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Glycan-mediated interactions play a crucial role in biology and medicine, influencing signalling, immune responses, and disease pathogenesis. However, the use of glycans in biosensing and diagnostics is limited by cross-reactivity, as certain glycan motifs can be recognised by multiple biologically distinct protein receptors. To address this specificity challenge, we report the enzymatic synthesis of a 150-member library of site-specifically fluorinated Lewis x analogues ('glycofluoroforms') using naturally occurring enzymes and fluorinated monosaccharides. Subsequent incorporation of a subset of these glycans into nanoparticles or a microarray revealed a striking spectrum of distinct binding intensities across different proteins that recognise Lewis x . Notably, we show that for two proteins with unique binding sites for Lewis x , glycofluoroforms exhibited enhanced binding to one protein, whilst reduced binding to the other, with selectivity governed by fluorination patterns. We finally showcase the potential diagnostic utility of this approach in glycofluoroform-mediated bacterial toxin detection by lateral flow., (© 2024. The Author(s).)

Background: Diabetes mellitus is associated with increased risk of ocular surface diseases in elderly. We consider neutrophil CD15 as a potential marker of ocular surface damage in type II diabetes mellitus patients., Aim: We aimed to evaluate expression of neutrophil CD15 and correlate it with results of conjunctival impression cytology and routine objective anterior ocular surface tests (TMH, NIBUT, LLT, MGD) in T2DM patients., Materials and Methods: We prospectively enrolled sixty type II diabetes mellitus patients (120 eyes) into a study group. The control group included forty (80 eyes) age- and sex-matched healthy individuals. All patients underwent comprehensive ophthalmological examination, and tear meniscus height test (TMH), noninvasive tear break-up time (NIBUT), lipid layer thickness measurement (LLT), Meibomian gland dysfunction evaluation (MGD), conjunctival impression cytology (CIC) and expression of CD15., Results: Abnormal Nelson's grades of squamous metaplasia (grades 2 and 3) were observed in 50% (60 eyes) of the study group, and 13.8 (11 eyes) of the control group. Fifteen patients with type II diabetes mellitus suffered from grade 3 squamous metaplasia. Nelson's grades of squamous metaplasia have shown a positive correlation with the level of CD15 expression either in the study and control groups (rs = 0.628, p  = <0.0001; rs = 0.746, p  < 0.0001; respectively)., Conclusions: The research shows significantly reduced values of routine objective ocular tests in type II diabetes mellitus patients in comparison to healthy participants older than 60 y.o. Increased CD15 in the peripheral blood is associated with the development of squamous metaplasia and may be used to evaluate the severity of ocular surface damage in type II diabetes mellitus patients., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Introduction: Chronic hyperglycemia affects neutrophil functions, leading to reduced pathogen killing and increased morbidity. This impairment has been directly linked to increased glycemia, however, how this specifically affects neutrophils metabolism and their differentiation in the bone marrow is unclear and difficult to study., Research Design and Methods: We used high-resolution respirometry to investigate the metabolism of resting and activated donor neutrophils, and flow cytometry to measure surface CD15 and CD11b expression. We then used HL-60 cells differentiated towards neutrophil-like cells in standard media and investigated the effect of doubling glucose concentration on differentiation metabolism. We measured the oxygen consumption rate (OCR), and the enzymatic activity of carnitine palmitoyl transferase 1 (CPT1) and citrate synthase during neutrophil-like differentiation. We compared the surface phenotype, functions, and OCR of neutrophil-like cells differentiated under both glucose concentrations., Results: Donor neutrophils showed significant instability of CD11b and OCR after phorbol 12-myristate 13-acetate stimulation at 3 hours post-enrichment. During HL-60 neutrophil-like cell differentiation, there was a significant increase in surface CD15 and CD11b expression together with the loss of mitochondrial mass. Differentiated neutrophil-like cells also exhibited higher CD11b expression and were significantly more phagocytic. In higher glucose media, we measured a decrease in citrate synthase and CPT1 activities during neutrophil-like differentiation., Conclusions: HL-60 neutrophil-like differentiation recapitulated known molecular and metabolic features of human neutrophil differentiation. Increased glucose concentrations correlated with features described in hyperglycemic donor neutrophils including increased CD11b and phagocytosis. We used this model to describe metabolic features of neutrophil-like cell differentiation in hyperglycemia and show for the first time the downregulation of CPT1 and citrate synthase activity, independently of mitochondrial mass., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Men who have sex with men (MSM) with HIV are at high risk for squamous intraepithelial lesion (SIL) and anal cancer. Identifying local immunological mechanisms involved in the development of anal dysplasia could aid treatment and diagnostics. Here, we studied 111 anal biopsies obtained from 101 MSM with HIV, who participated in an anal screening program. We first assessed multiple immune subsets by flow cytometry, in addition to histological examination, in a discovery cohort. Selected molecules were further evaluated by immunohistochemistry in a validation cohort. Pathological samples were characterized by the presence of resident memory T cells with low expression of CD103 and by changes in natural killer cell subsets, affecting residency and activation. Furthermore, potentially immunosuppressive subsets, including CD15+CD16+ mature neutrophils, gradually increased as the anal lesion progressed. Immunohistochemistry verified the association between the presence of CD15 in the epithelium and SIL diagnosis for the correlation with high-grade SIL. A complex immunological environment with imbalanced proportions of resident effectors and immune-suppressive subsets characterized pathological samples. Neutrophil infiltration, determined by CD15 staining, may represent a valuable pathological marker associated with the grade of dysplasia.

