RNA-Containing Cytoplasmic Inclusion Bodies in Ciliated Bronchial Epithelium Months to Years after Acute Kawasaki Disease

Kawasaki Disease (KD) is the most common cause of acquired heart disease in children in developed nations. The KD etiologic agent is unknown but likely to be a ubiquitous microbe that usually causes asymptomatic childhood infection, resulting in KD only in genetically susceptible individuals. KD synthetic antibodies made from prevalent IgA gene sequences in KD arterial tissue detect intracytoplasmic inclusion bodies (ICI) resembling viral ICI in acute KD but not control infant ciliated bronchial epithelium. The prevalence of ICI in late-stage KD fatalities and in older individuals with non-KD illness should be low, unless persistent infection is common.Lung tissue from late-stage KD fatalities and non-infant controls was examined by light microscopy for the presence of ICI. Nucleic acid stains and transmission electron microscopy (TEM) were performed on tissues that were strongly positive for ICI. ICI were present in ciliated bronchial epithelium in 6/7 (86%) late-stage KD fatalities and 7/27 (26%) controls ages 9-84 years (p = 0.01). Nucleic acid stains revealed RNA but not DNA within the ICI. ICI were also identified in lung macrophages in some KD cases. TEM of bronchial epithelium and macrophages from KD cases revealed finely granular homogeneous ICI.These findings are consistent with a previously unidentified, ubiquitous RNA virus that forms ICI and can result in persistent infection in bronchial epithelium and macrophages as the etiologic agent of KD

Differences in interferon sensitivity and biological properties of two related isolates of simian virus 5: A model for virus persistence

AbstractCPI+ and CPI− are two canine isolates of simian virus 5 (SV5). CPI+ was originally isolated from the cerebrospinal fluid of a dog with temporary posterior paralysis and CPI− was recovered at 12 days p.i. from the brain tissue of a dog experimentally infected with CPI+. We have previously shown that the V protein of SV5 blocks interferon (IFN) signalling by targeting STAT1 for degradation. Here we report that whilst CPI+ targets STAT1 for degradation, CPI− fails to and as a consequence, CPI+ blocks IFN signalling but CPI− does not. Three amino acid differences in the P/V N-terminal common domain of the V protein are responsible for the observed difference in the abilities of CPI+ and CPI− to block IFN signalling. In cells persistently infected with CPI− the virus may become repressed in response to IFN, under which circumstances virus glycoproteins are lost from the surface of infected cells and virus nucleocapsid proteins accumulate in cytoplasmic inclusion bodies. We suggest that in vivo cells infected with IFN-resistant viruses (in which there would be continuous virus protein synthesis) may be more susceptible to killing by cytotoxic T cells than cells infected with IFN-sensitive viruses (in which virus protein synthesis was repressed), and a model of virus persistence is put forward in which there is alternating selection of IFN-resistant and IFN-sensitive viruses depending upon the state of the adaptive immune response

The Prognostic Influence of BRAF Mutation and other Molecular, Clinical and Laboratory Parameters in Stage IV Colorectal Cancer

Our aim was to evaluate the predictive and prognostic influence of BRAF mutation and other molecular, clinical and laboratory parameters in stage IV colorectal cancer (CRC). 60 patients were included in this retrospective analysis, and 17 variables were examined for their relation with treatment response and survival. KRAS mutation was identified in 40.3 % of cases, BRAF and PIK3CA in 8.8 % and 10.5 % respectively. 29.8 % of patients responded to treatment. Median survival time was 14.3 months. Weight loss, fever, abdominal metastases, blood transfusion, hypoalbuminaimia, BRAF and PIK3CA mutations, CRP and DNA Index were associated with survival. In multivariate analysis, male patients had 3.8 times higher probability of response, increased DNA Index was inversely correlated with response and one unit raise of DNA Index augmented 6 times the probability of death. Our findings potentiate the prognostic role of BRAF, PIK3CA mutations and ploidy in advanced CRC. © 2016, Arányi Lajos Foundation

Relationships and host range of human, canine, simian and porcine isolates of Simian Virus 5 (Parainfluenza virus 5).

Sequence comparison of the V/P and F genes of 13 human, canine, porcine and simian isolates of simian virus 5 (SV5) revealed a surprising lack of sequence variation at both the nucleotide and amino acid levels (0-3%), even though the viruses were isolated over 30 years and originated from countries around the world. Furthermore, there were no clear distinguishing amino acid or nucleotide differences among the isolates that correlated completely with the species from which they were isolated. In addition, there was no evidence that the ability of the viruses to block interferon signalling by targeting STAT1 for degradation was confined to the species from which they were isolated. All isolates had an extended cytoplasmic tail in the F protein, compared with the original W3A and WR monkey isolates. Sequence analysis of viruses that were derived from human bone-marrow cells isolated in London in the 1980s revealed that, whilst they were related more closely to one another than to the other isolates, they all had identifying differences, suggesting that they were independent isolates. These results therefore support previous data suggesting that SV5 can infect humans persistently, although the relationship of SV5 to any human disease remains highly contentious. Given that SV5 has been isolated on multiple occasions from different species, it is proposed that the term simian virus 5 is inappropriate and suggested that the virus should be renamed parainfluenza. virus 5.</p

