Protein Haptenation and Its Role in Allergy.

Humans are exposed to numerous electrophilic chemicals either as medicines, in the workplace, in nature, or through use of many common cosmetic and household products. Covalent modification of human proteins by such chemicals, or protein haptenation, is a common occurrence in cells and may result in generation of antigenic species, leading to development of hypersensitivity reactions. Ranging in severity of symptoms from local cutaneous reactions and rhinitis to potentially life-threatening anaphylaxis and severe hypersensitivity reactions such as Stephen-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), all these reactions have the same Molecular Initiating Event (MIE), i.e. haptenation. However, not all individuals who are exposed to electrophilic chemicals develop symptoms of hypersensitivity. In the present review, we examine common chemistry behind the haptenation reactions leading to formation of neoantigens. We explore simple reactions involving single molecule additions to a nucleophilic side chain of proteins and complex reactions involving multiple electrophilic centers on a single molecule or involving more than one electrophilic molecule as well as the generation of reactive molecules from the interaction with cellular detoxification mechanisms. Besides generation of antigenic species and enabling activation of the immune system, we explore additional events which result directly from the presence of electrophilic chemicals in cells, including activation of key defense mechanisms and immediate consequences of those reactions, and explore their potential effects. We discuss the factors that work in concert with haptenation leading to the development of hypersensitivity reactions and those that may act to prevent it from developing. We also review the potential harnessing of the specificity of haptenation in the design of potent covalent therapeutic inhibitors.

Patients with naproxen-induced liver injury display T-cell memory responses toward an oxidative (S)-O-desmethyl naproxen metabolite but not the acyl glucuronide.

BACKGROUND: Exposure to nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen (IBU) and naproxen (NAP) is associated with idiosyncratic drug-induced liver injury (DILI). Carboxylate bioactivation into reactive metabolites (e.g., acyl glucuronides, AG) and resulting T-cell activation is hypothesized as causal for this adverse event. However, conclusive evidence supporting this is lacking. METHODS: In this work, we identify CD4+ and CD8+ T-cell hepatic infiltration in a biopsy from an IBU DILI patient. Lymphocyte transformation test and IFN-γ ELIspot, conducted on peripheral blood mononuclear cells (PBMCs) of patients with NAP-DILI, were used to explore drug-specific T-cell activation. T-cell clones (TCC) were generated and tested for drug specificity, phenotype/function, and pathways of T-cell activation. Cells were exposed to NAP, its oxidative metabolite 6-O-desmethyl NAP (DM-NAP), its AG or synthesized NAP-AG human-serum albumin adducts (NAP-AG adduct). RESULTS: CD4+ and CD8+ T-cells from patients expressing a range of different Vß receptors were stimulated to proliferate and secrete IFN-γ and IL-22 when exposed to DM-NAP, but not NAP, NAP-AG or the NAP-AG adduct. Activation of the CD4+ TCC was HLA-DQ-restricted and dependent on antigen presenting cells (APC); most TCC were activated with DM-NAP-pulsed APC, while fixation of APC blocked the T-cell response. Cross-reactivity was not observed with structurally-related drugs. CONCLUSION: Our results confirm hepatic T-cell infiltrations in NSAID-induced DILI, and show a T-cell memory response toward DM-NAP indicating an immune-mediated basis for the adverse event. Whilst bioactivation at the carboxylate group is widely hypothesized to be pathogenic for NSAID associated DILI, we found no evidence of this with NAP.

Head-to-Head Comparison of 68Ga-PSMA-11 with 68Ga-P137 in Patients with Suspected Prostate Cancer.