Background: An elevated neutrophil-to-lymphocyte ratio (NLR) in peripheral blood is an independent prognostic indicator of various cancers., Aims: In this study, we aimed to investigate the prognostic relevance of the intratumoral immune cell balance in gastric cancer., Methods and Results: The study included 82 patients who underwent curative resection for gastric cancer. The intratumoral cluster of differentiation (CD) 15- and CD8-positive cells were evaluated using immunohistochemical staining. Additionally, clinicopathological factors and prognoses were analyzed. Patients with high intratumoral CD15/CD8 ratios had significantly lower overall survival (OS) and relapse-free survival (RFS) compared to those with low CD15/CD8 ratios (p = .0026 and p < .0001, respectively). Additionally, a high CD15/CD8 ratio was associated with lymph node metastasis (p = .019). Patients with high NLR had a significantly lower RFS than those with low NLR (p = .0050). Multivariate analysis revealed that the intratumoral CD15/CD8 ratio, NLR, and venous invasion were independent prognostic indicators of RFS (CD15/CD8 ratio: p < .001, hazard ratio (HR) = 14.7, 95% confidence interval (CI) = 3.8-56.8; NLR: p = .010, HR = 5.4, 95% CI = 1.5-19.6; venous invasion: p = .005, HR = 7.4, 95% CI = 1.8-29.7)., Conclusion: In summary, we found that the intratumoral CD15/CD8 ratio is an independent prognostic factor following gastric cancer resection and its increase is associated with lymph node metastasis and microscopic lymph vessel invasion. Immunological evaluation with additional aspects of innate immunity may be useful in predicting cancer prognosis., (© 2024 The Author(s). Cancer Reports published by Wiley Periodicals LLC.)

Peripheral T-cell lymphomas (PTCL) are morphologically and biologically heterogeneous and a subset expresses CD30, including anaplastic large cell lymphomas (ALCL) and a minority of PTCL, not otherwise specified (PTCL, NOS). ALCL with ALK translocations (ALCL, ALK+) are readily identified by routine diagnostic methods, but differentiating ALCL without ALK translocation (ALCL, ALK-) and PTCL, NOS expressing CD30 (PTCL CD30+) can be challenging. Furthermore, rare PTCL co-express CD30 and CD15 (PTCL CD30+CD15+); some resemble ALCL, ALK- while others resemble classic Hodgkin lymphoma. To explore the relationship between PTCL CD30+CD15+ and ALCL, ALK-, we analysed 19 cases of PTCL with CD30 expression, previously diagnosed as ALCL, ALK- (nine cases) and PTCL CD30+CD15+ (10 cases) for DUSP22/IRF4 rearrangements, coding RNA expression and selected transcriptome analysis using the NanoString nCounter gene expression analysis platform. Unsupervised clustering showed no clear segregation between ALCL, ALK- and PTCL CD30+CD15+. Three cases previously classified as PTCL CD30+CD15+ showed DUSP22/IRF4 rearrangements, favouring a diagnosis of ALCL, ALK-. Our results suggest that cases previously designated PTCL CD30+CD15+, likely fall within the spectrum of ALCL, ALK-; additionally, a subset of ALCL, ALK- with DUSP22/IRF4 rearrangement expresses CD15, consistent with previous reports and expands the immunophenotypic spectrum of this lymphoma subgroup., (© 2024 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Melanoma incidence and mortality rates are historically higher for men than women. Although emerging studies have highlighted tumorigenic roles for the male sex hormone androgen and its receptor (AR) in melanoma, cellular and molecular mechanisms underlying these sex-associated discrepancies are poorly defined. Here, we delineate a previously undisclosed mechanism by which androgen-activated AR transcriptionally upregulates fucosyltransferase 4 (FUT4) expression, which drives melanoma invasiveness by interfering with adherens junctions (AJs). Global phosphoproteomic and fucoproteomic profiling, coupled with in vitro and in vivo functional validation, further reveal that AR-induced FUT4 fucosylates L1 cell adhesion molecule (L1CAM), which is required for FUT4-increased metastatic capacity. Tumor microarray and gene expression analyses demonstrate that AR-FUT4-L1CAM-AJs signaling correlates with pathological staging in melanoma patients. By delineating key androgen-triggered signaling that enhances metastatic aggressiveness, our findings help explain sex-associated clinical outcome disparities and highlight AR/FUT4 and its effectors as potential prognostic biomarkers and therapeutic targets in melanoma., (© 2024. The Author(s).)

