Expression and regulation of Toll-like receptor 4 in allergic disease

The prevalence of allergic diseases is increasing worldwide for unclear reasons. This sudden widespread increase, particularly in children, suggests causes that are not due to genetic makeup of individuals, but rather a change in environment and lifestyle factors. Growing epidemiological and immunological evidence supports the hygiene hypothesis, which states that decreased exposure to immune stimulatinginfections in early childhood is the cause of the rise in allergic diseases. Studies have shown that even bacterial components such as lipopolysaccharide (LPS) can have a protective effect. The receptor for LPS is Toll-like receptor 4 (TLR4), an innate immunological receptor, which may play an essential role in regulation of allergic disease. We investigated the expression of TLR4 in the nasal mucosa of allergic and non-allergic children and adults, the effects of LPS on allergic inflammation and the regulation of TLR4 expression. We hypothesized that LPS, through TLR4, could regulate allergic inflammation and that long-term allergic inflammation would limit these responses.Children and adults undergoing nasal surgery were recruited from the local hospitals. Nasal biopsies from the patients were explanted and cultured ex vivo with allergen and LPS. LPS could prevent allergen-induced Th2 cytokine production and inflammatory cell increases in explants from atopic children; this was through induction of Th1 cytokines and IL-10. TLR4 was also detected on CD4+CD25+ T cells. LPS did not provide the same protection in adults. Adults, especially atopic subjects, had less TLR4 immunopositive cells; hence, their reduced responsiveness to LPS. This suggests that factors could downregulate TLR4 in allergic adults. The U-937 monocytic cell line was used as an in vitro model to study the regulation of TLR4 by interleukin-4 (IL-4), a T-helper type 2 (Th2) cytokine involved in allergic inflammation. U-937 cells cultured with IL-4 had decreased LPS responsiveness, TLR4 protein surface expression, TLR4 mRNA expression and transcriptional activity of the upstream region of TLR4. This effect was tyrosine kinase and STAT6 dependent. A STAT6 binding site was determined in an area of the TLR4 gene necessary to mediate IL-4's inhibitory effects on TLR4 transcription. IL-4 was also determined to reduce TLR4 expression in PBMCs, especially in CD4+ T cells purified from the blood of children.As predicted in epidemiological studies and animal studies, LPS was shown to provide anti-inflammatory effects against allergen in human nasal tissue. LPS may directly stimulate regulatory T cells (CD25+CD4+) to produce anti-inflammatory cytokine production. The effects of LPS exposure may be lost due to reduced expression of TLR4 by inflammatory cells, and this may be caused by the allergic inflammation itself. Therefore, development of allergic inflammation can down-regulate anti-inflammatory mechanisms and promote long term chronic inflammation

A Case of Organizing Pneumonia Following Azacitidine Treatment for Myelodysplastic Syndrome

Organizing pneumonia (OP) is a lung pathology mainly affecting distal lung structures. Its etiology is often unknown, in which case it is termed cryptogenic organizing pneumonia (COP).  Of those cases of OP with an identified cause, the usual culprits include infections, medications, and radiation therapy. In this report, we present the case of a 73-year-old female on azacitidine –a pyrimidine analogue– used for treatment of myelodysplastic syndrome (MDS). The patient presented with fever, productive cough, and pleuritic chest pain. A CT of the chest, a bronchoalveolar lavage and a transthoracic biopsy were performed, and findings were consistent with OP, thought to be induced by azacitidine. The patient was treated with prednisone and subsequently showed significant improvement. Although rare, this case underlines the importance of considering OP in the context of non-resolving pulmonary infiltrates, particularly when there is a potentially relevant exposure, such as azacitidine

Integrating NGS-derived mutational profiling in the diagnosis of multiple lung adenocarcinomas