A single amino acid substitution in the V protein of SV5 differentiates its ability to block IFN signalling in human and murine cells

Previous work has demonstrated that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation (thereby blocking interferon [IFN] signaling) in human but not in murine cells. In murine BF cells, SV5 establishes a low-grade persistent infection in which the virus fluxes between active and repressed states in response to local production of IFN. Upon passage of persistently infected BF cells, virus mutants were selected that were better able to replicate in murine cells than the parental W3 strain of SV5 (wild type [wt]). Viruses with mutations in the Pk region of the N-terminal domain of the V protein came to predominate the population of viruses carried in the persistently infected cell cultures. One of these mutant viruses, termed SV5 mci-2, was isolated, Sequence analysis of the V/P gene of SV5 mci-2 revealed two nucleotide differences compared to wt SV5, only one of which resulted in an amino acid substitution (asparagine [N], residue 100, to aspartic acid [D]) in V. Unlike the protein of wt SV5, the V protein of SV5 mci-2 blocked IFN signaling in murine cells. Since the SV5 mci-2 virus had additional mutations in genes other than the V/P gene, a recombinant virus (termed rSV5-V/P N100D) was constructed that contained this substitution alone within the wt SV5 backbone to evaluate what effect the asparagine-to-aspartic-acid substitution in V had on the virus phenotype. In contrast to wt SV5, rSV5-V/P N100D blocked IFN signaling in murine cells. Furthermore, rSV5-V/P N100D virus protein synthesis in BF cells continued for significantly longer periods than that for wt SV5, However, even in cells infected with rSV5-V/P N100D, there was a late, but significant, inhibition in virus protein synthesis. Nevertheless, there was an increase in virus yield from BF cells infected with rSV5-V/P N100D compared to wt SV5, demonstrating a clear selective advantage to SV5 in being able to block IFN signaling in these cells.</p

Association between PD-L1 expression and driver gene mutations in non-small cell lung cancer patients: correlation with clinical data

Lung cancer is the leading cause of cancer death worldwide. Recently, promising therapies have emerged based on PD-1/PD-L1 immune checkpoint inhibitors, which have been approved even as frontline treatment for patients with non-small cell lung cancer (NSCLC). We examined the association between PD-L1 expression and clinicopathological parameters as well as overall survival in 220 NSCLC patients. PD-L1 expression was estimated by immunohistochemistry using 22C3 PharmDx Dako assay and was defined as high, if TPS was ≥ 50%, low, if TPS was 1%–49%, and absent, if TPS was &lt; 1%. EGFR mutations were detected by COBAS while KRAS and BRAF mutations by pyrosequencing. ROS1 and ALK rearrangements were estimated by immunohistochemistry with positive cases being confirmed by CISH and FISH, respectively. Data analysis was performed using SPSS v25.0. PD-L1 expression was positively correlated with KRAS mutations. Anti-PD-1 therapy (pembrolizumab) prolonged overall survival compared to any other treatment. This effect was more pronounced in KRAS-mutated cases compared to KRAS wild-type ones. Patients with positive PD-L1 expression – high or low – who had been treated with pembrolizumab, showed significant survival benefit compared to positive or negative PD-L1 expressors who did not receive immunotherapy. In multivariate analysis, PD-L1 status, stage and pembrolizumab treatment were independent variables for overall survival. PD-L1 expression (TPS ≥ 1%) by itself emerged as a poor prognostic factor, while treatment with pembrolizumab prolonged overall survival. KRAS mutations may affect tumour microenvironment and patient’s response to immunotherapy. Immune checkpoint inhibitors could represent an alternative therapeutic option particularly for KRAS-mutated NSCLC patients. Further investigation into this notion is warranted in order to validate this observation. © 2020, Springer-Verlag GmbH Germany, part of Springer Nature

MTHFR gene variants and non-MALT lymphoma development in primary Sjogren&apos;s syndrome

Primary Sjogren&apos;s syndrome (pSS) confers increased risk for non-Hodgkin lymphoma (NHL) development. Two common polymorphisms, the c. 677C &gt; T and c. 1298A &gt; C, of the methylene-tetrahydrofolate reductase (MTHFR) gene, an enzyme essential in DNA synthesis and methylation, have been associated with susceptibility to NHL. Herein, we tested the hypothesis that MTHFR variants contribute to pSS-related lymphomagenesis. 356 pSS patients, of whom 75 had MALT and 19 non-MALT NHL and 600 healthy controls were genotyped for the detection of MTHFR polymorphisms. DNA methylation levels were assessed by pyrosequencing of the LINE-1 retroelement promoter in DNA from 55 salivary gland tissues from pSS patients. DNA double-strand breaks were determined in peripheral blood mononuclear cells from 13 pSS patients, using comet assay. Αnalysis according to lymphoma subtype revealed increased frequency of c. 677C &gt; T TT genotype and T allele, as well as reduced prevalence of the c. 1298A &gt; C C allele in the pSS non-MALT group compared to controls and patients without NHL. MTHFR c. 677C &gt; T TT genotype was associated with reduced DNA methylation levels, while MTHFR c. 1298A &gt; C AC genotype with reduced DNA double-strand breaks levels. MTHFR variants may be involved in SS non-MALT NHL development, through contribution to defective DNA methylation and genomic instability. © 2017 The Author(s)