P137 is a novel oxalyldiaminopropionic acid-urea-based prostate-specific membrane antigen (PSMA) targeting agent. This study compared the uptake patterns of 68Ga-P137 and the FDA-approved PET tracer 68Ga-PSMA-11 for diagnosing prostate cancer (PCa). Sixteen patients suspected of PCa were scanned by 68Ga-PSMA-11 and 68Ga-P137 PET/CT, respectively, followed by prospective analysis. The tumor-to-background ratio was calculated using normal prostate tissue, blood pool, muscle, and urine as backgrounds. Pathology or follow-up results were used to analyze uptake patterns of benign/malignant lesions and various organs. Thirteen patients were diagnosed with PCa and three with benign prostate diseases (BPD). The number and location of primary lesions, lymph node metastasis (LNM) (n = 25), bone metastasis (n = 30), and liver metastasis (n = 3) detected by the two tracers were identical. Maximum standardized uptake value (SUVmax), tumor/normal prostate ratio, as well as semiquantitative miPSMA-ES and PRIMARY diagnostic scores (P all >0.05) showed similar uptake levels of primary lesions between 68Ga-P137 and 68Ga-PSMA-11. Compared to 68Ga-P137, the SUVmax of 68Ga-PSMA-11 was significantly higher for bone metastasis, LNM, and liver metastasis (14.9 ± 7.2 vs 9.1 ± 4.4, 14.4 ± 5.0 vs 7.5 ± 2.4, 13.9 ± 2.0 vs 8.8 ± 2.4, P all <0.05). One-hour postinjection, SUVmax of the duodenum (9.4 ± 2.1 vs 16.2 ± 6.1), kidney (19.4 ± 4.3 vs 45.6 ± 20.9), and urine (14.1 ± 7.1 vs 42.1 ± 25.9) were significantly lower for 68Ga-P137 than for 68Ga-PSMA-11 (P all <0.05), whereas the radioactivity accumulation of blood pool and muscle (3.9 ± 0.5 vs 1.6 ± 0.4, 1.0 ± 0.1 vs 0.6 ± 0.1, P all <0.05) of 68Ga-P137 was significantly higher than 68Ga-PSMA-11. The uptake level of 68Ga-P137 has no significant difference from that of 68Ga-PSMA-11 in prostate primary lesions, and their imaging performances are visually equivalent for both primary and metastatic lesions, despite a higher blood pool and muscle background and a lower uptake in metastatic lesions. Due to the lower urine excretion of 68Ga-P137, primary prostate lesions near the urine can potentially be displayed clearer than 68Ga-PSMA-11.

A blinded in vitro analysis of the intrinsic immunogenicity of hepatotoxic drugs: implications for preclinical risk assessment.

In vitro preclinical drug-induced liver injury (DILI) risk assessment relies largely on the use of hepatocytes to measure drug-specific changes in cell function or viability. Unfortunately, this does not provide indications toward the immunogenicity of drugs and/or the likelihood of idiosyncratic reactions in the clinic. This is because the molecular initiating event in immune DILI is an interaction of the drug-derived antigen with MHC proteins and the T-cell receptor. This study utilized immune cells from drug-naïve donors, recently established immune cell coculture systems and blinded compounds with and without DILI liabilities to determine whether these new methods offer an improvement over established assessment methods for the prediction of immune-mediated DILI. Ten blinded test compounds (6 with known DILI liabilities; 4 with lower DILI liabilities) and 5 training compounds, with known T-cell-mediated immune reactions in patients, were investigated. Naïve T-cells were activated with 4/5 of the training compounds (nitroso sulfamethoxazole, vancomycin, Bandrowski's base, and carbamazepine) and clones derived from the priming assays were activated with drug in a dose-dependent manner. The test compounds with DILI liabilities did not stimulate T-cell proliferative responses during dendritic cell-T-cell coculture; however, CD4+ clones displaying reactivity were detected toward 2 compounds (ciprofloxacin and erythromycin) with known liabilities. Drug-responsive T-cells were not detected with the compounds with lower DILI liabilities. This study provides compelling evidence that assessment of intrinsic drug immunogenicity, although complex, can provide valuable information regarding immune liabilities of some compounds prior to clinical studies or when immune reactions are observed in patients.

Why drug exposure is frequently associated with T-cell mediated cutaneous hypersensitivity reactions.

