis of FimH and fimH identified no signs of distinct adaptation of UTI isolates compared to faecal-only isolates not causing UTI. In summary, the results showed that (i) healthy women who had never previously had UTI carried faecal E. coli which were overall closely related to UTI and faecal isolates from UTI patients; (ii) UTI isolates do not cluster separately from faecal-only isolates based on SNP analysis; and (iii) 22 gene families of a genomic island, putative T6SS proteins as well as specific metabolism and virulence associated proteins were significantly more common in UTI isolates compared to faecal-only isolates and (iv) evolution of fimH for these isolates was not linked to the clinical source of the isolates, apart from the mutation combination N70S/S78N, which was correlated to UTI isolates of phylogroup B2. Combined, these findings illustrate that faecal and UTI isolates, as well as faecal-only and faecal-UTI isolates, are closely related and can only be distinguished, if at all, by their accessory genome., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

In microbial food safety, molecular methods such as quantitative PCR (qPCR) and next-generation sequencing (NGS) of bacterial isolates can potentially be replaced by diagnostic shotgun metagenomics. However, the methods for pre-analytical sample preparation are often optimized for qPCR, and do not necessarily perform equally well for qPCR and sequencing. The present study investigates, through screening of methods, whether qPCR can be used as an indicator for the optimization of sample preparation for NGS-based shotgun metagenomics with a diagnostic focus. This was used on human fecal samples spiked with 10³ or 10⁶ colony-forming units (CFU)/g Campylobacter jejuni , as well as porcine fecal samples spiked with 10³ or 10⁶ CFU/g Salmonella typhimurium . DNA was extracted from the samples using variations of two widely used kits. The following quality parameters were measured: DNA concentration, qPCR, DNA fragmentation during library preparation, amount of DNA available for sequencing, amount of sequencing data, distribution of data between samples in a batch, and data insert size; none showed any correlation with the target ratio of the spiking organism detected in sequencing data. Surprisingly, diagnostic metagenomics can have better detection sensitivity than qPCR for samples spiked with 10³ CFU/g C. jejuni . The study also showed that qPCR and sequencing results may be different due to inhibition in one of the methods. In conclusion, qPCR cannot uncritically be used as an indicator for the optimization of sample preparation for diagnostic metagenomics., Competing Interests: The authors declare no conflict of interest.

Most Staphylococcus aureus isolates can cause invasive disease given the right circumstances, but it is unknown if some isolates are more likely to cause severe infections than others. S. aureus bloodstream isolates from 120 patients with definite infective endocarditis and 121 with S. aureus bacteraemia without infective endocarditis underwent whole-genome sequencing. Genome-wide association analysis was performed using a variety of bioinformatics approaches including SNP analysis, accessory genome analysis and k-mer based analysis. Core and accessory genome analyses found no association with either of the two clinical groups. In this study, the genome sequences of S. aureus bloodstream isolates did not discriminate between bacteraemia and infective endocarditis. Based on our study and the current literature, it is not convincing that a specific S. aureus genotype is clearly associated to infective endocarditis in patients with S. aureus bacteraemia.

This report describes one Salmonella isolate harbouring both mcr-1 and mcr-3. We also found nine other Salmonella isolates positive for the plasmid-borne colistin resistance gene, mcr-3. The strains were isolated from patients in Denmark between 2009 and 2017 and five of the patients had travelled to Asia. In addition to mcr-3, all strains were found positive for bla TEM-1 , strA, strB, sul2 and tet(A) or tet(B), and most strains were positive for bla CTX-M-55 and qnrS., (This article is copyright of The Authors, 2017.)

