In acute and chronic inflammation, neutrophils and platelets, both of which promote monocyte recruitment, are often activated simultaneously. We investigated how secretory products of neutrophils and ...platelets synergize to enhance the recruitment of monocytes. We found that neutrophil-borne human neutrophil peptide 1 (HNP1, α-defensin) and platelet-derived CCL5 form heteromers. These heteromers stimulate monocyte adhesion through CCR5 ligation. We further determined structural features of HNP1-CCL5 heteromers and designed a stable peptide that could disturb proinflammatory HNP1-CCL5 interactions. This peptide attenuated monocyte and macrophage recruitment in a mouse model of myocardial infarction. These results establish the in vivo relevance of heteromers formed between proteins released from neutrophils and platelets and show the potential of targeting heteromer formation to resolve acute or chronic inflammation.
•We prove in genotype-matched complement-mediated aHUS cohorts that C5 inhibition results in a statistical improvement in ESKD-free survival.•We demonstrate that biallelic pathogenic mutations in ...EXOSC3 cause eculizumab nonresponsive aHUS.
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Historically, the majority of patients with complement-mediated atypical hemolytic uremic syndrome (CaHUS) progress to end-stage kidney disease (ESKD). Single-arm trials of eculizumab with a short follow-up suggested efficacy. We prove, for the first time to our knowledge, in a genotype matched CaHUS cohort that the 5-year cumulative estimate of ESKD-free survival improved from 39.5% in a control cohort to 85.5% in the eculizumab-treated cohort (hazard ratio, 4.95; 95% confidence interval CI, 2.75-8.90; P = .000; number needed to treat, 2.17 95% CI, 1.81-2.73). The outcome of eculizumab treatment is associated with the underlying genotype. Lower serum creatinine, lower platelet count, lower blood pressure, and younger age at presentation as well as shorter time between presentation and the first dose of eculizumab were associated with estimated glomerular filtration rate >60 ml/min at 6 months in multivariate analysis. The rate of meningococcal infection in the treated cohort was 550 times greater than the background rate in the general population. The relapse rate upon eculizumab withdrawal was 1 per 9.5 person years for patients with a pathogenic mutation and 1 per 10.8 person years for those with a variant of uncertain significance. No relapses were recorded in 67.3 person years off eculizumab in those with no rare genetic variants. Eculizumab was restarted in 6 individuals with functioning kidneys in whom it had been stopped, with no individual progressing to ESKD. We demonstrated that biallelic pathogenic mutations in RNA-processing genes, including EXOSC3, encoding an essential part of the RNA exosome, cause eculizumab nonresponsive aHUS. Recessive HSD11B2 mutations causing apparent mineralocorticoid excess may also present with thrombotic microangiopathy.
In this month’s CME article, Brocklebank et al present detailed clinical characteristics and complete genetic analyses of a large United Kingdom cohort of patients with suspected complement-associated atypical hemolytic uremic syndrome (CaHUS) treated with eculizumab. The authors demonstrate superiority over historical therapies in genotype-matched patients with CaHUS, document that outcome after eculizumab cessation is genotype-dependent, and show that relapse is very unlikely if no pathogenic gene variant is present. The data also reveal a new causative genetic lesion for eculizumab-refractory CaHUS and provide a benchmark that should prove useful as additional options for CaHUS treatment emerge.
Abstract
Background
Hepatitis E virus (HEV) is a major cause of acute viral hepatitis. Better understanding of HEV subtypes involved in hepatitis E infections is essential. Investigation of sources ...and routes of transmission and the identification of potential clusters/outbreaks rely upon molecular typing of viral strains. A study was carried out to evaluate the ability of laboratories to undertake molecular typing with genotype and subtype determination.
Methods
A blinded panel of 11 different Orthohepevirus A strains was distributed to 28 laboratories performing HEV sequence analysis. Laboratories used their routine HEV sequencing and genotyping methods.
Results
Results were returned by 25 laboratories. Overall, 93% samples were assigned to the correct genotype and 81% were assigned to the correct subtype. Fragments amplified for typing ranged in size and the sequencing assays targeted both the structural and non-structural protein-coding regions. There was good agreement between the reported sequences where methods targeted overlapping fragments. In some cases, incorrect genotypes/subtypes were reported, including those not contained in the panel, and in one case, a genotype was reported for a blinded control sample containing Zika virus; collectively these data indicate contamination problems.
Conclusions
In general, identification of genotypes was good; however, in a small number of cases, there was a failure to generate sequences from some of the samples. There was generally broad agreement between the use of online typing tools such as the one provided by HEVnet and curated lists of published HEV reference sequences; however, going forward harmonization between these resources is essential.
Domain swapping is a widespread oligomerization process that is observed in a large variety of protein families. In the large superfamily of substrate‐binding proteins, non‐monomeric members have ...rarely been reported. The arginine‐binding protein from Thermotoga maritima (TmArgBP), a protein endowed with a number of unusual properties, presents a domain‐swapped structure in its dimeric native state in which the two polypeptide chains mutually exchange their C‐terminal helices. It has previously been shown that mutations in the region connecting the last two helices of the TmArgBP structure lead to the formation of a variety of oligomeric states (monomers, dimers, trimers and larger aggregates). With the aim of defining the structural determinants of domain swapping in TmArgBP, the monomeric form of the P235GK mutant has been structurally characterized. Analysis of this arginine‐bound structure indicates that it consists of a closed monomer with its C‐terminal helix folded against the rest of the protein, as typically observed for substrate‐binding proteins. Notably, the two terminal helices are joined by a single nonhelical residue (Gly235). Collectively, the present findings indicate that extending the hinge region and conferring it with more conformational freedom makes the formation of a closed TmArgBP monomer possible. On the other hand, the short connection between the helices may explain the tendency of the protein to also adopt alternative oligomeric states (dimers, trimers and larger aggregates). The data reported here highlight the importance of evolutionary control to avoid the uncontrolled formation of heterogeneous and potentially harmful oligomeric species through domain swapping.
The crystal structure of the P235GK mutant of the arginine‐binding protein from Thermotoga maritima in its monomeric state provides clues to the structural determinants of the domain swapping observed in the wild‐type protein.
SLC5A8, proposed as a thyroid apical iodide transporter, was recently defined as a Na+-coupled transporter of short-chain fatty acid. To document the expression pattern of SLC5A8 in the thyroid, we ...analyzed the regulation of its expression in normal human thyrocytes in culture and in tissues with distinct functional activity. To determine whether SLC5A8 expression is altered in all thyroid carcinomas or only in particular subtypes, we investigated the level of its expression in a series of 50 hypofunctioning tumors. SLC5A8 expression was studied at the transcript level and compared with that of SLC26A4 or Pendrin and SLC5A5 or Na+/iodide symporter. SLC5A8 expression, unlike that of SLC5A5 and SLC26A4, was not regulated by TSH in normal human thyrocytes in culture and was not related to the functional state of thyroid tissue; toxic adenomas and adjacent resting tissues exhibited the same SLC5A8 transcript content. SLC5A8 expression was selectively down-regulated (40-fold) in papillary thyroid carcinomas of classical form (PTC-cf.). Methylation-specific PCR analyses showed that SLC5A8 was methylated in 90% of PTC-cf. and in about 20% of other papillary thyroid carcinomas. In a series of 52 PTC-cf., a low SLC5A8 expression was highly significantly associated with the presence of BRAF T1796A mutation. These data identify a relationship between the methylation-associated silencing of the tumor-suppressor gene SLC5A8 and the T1796A point mutation of the BRAF gene in the PTC-cf. subtype of thyroid carcinomas.
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