Cutaneous hypersensitivity reactions represent the most common manifestation of drug allergy seen in the clinic, with 25% of all adverse drug reactions appearing in the skin. The severity of cutaneous eruptions can vastly differ depending on the cellular mechanisms involved from a minor, self-resolving maculopapular rash to major, life-threatening pathologies such as the T-cell mediated bullous eruptions, i.e., Stevens Johnson syndrome/toxic epidermal necrolysis. It remains a significant question as to why these reactions are so frequently associated with the skin and what factors polarise these reactions towards more serious disease states. The barrier function which the skin performs means it is constantly subject to a barrage of danger signals, creating an environment that favors elicitation. Therefore, a critical question is what drives the expansion of cutaneous lymphocyte antigen positive, skin homing, T-cell sub-populations in draining lymph nodes. One answer could be the heterologous immunity hypothesis whereby tissue resident memory T-cells that express T-cell receptors (TCRs) for pathogen derived antigens cross-react with drug antigen. A significant amount of research has been conducted on skin immunity in the context of contact allergy and the role of tissue specific antigen presenting cells in presenting drug antigen to T-cells, but it is unclear how this relates to epitopes derived from circulation. Studies have shown that the skin is a metabolically active organ, capable of generating reactive drug metabolites. However, we know that drug antigens are displayed systemically so what factors permit tolerance in one part of the body, but reactivity in the skin. Most adverse drug reactions are mild, and skin eruptions tend to be visible to the patient, whereas minor organ injury such as transient transaminase elevation is often not apparent. Systemic hypersensitivity reactions tend to have early cutaneous manifestations, the progression of which is halted by early diagnosis and treatment. It is apparent that the preference for cutaneous involvement of drug hypersensitivity reactions is multi-faceted, therefore this review aims to abridge the findings from literature on the current state of the field and provide insight into the cellular and metabolic mechanisms which may contribute to severe cutaneous adverse reactions.

HLA-C*15:02 and epidermal growth factor receptor inhibitor-induced erosive pustular dermatosis of the scalp.

Epidermal growth factor receptor inhibitors (EGFRIs) are widely used to treat various types of malignancies. One of the common adverse reactions is cutaneous toxicity, mostly presenting as acneiform eruptions, paronychia and xerosis. Erosive pustular dermatosis of the scalp (EPDS) is a rare cutaneous adverse reaction that develops during treatment with EGFRIs. The pathogenesis of EGFRI-induced EPDS is poorly understood. Here we present three cases of EPDS induced by EGFRIs. The proteins LTA4H (leukotriene A-4 hydrolase), METAP1 (methionine aminopeptidase 1), BID (BH3-interacting domain death agonist), SMAD1 (mothers against decapentaplegic homologue), PRKRA (interferon-inducible double-stranded RNA-dependent protein kinase activator A), YES1 (tyrosine-protein kinase Yes) and EGFL7 (epidermal growth factor-like protein 7) were significantly upregulated in EGFRI-stimulated peripheral blood mononuclear cell cultures, and validated in the lesions. All of the proteins colocalized with CD4+ and CD8+ T-cell expression. Next-generation-based human leucocyte antigen (HLA) typing showed all patients carried HLA-C*15:02, and modelling studies showed that afatinib and erlotinib bound well within the E/F binding pockets of HLA-C*15:02. Moreover, T cells were preferentially activated by EGFRIs in individuals carrying HLA-C*15:02. The case series revealed that EGFRI-induced EPDS may be mediated by drug-specific T cells.

PI-RADS-Based Segmented Threshold of PSMA-PET SUVmax Is Better than Traditional Fixed Threshold for Diagnosing Clinically Significant Prostate Cancer Especially for PI-RADS 3 Lesions.

OBJECTIVES: Our purpose was to compare the performance of prostate-specific membrane antigen (PSMA)-positron emission tomography (PET) traditional fixed threshold (FT) and newly established Prostate Imaging Reporting and Data System (PI-RADS)-based segmented threshold (ST) for diagnosing clinically significant prostate cancer (csPCa). METHODS: The study retrospectively included 218 patients who underwent multiparametric magnetic resonance imaging (mpMRI) and PSMA-PET examination for suspected prostate cancer (PCa) from January 2018 to November 2021. Lesions with Gleason score ≥ 3 + 4 were diagnosed as csPCa. In PSMA-PET maximum standardized uptake value (SUVmax), the FT for all the lesions and STs for lesions with different PI-RADS score for diagnosing csPCa were determined by receiver operating characteristic (ROC) curves analysis and compared with Z test. The McNemar test was used to compare sensitivity and specificity. RESULTS: Among the 218 patients, there were 113 csPCa and 105 non-csPCa. The PSMA-PET FT was SUVmax > 5.3 (area under the curve, AUC = 0.842) and STs for PI-RADS 3/4/5 were SUVmax > 4.2/5.7/6.0 (AUCs = 0.870/0.867/0.882), respectively. The AUC of PSMA-PET ST was higher than that of PSMA-PET FT (0.872 vs. 0.842), especially for PI-RADS 3 (0.870 vs. 0.653). Multimodality diagnostic criteria combining PSMA-PET ST and PI-RADS scores of mpMRI was established and its AUC was higher than that of PSMA-PET ST (0.893 vs. 0.872) and significantly higher than that of PSMA-PET FT (0.893 vs. 0.842) with an improvement in sensitivity (93% vs. 78%, p < 0.05) without significantly sacrificing specificity (86% vs. 91%, p > 0.05). CONCLUSIONS: For diagnosing csPCa, PI-RADS-based PSMA-PET segmented threshold achieved better performance than traditional fixed threshold, especially for PI-RADS 3 lesions. Multimodality diagnostic criteria demonstrated higher diagnostic performance than segmented threshold and significantly better than PSMA-PET fixed threshold for detecting csPCa.