Background: The development of diagnostic metagenomics is driven by the need for universal, culture-independent methods for detection and characterization of pathogens to substitute the time-consuming, organism-specific, and often culture-based laboratory procedures for epidemiological source-tracing. Some of the challenges in diagnostic metagenomics are, that it requires a great next-generation sequencing depth and unautomated data analysis., Results: DNA from human fecal samples spiked with 7.75 × 10 1 -7.75 × 10 7 colony forming unit (CFU)/ml Campylobacter jejuni and chicken fecal samples spiked with 1 × 10 2 -1 × 10 6  CFU/g Campylobacter jejuni was sequenced and data analysis was done by the metagenomic tools Kraken and CLARK. More hits were obtained at higher spiking levels, however with no significant linear correlations (human samples p = 0.12, chicken samples p = 0.10). Therefore, no definite detection limit could be determined, but the lowest spiking levels found positive were 7.75 × 10 4  CFU/ml in human feces and 10 3  CFU/g in chicken feces. Eight human clinical fecal samples with estimated Campylobacter infection loads from 9.2 × 10 4 -1.0 × 10 9  CFU/ml were analyzed using the same methods. It was possible to detect Campylobacter in all the clinical samples., Conclusions: Sensitivity in diagnostic metagenomics is improving and has reached a clinically relevant level. There are still challenges to overcome before real-time diagnostic metagenomics can replace quantitative polymerase chain reaction (qPCR) or culture-based surveillance and diagnostics, but it is a promising new technology.

Background: Listeriosis is a serious foodborne infection. Outbreaks of listeriosis occur rarely, but have often proved difficult to solve. In June 2014, we detected and investigated a listeriosis outbreak in Denmark using patient interviews and whole-genome sequencing (WGS)., Methods: We performed WGS on Listeria monocytogenes isolates from patients and available isolates from ready-to-eat foods and compared them using single-nucleotide polymorphism (SNP) analysis. Case patients had L. monocytogenes with ≤3 SNPs (the outbreak strain) isolated in September 2013-December 2014. Through interviews, we established case patients' food and clinical histories. Food production facilities were inspected and sampled, and we performed trace-back/trace-forward of food delivery chains., Results: In total, 41 cases were identified; 17 deaths occurred (41%). An isolate from a delicatessen meat (spiced meat roll) from company A was identical to the outbreak strain. Half of the patients were infected while hospitalized/institutionalized; institutions were supplied food by company A. The outbreak strain was repeatedly isolated from further samples taken within this company and within companies in its distribution chain. Products from company A were traced and recalled from >6000 food establishments, after which the outbreak ended., Conclusions: Ready-to-eat spiced meat roll from a single production facility caused this outbreak. The product, served sliced and cold, is popular among the elderly; serving it at hospitals probably contributed to the high case-fatality rate. WGS used for patient isolates and isolates from food control inspections, coupled with routine epidemiological follow-up, was instrumental in swiftly locating the source of infections, preventing further illnesses and deaths., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Denmark has a high incidence of invasive listeriosis (0.9 cases/100,000 population in 2012). We analyzed patient data, clinical outcome, and trends in pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of Listeria monocytogenes strains isolated in Denmark during 2002-2012. We performed 2-enzyme PFGE and serotyping on 559 isolates and MLST on 92 isolates and identified some correlation between molecular type and clinical outcome and patient characteristics. We found 178 different PFGE types, but isolates from 122 cases belonged to just 2 closely related PFGE types, clonal complex 8 and sequence type 8. These 2 types were the main cause of a peak in incidence of invasive listeriosis during 2005-2009, possibly representing an outbreak or the presence of a highly prevalent clone. However, current typing methods could not fully confirm these possibilities, highlighting the need for more refined discriminatory typing methods to identify outbreaks within frequently occurring L. monocytogenes PFGE types.

Unlabelled: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations., Importance: With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA., (Copyright © 2014 Stegger et al.)

It has been hypothesized that, in aggregate, rare variants in coding regions of genes explain a substantial fraction of the heritability of common diseases. We sequenced the exomes of 1,000 Danish cases with common forms of type 2 diabetes (including body mass index > 27.5 kg/m(2) and hypertension) and 1,000 healthy controls to an average depth of 56×. Our simulations suggest that our study had the statistical power to detect at least one causal gene (a gene containing causal mutations) if the heritability of these common diseases was explained by rare variants in the coding regions of a limited number of genes. We applied a series of gene-based tests to detect such susceptibility genes. However, no gene showed a significant association with disease risk after we corrected for the number of genes analyzed. Thus, we could reject a model for the genetic architecture of type 2 diabetes where rare nonsynonymous variants clustered in a modest number of genes (fewer than 20) are responsible for the majority of disease risk., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Staphylococcus aureus ST291 has been reported as a homologue recombinant double locus variant of the livestock associated S. aureus ST398. However, whole genome sequencing show that ST291 is a unique genetic lineage with highly variable content within its accessory genome compared to both human and livestock associated genome sequenced CC398s.

