Improving quality control in the routine practice for histopathological interpretation of gastrointestinal endoscopic biopsies using artificial intelligence.

BACKGROUND: Colorectal and gastric cancer are major causes of cancer-related deaths. In Korea, gastrointestinal (GI) endoscopic biopsy specimens account for a high percentage of histopathologic examinations. Lack of a sufficient pathologist workforce can cause an increase in human errors, threatening patient safety. Therefore, we developed a digital pathology total solution combining artificial intelligence (AI) classifier models and pathology laboratory information system for GI endoscopic biopsy specimens to establish a post-analytic daily fast quality control (QC) system, which was applied in clinical practice for a 3-month trial run by four pathologists. METHODS AND FINDINGS: Our whole slide image (WSI) classification framework comprised patch-generator, patch-level classifier, and WSI-level classifier. The classifiers were both based on DenseNet (Dense Convolutional Network). In laboratory tests, the WSI classifier achieved accuracy rates of 95.8% and 96.0% in classifying histopathological WSIs of colorectal and gastric endoscopic biopsy specimens, respectively, into three classes (Negative for dysplasia, Dysplasia, and Malignant). Classification by pathologic diagnosis and AI prediction were compared and daily reviews were conducted, focusing on discordant cases for early detection of potential human errors by the pathologists, allowing immediate correction, before the pathology report error is conveyed to the patients. During the 3-month AI-assisted daily QC trial run period, approximately 7-10 times the number of slides compared to that in the conventional monthly QC (33 months) were reviewed by pathologists; nearly 100% of GI endoscopy biopsy slides were double-checked by the AI models. Further, approximately 17-30 times the number of potential human errors were detected within an average of 1.2 days. CONCLUSIONS: The AI-assisted daily QC system that we developed and established demonstrated notable improvements in QC, in quantitative, qualitative, and time utility aspects. Ultimately, we developed an independent AI-assisted post-analytic daily fast QC system that was clinically applicable and influential, which could enhance patient safety.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.

SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19. In Brief: LY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated. Highlights: LY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variantsNo loss of potency against currently circulating variantsBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID databaseBreadth of neutralizing activity and potency supports clinical development.

Screening Antibodies Raised against the Spike Glycoprotein of SARS-CoV-2 to Support the Development of Rapid Antigen Assays.

Severe acute respiratory coronavirus-2 (SARS-CoV-2) is a novel viral pathogen and therefore a challenge to accurately diagnose infection. Asymptomatic cases are common and so it is difficult to accurately identify infected cases to support surveillance and case detection. Diagnostic test developers are working to meet the global demand for accurate and rapid diagnostic tests to support disease management. However, the focus of many of these has been on molecular diagnostic tests, and more recently serologic tests, for use in primarily high-income countries. Low- and middle-income countries typically have very limited access to molecular diagnostic testing due to fewer resources. Serologic testing is an inappropriate surrogate as the early stages of infection are not detected and misdiagnosis will promote continued transmission. Detection of infection via direct antigen testing may allow for earlier diagnosis provided such a method is sensitive. Leading SARS-CoV-2 biomarkers include spike protein, nucleocapsid protein, envelope protein, and membrane protein. This research focuses on antibodies to SARS-CoV-2 spike protein due to the number of monoclonal antibodies that have been developed for therapeutic research but also have potential diagnostic value. In this study, we assessed the performance of antibodies to the spike glycoprotein, acquired from both commercial and private groups in multiplexed liquid immunoassays, with concurrent testing via a half-strip lateral flow assays (LFA) to indicate antibodies with potential in LFA development. These processes allow for the selection of pairs of high-affinity antispike antibodies that are suitable for liquid immunoassays and LFA, some of which with sensitivity into the low picogram range with the liquid immunoassay formats with no cross-reactivity to other coronavirus S antigens. Discrepancies in optimal ranking were observed with the top pairs used in the liquid and LFA formats. These findings can support the development of SARS-CoV-2 LFAs and diagnostic tools.

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

Indoxyl Sulfate-Mediated Metabolic Alteration of Transcriptome Signatures in Monocytes of Patients with End-Stage Renal Disease (ESRD).

End-stage renal disease (ESRD) is the final stage of chronic kidney disease, which is increasingly prevalent worldwide and is associated with the progression of cardiovascular disease (CVD). Indoxyl sulfate (IS), a major uremic toxin, plays a key role in the pathology of CVD via adverse effects in endothelial and immune cells. Thus, there is a need for a transcriptomic overview of IS responsive genes in immune cells of ESRD patients. Here, we investigated IS-mediated alterations in gene expression in monocytes from ESRD patients. Transcriptomic analysis of ESRD patient-derived monocytes and IS-stimulated monocytes from healthy controls was performed, followed by analysis of differentially expressed genes (DEGs) and gene ontology (GO). We found that 148 upregulated and 139 downregulated genes were shared between ESRD patient-derived and IS-stimulated monocytes. Interaction network analysis using STRING and ClueGo suggests that mainly metabolic pathways, such as the pentose phosphate pathway, are modified by IS in ESRD patient-derived monocytes. These findings were confirmed in IS-stimulated monocytes by the increased mRNA expression of genes including G6PD, PGD, and TALDO1. Our data suggest that IS causes alteration of metabolic pathways in monocytes of ESRD patients and, thus, these altered genes may be therapeutic targets.