Activation of Human CD8+ T Cells with Nitroso Dapsone-Modified HLA-B*13:01-Binding Peptides.

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01-restricted nitroso dapsone-modified Pep1- (n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele-restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.

Detection of Hepatic Drug Metabolite-Specific T-Cell Responses Using a Human Hepatocyte, Immune Cell Coculture System.

Drug-responsive T-cells are activated with the parent compound or metabolites, often via different pathways (pharmacological interaction and hapten). An obstacle to the investigation of drug hypersensitivity is the scarcity of reactive metabolites for functional studies and the absence of coculture systems to generate metabolites in situ. Thus, the aim of this study was to utilize dapsone metabolite-responsive T-cells from hypersensitive patients, alongside primary human hepatocytes to drive metabolite formation, and subsequent drug-specific T-cell responses. Nitroso dapsone-responsive T-cell clones were generated from hypersensitive patients and characterized in terms of cross-reactivity and pathways of T-cell activation. Primary human hepatocytes, antigen-presenting cells, and T-cell cocultures were established in various formats with the liver and immune cells separated to avoid cell contact. Cultures were exposed to dapsone, and metabolite formation and T-cell activation were measured by LC-MS and proliferation assessment, respectively. Nitroso dapsone-responsive CD4+ T-cell clones from hypersensitive patients were found to proliferate and secrete cytokines in a dose-dependent manner when exposed to the drug metabolite. Clones were activated with nitroso dapsone-pulsed antigen-presenting cells, while fixation of antigen-presenting cells or omission of antigen-presenting cells from the assay abrogated the nitroso dapsone-specific T-cell response. Importantly, clones displayed no cross-reactivity with the parent drug. Nitroso dapsone glutathione conjugates were detected in the supernatant of hepatocyte immune cell cocultures, indicating that hepatocyte-derived metabolites are formed and transferred to the immune cell compartment. Similarly, nitroso dapsone-responsive clones were stimulated to proliferate with dapsone, when hepatocytes were added to the coculture system. Collectively, our study demonstrates the use of hepatocyte immune cell coculture systems to detect in situ metabolite formation and metabolite-specific T-cell responses. Similar systems should be used in future diagnostic and predictive assays to detect metabolite-specific T-cell responses when synthetic metabolites are not available.

Identification of flucloxacillin-modified hepatocellular proteins: implications in flucloxacillin-induced liver injury.

Flucloxacillin is a ß-lactam antibiotic associated with a high incidence of drug-induced liver injury. Although expression of HLA-B*57:01 is associated with increased susceptibility, little is known of the pathological mechanisms involved in the induction of the clinical phenotype. Irreversible protein modification is suspected to drive the reaction through the provision of flucloxacillin-modified peptides that are presented to T-cells by the protein encoded by the risk allele. In this study, we have shown that flucloxacillin binds to multiple proteins within human primary hepatocytes, including major hepatocellular proteins (hemoglobin and albumin) and mitochondrial proteins. Inhibition of membrane transporters multidrug resistance-associated protein 2 (MRP2) and P-glycoprotein (P-gp) appeared to reduce the levels of covalent binding. A diverse range of proteins with different functions was found to be targeted by flucloxacillin, including adaptor proteins (14-3-3), proteins with catalytic activities (liver carboxylesterase 1, tRNA-splicing endonuclease subunit Sen2, All-trans-retinol dehydrogenase ADH1B, Glutamate dehydrogenase 1 mitochondrial, Carbamoyl-phosphate synthase [ammonia] mitochondrial), and transporters (hemoglobin, albumin, and UTP-glucose-1-phosphate uridylyltransferase). These flucloxacillin-modified intracellular proteins could provide a potential source of neoantigens for HLA-B*57:01 presentation by hepatocytes. More importantly, covalent binding to critical cellular proteins could be the molecular initiating events that lead to flucloxacillin-induced cholestasis Data are available via ProteomeXchange with identifier PXD038581.