Nearly 20 years of experience at Severance Hospital has shown that utilizing exchange donors increases the donor pool safely, with outcomes comparable to living related donor grafts. The exchange donor program is invaluable for incompatible donor-recipient pairs to consecutively proceed to transplantation. Recently, newer desensitization protocols have been devised to approach incompatible donor-recipient pairs, but not without risks. These desensitization protocols may be an alternative when confronting the limitations in the exchange program. Therefore, the exchange program and the desensitization protocols should be complementary, not competing strategies and centers should weigh the merits and limitations of each protocol in each incompatible donor-recipient pair to select the optimal method for a safe and successful transplantation.

Exchange donor programs and desensitization protocols are available options for ABO-incompatible and positive cross-match living donor kidney transplantations. Patient and donor blood types, the degree of sensitization, and DSA titers should be considered to achieve successful kidney transplantation in recipients with incompatible living donors. Highly sensitized blood type O patients with ABO-incompatible living donors, or highly sensitized patients with a positive cross-match (with low DSA titer) to their AB donors should be considered for a desensitization protocol as a first priority. Narrowly sensitized patients with a positive cross-match (and high DSA titer) should be considered for an exchange donor program if possible. Patients with positive cross-match O donors can be easily matched and benefit from an exchange program. Exchange donor programs and desensitization protocols are complementary strategies to overcome HLA and ABO antibody barriers.

Background: The shortage of donor organs is one of the major barriers to transplantation worldwide. After the success of the direct exchange donor (swap) program in Korea since 1991, we have developed a swap-around program. However, reports on the long-term outcomes of exchange donor programs are scarce., Methods: From September 1995 to September 2006, we performed 1193 cases of renal transplantation, including 398 cases from living-unrelated donors. The living-unrelated donors included 129 exchange donors and 269 nonexchange donors. We compared the outcomes of the exchange program with that of the nonexchange program, and examined the merits and limitations of the exchange program., Results: The reasons for the exchange program were ABO incompatibility (n=84, 65.1%), human leukocyte antigen mismatching beyond our criteria (n=39, 30.2%), or positive lymphocyte crossmatch (n=6, 4.7%). The overall 10-year graft survival (86.3%) of exchange transplantation was comparable with that of nonexchange (82.3%) or one- haplotype matched living-related (81.2%) transplantation (P=0.2994). In multivariate analysis, exchange versus nonexchange donors did not affect graft survival. The proportion of blood-type O donors was much lower in the exchange group (29.5%) than in the nonexchange group (42.4%; P=0.026). Blood-type O kidneys were preferentially allocated to blood-type O recipients (78.9%) in the exchange group as compared with the nonexchange group (54.4%; P=0.007)., Conclusion: We achieved excellent outcomes by using a donor exchange program as an option to reduce the donor organ shortage. However, the exchange donor program has no added benefit for blood-type O recipients.

We describe the design and evaluate the use of a high-density oligonucleotide microarray covering seven sequenced Escherichia coli genomes in addition to several sequenced E. coli plasmids, bacteriophages, pathogenicity islands, and virulence genes. Its utility is demonstrated for comparative genomic profiling of two unsequenced strains, O175:H16 D1 and O157:H7 3538 (Deltastx(2)::cat) as well as two well-known control strains, K-12 W3110 and O157:H7 EDL933. By using fluorescently labeled genomic DNA to query the microarrays and subsequently analyze common virulence genes and phage elements and perform whole-genome comparisons, we observed that O175:H16 D1 is a K-12-like strain and confirmed that its phi3538 (Deltastx(2)::cat) phage element originated from the E. coli 3538 (Deltastx(2)::cat) strain, with which it shares a substantial proportion of phage elements. Moreover, a number of genes involved in DNA transfer and recombination was identified in both new strains, providing a likely explanation for their capability to transfer phi3538 (Deltastx(2)::cat) between them. Analyses of control samples demonstrated that results using our custom-designed microarray were representative of the true biology, e.g., by confirming the presence of all known chromosomal phage elements as well as 98.8 and 97.7% of queried chromosomal genes for the two control strains. Finally, we demonstrate that use of spatial information, in terms of the physical chromosomal locations of probes, improves the analysis.

