Rare Thyroid Diseases Mimicking or Concealing Primitive Thyroid Neoplasms: A Call for a "Check and Double-Check" Clinical Mindset.

Clinical endocrinologists encounter in their practice patients with thyroid diseases on a daily basis. Still, diagnosis of rare structural thyroid disorders can be quite challenging. In some instances, they do not only impersonate but can also conceal, other conditions such as thyroid carcinomas. We describe a series of patients with structural thyroid disorders including 1) anaplastic thyroid carcinoma initially presenting with features of thyroid abscess; 2) unicentric hyaline vascular Castleman's disease of the thyroid embedded in a stroma of papillary thyroid carcinoma; and 3) primary thyroid lymphoma with a rapid and fulminant evolution. The common challenge in the diagnosis of these cases lies in both their low incidence and their complex presentation. We use the presentation of these cases to raise the attention related to their identification. We highlight the need for precision diagnosis to enable a patient-tailored management approach and improve patient outcomes.

Phase II Trial Assessing the Repeatability and Tumor Uptake of [68Ga]Ga-HER2 Single-Domain Antibody PET/CT in Patients with Breast Carcinoma.

Human epidermal growth factor receptor 2 (HER2) status is used for decision-making in breast carcinoma treatment. The status is obtained through immunohistochemistry or in situ hybridization. These two methods have the disadvantage of necessitating tissue sampling, which is prone to error due to tumor heterogeneity or interobserver variability. Whole-body imaging might be a solution to map HER2 expression throughout the body. Methods: Twenty patients with locally advanced or metastatic breast carcinoma (5 HER2-positive and 15 HER2-negative patients) were included in this phase II trial to assess the repeatability of uptake quantification and the extended safety of the [68Ga]Ga-NOTA-anti-HER2 single-domain antibody (sdAb). The tracer was injected, followed by a PET/CT scan at 90 min. Within 8 d, the procedure was repeated. Blood samples were taken for antidrug antibody (ADA) assessment and liquid biopsies. On available tissues, immunohistochemistry, in situ hybridization, and mass spectrometry were performed to determine the correlation of HER2 status with uptake values measured on PET. If relevant preexisting [18F]FDG PET/CT images were available (performed as standard of care), a comparison was made. Results: With a repeatability coefficient of 21.8%, this imaging technique was repeatable. No clear correlation between PET/CT uptake values and pathology could be established, as even patients with low levels of HER2 expression showed moderate to high uptake. Comparison with [18F]FDG PET/CT in 16 patients demonstrated that in 7 patients, [68Ga]Ga-NOTA-anti-HER2 shows interlesional heterogeneity within the same patient, and [18F]FDG uptake did not show the same heterogeneous uptake in all patients. In some patients, the extent of disease was clearer with the [68Ga]Ga-NOTA-anti-HER2-sdAb. Sixteen adverse events were reported but all without a clear relationship to the tracer. Three patients with preexisting ADAs did not show adverse reactions. No new ADAs developed. Conclusion: [68Ga]Ga-NOTA-anti-HER2-sdAb PET/CT imaging shows similar repeatability to [18F]FDG. It is safe for clinical use. There is tracer uptake in cancer lesions, even in patients previously determined to be HER2-low or -negative. The tracer shows potential in the assessment of interlesional heterogeneity of HER2 expression. In a subset of patients, [68Ga]Ga-NOTA-anti-HER2-sdAb uptake was seen in lesions with no or low [18F]FDG uptake. These findings support further clinical development of [68Ga]Ga-NOTA-anti-HER2-sdAb as a PET/CT tracer in breast cancer patients.

Comparison of Omentum and Subcutis as Implant Sites for Device-Encapsulated Human iPSC-Derived Pancreatic Endoderm in Nude Rats.