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection.

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection. ONE SENTENCE SUMMARY: LY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.

Indoxyl sulfate (IS)-mediated immune dysfunction provokes endothelial damage in patients with end-stage renal disease (ESRD).

Progressive renal failure causes uremia-related immune dysfunction, which features a chronic inflammatory milieu. Given the central role of end-stage renal disease (ESRD)-related immune dysfunction in the pathogenesis of cardiovascular diseases (CVDs), much attention has been focused on how uremic toxins affect cellular immunity and the mechanisms underlying pathogenesis of atherosclerosis in ESRD patients. Here, we investigated the characteristics of monocytes and CD4+ T cells in ESRD patients and the immune responses induced by indoxyl sulfate (IS), a key uremic toxin, in order to explore the pathogenic effects of these cells on vascular endothelial cells. In ESRD patients, monocytes respond to IS through the aryl hydrocarbon receptor (AhR) and consequently produce increased levels of TNF-α. Upon stimulation with TNF-α, human vascular endothelial cells produce copious amounts of CX3CL1, a chemokine ligand of CX3CR1 that is highly expressed on CD4+CD28-T cells, the predominantly expanded cell type in ESRD patients. A migration assay showed that CD4+CD28- T cells were preferentially recruited by CX3CL1. Moreover, activated CD4+CD28- T cells exhibited cytotoxic capability allowing for the induction of apoptosis in HUVECs. Our findings suggest that in ESRD, IS-mediated immune dysfunction may cause vascular endothelial cell damage and thus, this toxin plays a pivotal role in the pathogenesis of CVD.

Expansion of CD8(+) T cells lacking the IL-6 receptor α chain in patients with coronary artery diseases (CAD).

BACKGROUND AND AIMS: The pathogenesis of coronary artery disease (CAD) is closely associated with chronic inflammatory processes. CD8(+) T cells are a key participant in the pathogenesis of atherosclerosis, the major cause of CAD; however, it remains unclear which CD8(+) T-cell subset is responsible. We investigated the immunological features of CD8(+) T cells expressing low and high levels of the IL-6 receptor α chain (IL-6Rα), a cytokine known to play a key role in cardiovascular diseases. METHODS: The expression of IL-6Rα on CD8(+) T cells and its association with plasma levels of soluble components of the IL-6/IL-6Rs as well as with clinical parameters were analyzed using FACS analysis and ELISA of CAD patients and age-matched healthy controls (HCs). Immunological characteristics of CD8(+) T cells expressing low and high levels of IL-6Rα (CD8(+)IL-6Rα(low or high)) were examined by in vitro culture and intracellular FACS analysis. RESULTS: CAD patients had higher frequencies of circulating CD8(+)IL-6Rα(low) effector memory (EM) T cells compared with HCs (median frequency; 74.59% vs. 60.09%, p = 0.0158). Expanded CD8(+)IL-6Rα(low) T cells positively correlated with the frequency of senescent, cytotoxic CD8(+)CD57(+) T cells (r = 0.6655, p < 0.0001) and plasma IL-6 level (r = 0.3995, p = 0.0432) in CAD patients. Loss of IL-6Rα expression on CD8(+) T cells was induced by the combination of IL-6 and IL-15 with accompanying TCR-independent proliferation (p = 0.0101). Moreover, these CD8(+)IL-6Rα(low) T cells had features of type 1 cytotoxic CD8(+) T cells. CONCLUSIONS: Our findings suggest the possible involvement of expanded CD8(+)IL-6Rα(low) EM T cells in CAD through their pro-inflammatory and highly cytotoxic capacities.

The prognosis of patients with lung cancer admitted to the medical intensive care unit.