Clinical and genomic features in patients with second primary glioblastoma following first primary renal cell carcinoma.

PURPOSE: To explore the potential pathogenesis and clinical features of second primary glioblastoma (spGBM) following first primary renal cell carcinoma (fpRCC). METHODS: Patients with spGBM after fpRCC were enrolled from our institution and the SEER dataset. Sanger sequencing, whole genome sequencing, and immunehistochemistry were used to detect molecular biomarkers. RESULTS: Four and 122 cases from our institution and the SEER dataset, respectively, were collected with an overall median age of 69 years at spGBM diagnosis following fpRCC. The median interval time between fpRCC and spGBM was 50.7 months and 4 years, for the four and 122 cases respectively. The median overall survival time was 11.2 and 6.0 months for the two datasets. In addition, spGBM patients of younger age (< 75 years) or shorter interval time (< 1 year) had favorable prognosis (p = 0.081 and 0.05, respectively). Moreover, the spGBM cases were molecularly classified as TERT only paired with TP53 mutation, PIK3CA mutation, EGFR alteration, low tumor mutation burden, and stable microsatellite status. CONCLUSIONS: This is the first study to investigate the pathogenesis and clinical features of spGBM following spRCC. We found that spGBMs are old-age related, highly malignant, and have short survival time. Moreover, they might be misdiagnosed and treated as brain metastases from RCC. Thus, the incidence of spGBMs after fpRCC is underestimated. Further studies are needed to investigate the underlying molecular mechanisms and clinical biomarkers for the development of spGBM following fpRCC.

Activation of tolvaptan-responsive T-cell clones with the structurally-related mozavaptan.

Tolvaptan is an effective drug for the treatment of autosomal dominant polycystic kidney disease, but its use is associated with a significant risk of T-cell-mediated liver injury in a small number of patients. An important clinical conundrum following the contraindication of tolvaptan is whether administration of agents of similar pharmacological action and structure will be tolerated. Herein, we addressed this question through the exposure of tolvaptan-responsive T-cell clones to similar pharmaceutical agents. Whilst lixivaptan and conivaptan did not activate tolvaptan-responsive T-cells, mozavaptan evoked proliferative responses comparable with tolvaptan itself, indicating that there may be collateral immunological intolerance to this compound as a product of sensitization to tolvaptan.

Coronal T2-weighted imaging improves the measurement accuracy of the subarachnoid space in infants: A descriptive study.

Background: The subarachnoid space width (SASw) is part of crucial neuroimaging criteria for the diagnosis of subarachnoid space enlargement in infants. In addition to indicating the presence of these diseases, SASw can be used to assess their severity. Therefore, it is important to be able to measure the SASw accurately. Aim: This study aimed to compare the accuracy of measurements made from axial and coronal T2-weighted imaging (T2WI) and to establish a consentaneous measurement scheme of SASw in infants. Methods: A total of 63 infants (31 males and 32 females) aged 4 days to 24 months were enrolled in this study. The supratentorial subarachnoid space volume (SASv) and corrected SASv (cSASv) were used as the gold standard reference. The SASw (including interhemispheric width and bilateral frontal craniocortical width) was measured on axial and coronal T2WI. The intra- and inter-observer reproducibility and agreement of the SASw were assessed by the intraclass correlation coefficient (ICC) and Bland-Altman analysis. A paired t-test was used to compare SASw measured on axial and coronal images. The accuracy of SASw measurements made from axial and coronal T2WI was evaluated by the relationships between the SASw and supratentorial SASv and between the SASw and supratentorial cSASv, and the relationships were examined by multivariate linear regression. Results: The intra- and inter-observer ICC values of the three SASw measurements were greater on coronal T2WI than on axial T2WI. Bland-Altman analysis confirmed that the SASw values measured on coronal T2WI had better intra- and inter-observer agreement than axial T2WI. According to the multivariate linear regression results, model 4 (the SASw measured in coronal T2WI) was the best predictor of supratentorial cSASv (R2 = 0.755). Conclusions: The SASw measured on coronal T2WI was more repeatable and accurate than axial T2WI and was more representative of the actual cerebrospinal fluid accumulation in the supratentorial subarachnoid space. Relevance for Patients: The SASw has been found to be a simple and essential substitution for supratentorial SASv, which can be measured on both axial T2WI passing through the bodies of the bilateral ventricles and coronal T2WI at the level of the foramen of Monro. The SASw measured on coronal T2WI was more beneficial to the diagnosis and severity assessment of subarachnoid space enlargement in infants.