MICROABSTRACT: Integration of Next Generation Sequencing (NGS) information for use in distinguishing between Multiple Primary Lung Cancer and intrapulmonary metastasis was evaluated. We used a probabilistic model, comprehensive histologic assessment and NGS to classify patients. Integrating NGS data confirmed initial diagnosis (n = 41), revised the diagnosis (n = 12), while resulted in non-informative data (n = 8). Accuracy of diagnosis can be significantly improved with integration of NGS data. BACKGROUND: Distinguishing between multiple primary lung cancers (MPLC) and intrapulmonary metastases (IPM) is challenging. The goal of this study was to evaluate how Next Generation Sequencing (NGS) information may be integrated in the diagnostic strategy. PATIENTS AND METHODS: Patients with multiple lung adenocarcinomas were classified using both the comprehensive histologic assessment and NGS. We computed the joint probability of each pair having independent mutations by chance (thus being classified as MPLC). These probabilities were computed using the marginal mutation rates of each mutation, and the known negative dependencies between driver genes and different gene loci. With these NGS-driven data, cases were re-classified as MPLC or IPM. RESULTS: We analyzed 61 patients with a total of 131 tumors. The most frequent mutation was KRAS (57.3%) which occured at a rate higher than expected (p < 0.001) in lung cancer. No mutation was detected in 25/131 tumors (19.1%). Discordant molecular findings between tumor sites were found in 46 patients (75.4%); 11 patients (18.0%) had concordant molecular findings, and 4 patients (6.6%) had concordant molecular findings at 2 of the 3 sites. After integration of the NGS data, the initial diagnosis was confirmed for 41 patients (67.2%), the diagnosis was revised for 12 patients (19.7%) or was considered as non-informative for 8 patients (13.1%). CONCLUSION: Integrating the information of NGS data may significantly improve accuracy of diagnosis and staging

Specific detection of methionine 27 mutation in histone 3 variants (H3K27M) in fixed tissue from high-grade astrocytomas

Studies in pediatric high-grade astrocytomas (HGA) by our group and others have uncovered recurrent somatic mutations affecting highly conserved residues in histone 3 (H3) variants. One of these mutations leads to analogous p.Lys27Met (K27M) mutations in both H3.3 and H3.1 variants, is associated with rapid fatal outcome, and occurs specifically in HGA of the midline in children and young adults. This includes diffuse intrinsic pontine gliomas (80 %) and thalamic or spinal HGA (>90 %), which are surgically challenging locations with often limited tumor material available and critical need for specific histopathological markers. Here, we analyzed formalin-fixed paraffin-embedded tissues from 143 pediatric HGA and 297 other primary brain tumors or normal brain. Immunohistochemical staining for H3K27M was compared to tumor genotype, and also compared to H3 tri-methylated lysine 27 (H3K27me3) staining, previously shown to be drastically decreased in samples carrying this mutation. There was a 100 % concordance between genotype and immunohistochemical analysis of H3K27M in tumor samples. Mutant H3K27M was expressed in the majority of tumor cells, indicating limited intra-tumor heterogeneity for this specific mutation within the limits of our dataset. Both H3.1 and H3.3K27M mutants were recognized by this antibody while non-neoplastic elements, such as endothelial and vascular smooth muscle cells or lymphocytes, did not stain. H3K27me3 immunoreactivity was largely mutually exclusive with H3K27M positivity. These results demonstrate that mutant H3K27M can be specifically identified with high specificity and sensitivity using an H3K27M antibody and immunohistochemistry. Use of this antibody in the clinical setting will prove very useful for diagnosis, especially in the context of small biopsies in challenging midline tumors and will help orient care in the context of the extremely poor prognosis associated with this mutation. Electronic supplementary material The online version of this article (doi:10.1007/s00401-014-1337-4) contains supplementary material, which is available to authorized users

Local Induction of a Specific Th1 Immune Response by Allergen Linked Immunostimulatory DNA in the Nasal Explants of Ragweed- Allergic Subjects