Introduction: Upon analysis of the risk factors affecting renal graft survival and function, the time-dependent effects of each risk factor should be differentiated from their net effects. To evaluate the chronologically different impacts of risk factors on graft renal function, we reviewed 390 recipients who received a kidney from 1-haplotype-matched living-related donors., Materials and Methods: Until 5-yr post-transplantation (TX), yearly serum creatinine (Scr), 24-h urinary excretion of protein, and their yearly changes were compared by the episodes of acute rejection within 1 yr, the kidney weight to recipient body weight (KW/BW) ratio, the donor/recipient (D/R) age ratio, and the D/R gender pairing. The Kaplan-Meier method, Cox proportional hazard model, ANOVA, and repeated measures ANOVA were each applied for different purposes., Results: Only the episodes of acute rejection were a significant risk factor affecting graft survival. The episodes of acute rejection, KW/BW ratio, D/R age ratio, and D/R gender pairing consistently and independently had significant influences on Scr. Recipients having the lowest KW/BW ratio (first quartile) or the highest D/R age ratio (fourth quartile) had rapid increments of Scr after 4-yr post-TX. After 3-yr post-TX, there were significant correlations between the number of non-immunologic risk factors present and the yearly changes in Scr., Conclusions: Non-immunologic factors had a detrimental effect on renal graft function, especially after 3-yr post-TX. If immunologic risks seem to be similar, size matching, age, and gender pairing should be considered for better long-term graft function in renal TX recipients.

Genome analyses of the plant pathogens Pseudomonas syringae pv. tomato DC3000, pv. syringae B728a and pv. phaseolicola 1448A reveal fewer extracytoplasmic function (ECF) sigma factors than in related Pseudomonads with different lifestyles. We highlight the presence of a P. syringae-specific ECF sigma factor that is an interesting target for future studies because of its potential role in the adaptation of P. syringae to its specialized phytopathogenic lifestyle.

We have devised a new surgical method of video-assisted minilaparotomy surgery-live donor nephrectomy (VAMS-LDN), which is a hybridized form of laparoscopic and open surgeries that combines the advantages of both. We present the findings of our series of 239 consecutive patients. Since 1993 we have performed 239 successful VAMS-LND. All 239 healthy kidney donors' characteristics and their postoperative courses were retrospectively reviewed and the data were compared to 95 open donor nephrectomies performed during the same period. The mean age and weight of the patients were 37.9 +/- 11.0 years and 62.4 +/- 7.9 kg, respectively. The mean operating time was 154 +/- 41 minutes, which was similar to open donor nephrectomy but shorter than laparoscopic donor nephrectomy. There were no major intraoperative complications except two tears to lumbar veins which required transfusion. The mean warm ischemic time was 2.1 +/- 0.7 minutes, which was equal to open donor nephrectomy. The patients experienced less postoperative pain and recovered quicker than the open donor nephrectomy patients. VAMS-LDN is a safe and minimally invasive technique for live donor nephrectomy, incorporating advantages of both conventional open and laparoscopic methods. We suggest that VAMS-LDN is a viable option for living donor kidney transplantation.

Vascular smooth muscle cell (VSMC) proliferation is the major pathologic feature associated with chronic allograft nephropathy, and mycophenolic acid (MPA) inhibits VSMC proliferation. Since the role of inosine monophosphate dehydrogenase (IMPDH)-dependent de novo guanosine synthesis is limited in VSMCs, we examined the effects of MPA on platelet-derived growth factor (PDGF)-induced cellular ROS and mitogen-activated protein kinases (MAPK) activation in VSMCs. Primary cultured rat VSMCs were stimulated with PDGF-BB in the presence or absence of MPA. Cell proliferation was assessed by [3H]-thymidine incorporation, ROS by flow cytometry and MAPK activation by Western blot analysis. PDGF increased cell proliferation, cellular ROS and extracellular-regulated protein kinase (ERK) 1/2 and p38 MAPK activation by 3.4-, 1.6-, 3.3- and 3.9-fold, respectively. MPA at above 1 muM inhibited PDGF-induced cellular ROS and ERK 1/2 and p38 MAPK activation, as well as proliferation. Structurally different anti-oxidants and inhibitor of ERK or p38 MAPK blocked PDGF-induced proliferation. Anti-oxidants also inhibited ERK 1/2 and p38 MAPK activation. Exogenous guanosine partially recovered the inhibitory effect of MPA on VSMC proliferation. These results suggest that MPA may inhibit PDGF-induced VSMC proliferation partially through inhibiting cellular ROS, and subsequent ERK 1/2 and p38 MAPK activation in addition to inhibiting IMPDH.