Subcutaneous implants of device-encapsulated stem cell-derived pancreatic endoderm (PE) can establish a functional beta cell mass (FBM) with metabolic control in immune-compromised mice. In a study with human-induced pluripotent stem cell-PE, this outcome was favored by a preformed pouch which allowed lesion-free insertion of devices in a pre-vascularized site. This was not reproduced in nude rats, known to exhibit a higher innate reactivity than mice and therefore relevant as preclinical model: a dense fibrotic capsule formed around subcutis (SC) implants with virtually no FBM formation. Placement in omentum (OM) of nude rats provided a less fibrous, better vascularized environment than SC. It resulted in less donor cell loss (56% recovery at post-transplant-PT week 3 versus 16% in SC) allowing FBM-formation. At PT week 30, 6/13 OM-recipients exhibited glucose-induced plasma hu-C-peptide to 0.1-0.4 ng/ml, versus 0/8 in SC-recipients. These levels are more than 10-fold lower than in a state of metabolic control. This shortcoming is not caused by inadequate glucose responsiveness of the beta cells but by their insufficient number. The size of the formed beta cell mass (0.4 ± 0.2 µl) was lower than that reported in mice receiving the same cell product subcutaneously; the difference is attributed to a lower expansion of pancreatic progenitor cells and to their lower degree of differentiation to beta cells. This study in the nude rat model demonstrates that OM provides a better environment for formation of beta cells in device-encapsulated PE-implants than SC. It also identified targets for increasing their dose-efficacy.

Undetectable viral RNA in follicular fluid, cumulus cells, and endometrial tissue samples in SARS-CoV-2-positive women.

OBJECTIVE: To study the presence of viral RNA in the follicular fluid, cumulus cells, and endometrial tissue samples in SARS-CoV-2-positive women undergoing assisted reproductive technology (ART). DESIGN: Prospective, single-center, observational study. SETTING: Tertiary hospital. PATIENT(S): A total of 16 patients undergoing transvaginal oocyte retrieval who had a positive SARS-CoV-2 RNA test <48 hours before the procedure. All patients underwent the retrieval between September 2020 and June 2021 and used in vitro fertilization or intracytoplasmic sperm injection. All embryos were vitrified to avoid conception during SARS-CoV-2 infection. INTERVENTION(S): Follicular fluid aspirated during oocyte retrieval, cumulus cells, and endometrial samples were analyzed for SARS-CoV-2 RNA using the RealStar SARS-CoV-2 RT-PCR-Kit1.0. MAIN OUTCOME MEASURE(S): The primary outcome parameter was the detection of viral RNA in the follicular fluid, cumulus cells, and endometrial cells. Fertilization rate, embryo developmental potential, and clinical outcome after frozen embryo transfer were secondary outcome parameters. RESULT(S): Samples from 16 patients were analyzed. Cycle threshold values of <40 were considered positive. All samples were negative for SARS-CoV-2 viral RNA. No inflammatory lesions of the endometrium were identified histologically. Fertilization rate, embryo development, and clinical outcomes after embryo transfer were reassuring. CONCLUSION(S): In women infected with SARS-CoV-2 who underwent ART, viral RNA was undetectable in the follicular fluid, cumulus cells, and endometrium. Caution is warranted in view of the small sample size, and the risk of SARS-CoV-2 affecting the embryo via ART cannot be ruled out. Adequate counseling of women and couples undergoing ART is crucial in parallel with further research on the effect of exposure of the early human embryo to SARS-CoV-2. CLINICAL TRIAL REGISTRATION NUMBER: NCT04425317.

Assessing Tumor-Infiltrating Lymphocytes in Breast Cancer: A Proposal for Combining Immunohistochemistry and Gene Expression Analysis to Refine Scoring.

Scoring of tumor-infiltrating lymphocytes (TILs) in breast cancer specimens has gained increasing attention, as TILs have prognostic and predictive value in HER2+ and triple-negative breast cancer. We evaluated the intra- and interrater variability when scoring TILs by visual inspection of hematoxylin and eosin-stained tissue sections. We further addressed whether immunohistochemical staining of these sections for immune cell surface markers CD45, CD3, CD4, and CD8 and combination with nanoString nCounter® gene expression analysis could refine TIL scoring. Formalin-fixed paraffin-embedded and fresh-frozen core needle biopsies of 12 female and treatment-naive breast cancer patients were included. Scoring of TILs was performed twice by three independent pathologists with a washout period of 3 days. Increasing intra- and interrater variability was observed with higher TIL numbers. The highest reproducibility was observed on tissue sections stained for CD3 and CD8. The latter TIL scores correlated well with the TIL scores obtained through nanoString nCounter® gene expression analysis. Gene expression analysis also revealed 104 and 62 genes that are positively and negatively related to both TIL scores. In conclusion, integration of immunohistochemistry and gene expression analysis is a valuable strategy to refine TIL scoring in breast tumors.