Background: Allergen immunotherapy is effective in allergic individuals however efforts are being made to improve its safety, convenience, and efficacy. It has recently been demonstrated that allergen-linked immunostimulatory DNA (ISS) is effective in stimulating an allergen-specific Th1 response with decreased allergenicity. The objective of this study is to investigate whether ISS linked to purified ragweed allergen Amb-a-1 (AIC) can inhibit local allergen-specific Th2 and induce allergen-specific Th1 responses in explanted nasal mucosa of ragweed-sensitive subjects. In addition, we set out to determine whether AIC is more effective compared to stimulation with unlinked Amb a 1 and ISS. Methods: Tissue from ragweed-sensitive patients (n=12) was cultured with whole ragweed allergen (RW), Amb-a-1, AIC, Amb-a-1 and ISS (unlinked), or tetanus toxoid (TT) for 24 hours. IL-4, −5, −13, TNF-α and IFN-γ mRNA-positive cells were visualized by in situ hybridization and T cells, B cells and neutrophils were enumerated using immunocytochemistry. Results: RW or Amb-a-1 increased the number of IL-4, IL-5, and IL-13 mRNA+cells in the tissue compared to medium alone. AIC had similar cytokine mRNA reactivity as control tissue. AIC and TT increased IFNγ-mRNA expression. Unlinked Amb-a-1 and ISS showed similar effects to AIC, however this response was weaker. The number of TNF mRNA+ cells, T cells, B cells and neutrophils remained unchanged. Conclusions: AIC is effective in stimulating a local allergen-specific Th1- and abolishing Th2-cytokine mRNA reactivity in the nose and may be considered as a strong candidate for an improved approach to immunotherapy in ragweed-sensitive individuals

An integrated virtual pathology education platform developed using Microsoft Power Apps and Microsoft Teams

The transition towards digital pathology and an extensive selection of video conferencing platforms have helped provide continuity to education even during the COVID-19 pandemic. Innovative approaches for pathology education, will likely persist beyond the pandemic, as they have powerful didactic potential. While there is a wide selection of software for use as educational tools, an environment to access all resources with ease is clearly lacking. In this technical note, we highlight our customized educational applications built using a low-code approach. Our applications, developed with Microsoft Power Apps, serve both educational and examination purposes and are launched using Microsoft Teams. Building applications using a low-code approach has made our applications very specific to our use and enabled daily distanced education. Combined with existing features on Teams, such as file sharing, meeting scheduling, and messaging, the applications serve as a unique and customizable pathology educational platform

Routine Clinically Detected Increased ROS1 Transcripts Are Related With ROS1 Expression by Immunohistochemistry and Associated With EGFR Mutations in Lung Adenocarcinoma

Introduction: Translocations of the ROS1 gene were found to drive tumorigenesis in 1% to 2% of lung adenocarcinoma. In clinical practice, ROS1 rearrangements are often screened by immunohistochemistry (IHC) before confirmation with either fluorescence in situ hybridization or molecular techniques. This screening test leads to a non-negligible number of cases that have equivocal or positive ROS1 IHC, without ROS1 translocation. Methods: In this study, we have analyzed retrospectively 1021 cases of nonsquamous NSCLC having both ROS1 IHC and molecular analysis using next-generation sequencing. Results: ROS1 IHC was negative in 938 cases (91.9%), equivocal in 65 cases (6.4%), and positive in 18 cases (1.7%). Among these 83 equivocal or positive cases, only two were ROS1 rearranged, leading to a low predictive positive value of the IHC test (2%). ROS1-positive IHC was correlated with an increased mRNA ROS1 transcripts. Moreover, we have found a mean statistically significant relationship between ROS1 expression and EGFR gene mutations, suggesting a crosstalk mechanism between these oncogenic driver molecules. Conclusion: This study demonstrates that ROS1 IHC represents true ROS1 mRNA expression, and raises the question of a potential benefit of combined targeted therapy in EGFR-mutated NSCLC