Information is limited regarding the histological features related to chronic graft dysfunction and failure in patients with IgA nephropathy developing after renal transplantation. The prevalence and significance of glomerular crescents in post-transplant IgAN including recurrent, de novo and transmitted forms (TxIgAN), were studied. Renal morphology was evaluated in 71 patients of TxIgAN, obtained at more than 6 months post-transplant, and compared with regard to the presence (C-TxIgAN) or absence (N-TxIgAN) of glomerular crescents. Crescents were demonstrated in 12 samples of 10 patients (14.1%). The percentages of crescents were from 4.8% to 83.3% (median, 28.6%) in each sample. Ten samples of C-TxIgAN had cellular to fibrocellular crescents, and four of these were associated with diffuse mesangial proliferation. Serum creatinine levels and the frequency of nephrotic range proteinuria at the time of biopsy and the degree of interstitial inflammation were significantly different in the two groups. Graft survival after allograft biopsies was significantly lower in C-TxIgAN (P = 0.0017). Chronic rejection was a major cause of graft loss in N-TxIgAN (31.8%), whereas TxIgAN was the major cause in C-TxIgAN (66.7%). In conclusion, the current study suggests that glomerular crescents are not rare and that they are responsible for chronic graft dysfunction in TxIgAN patients.

The assessment of the adenosine deaminase activity (ADA) in the pleural effusion is used for the diagnosis of tuberculous pleural effusion (TPE). To examine whether the procedure can be applied to immunocompromised patients, we analyzed the ADA activity in the pleural fluid of renal transplant recipients. We studied 23 renal transplant patients with TPE (21 men and 2 women; the mean age, 33 years). They were treated at the Yonsei University Hospital between January 1985 and December 2001. Patients with granuloma in the pleural biopsy specimen or positive for Mycobacterium tuberculosis in the pleural fluid culture were recruited. The ADA activity in the pleural effusion of 23 renal transplant patients with TPE was compared with 23 immunocompetent patients with TPE. The mean ADA activity was 69.5 +/- 4.6 in renal transplant patients and 65.0 +/- 4.9 U/L in immunocompetent patients. Applying the 40 U/L cut-off point, the positivity of ADA was 91.3% in renal transplant patients, and 86.9% in immunocompetent patients. We thus concluded that the measurement of ADA in the pleural fluid is a useful means in the diagnosis of TPE in renal transplant patients.

Posttransplant lymphoproliferative disorder (PTLD) is a serious complication of organ transplantation. PTLD is the disorder arising from the combined effects of Epstein-Barr virus associated lymphoid proliferation with the disruption of the normal immune control by the cytotoxic T cells. The treatment for PTLD is one of the most controversial topics in solid organ transplantation. It is well known that the initial management of PTLD is a reduction of immunosuppression. Early diagnosis and the early reduction in immunosuppression are essential even for monomorphic lymphoma. We report here on a case of the complete resolution of PTLD (diffuse large B cell lymphoma) which occurred after a drastic reduction of immunosuppression in a renal transplant recipient.

Background: Minimally invasive, living-donor nephrectomy (LDN) is an attractive procedure for the donor in kidney transplantation (KTx). Its advantages include better cosmesis, shorter hospital stay, and rapid recovery. The most commonly performed, minimally invasive nephrectomy is done laparoscopically. However, the technical challenges, a steep learning curve for the surgeon, the risk of impaired early graft function, and the high cost of the procedure, have prevented minimally invasive LDN from gaining wide acceptance. To overcome these problems, we have developed a new surgical procedure named video-assisted minilaparotomy (VAM) for LDN. VAM-LDN is performed entirely with a small retrieval incision. Moreover, it does not require the induction of pneumoperitoneum, thereby avoiding potential vascular and renal complications., Methods: We evaluated the outcome of transplant recipients receiving kidneys with the VAM-LDN procedure by retrospectively comparing the surgical outcomes of patients who underwent KTx with the conventional open nephrectomy (group I, n=82) and VAM-LDN (group II, n=70) procedures from March 1, 1997, to June 30, 2002, at our institution. We compared postoperative complications, patient and graft survival, and graft functions between these two groups during a 12-month follow-up period., Results: There were no differences in demographic data, ABO compatibility, degree of human leukocyte antigen matching, or method of immunosuppression between the two groups (P >0.05). No significant difference was observed in complications such as delayed graft function, acute rejection, ureter complication, graft failure, or patient's mortality. There was no difference in graft function between the two groups, as determined by serum creatinine level measured during the 12-month follow-up., Conclusion: The short-term recipient outcome was favorable in patients who underwent KTx with the VAM-LDN procedure.