Efficacy of a Flat Low Dose of Nivolumab in Advanced Cancer: A Retrospective Case Series.

BACKGROUND/AIM: Immunotherapy with PD-1/PDL1 blocking monoclonal antibodies has improved survival compared to the standard-of-care chemotherapy for several malignancies at different stages of these malignancies. Due to several reasons, many cancer patients in medical need have no access to these drugs. In this study, we aimed to investigate whether a low dose of nivolumab could also lead to a therapeutic response. PATIENTS AND METHODS: Patients with advanced cancer were treated with a flat low dose of 10 mg of nivolumab IV every two weeks at no drug cost. RESULTS: Disease control was noted in nine of the 18 patients. Two patients achieved complete remission, two had prolonged partial remission, and five had stable disease, of these only two experienced adverse events. CONCLUSION: A flat low dose of nivolumab may have clinical activity and is a cheap therapeutic option in patients in medical need for whom standard-dose immune checkpoint inhibitors are not accessible for any reason.

Inter-observer agreement for the histological diagnosis of invasive lobular breast carcinoma.

Invasive lobular breast carcinoma (ILC) is the second most common breast carcinoma (BC) subtype and is mainly driven by loss of E-cadherin expression. Correct classification of BC as ILC is important for patient treatment. This study assessed the degree of agreement among pathologists for the diagnosis of ILC. Two sets of hormone receptor (HR)-positive/HER2-negative BCs were independently reviewed by participating pathologists. In set A (61 cases), participants were provided with hematoxylin/eosin (HE)-stained sections. In set B (62 cases), participants were provided with HE-stained sections and E-cadherin immunohistochemistry (IHC). Tumor characteristics were balanced. Participants classified specimens as non-lobular BC versus mixed BC versus ILC. Pairwise inter-observer agreement and agreement with a pre-defined reference diagnosis were determined with Cohen's kappa statistics. Subtype calls were correlated with molecular features, including CDH1/E-cadherin mutation status. Thirty-five pathologists completed both sets, providing 4,305 subtype calls. Pairwise inter-observer agreement was moderate in set A (median κ = 0.58, interquartile range [IQR]: 0.48-0.66) and substantial in set B (median κ = 0.75, IQR: 0.56-0.86, p < 0.001). Agreement with the reference diagnosis was substantial in set A (median κ = 0.67, IQR: 0.57-0.75) and almost perfect in set B (median κ = 0.86, IQR: 0.73-0.93, p < 0.001). The median frequency of CDH1/E-cadherin mutations in specimens classified as ILC was 65% in set A (IQR: 56-72%) and 73% in set B (IQR: 65-75%, p < 0.001). Cases with variable subtype calls included E-cadherin-positive ILCs harboring CDH1 missense mutations, and E-cadherin-negative ILCs with tubular elements and focal P-cadherin expression. ILCs with trabecular growth pattern were often misclassified as non-lobular BC in set A but not in set B. In conclusion, subtyping of BC as ILC achieves almost perfect agreement with a pre-defined reference standard, if assessment is supported by E-cadherin IHC. CDH1 missense mutations associated with preserved E-cadherin protein expression, E- to P-cadherin switching in ILC with tubular elements, and trabecular ILC were identified as potential sources of discordant classification.

Discovery and 3D imaging of a novel ΔNp63-expressing basal cell type in human pancreatic ducts with implications in disease.

OBJECTIVE: The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas. DESIGN: We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed. RESULTS: In normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme. CONCLUSION: In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models.

Overcoming the Challenges of High Quality RNA Extraction from Core Needle Biopsy.

