Cardiovascular pathology in 2 young adults with sudden, unexpected death due to coronary aneurysms from Kawasaki disease in childhood.

PurposeCoronary artery aneurysms (CAA) may remain silent after Kawasaki disease (KD) until adulthood when myocardial ischemia can lead to sudden death. We postulated that there would be young adults with sudden, unexpected death due to CAA from KD who would have a state-mandated autopsy performed by the San Diego County Medical Examiner's Office (SDCMEO).MethodsWe reviewed all autopsy cases <35years of age from 1997 to 2012 at the SDCMEO with a cardiovascular cause of death (n=154).ResultsWe found 2 cases meeting inclusion criteria. Case 1 was a 22-year-old Korean male with chronic ischemic changes due to a partially occluded and diffusely calcified 15mm aneurysm at the bifurcation of the left main coronary artery. Interview of the mother revealed that this patient had been diagnosed with KD complicated by giant aneurysms at age two years. Case 2 was a 30-year-old Hispanic male with myocardial infarction due to thrombosis of a calcified left anterior descending artery aneurysm. Histologic findings included diffuse myocardial fibrosis and a recanalized aneurysm in the right coronary artery. Interview of the family revealed a KD-compatible illness in childhood. Immunohistochemical staining showed expression of transforming growth factor β pathway molecules in the aneurysmal arterial wall.ConclusionsIn a medical examiner's office serving a population of approximately 3 million people, 2 of 154 (1.3%) cardiovascular deaths in persons <35years were attributed to cardiovascular complications of KD in childhood. Antecedent KD should be considered in the evaluation of all cases of sudden, unexpected death in young adults

Autoreactive T effector memory differentiation mirrors β cell function in type 1 diabetes

In type 1 diabetes, cytotoxic CD8+ T cells with specificity for β cell autoantigens are found in the pancreatic islets, where they are implicated in the destruction of insulin-secreting β cells. In contrast, the disease relevance of β cell–reactive CD8+ T cells that are detectable in the circulation, and their relationship to β cell function, are not known. Here, we tracked multiple, circulating β cell–reactive CD8+ T cell subsets and measured β cell function longitudinally for 2 years, starting immediately after diagnosis of type 1 diabetes. We found that change in β cell–specific effector memory CD8+ T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8+ T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer-specific protein of 37 kDa, and CD16, and reduced expression of CD28) compared with their CD57– counterparts, and network association modeling indicated that the dynamics of β cell–reactive CD57+ effector memory CD8+ T cell subsets were strongly linked. Thus, coordinated changes in circulating β cell–specific CD8+ T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy

<i>Pichia pastoris</i>-Expressed Dengue 2 Envelope Forms Virus-Like Particles without Pre-Membrane Protein and Induces High Titer Neutralizing Antibodies

<div><p>Dengue is a mosquito-borne viral disease with a global prevalence. It is caused by four closely-related dengue viruses (DENVs 1–4). A dengue vaccine that can protect against all four viruses is an unmet public health need. Live attenuated vaccine development efforts have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This has emphasized the need to explore non-replicating dengue vaccine options. Virus-like particles (VLPs) which can elicit robust immunity in the absence of infection offer potential promise for the development of non-replicating dengue vaccine alternatives. We have used the methylotrophic yeast <i>Pichia pastoris</i> to develop DENV envelope (E) protein-based VLPs. We designed a synthetic codon-optimized gene, encoding the N-terminal 395 amino acid residues of the DENV-2 E protein. It also included 5’ pre-membrane-derived signal peptide-encoding sequences to ensure proper translational processing, and 3’ 6× His tag-encoding sequences to facilitate purification of the expressed protein. This gene was integrated into the genome of <i>P. pastoris</i> host and expressed under the alcohol oxidase 1 promoter by methanol induction. Recombinant DENV-2 protein, which was present in the insoluble membrane fraction, was extracted and purified using Ni²⁺-affinity chromatography under denaturing conditions. Amino terminal sequencing and detection of glycosylation indicated that DENV-2 E had undergone proper post-translational processing. Electron microscopy revealed the presence of discrete VLPs in the purified protein preparation after dialysis. The E protein present in these VLPs was recognized by two different conformation-sensitive monoclonal antibodies. Low doses of DENV-2 E VLPs formulated in alum were immunogenic in inbred and outbred mice eliciting virus neutralizing titers >1∶1200 in flow cytometry based assays and protected AG129 mice against lethal challenge (<i>p</i><0.05). The formation of immunogenic DENV-2 E VLPs in the absence of pre-membrane protein highlights the potential of <i>P. pastoris</i> in developing non-replicating, safe, efficacious and affordable dengue vaccine.</p></div

Characterization of DENV-2-specific antibodies elicited by DENV-2 E VLPs.

<p>(A) Analysis of virus-specific antibody titers in anti-DENV-2 E antisera (blue bars) and mock-immune sera (black bars) in indirect ELISAs using infectious DENVs as coating antigen. (B) Determination of virus-neutralizing antibody titers using FACS neutralization assay. Serial dilutions of anti-DENV-2 E antisera were tested for their capacity to neutralize infectivity of all four DENV serotypes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064595#pone.0064595-Kraus1" target="_blank">[30]</a>. The vertical axis denotes the serum dilution corresponding to 50% neutralization (FNT₅₀ titre) of virus infectivity. Murine sera used in experiments shown in panels A and B were from Balb/C mice; the Arabic numerals along the x-axis, in both these panels, indicate DENV serotype. (C) Determination of protective efficacy of DENV-2 E VLP immunization. AG129 mice were either mock-immunized (black curve, n = 4) or immunized with DENV-2 E VLPs (blue curve, n = 6) and challenged with a virulent strain of DENV-2. The mice were monitored daily (up to 18 days post challenge) for mortality and the resultant data plotted as Kaplan-Meir survival curves.</p