Shared Clavulanate and Tazobactam Antigenic Determinants Activate T-Cells from Hypersensitive Patients.

ß-Lactamase inhibitors such as clavulanic acid and tazobactam were developed to overcome ß-lactam antibiotic resistance. Hypersensitivity reactions to these drugs have not been studied in detail, and the antigenic determinants that activate T-cells have not been defined. The objectives of this study were to (i) characterize clavulanate- and tazobactam-responsive T-cells from hypersensitive patients, (ii) explore clavulanate and tazobactam T-cell crossreactivity, and (iii) define the antigenic determinants that contribute to T-cell reactivity. Antigen specificity, pathways of T-cell activation, and crossreactivity with clavulanate- and tazobactam-specific T-cell clones were assessed by proliferation and cytokine release assays. Antigenic determinants were analyzed by mass spectrometry-based proteomics methods. Clavulanate- and tazobactam-responsive CD4+ T-cell clones were stimulated to proliferate and secrete IFN-γ in an MHC class II-restricted and dose-dependent manner. T-cell activation with clavulanate- and tazobactam was dependent on antigen presenting cells because their fixation prevented the T-cell response. Strong crossreactivity was observed between clavulanate- and tazobactam-T-cells; however, neither drug activated ß-lactam antibiotic-responsive T-cells. Mass spectrometric analysis revealed that both compounds form multiple antigenic determinants with lysine residues on proteins, including an overlapping aldehyde and hydrated aldehyde adduct with mass additions of 70 and 88 Da, respectively. Collectively, these data show that although clavulanate and tazobactam are structurally distinct, the antigenic determinants formed by both drugs overlap, which explains the observed T-cell cross-reactivity.

Improved interobserver agreement on nodule type and Lung-RADS classification of subsolid nodules using computer-aided solid component measurement.

PURPOSE: The Lung CT Screening Reporting and Data System (Lung-RADS) classification of subsolid nodules (SSNs) can be challenging due to limited interobserver agreement in determining the type and size of the nodule. Our study aimed to assess the effect of a computer-aided method on the interobserver agreement of Lung-RADS classification for SSNs. MATERIALS AND METHODS: This study consisted of 156 SSNs in 121 patients who underwent initial CT screening for lung cancer. Three independent readers determined the nodule type and measured the size of the entire nodule as well as the solid component, first without and then assisted by a semi-automated computer-aided tool. They assigned to each nodule the corresponding Lung-RADS 1.1 category. Agreement in size measurements was assessed by intraclass correlation coefficient (ICC) and Bland-Altman indexes, while agreement in nodule type and Lung-RADS was determined using Fleiss kappa statistics. The relationship between final diagnosis of the nodules and Lung-RADS classifications was also evaluated. RESULTS: Among the 156 nodules, manual size measurement reached an ICC of 0.994, and 48 nodules contained solid component measured by all the three readers both manually and semi-automatically. ICCs for the solid component measurement were 0.952, 0.997 and 0.996 for manual diameter, semi- automated diameter and volume measurement, respectively. Bias and 95% limits of agreement for average diameter of solid component were smaller with semi-automated measurements than with manual measurements. Kappa values of semi-automated assessment for nodule type (0.974) and Lung-RADS classification (0.958 for diameter and 0.952 for volume) were higher than with the manual measurements (0.783 for nodule type and 0.652 for Lung-RADS classification). Compared to manual work, the semi-automated assessment identified more 4B nodules among the 26 pathologically confirmed invasive adenocarcinomas (IACs). CONCLUSION: Semi-automated assessment could improve the interobserver agreement of nodule type and Lung-RADS classification for SSNs, and be inclined to classify SSNs corresponding to pathologically confirmed IACs into higher risk categories.