The expression of one or more of a small number of molecules, typically cell surface-associated antigens, or transcription factors, is widely used for identifying pluripotent stem cells (PSCs) or for monitoring their differentiation. However, none of these marker molecules are uniquely expressed by PSCs and all are expressed by stem cells that have lost the ability to differentiate. Consequently, none are indicators of pluripotency, per se. Here we summarize the nature and characteristics of several markers that are in wide use, including the cell surface antigens, stage-specific embryonic antigen (SSEA)-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, GCTM2, and the transcription factors POUF5/OCT4, NANOG, and SOX2, highlighting issues that must be considered when interpreting data about their expression on putative PSCs., Competing Interests: Declaration of interests P.W.A. receives royalties from the Wistar Institute from sales of the TRA series of antibodies and is a member of the SAB of TreeFrog Therapeutics., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Tumor cells use adhesion molecules like CD15 or sialylCD15 (sCD15) for metastatic spreading. We analyzed the expression of CD15 and sCD15 in clear cell renal cell carcinoma (ccRCC) regarding prognosis., Methods: A tissue microarray containing tissue specimens of 763 patients with ccRCC was immunohistochemically stained for CD15 and sCD15, their expression quantified using digital image analysis, and the impact on patients' survival analyzed. The cell lines 769p and 786o were stimulated with CD15 or control antibody in vitro and the effects on pathways activating AP-1 and tumor cell migration were examined., Results: ccRCC showed a broad range of CD15 and sCD15 expression. A high CD15 expression was significantly associated with favorable outcome (p &lt; 0.01) and low-grade tumor differentiation (p &lt; 0.001), whereas sCD15 had no significant prognostic value. Tumors with synchronous distant metastasis had a significantly lower CD15 expression compared to tumors without any (p &lt; 0.001) or with metachronous metastasis (p &lt; 0.01). Tumor cell migration was significantly reduced after CD15 stimulation in vitro, but there were no major effects on the activating pathways of AP-1., Conclusion: CD15, but not sCD15, qualifies as a biomarker for risk stratification and as an interesting novel target in ccRCC. Moreover, the data indicate a contribution of CD15 to metachronous metastasis. Further research is warranted to decipher the intracellular pathways of CD15 signaling in ccRCC in order to characterize the CD15 effects on ccRCC more precisely., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Inflammation is a hallmark of cancer 1 . In patients with cancer, peripheral blood myeloid expansion, indicated by a high neutrophil-to-lymphocyte ratio, associates with shorter survival and treatment resistance across malignancies and therapeutic modalities 2-5 . Whether myeloid inflammation drives progression of prostate cancer in humans remain unclear. Here we show that inhibition of myeloid chemotaxis can reduce tumour-elicited myeloid inflammation and reverse therapy resistance in a subset of patients with metastatic castration-resistant prostate cancer (CRPC). We show that a higher blood neutrophil-to-lymphocyte ratio reflects tumour myeloid infiltration and tumour expression of senescence-associated mRNA species, including those that encode myeloid-chemoattracting CXCR2 ligands. To determine whether myeloid cells fuel resistance to androgen receptor signalling inhibitors, and whether inhibiting CXCR2 to block myeloid chemotaxis reverses this, we conducted an investigator-initiated, proof-of-concept clinical trial of a CXCR2 inhibitor (AZD5069) plus enzalutamide in patients with metastatic CRPC that is resistant to androgen receptor signalling inhibitors. This combination was well tolerated without dose-limiting toxicity and it decreased circulating neutrophil levels, reduced intratumour CD11b + HLA-DR lo CD15 + CD14 - myeloid cell infiltration and imparted durable clinical benefit with biochemical and radiological responses in a subset of patients with metastatic CRPC. This study provides clinical evidence that senescence-associated myeloid inflammation can fuel metastatic CRPC progression and resistance to androgen receptor blockade. Targeting myeloid chemotaxis merits broader evaluation in other cancers., (© 2023. The Author(s).)

Immune checkpoint blockade (ICB) benefits some patients with triple-negative breast cancer, but what distinguishes responders from non-responders is unclear 1 . Because ICB targets cell-cell interactions 2 , we investigated the impact of multicellular spatial organization on response, and explored how ICB remodels the tumour microenvironment. We show that cell phenotype, activation state and spatial location are intimately linked, influence ICB effect and differ in sensitive versus resistant tumours early on-treatment. We used imaging mass cytometry 3 to profile the in situ expression of 43 proteins in tumours from patients in a randomized trial of neoadjuvant ICB, sampled at three timepoints (baseline, n = 243; early on-treatment, n = 207; post-treatment, n = 210). Multivariate modelling showed that the fractions of proliferating CD8 + TCF1 + T cells and MHCII + cancer cells were dominant predictors of response, followed by cancer-immune interactions with B cells and granzyme B + T cells. On-treatment, responsive tumours contained abundant granzyme B + T cells, whereas resistant tumours were characterized by CD15 + cancer cells. Response was best predicted by combining tissue features before and on-treatment, pointing to a role for early biopsies in guiding adaptive therapy. Our findings show that multicellular spatial organization is a major determinant of ICB effect and suggest that its systematic enumeration in situ could help realize precision immuno-oncology., (© 2023. The Author(s).)