AIMS: Lung cancer remains the leading cause of cancer mortality worldwide. Despite their poor prognosis, patients with lung cancer are increasingly being admitted to the medical intensive care unit (MICU) for treatment of critical illnesses. The aim of this study was to assess the outcome of patients with lung cancer who are admitted to an MICU and to identify the measurable predictors of their MICU outcome. METHODS: We conducted retrospective analysis on 97 patients with lung cancer admitted to the MICU between 2007 and 2011. RESULTS: The mean age ± standard deviation was 71.8 ± 6.8 years. Of the 97 patients (82 male), 73 patients (75%) had non-small cell lung cancer stage IIIB, IV and 24 patients (25%) had small cell lung cancer. The intensive care unit mortality and in-hospital mortality rates were 53.6 and 61.8%. The main reasons for MICU admission were pneumonia (n = 51) and complication of cancer management (n = 45). The predictors of poor MICU outcome were history of diabetes mellitus (P = 0.028), Acute Physiology and Chronic Health Evaluation II score (P = 0.018), need for mechanical ventilation (P = 0.014), use of vasoactive agents (P < 0.0001), the presence of acute renal failure (P < 0.0001) and presence of multiorgan failure (P < 0.0001). CONCLUSIONS: We found that in-hospital mortality was not influenced by age, sex or performance status score of patients with lung cancer but increased with the severity of organ failure at MICU admission.

Inhibition of interleukin-1ß-mediated interleukin-1 receptor-associated kinase 4 phosphorylation by zinc leads to repression of memory T helper type 17 response in humans.

Zinc is an essential trace element that plays pivotal roles in multiple facets of the immune system. Besides its catalytic and structural roles, zinc also functions as an intracellular signalling molecule, and changes in zinc levels can cause both direct and indirect modulation of immune responses. Further, cytoplasmic levels of bioavailable zinc in immune cells are largely influenced by many extracellular stimuli. Here we provide evidence that zinc represses memory T helper type 17 responses in humans by inhibiting interleukin-1ß (IL-1ß)-mediated signal. In vitro zinc treatment of CD4(+) T cells in the presence of activated monocytes inhibited interferon-γ-producing cells and IL-17-producing cells, but not IL-4-producing cells. Of note, production of IL-17(+) cells from memory CD4(+) T cells, which is significantly up-regulated by lipopolysaccharide-stimulated monocytes, was preferentially repressed by zinc. Increased cytoplasmic zinc in T cells suppressed IL-1ß signalling through repression of phosphorylation of IL-1 receptor-associated kinase 4 (IRAK4), so leading to an inhibitory effect on T helper type 17 responses facilitated by monocyte-derived IL-1ß in humans. These findings suggest that extracellular zinc bioavailability may affect memory CD4(+) T-cell responses by modulating the zinc-mediated signalling pathway.

Pemetrexed induces apoptosis in malignant mesothelioma and lung cancer cells through activation of reactive oxygen species and inhibition of sirtuin 1.

Pemetrexed is a multitargeted antifolate used for the treatment of malignant mesothelioma and non-small cell lung cancer (NSCLC). However, the mechanism by which pemetrexed induces apoptosis remains unclear. In the present study, we investigated the involvement of reactive oxygen species (ROS) and sirtuin 1 (SIRT1) in pemetrexed-induced apoptosis in MSTO-211 malignant mesothelioma cells and A549 NSCLC cells. Pemetrexed enhanced caspase-dependent apoptosis, induced intracellular ROS generation, and downregulated SIRT1 in the MSTO-211 and A549 cells. Pemetrexed-induced apoptosis, which was prevented by pretreatment with N-acetyl-cysteine (NAC), was mediated by effects on the mitochondria, including mitochondrial membrane potential transition (MPT) and cytosolic release of cytochrome c, and also involved regulation of SIRT1 expression. Interference with SIRT1 expression using siRNA enhanced pemetrexed-induced apoptosis through mitochondrial dysfunction and ROS generation, whereas resveratrol, an activator of SIRT1, protected against pemetrexed-induced apoptosis. These results show that pemetrexed induces apoptosis in MSTO-211 mesothelioma cells and A549 NSCLC cells through mitochondrial dysfunction mediated by ROS accumulation and SIRT1 downregulation.

Enhanced apoptosis by pemetrexed and simvastatin in malignant mesothelioma and lung cancer cells by reactive oxygen species-dependent mitochondrial dysfunction and Bim induction.

Pemetrexed is a multitarget antifolate currently used for the treatment of malignant mesothelioma and non-small cell lung cancer (NSCLC). Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors used primarily for hyperlidpidemia, have been studied for their antiproliferative and pro-apoptotic effects. However, the effects of simvastatin on pemetrexed-induced apoptosis have not been investigated. In this study, we investigated whether combination treatment with pemetrexed and simvastatin potentiates the apoptotic activity above that is seen with either drug alone in malignant mesothelioma and NSCLC cells. We found that the combination of pemetrexed and simvastatin induced more extensive caspase-dependent apoptosis than either drug alone in malignant mesothelioma cells (MSTO-211) or NSCLC cells (A549). In addition, reactive oxygen species (ROS) generation in cells treated with both pemetrexed and simvastatin was markedly increased compared to cells treated with either pemetrexed or simvastatin alone. Combination treatment also increased the loss of mitochondrial membrane potential, increased cytosolic release of cytochrome c, and altered expression of inhibitor of apoptosis proteins (IAP) and B-cell lymphoma-2 (Bcl-2) families of apoptosis related proteins. On the other hand, pretreatment with N-acetylcysteine (NAC) prevented apoptosis and mitochondrial dysfunction by pemetrexed and simvastatin. In addition, Bim siRNA conferred protection against apoptosis induced by pemetrexed and simvastatin. These results suggest that combination of pemetrexed and simvastatin potentiates their apoptotic activity beyond that of either drug alone in malignant mesothelioma and lung cancer cells. This activity is mediated through ROS-dependent mitochondrial dysfunction and Bim induction.