The use of gene expression profiling (GEP) in cancer management is rising, as GEP can be used for disease classification and diagnosis, tailoring treatment to underlying genetic determinants of pharmacological response, monitoring of therapy response, and prognosis. However, the reliability of GEP heavily depends on the input of RNA in sufficient quantity and quality. This highlights the need for standard procedures to ensure best practices for RNA extraction from often small tumor biopsies with variable tissue handling. We optimized an RNA extraction protocol from fresh-frozen (FF) core needle biopsies (CNB) from breast cancer patients and from formalin-fixed paraffin-embedded (FFPE) tissue when FF CNB did not yield sufficient RNA. Methods to avoid ribonucleases andto homogenize or to deparaffinize tissues and the impact of tissue composition on RNA extraction were studied. Additionally, RNA's compatibility with the nanoString nCounter® technology was studied. This technology platform enables GEP using small RNA fragments. After optimization of the protocol, RNA of high quality and sufficient quantity was obtained from FF CNB in 92% of samples. For the remaining 8% of cases, FFPE material prepared by the pathology department was used for RNA extraction. Both resulting RNA end products are compatible with the nanoString nCounter® technology.

Evaluation of an online training tool for scoring programmed cell death ligand-1 (PD-L1) diagnostic tests for lung cancer.

BACKGROUND: Numerous studies indicate that higher tumour programmed cell death ligand-1 (PD-L1) expression is associated with greater response to anti-programmed cell death-1 (PD-1)/PD-L1 immunotherapy in non-small cell lung cancer (NSCLC). In the era of precision medicine, there is a need to provide reliable, standardised training for pathologists to improve their accuracy of interpretation and scoring, as the results are used directly to inform clinical decisions. Here we present findings regarding reader reproducibility of PD-L1 tumour cell (TC) staining scoring for NSCLC using a PD-L1 e-trainer tool as part of a PD-L1 immunohistochemistry reader training course. METHODS: The PD-L1 training course was developed based on the use of VENTANA PD-L1 (SP263) and Dako PD-L1 IHC PharmDx 22C3 stained NSCLC samples in combination with a PD-L1 e-trainer tool. Five-hundred formalin-fixed, paraffin-embedded archival samples were obtained from commercial sources and stained for PD-L1. Slides were scored by two expert pathologists, then scanned to produce digital images and re-scored. Thirty-three cases were selected and sorted into three sets: a training set and two self-assessment tests (pre-test and 'competence' test). Participants (all selected board-certified pathologists) received face-to-face training including use of an e-trainer tool. Statistical analyses were performed using the competence test set. Overall percentage agreement (OPA) was assessed between the participant pathologists' registered scores and the reference scores assigned by expert pathologists at clinically relevant PD-L1 cut-offs (≥1%, ≥25% and ≥ 50%). RESULTS: Seven sessions were held and 69 participant pathologists completed the training. Inter-reader concordance indicated high OPA (85-95%) for PD-L1 TC scoring at clinically relevant cut-offs, with Fleiss' Kappa > 0.5. CONCLUSIONS: Use of this web-based training tool incorporated into classroom-style training was associated with an overall moderately good level of inter-reader reproducibility at key cut-offs for TC PD-L1 expression testing in NSCLC. Overall, the online training tool offers a means of standardised training for practising pathologists in a clinical setting.

Evaluation of Distance Metrics and Spatial Autocorrelation in Uniform Manifold Approximation and Projection Applied to Mass Spectrometry Imaging Data.