Induced pluripotent stem cells (iPSCs) can differentiate into all types of cells and can be used in livestock for research on biological development, genetic breeding, and in vitro genetic resource conservation. The Bactrian camel is a large domestic animal that inhabits extreme environments and holds value in the treatment of various diseases and the development of the local economy. Therefore, we transferred four mouse genes ( Oct4 , Sox2 , Klf4 , and c- Myc ) into Bactrian camel fetal fibroblasts (BCFFs) using retroviruses with a large host range to obtain Bactrian camel induced pluripotent stem cells (bciPSCs). They were comprehensively identified based on cell morphology, pluripotency gene and marker expression, chromosome number, transcriptome sequencing, and differentiation potential. The results showed the pluripotency of bciPSCs. However, unlike stem cells of other species, late formation of stem cell clones was observed; moreover, the immunofluorescence of SSEA1, SSEA3, and SSEA4 were positive, and teratoma formation took four months. These findings may be related to the extremely long gestation period and species specificity of Bactrian camels. By mining RNA sequence data, 85 potential unique pluripotent genes of Bactrian camels were predicted, which could be used as candidate genes for the production of bciPSC in the future. Among them, ASF1B , DTL , CDCA5 , PROM1 , CYTL1 , NUP210 , Epha3 , and SYT13 are more attractive. In conclusion, we generated bciPSCs for the first time and obtained their transcriptome information, expanding the iPSC genetic information database and exploring the applicability of iPSCs in livestock. Our results can provide an experimental basis for Bactrian camel ESC establishment, developmental research, and genetic resource conservation.

Background: Expression of cluster of differentiation (CD) antigens changes according to disease status and inflammation. Profiles of CD antigens expression in gastric cancer patients are different based on the status of H. pylori infection., Aims: We conducted this study to profile CD antigen markers in gastric adenocarcinoma cells (AGS cell line) infected with distinct cytotoxin-associated gene A (cagA) genotypes of H. pylori clinical isolates., Methods: The AGS cells were infected with H. pylori isolates with different cagA genotypes, and CD antigens expression was determined using DotScan™ antibody microarray. Formation of "hummingbird" phenotype was determined, and the percentage was calculated., Results: H. pylori strains harboring cagA upregulated the expression of CD antigen involved in cancer stem cell formation (CD55), but downregulated CD antigens involved in immune regulation (CD40 and CD186) and cell adhesion (CD44). CD54 (neutrophil adhesion) and CD71 (iron transfer) were highly downregulated in the gastric cells infected with Western cagA isolates compared with East Asian isolates. CD antigen expression was different in the cells infected with H. pylori harboring different CagA EPIYA (Glu-Pro-Ile-Tyr-Ala) numbers, in which higher repression of CD54 and CD15 (Lewis x antigen) were observed in the isolate with the highest number of EPIYA motif. Furthermore, higher downregulation of CD15 was observed in the infected gastric cells with high percentage of "hummingbird" phenotype than that of low percentage of "hummingbird" phenotype., Conclusion: Our study demonstrated the critical roles of CD antigens in the CagA pathogenesis and should be investigated further., (© 2022 John Wiley & Sons Ltd.)

The conservation of Taiwan Country chicken (TCC) is important due to concerns for the local breed's adaptability to the area and disease resistance. Furthermore, the genetic resource base of native chickens can be used to improve egg and meat production efficiency in commercial TCC. As the embryonic stem cells (ESCs) hold great potential for regenerative medicine and species conservation, the aims of this study were to isolate and characterize ESCs of TCC. The blastodermal cells (BCs) were isolated from the zona pellucida of stage X chicken embryos and cultured in conditioned medium for the proliferation and maintenance of BCs in vitro . The quantitative real-time polymerase chain reaction (qPCR) results showed that POUV , SOX2 and NANOG were expressed in the putative ESCs. In addition, the expression of pluripotent markers, SSEA-1 and SSEA-4, was detected. The DiI-stained ESCs were injected into the dorsal aorta of the E3.5 recipient fetuses soon after staining and the injected embryos were continuously incubated and checked on day 7 of incubation. It was shown that some DiI-positive cells were found in the 7-d-old chimeric embryos. The results demonstrated that some pluripotent cells existed in the cultured BCs for the production of germline chimeric embryos from TCC.