Clostridium difficile Infection in lung cancer patients.

Clostridium difficile infection (CDI) is a common nosocomial infection. Lung cancer patients have a high risk of developing CDI because of continuous antibiotic treatment or chemotherapy, prolonged hospitalization, and general weakness. This study aimed to analyze predisposing or associated risk factors for CDI in lung cancer patients receiving chemotherapy. This study was a retrospective review of 188 lung cancer patients who were admitted to the Wonkwang University Hospital between 2008 and 2009. Of the 188 patients, 44 were diagnosed with CDI. The albumin levels were significantly lower and the performance status (PS) score was significantly higher in lung cancer patients with CDI than in those without CDI (P < 0.05). In conclusion, clinicians should consider the possibility of CDI occurrence in lung cancer patients receiving chemotherapy, particularly in those with low albumin levels and high PS scores, because most lung cancer patients have a high risk of developing CDI.

Primary malignant fibrous histiocytoma of the pleura.

Malignant fibrous histiocytoma, a type of sarcoma, is a malignant neoplasm with uncertain origin that arises in both the soft tissues and the bone. The occurrence of primary malignant fibrous histiocytoma of the pleura is extremely rare. We report a case of a 65-year-old Korean man who is being diagnosed with primary malignant fibrous histiocytoma of the pleura.

Diagnostic value and prognostic significance of pleural C-reactive protein in lung cancer patients with malignant pleural effusions.

PURPOSE: C-reactive protein (CRP) has been implicated in various inflammatory and advanced malignant states. Increased serum CRP (s-CRP) levels have been shown to be associated with independent prognostic factors for survival in patients with advanced lung cancer. However, only few studies have focused on the role of CRP in pleural effusions. This study aimed to evaluate the diagnostic and prognostic value of pleural CRP (p-CRP) in lung cancer patients with malignant pleural effusion (MPE). MATERIALS AND METHODS: Pleural effusion (PE) samples were collected from patients with MPE (68 lung cancers; 12 extrathoracic tumors), and from 68 patients with various benign conditions (31 with pneumonia; 37 with tuberculosis). Concentrations of p- and s-CRP were measured by enzyme-linked immunosorbent assay. CRP level in pleural fluid and its association with survival were examined. RESULTS: p-CRP levels correlated with s-CRP levels (r=0.82, p<0.0001). For the differential diagnosis of MPE and benign PE, the area under the receiver operating characteristic curve was greater for p-CRP (0.86) than for s-CRP (0.77). High p-CRP expression significantly correlated with shorter overall survival (p=0.006). P-CRP was independent prognostic factor significantly associated with overall survival on multivariated analysis (p=0.0001). The relative risk of death for lung cancer patients with high p-CRP levels was 3.909 (95% confidence interval, 2.000-7.639). CONCLUSION: P-CRP is superior to s-CRP in determining pleural fluid etiology. Quantitative measurement of p-CRP might be a useful complementary diagnostic and prognostic test for lung cancer patients with MPE.

Inhibition of LPS binding to MD-2 co-receptor for suppressing TLR4-mediated expression of inflammatory cytokine by 1-dehydro-10-gingerdione from dietary ginger.

Myeloid differentiation protein 2 (MD-2) is a co-receptor of toll-like receptor 4 (TLR4) for innate immunity. Here, we delineated a new mechanism of 1-dehydro-10-gingerdione (1D10G), one of pungent isolates from ginger (Zingiber officinale), in the suppression of lipopolysaccharide (LPS)-induced gene expression of inflammatory cytokines. 1D10G inhibited LPS binding to MD-2 with higher affinity than gingerol and shogaol from dietary ginger. Moreover, 1D10G down-regulated TLR4-mediated expression of nuclear factor-κB (NF-κB) or activating protein 1 (AP1)-target genes such as tumor necrosis factor α (TNF-α) and interleukin-1ß, as well as those of interferon (IFN) regulatory factor 3 (IRF3)-target IFN-ß gene and IFN-γ inducible protein 10 (IP-10) in LPS-activated macrophages. Taken together, MD-2 is a molecular target in the anti-inflammatory action of 1D10G.