In this work, uniform manifold approximation and projection (UMAP) is applied for nonlinear dimensionality reduction and visualization of mass spectrometry imaging (MSI) data. We evaluate the performance of the UMAP algorithm on MSI data sets acquired in mouse pancreas and human lymphoma samples and compare it to those of principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), and the Barnes-Hut (BH) approximation of t-SNE. Furthermore, we compare different distance metrics in (BH) t-SNE and UMAP and propose the use of spatial autocorrelation as a means of comparing the resulting low-dimensional embeddings. The results indicate that UMAP is competitive with t-SNE in terms of visualization and is well-suited for the dimensionality reduction of large (>100 000 pixels) MSI data sets. With an almost fourfold decrease in runtime, it is more scalable in comparison with the current state-of-the-art: t-SNE or the Barnes-Hut approximation of t-SNE. In what seems to be the first application of UMAP to MSI data, we assess the value of applying alternative distance metrics, such as the correlation, cosine, and the Chebyshev metric, in contrast to the traditionally used Euclidean distance metric. Furthermore, we propose "histomatch" as an additional custom distance metric for the analysis of MSI data.

Validated programmed cell death ligand 1 immunohistochemistry assays (E1L3N and SP142) reveal similar immune cell staining patterns in melanoma when using the same sensitive detection system.

AIMS: Tumour cell and/or immune cell programmed cell death ligand 1 (PD-L1) expression is considered as a potential biomarker for anti-PD1 and anti-PD-L1 immunotherapy. Currently, different PD-L1 assays are used. This study aims to compare the staining patterns of two PD-L1 antibody clones in melanoma metastases and correlate them with PD-L1 mRNA expression. METHODS AND RESULTS: The immunohistochemistry assays were optimized and validated independently on a Ventana Benchmark Ultra (Ventana Medical Systems Inc., Tucson, AZ, USA) (E1L3N) and XT (SP142), using the same detection system. In total, 46 melanoma metastases were stained with both validated immunohistochemistry assays. Stained slides were digitized for qualitative and semi-quantitative evaluation; done by pathologist and semi-automated software analysis. A subset of 21 melanoma metastases was selected for quantification of the PD-L1 mRNA expression. Accuracy and precision criteria were met for both assays. PD-L1 protein and mRNA expression showed remarkably good Spearman's coefficients of 0.90 (E1L3N) and 0.87 (SP142). Despite the remarkable correlation between both PD-L1 assays in expression patterns and quantification values (ρ > 0.90), E1L3N showed significantly more tumour cell staining than SP142. CONCLUSIONS: E1L3N and SP142 IHC assays were optimized and validated successfully and independently for sensitive and accurate PD-L1 detection. Concordance was best for immune cell scoring, while E1L3N tended to detect more tumour cells. Determination of the clinically relevant cut-off values for immune cell versus tumour cell detection requires further research.

Development and Validation of a Histological Method to Measure Microvessel Density in Whole-Slide Images of Cancer Tissue.

Despite all efforts made to develop predictive biomarkers for antiangiogenic therapies, no unambiguous markers have been identified so far. This is due to among others the lack of standardized tests. This study presents an improved microvessel density quantification method in tumor tissue based on stereological principles and using whole-slide images. Vessels in tissue sections of different cancer types were stained for CD31 by an automated and validated immunohistochemical staining method. The stained slides were digitized with a digital slide scanner. Systematic, uniform, random sampling of the regions of interest on the whole-slide images was performed semi-automatically with the previously published applications AutoTag and AutoSnap. Subsequently, an unbiased counting grid was combined with the images generated with these scripts. Up to six independent observers counted microvessels in up to four cancer types: colorectal carcinoma, glioblastoma multiforme, ovarian carcinoma and renal cell carcinoma. At first, inter-observer variability was found to be unacceptable. However, after a series of consensus training sessions and interim statistical analysis, counting rules were modified and inter-observer concordance improved considerably. Every CD31-positive object was counted, with exclusion of suspected CD31-positive monocytes, macrophages and tumor cells. Furthermore, if interconnected, stained objects were considered a single vessel. Ten regions of interest were sufficient for accurate microvessel density measurements. Intra-observer and inter-observer variability were low (intraclass correlation coefficient > 0.7) if the observers were adequately trained.

Predictive tissue biomarkers for bevacizumab-containing therapy in metastatic colorectal cancer: an update.