Sialyl 6-sulfo Lewis X (6-sulfo sLe X ) and its derivative sialyl 6-sulfo N -acetyllactosamine (LacNAc) are sialylated and sulfated glycans of sialomucins found in the high endothelial venules (HEVs) of secondary lymphoid organs. A component of 6-sulfo sLe X present in the core 1-extended O -linked glycans detected by the MECA-79 antibody was previously shown to exist in the lymphoid aggregate vasculature and bronchial mucosa of allergic and asthmatic lungs. The components of 6-sulfo sLe X in pulmonary tissues under physiological conditions remain to be analyzed. The CL40 antibody recognizes 6-sulfo sLe X and sialyl 6-sulfo LacNAc in O -linked and N -linked glycans, with absolute requirements for both GlcNAc-6-sulfation and sialylation. Immunostaining of normal mouse lungs with CL40 was performed and analyzed. The contribution of GlcNAc-6- O -sulfotransferases (GlcNAc6STs) to the synthesis of the CL40 epitope in the lungs was also elucidated. Here, we show that the expression of the CL40 epitope was specifically detected in the mesothelin-positive mesothelium of the pulmonary pleura. Moreover, GlcNAc6ST2 (encoded by Chst4 ) and GlcNAc6ST3 (encoded by Chst5 ), but not GlcNAc6ST1 (encoded by Chst2 ) or GlcNAc6ST4 (encoded by Chst7 ), are required for the synthesis of CL40-positive glycans in the lung mesothelium. Furthermore, neither GlcNAc6ST2 nor GlcNAc6ST3 is sufficient for in vivo expression of the CL40 epitope in the lung mesothelium, as demonstrated by GlcNAc6ST1/3/4 triple-knock-out and GlcNAc6ST1/2/4 triple-knock-out mice. These results indicate that CL40-positive sialylated and sulfated glycans are abundant in the pleural mesothelium and are synthesized complementarily by GlcNAc6ST2 and GlcNAc6ST3, under physiological conditions in mice.

N-glycans are diversified by a panel of glycosyltransferases in the Golgi, which are supposed to modify various glycoproteins in promiscuous manners, resulting in unpredictable glycosylation profiles in general. In contrast, our previous study showed that fucosyltransferase 9 (FUT9) generates Lewis X glycotopes primarily on lysosome-associated membrane protein 1 (LAMP-1) in neural stem cells. Here, we demonstrate that a contiguous 29-amino acid sequence in the N-terminal domain of LAMP-1 is responsible for promotion of the FUT9-catalyzed Lewis X modification. Interestingly, Lewis X modification was induced on erythropoietin as a model glycoprotein both in vitro and in cells, just by attaching this sequence to its C-terminus. Based on these results, we conclude that the amino acid sequence from LAMP-1 functions as a "Lewis X code", which is deciphered by FUT9, and can be embedded into other glycoproteins to evoke a Lewis X modification, opening up new possibilities for protein engineering and cell engineering., (© 2022. The Author(s).)

Structural changes known as airway remodeling characterize chronic/severe asthma and contribute to lung dysfunction. We previously reported that neonatal SSEA-1 + pulmonary stem/progenitor cells (PSCs) ameliorated airway inflammation in asthmatic mice. However, the molecular mechanisms by which endogenous SSEA-1 + PSC of adult mice afford beneficial effects in alveolar homeostasis and lung repair after allergen challenge remain incompletely understood. To analyze the expression profile and clarify the biological significance of endogenous adult lung SSEA-1 + cells in asthmatic mice. Lung SSEA-1 + cells and circulating SSEA-1 + cells in peripheral blood were determined by confocal microscopy and cytometric analysis. GFP chimeric mice were used to trace cell lineage in vivo. The roles of circulating SSEA-1 + cells were verified in ovalbumin-induced and house dust mite-induced allergic asthmatic models. In asthmatic mice, endogenous lung SSEA-1 + cells almost disappeared; however, a unique population of circulating SSEA-1 + cells was enriched after the challenge phase. In asthmatic mice, adoptive transfer of circulating SSEA-1 + cells had a specific homing preference for the lung in response to inhaled antigen through upregulating CXCR7-CXCL11 chemokine axis. Circulating SSEA-1 + cells can transdifferentiate in the alveolar space and ameliorate lung inflammation and structural damage through inhibiting the infiltration of inflammatory cells into peribronchovascular and goblet cell hyperplasia areas, reducing the thickened smooth muscle layers and PAS-positive mucus-containing goblet cells. Reinforcing bone marrow-derived circulating SSEA-1 + cells from peripheral blood into lung tissue which create a rescue mechanism in maintaining alveolar homeostasis and tissue repair to mediate lung protection for emergency responses after allergen challenge in asthmatic conditions., (© 2022. The Author(s).)