Background/aims: Little is known of the characteristics of gastric adenocarcinoma after renal transplantation. This study was performed to find out the incidence and clinicopathological features of gastric adenocarcinoma after renal transplantation in an endemic area for gastric cancer., Methodology: Between April 1979 and March 2001, fourteen gastric adenocarcinoma patients (0.7%) out of 2000 renal transplant recipients in a single institute were retrospectively reviewed., Results: Gastric adenocarcinoma was identified in 14 recipients (8 males and 6 females; mean age 47.6 years) about 60 months after renal transplantation. Four patients had early gastric cancer; ten patients had cancer in an advanced stage. Eleven patients underwent surgical resection, while three with distant metastasis were treated symptomatically. There was no postoperative mortality. Seven patients survived without evidence of recurrence, whereas four died of recurrence and three of gastric cancer progression., Conclusions: Renal transplant recipients are at increased risk of gastric adenocarcinoma, the most common malignancy in Korea. With curative surgery, favorable prognosis can be anticipated when the diagnosis is made at an early stage. Regular endoscopic examination for early diagnosis is recommended during the follow-up period after renal transplantation in a gastric cancer endemic area.

Polyomavirus (PV) nephropathy is a rare cause of graft dysfunction, but it may accompany acute rejection (AR), resulting in complications with respect to its diagnosis and treatment. To examine the validity of tubulitis and inflammatory phenotype in the diagnosis of concurrent AR, we reviewed the renal histology of ten biopsy samples from nine patients with PV nephropathy, and the immunohistochemistry from eight samples. Tubulitis was present in seven patients and was associated with AR in six. The degrees of tubulitis and interstitial inflammation were higher in biopsy samples with AR than in those without, but the degree of tubulitis was not related to the degree of interstitial inflammation. Virally infected cells were rare in the samples with no, or mild, tubulitis, but did not increase with the degree of interstitial inflammation. Immuno-phenotyping of inflammatory cells did not show any T-cell dominance in AR: T cells were dominant over B cells in three of six samples with AR and both samples without AR. Although the degrees of tubulitis and interstitial inflammation were higher in the AR subjects, the presence of tubulitis or inflammatory phenotype was not helpful in the diagnosis of concurrent AR. Further studies will be required to find a better marker for coexisting AR in patients with PV nephropathy and to establish strategies for treatment.

Introduction: Although graft dysfunction has been increasingly reported in post-transplant IgA nephropathy (Tx-IgAN), intragraft morphological changes have been largely overlooked. We evaluated glomerular changes in Tx-IgAN to identify the histological features pertaining to significant proteinuria and therapeutic response to enalapril., Materials and Methods: Fifty-four renal allograft biopsies, diagnosed as Tx-IgAN at a median of 46 months after transplantation, were the subject of the study. In 10 patients, glomerular morphometry was performed. In 14 patients who have been treated with enalapril for more than 12 months, we correlated the therapeutic response to enalapril with allograft histology., Results: No uniform pattern was found in the glomeruli of Tx-IgAN. The glomerular mesangium was mostly indistinct. Interstitial fibrosis was negative or mild in 88.9%. By morphometry, the glomerular tuft areas and mesangial areas were significantly larger in Tx-IgAN than those of the normal native kidney (p < 0.05), but were not different from transplant cases without glomerulonephritis. Proteinuria of >/=1 g/24 h was correlated with glomerulosclerosis, interstitial fibrosis and interstitial inflammation at time of biopsy (p < 0.005). The presence of segmental sclerosis (SS) correlated well with the amount of 24-h proteinuria (p < 0.001). After treatment with enalapril, the amount of proteinuria reduced in 64.3%. Therapeutic response to enalapril tended to be less effective in patients having SS (28.6 versus 71.4%), but this finding did not reach a statistical significance., Conclusions: Significant proteinuria was associated with advanced chronic injury, especially with the presence of SS in Tx-IgAN, but anti-proteinuric effect of enalapril was not affected by graft histology. It remains to be clarified whether glomerular mesangial expansion plays a role in graft dysfunction in a subset of Tx-IgAN showing prominent mesangial changes.