Bevacizumab is the first anti-angiogenic agent approved for the treatment of metastatic colorectal cancer. The need for patient selection before initiating therapy necessitates the study of various proteins expressed in metastatic colorectal cancer tissue as candidate predictive markers. Immunohistochemistry is a valuable, commonly available and cost-effective method to assess predictive biomarkers. However, it is subject to variations and therefore requires rigorous protocol standardizations. Furthermore, validated quantification methodologies to study these angiogenic elements have to be applied. Based on their function in tumor angiogenesis and their relation to the mechanism of action of bevacizumab, protein markers were divided in four groups: VEGF A-signaling proteins; other relevant angiogenesis factors; factors regarding the tumor microenvironment and tumor intrinsic markers. Conceivably, nimbly selecting a small but relevant group of therapy-guided patients by the appropriate combination of predictive biomarkers may confer great value to this angiogenic inhibitor.

Design of an Optimized Wilms' Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo.

Tumor antigen-encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K.Molecular Therapy-Nucleic Acids (2013) 2, e134; doi:10.1038/mtna.2013.54; published online 19 November 2013.

Nanobody-based targeting of the macrophage mannose receptor for effective in vivo imaging of tumor-associated macrophages.

Tumor-associated macrophages (TAM) are an important component of the tumor stroma and exert several tumor-promoting activities. Strongly pro-angiogenic TAMs that reside in hypoxic tumor areas highly express macrophage mannose receptor (MMR, CD206). In this study, we targeted MMR+ TAMs using nanobodies, which are single-domain antigen-binding fragments derived from Camelidae heavy-chain antibodies. MMR-specific nanobodies stained TAMs in lung and breast tumor single-cell suspensions in vitro, and intravenous injection of 99mTc-labeled anti-MMR nanobodies successfully targeted tumor in vivo. Retention of the nanobody was receptor-specific and absent in MMR-deficient mice. Importantly, co-injection of excess unlabeled, bivalent anti-MMR nanobodies reduced nanobody accumulation in extratumoral organs to background levels, without compromising tumor uptake. Within tumors, the 99mTc-labeled nanobodies specifically labeled MMR+ TAMs, as CCR2-deficient mice that contain fewer TAMs showed significantly reduced tumor uptake. Further, anti-MMR nanobodies accumulated in hypoxic regions, thus targeting pro-angiogenic MMR+ TAMs. Taken together, our findings provide preclinical proof of concept that anti-MMR nanobodies can be used to selectively target and image TAM subpopulations in vivo.

A dissociated glucocorticoid receptor modulator reduces airway hyperresponsiveness and inflammation in a mouse model of asthma.

The glucocorticoid receptor (GR) is a transcription factor able to support either target gene activation via direct binding to DNA or gene repression via interfering with the activity of various proinflammatory transcription factors. An improved therapeutic profile for combating chronic inflammatory diseases has been reported through selectively modulating the GR by only triggering its transrepression function. We have studied in this paper the activity of Compound A (CpdA), a dissociated GR modulator favoring GR monomer formation, in a predominantly Th2-driven asthma model. CpdA acted similarly to the glucocorticoid dexamethasone (DEX) in counteracting OVA-induced airway hyperresponsiveness, recruitment of eosinophils, dendritic cells, neutrophils, B and T cells, and macrophages in bronchoalveolar lavage fluid, lung Th2, Tc2, Th17, Tc17, and mast cell infiltration, collagen deposition, and goblet cell metaplasia. Both CpdA and DEX inhibited Th2 cytokine production in bronchoalveolar lavage as well as nuclear translocation of NF-κB and its subsequent recruitment onto the IκBα promoter in the lung. By contrast, DEX but not CpdA induces expression of the GR-dependent model gene MAPK phosphatase 1 in the lung, confirming the dissociative action of CpdA. Mechanistically, we demonstrate that CpdA inhibited IL-4-induced STAT6 translocation and that GR is essential for CpdA to mediate chemokine repression. In conclusion, we clearly show in this study the anti-inflammatory effect of CpdA in a Th2-driven asthma model in the absence of transactivation, suggesting a potential therapeutic benefit of this strategy.

Pulmonary lymphoid neogenesis in idiopathic pulmonary arterial hypertension.