The phenotype of glioma-initiating cells (GIC) is modulated by cell-intrinsic and cell-extrinsic factors. Phenotypic heterogeneity and plasticity of GIC is an important limitation to therapeutic approaches targeting cancer stem cells. Plasticity also presents a challenge to the identification, isolation, and propagation of purified cancer stem cells. Here we use a barcode labelling approach of GIC to generate clonal populations over a number of passages, in combination with phenotyping using the established stem cell markers CD133, CD15, CD44, and A2B5. Using two cell lines derived from isocitrate dehydrogenase (IDH)-wildtype glioblastoma, we identify a remarkable heterogeneity of the phenotypes between the cell lines. During passaging, clonal expansion manifests as the emergence of a limited number of barcoded clones and a decrease in the overall number of clones. Dual-labelled GIC are capable of forming traceable clonal populations which emerge after as few as two passages from mixed cultures and through analyses of similarity of relative proportions of 16 surface markers we were able to pinpoint the fate of such populations. By generating tumour organoids we observed a remarkable persistence of dominant clones but also a significant plasticity of stemness marker expression. Our study presents an experimental approach to simultaneously barcode and phenotype glioma-initiating cells to assess their functional properties, for example to screen newly established GIC for tumour-specific therapeutic vulnerabilities., (© 2021. The Author(s).)

Lewis x functions as an adhesion molecule in glycolipids and glycoproteins since it mediates homophilic and heterophilic attachment of normal and tumoral cells. During malignancy, altered glycosylation is a frequent event; accumulating data support the expression of Lewis x in tumors although controversial results have been described including its relationship with patient survival. This report has been developed as an introduction to the relationship between Lewis x expression and breast cancer and head and neck squamous cell carcinoma (HNSCC). Results obtained in our laboratory are presented in the context of the literature.

Infection with the SARS-CoV2 virus can vary from asymptomatic, or flu-like with moderate disease, up to critically severe. Severe disease, termed COVID-19, involves acute respiratory deterioration that is frequently fatal. To understand the highly variable presentation, and identify biomarkers for disease severity, blood RNA from COVID-19 patient in an intensive care unit was analyzed by whole transcriptome RNA sequencing. Both SARS-CoV2 infection and the severity of COVID-19 syndrome were associated with up to 25-fold increased expression of neutrophil-related transcripts, such as neutrophil defensin 1 (DEFA1), and 3-5-fold reductions in T cell related transcripts such as the T cell receptor (TCR). The DEFA1 RNA level detected SARS-CoV2 viremia with 95.5% sensitivity, when viremia was measured by ddPCR of whole blood RNA. Purified CD15+ neutrophils from COVID-19 patients were increased in abundance and showed striking increases in nuclear DNA staining by DAPI. Concurrently, they showed >10-fold higher elastase activity than normal controls, and correcting for their increased abundance, still showed 5-fold higher elastase activity per cell. Despite higher CD15+ neutrophil elastase activity, elastase activity was extremely low in plasma from the same patients. Collectively, the data supports the model that increased neutrophil and decreased T cell activity is associated with increased COVID-19 severity, and suggests that blood DEFA1 RNA levels and neutrophil elastase activity, both involved in neutrophil extracellular traps (NETs), may be informative biomarkers of host immune activity after viral infection., Competing Interests: TM and IT have an equity interest in True Bearing Diagnostics, Inc., a diagnostics company developing RNA biomarkers for various diseases, including coronary artery disease and internal infections. TM is seeking patent protection for technology related to the current studies. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The other authors declare there are no competing interests.

Purpose: The identification of prognostically and therapeutically relevant molecular markers is fundamental to the further development of personalised therapies in brain tumours. Current therapeutic options for the treatment of gliomas rely mainly on surgical resection and the inhibition of tumour cell proliferation by irradiation and chemotherapy. Glioma stem cells are a subpopulation of proliferating tumour cells that have self-renewal capacity and can give rise to heterogeneous cells that comprise the tumour and are thought to play a role in the resistance of gliomas to therapy. The aim of this study was to evaluate the expression of markers of glioma stem cells and differentiated glial cells in proliferating glioma cells in comparison to the overall expression of the respective markers in the tumour tissue., Methods: Tissue microarrays were assembled from specimen of pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, and anaplastic ependymoma. These were immunohistochemically double stained with antibodies against the proliferation-associated antigen Ki67 and marker proteins for glioma stem cells (CD133, Nestin, Musashi, CD15, CD44), and differentiated glioma cells (GFAP, MAP2c)., Results: The expression of both glial and glioma stem cell markers differs between proliferating and non-proliferating glioma cells. Furthermore, the proliferating cells in the different glial tumour entities show a different expression profile., Conclusion: Further analysis of marker expression in proliferating glioma cells and correlation with clinical outcome and susceptibility to irradiation and chemotherapy might help establish new biomarkers and therapies for glioma., (© 2021. The Author(s).)