Identification of prognostic biomarkers for breast cancer brain metastases based on the bioinformatics analysis.

PURPOSE: The prognosis of breast cancer (BC) patients who develop into brain metastases (BMs) is very poor. Thus, it is of great significance to explore the etiology of BMs in BC and identify the key genes involved in this process to improve the survival of BC patients with BMs. PATIENTS AND METHODS: The gene expression data and the clinical information of BC patients were downloaded from TCGA and GEO database. Differentially expressed genes (DEGs) in TCGA-BRCA and GSE12276 were overlapped to find differentially expressed metastatic genes (DEMGs). The protein-protein interaction (PPI) network of DEMGs was constructed via STRING database. ClusterProfiler R package was applied to perform the gene ontology (GO) enrichment analysis of DEMGs. The univariate Cox regression analysis and the Kaplan-Meier (K-M) curves were plotted to screen DEMGs associated with the overall survival and the metastatic recurrence survival, which were identified as the key genes associated with the BMs in BC. The immune infiltration and the expressions of immune checkpoints for BC patients with brain relapses and BC patients with other relapses were analyzed respectively. The correlations among the expressions of key genes and the differently infiltrated immune cells or the differentially expressed immune checkpoints were calculated. The gene set enrichment analysis (GSEA) of each key gene was conducted to investigate the potential mechanisms of key genes involved in BC patients with BMs. Moreover, CTD database was used to predict the drug-gene interaction network of key genes. RESULTS: A total of 154 DEGs were identified in BC patients at M0 and M1 in TCGA database. A total of 667 DEGs were identified in BC patients with brain relapses and with other relapses. By overlapping these DEGs, 17 DEMGs were identified, which were enriched in the cell proliferation related biological processes and the immune related molecular functions. The univariate Cox regression analysis and the Kaplan-Meier curves revealed that CXCL9 and GPR171 were closely associated with the overall survival and the metastatic recurrence survival and were identified as key genes associated with BMs in BC. The analyses of immune infiltration and immune checkpoint expressions showed that there was a significant difference of the immune microenvironment between brain relapses and other relapses in BC. GSEA indicated that CXCL9 and GPR171 may regulate BMs in BC via the immune-related pathways. CONCLUSION: Our study identified the key genes associated with BMs in BC patients and explore the underlying mechanisms involved in the etiology of BMs in BC. These findings may provide a promising approach for the treatments of BC patients with BMs.

Checkpoint Inhibition Reduces the Threshold for Drug-Specific T-Cell Priming and Increases the Incidence of Sulfasalazine Hypersensitivity.

An emerging clinical issue associated with immune-oncology agents is the collateral effects on the tolerability of concomitant medications. One report of this phenomenon was the increased incidence of hypersensitivity reactions observed in patients receiving concurrent immune checkpoint inhibitors (ICIs) and sulfasalazine (SLZ). Thus, the aim of this study was to characterize the T cells involved in the pathogenesis of such reactions, and recapitulate the effects of inhibitory checkpoint blockade on de-novo priming responses to compounds within in vitro platforms. A regulatory competent human dendritic cell/T-cell coculture assay was used to model the effects of ICIs on de novo nitroso sulfamethoxazole- and sulfapyridine (SP) (the sulfonamide component of SLZ) hydroxylamine-specific priming responses. The role of T cells in the pathogenesis of the observed reactions was explored in 3 patients through phenotypic characterization of SP/sulfapyridine hydroxylamine (SPHA)-responsive T-cell clones (TCC), and assessment of cross-reactivity and pathways of T-cell activation. Augmentation of the frequency of responding drug-specific T cells and intensity of the T-cell response was observed with PD-1/PD-L1 blockade. Monoclonal populations of SP- and SPHA-responsive T cells were isolated from all 3 patients. A core secretory effector molecule profile (IFN-γ, IL-13, granzyme B, and perforin) was identified for SP and SPHA-responsive TCC, which proceeded through Pi and hapten mechanisms, respectively. Data presented herein provides evidence that drug-responsive T cells are effectors of hypersensitivity reactions observed in oncology patients administered ICIs and SLZ. Perturbation of drug-specific T-cell priming is a plausible explanation for clinical observations of how an increased incidence of these adverse events is occurring.

Identification of MicroRNAs as potential biomarkers for detecting ischemic stroke.