RATIONALE: Patients with idiopathic pulmonary arterial hypertension (IPAH) present circulating autoantibodies against vascular wall components. Pathogenic antibodies may be generated in tertiary (ectopic) lymphoid tissues (tLTs). OBJECTIVES: To assess the frequency of tLTs in IPAH lungs, as compared with control subjects and flow-induced PAH in patients with Eisenmenger syndrome, and to identify local mechanisms responsible for their formation, perpetuation, and function. METHODS: tLT composition and structure were studied by multiple immunostainings. Cytokine/chemokine and growth factor expression was quantified by real-time polymerase chain reaction and localized by immunofluorescence. The systemic mark of pulmonary lymphoid neogenesis was investigated by flow cytometry analyses of circulating lymphocytes. MEASUREMENTS AND MAIN RESULTS: As opposed to lungs from control subjects and patients with Eisenmenger syndrome, IPAH lungs contained perivascular tLTs, comprising B- and T-cell areas with high endothelial venules and dendritic cells. Lymphocyte survival factors, such as IL-7 and platelet-derived growth factor-A, were expressed in tLTs as well as the lymphorganogenic cytokines/chemokines, lymphotoxin-α/-ß, CCL19, CCL20, CCL21, and CXCL13, which might explain the depletion of circulating CCR6(+) and CXCR5(+) lymphocytes. tLTs were connected with remodeled vessels via an ER-TR7(+) stromal network and supplied by lymphatic channels. The presence of germinal center centroblasts, follicular dendritic cells, activation-induced cytidine deaminase, and IL-21(+)PD1(+) follicular helper T cells in tLTs together with CD138(+) plasma cell accumulation around remodeled vessels in areas of immunoglobulin deposition argued for local immunoglobulin class switching and ongoing production. CONCLUSIONS: We highlight the main features of lymphoid neogenesis specifically in the lungs of patients with IPAH, providing new evidence of immunological mechanisms in this severe condition.

The ubiquitin-editing protein A20 prevents dendritic cell activation, recognition of apoptotic cells, and systemic autoimmunity.

Dendritic cells (DCs) regulate both immunity and tolerance. Here we have shown that the ubiquitin editing enzyme A20 (Tnfaip3) determines the activation threshold of DCs, via control of canonical NF-κB activation. Tnfaip3(fl/fl)Cd11c-cre(+) mice lacking A20 in DCs demonstrated spontaneous proliferation of conventional and double-negative T cells, their conversion to interferon-γ (IFN-γ)-producing effector cells, and expansion of plasma cells. They developed ds-DNA antibodies, nephritis, the antiphospholipid syndrome, and lymphosplenomegaly-features of systemic lupus erythematosus-and extramedullary hematopoiesis. A20-deficient DCs were resistant to apoptosis, caused by increased sensitivity to CD40L and RANKL prosurvival signals and upregulation of antiapoptotic proteins Bcl-2 and Bcl-x. They captured injected apoptotic cells more efficiently, resisted the inhibitory effects of apoptotic cells, and induced self-reactive effector lymphocytes. Because genetic polymorphisms in TNFAIP3 are associated with human autoimmune disorders, these findings identify A20-mediated control of DC activation as a crucial checkpoint in the development of systemic autoimmunity.

Postextubation obstructive pseudomembranes: a case series and review of a rare complication after endotracheal intubation.

Endotracheal intubation obviously may be life-saving, but it may also lead to complications, including those related to damage of the airways. Superficial damage of the trachea at the site of the endotracheal cuff may trigger the formation of an obstructive fibrinous tracheal pseudomembrane (OFTP). Shortly after extubation, this clot, consisting of fibrin, leucocytes, and necrotic epithelium, can cause stridor due to adherence to the tracheal wall and obstruction of the airway. In most cases, the lesion is easily removed by rigid or fiberoptic bronchoscopy and virtually never leads to permanent damage. The study consisted of case series and review of the literature. This report describes a series of five adult cases and reviews all 19 other previously described cases. A careful analysis of all reported cases, however, did not highlight a simple predisposing factor or illness. It is important to consider OFTP in the differential diagnosis of stridor and respiratory insufficiency in the postextubation period.