The difficulty in treatment of glioblastoma is a consequence of its natural infiltrative growth and the existence of a population of therapy-resistant glioma cells that contribute to growth and recurrence. To identify cells more likely to have these properties, we examined the expression in tumor specimens of several protein markers important for glioma progression including the intermediate filament protein, Nestin (NES), a glucose transporter (Glut1/SLC2A1), the glial lineage marker, glial fibrillary acidic protein, and the proliferative indicator, Ki-67. We also examined the expression of von Willebrand factor, a marker for endothelial cells as well as the macrophage/myeloid markers CD163 and CD15. Using a multicolor immunofluorescence and hematoxylin and eosin staining approach with archival formalin-fixed, paraffin embedded tissue from primary, recurrent, and autopsy IDH1 wildtype specimens combined with high-resolution tissue image analysis, we have identified highly proliferative NES(+)/Glut1(-) cells that are preferentially perivascular. In contrast, Glut1(+)/NES(-) cells are distant from blood vessels, show low proliferation, and are preferentially located at the borders of pseudopalisading necrosis. We hypothesize that Glut1(+)/NES(-) cells would be naturally resistant to conventional chemotherapy and radiation due to their low proliferative capacity and may act as a reservoir for tumor recurrence., (© 2021 American Association of Neuropathologists, Inc. All rights reserved.)

Introduction: Protective host responses in those exposed to or infected with tuberculosis (TB) is thought to require a delicate balance between pro-inflammatory and regulatory immune responses. Myeloid-derived suppressor cells (MDSCs), regulatory cells that dampen T-cell function, have been described in cancer and other infectious diseases but there are limited data on their role in TB., Methods: Peripheral blood was obtained from patients with active pulmonary TB and participants with presumed latent TB infection (LTBI) from Cape Town, South Africa. MDSC frequency was ascertained by flow cytometry. Purified MDSCs were used to assess (i) their suppressive effect on T-cell proliferation using a Ki67 flow cytometric assay and (ii) their effect on mycobacterial containment by co-culturing with H37 Rv -infected monocyte-derived macrophages and autologous pre-primed effector T-cells with or without MDSCs. Mycobacterial containment was measured by plating colony forming units (CFU)., Results: MDSCs (CD15 + HLA-DR - CD33 + ) had significantly higher median frequencies (IQR) in patients with active TB (n=10) versus LTBI (n= 10) [8.2% (6.8-10.7) versus 42.2% (27-56) respectively; p=0.001]. Compared to MDSC-depleted peripheral blood mononuclear and effector T cell populations, dilutions of purified MDSCs isolated from active TB patients suppressed T-cell proliferation by up to 72% (n=6; p=0.03) and significantly subverted effector T-cell-mediated containment of H37 Rv in monocyte-derived macrophages (n=7; 0.6% versus 8.5%; p=0.02)., Conclusion: Collectively, these data suggest that circulating MDSCs are induced during active TB disease and can functionally suppress T-cell proliferation and subvert mycobacterial containment. These data may inform the design of vaccines and immunotherapeutic interventions against TB but further studies are required to understand the mechanisms underpinning the effects of MDSCs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Davids, Pooran, Smith, Tomasicchio and Dheda.)

Background: Lewis antigens such as Sialyl Lewis A (sLeA), Sialyl Lewis X (sLeX), Lewis X (LeX), and Lewis Y (LeY) are a class of carbohydrate molecules that are known to mediate adhesion between tumor cells and endothelium by interacting with its selectin ligands. However, their potential role in miscarriage remains enigmatic. This study aims to analyze the expression pattern of sLeA, sLeX, LeX, and LeY in the placental villi tissue of patients with a medical history of unexplained miscarriages., Methods: Paraffin-embedded slides originating from placental tissue were collected from patients experiencing a miscarriage early in their pregnancy (6-13 weeks). Tissues collected from spontaneous (n = 20) and recurrent (n = 15) miscarriages were analyzed using immunohistochemical and immunofluorescent staining. Specimens obtained from legally terminated normal pregnancies were considered as control group (n = 18). Assessment of villous vessel density was performed in another cohort (n = 10 each group) of gestation ages-paired placenta tissue. Protein expression was evaluated with Immunoreactive Score (IRS). Statistical analysis was performed by using Graphpad Prism 8., Results: Expression of sLeA, sLeX, LeX, and LeY in the syncytiotrophoblast was significantly upregulated in the control group compared with spontaneous and recurrent miscarriage groups. However, no prominent differences between spontaneous and recurrent miscarriage groups were identified. Potential key modulators ST3GAL6 and NEU1 were found to be significantly downregulated in the recurrent miscarriage group and upregulated in the spontaneous group, respectively. Interestingly, LeX and LeY expression was also detected in the endothelial cells of villous vessels in the control group but no significant expression in miscarriage groups. Furthermore, assessment of villous vessel density using CD31 found significantly diminished vessels in all size groups of villi (small villi <200 µm, P = 0.0371; middle villi between 200 and 400 µm, P = 0.0010 and large villi >400 µm, P = 0.0003). Immunofluorescent double staining also indicated the co-localization of LeX/Y and CD31., Conclusions: The expression of four mentioned carbohydrate Lewis antigens and their potential modulators, ST3GAL6 and NEU1, in the placenta of patients with miscarriages was significantly different from the normal pregnancy. For the first time, their expression pattern in the placenta was illustrated, which might shed light on a novel understanding of Lewis antigens' role in the pathogenesis of miscarriages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors (UJ)., (Copyright © 2021 Ma, Yang, Peng, Kuhn, Chelariu-Raicu, Mahner, Jeschke and von Schönfeldt.)