BACKGROUND: Increasing epidemic of ischemic stroke (IS) makes it urgent to understand the pathogenesis and regulatory mechanism, previous studies have described microRNAs (miRNAs) is part of the brain's response to ischemia. OBJECTIVE: The aim of this study was to screen potential biomarkers for the prediction and novel treatment of IS. METHODS: Differentially expressed miRNAs were screened from three newly diagnosed IS patients and three controls by RNA sequencing technology. Furthermore, target prediction databases were then used to analysis the target genes of different expressed miRNAs, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database were used to identify the functions and the main biochemical and signal pathways of differentially expressed target genes. RESULTS: Our results revealed that 27 miRNAs were differentially expressed in IS, among which, hsa-miR-659-5p was the most highly increased and was first found to be associated with IS. In addition, KEGG pathway analyses showed that differentially expressed miRNAs were mainly significantly enriched in lysosome pathway, cytokine-cytokine receptor interaction pathway, spliceosome pathway, base excision repair pathway. CONCLUSIONS: miRNAs were involved in IS pathogenesis, and hsa-miR-659-5p, hsa-miR-151a-3p and hsa-miR-29c-5p as the three highest |log2FoldChange| regulation in this study, which may be the biomarkers of IS and need further study.

Unrepresentative big surveys significantly overestimated US vaccine uptake.

Surveys are a crucial tool for understanding public opinion and behaviour, and their accuracy depends on maintaining statistical representativeness of their target populations by minimizing biases from all sources. Increasing data size shrinks confidence intervals but magnifies the effect of survey bias: an instance of the Big Data Paradox1. Here we demonstrate this paradox in estimates of first-dose COVID-19 vaccine uptake in US adults from 9 January to 19 May 2021 from two large surveys: Delphi-Facebook2,3 (about 250,000 responses per week) and Census Household Pulse4 (about 75,000 every two weeks). In May 2021, Delphi-Facebook overestimated uptake by 17 percentage points (14-20 percentage points with 5% benchmark imprecision) and Census Household Pulse by 14 (11-17 percentage points with 5% benchmark imprecision), compared to a retroactively updated benchmark the Centers for Disease Control and Prevention published on 26 May 2021. Moreover, their large sample sizes led to miniscule margins of error on the incorrect estimates. By contrast, an Axios-Ipsos online panel5 with about 1,000 responses per week following survey research best practices6 provided reliable estimates and uncertainty quantification. We decompose observed error using a recent analytic framework1 to explain the inaccuracy in the three surveys. We then analyse the implications for vaccine hesitancy and willingness. We show how a survey of 250,000 respondents can produce an estimate of the population mean that is no more accurate than an estimate from a simple random sample of size 10. Our central message is that data quality matters more than data quantity, and that compensating the former with the latter is a mathematically provable losing proposition.

MiR-204 suppresses the progression of acute myeloid leukemia through HGF/c-Met pathway.

Acute myeloid leukemia (AML) was confirmed to be associated with hematopoietic insufficiency, as well as abnormal proliferation, differentiation or survival of myeloid progenitors. Multiple studies reported that microRNA-204 (miR-204) and Hepatocyte growth factor (HGF) played important roles in types of cancers. However, the potential molecular regulatory mechanism between miR-204 and HGF in AML remains to be further defined. Real-time PCR (RT-PCR) was adopted to detect the expression of miR-204 and HG. Relative protein levels were detected by western blot assay. The viability, cell cycle, apoptosis, migration, and invasion were analyzed by MTT, flow cytometry, and transwell assays. Moreover, the target relationship between miR-204 and HGF was predicted by MiRcode website and confirmed by luciferase reporter, RNA pull-down, and western blot assays. Our data suggested that miR-204 was downregulated in AML serum samples and cells. MiR-204 overexpression repressed cell proliferation, migration, invasion, and induced cell apoptosis in AML cells. HGF was upregulated in AML samples and cells, and HGF knockdown inhibited the malignancy of AML cells. In addition, HGF was directly targeted by miR-204. HGF overexpression reversed the effects of miR-204 mimic on AML cell proliferation, apoptosis, migration, and invasion. Besides, miR-204 regulated the c-Met signaling by targeting HGF, thereby regulating the downstream protein levels related to cell proliferation, apoptosis, migration, and invasion in AML cells. In conclusion, miR-204 could regulate AML progression through regulating the HGF/c-Met pathway.