1. Long-term graft survival has been markedly improved after the introduction of CsA microemulsion and FK-506 as our main immunosuppressants, and the use of triple maintenance immunosuppression including MMF in living-donor kidney transplantation (KTX) at Younsei University. 2. The risk factors affecting long-term graft survival in living-donor KTX were recipient and donor age, type of immunosuppression including regimen, presence of pretransplant diabetes or hepatitis B, and the development of acute rejection after KTX. 3. The long-term graft survival rate with living-unrelated donor KTX was comparable to that with HLA-haploidentical living-related donor KTX. 4. Because of the striking disparity between organ donation and the increasing demand for KTX, distant relatives, living unrelated donors (including swap donors) should be considered as an alternative approach to increasing the number of available donors when accompanied by a careful evaluation process. 5. We recommend the use of minimally invasive approaches to donor nephrectomy to increase the rate of living donor donation. 6. We recommend negative lymphocyte crossmatch conversion protocols for patients with a positive crossmatch against their potential living donor.

Background: We demonstrated that higher donor kidney weight-to-recipient body weight (KW/BW) ratio showed better graft function in acute rejection-free renal recipients., Methods: We investigated the impacts of KW/BW ratios on the graft function including acute rejection and donor's age in 259 live-donor renal recipients. Renal parameters were measured yearly. Correlations between the variables and each parameter were assessed by mixed regression and analysis of variance., Results: Renal function showed a positive correlation with the KW/BW ratio, but an inverse correlation with the rejection episodes and donor's age. The regression slope for serum creatinine or creatinine clearance by these covariants was consistent each year. On comparing the lower KW/BW ratios (<3.0) with higher ratios (> or =4.5), the higher was associated with better graft function. Increased donor's age was associated with worse graft function., Conclusions: KW/BW ratio, donor's age, and the number of acute rejections are independent covariants for graft function.

Purpose: There is still no reliable method of preventing or treating chronic rejection or transplant vascular sclerosis. A recent in vitro cell culture study showed that carvedilol significantly inhibited the proliferation of vascular smooth muscle cells (VSMCs); however, the effect of carvedilol in the presence of cyclosporine (CsA) has not yet been reported. Using in vitro cultured VSMCs, we measured the antiproliferative activity of carvedilol alone and in combination with CsA., Methods: Growth-arrested cultured VSMCs from the thoracic aorta of Sprague-Dawley rats were exposed to platelet-derived growth factor (PDGF)-BB, endothelin (ET)-I, and angiotensin (ANG)-II. Carvedilol (1 and 10 microM) and/or CsA (100 nM) were added as test drugs. DNA synthesis was assessed by measuring the incorporated [3H]thymidine activity, and the percentage of inhibition in the presence of the test drugs was determined., Results: Compared with the PDGF-stimulated control, DNA synthesis decreased significantly to 60.3% +/- 10.4% and 18.3% +/- 5.9% in the presence of 1 and 10 microM of carvedilol, respectively (P < 0.05, each). Carvedilol significantly inhibited the activity of VSMCs stimulated by ET-1 and ANG-II. The IC50 of carvedilol was 1-10 microM. CsA only inhibited VSMCs significantly in the PDGF-stimulated subgroup. The addition of CsA in the presence of carvedilol did not affect the inhibitory activity of carvedilol. The pattern of inhibition in the combined group was uniform and similar to that of the carvedilol alone group, regardless of the stimulator used., Conclusion: Carvedilol significantly inhibited the DNA synthesis of VSMCs regardless of the kind of stimulators examined, even in the presence of CsA. These results indicate that carvedilol has the unique potential to inhibit the development of transplant vascular sclerosis in hypertensive transplant recipients under CsA-based immunosuppression.