Objective: Juvenile idiopathic arthritis (JIA) is the most common rheumatic disease in childhood. The predominant subtypes, oligoarticular and polyarticular JIA, are traditionally considered to be autoimmune diseases with a central role for T cells and autoantibodies. Mounting evidence suggests an important role for neutrophils in JIA pathogenesis. We undertook this study to investigate the phenotypic features of neutrophils present in the blood and inflamed joints of patients., Methods: JIA synovial fluid (SF) and parallel blood samples from JIA patients and healthy children were collected. SF-treated neutrophils from healthy donors and pleural neutrophils from patients with pleural effusion were investigated as controls for SF exposure and extravasation. Multicolor flow cytometry panels allowed for in-depth phenotypic analysis of neutrophils, focusing on the expression of adhesion molecules, activation, and maturation markers and chemoattractant receptors. Multiplex technology was used to quantify cytokines in plasma and SF., Results: SF neutrophils displayed an activated, hypersegmented phenotype with decreased CD62L expression, up-regulation of adhesion molecules CD66b, CD11b, and CD15, and down-regulation of CXCR1/2. An elevated percentage of CXCR4-positive neutrophils was detected in SF from patients. Pleural neutrophils showed less pronounced maturation differences. Strikingly, significant percentages of SF neutrophils showed a profound up-regulation of atypical neutrophil markers, including CXCR3, intercellular adhesion molecule 1, and HLA-DR., Conclusion: Our data show that neutrophils in inflamed joints of JIA patients have an activated phenotype. This detailed molecular analysis supports the notion that a complex intertwining between these innate immune cells and adaptive immune events drives JIA., (© 2020, American College of Rheumatology.)

Background: Endogenous pulmonary stem cells (PSCs) play an important role in lung development and repair; however, little is known about their role in bronchopulmonary dysplasia (BPD). We hypothesize that an endogenous PSC marker stage-specific embryonic antigen-1 (SSEA-1) and its enzyme, α1,3-fucosyltransferase IX (FUT9) play an important role in decreasing inflammation and restoring lung structure in experimental BPD., Methods: We studied the expression of SSEA-1, and its enzyme FUT9, in wild-type (WT) C57BL/6 mice, in room air and hyperoxia. Effects of intraperitoneal administration of recombinant human FUT9 (rhFUT9) on lung airway and parenchymal inflammation, alveolarization, and apoptosis were evaluated., Results: On hyperoxia exposure, SSEA-1 significantly decreased at postnatal day 14 in hyperoxia-exposed BPD mice, accompanied by a decrease in FUT9. BPD and respiratory distress syndrome (RDS) in human lungs showed decreased expression of SSEA-1 as compared to their term controls. Importantly, intraperitoneal administration of FUT9 in the neonatal BPD mouse model resulted in significant decrease in pulmonary airway (but not lung parenchymal) inflammation, alveolar-capillary leakage, alveolar simplification, and cell death in the hyperoxia-exposed BPD mice., Conclusions: An important role of endogenous PSC marker SSEA-1 and its enzyme FUT9 is demonstrated, indicating early systemic intervention with FUT9 as a potential therapeutic option for BPD., Impact: Administration of rhFUT9, an enzyme of endogenous stem cell marker SSEA-1, reduces pulmonary airway (but not lung parenchymal) inflammation, alveolar-capillary leak and cell death in the BPD mouse model. SSEA-1 is reported for the first time in experimental BPD models, and in human RDS and BPD. rhFUT9 treatment ameliorates hyperoxia-induced lung injury in a developmentally appropriate BPD mouse model. Our results have translational potential as a therapeutic modality for BPD in the developing lung.

Mesenchymal stem cells (MSCs) isolated from adipose tissue (adipose-derived stem cells [ADSCs]) are considered one of the most promising cell types for applications in regenerative medicine. However, the regenerative potency of ADSCs may vary because of heterogeneity. Long-term trypsin treatment (LTT) is known to significantly concentrate multilineage-differentiating stress-enduring (Muse) cells from human MSCs. In this study, we aimed to generate cells with high stem cell potency from canine ADSCs using LTT. After 16 h of treatment with trypsin, surviving ADSCs (LTT-tolerant cells) had significantly enhanced expression of stage-specific embryonic antigen (SSEA)-1, a mouse embryonic stem cell marker, and fucosyltransferase 9, one of several fucosyltransferases for SSEA-1 biosynthesis. However, LTT-tolerant cells did not enhance the expression of SSEA-3, a known human Muse cell marker. LTT-tolerant cells, however, showed significantly higher self-renewal capacity in the colony-forming unit fibroblast assay than ADSCs. In addition, the LTT-tolerant cells formed cell clusters similar to embryoid bodies and expressed undifferentiated markers. Moreover, these cells differentiated into cells of all three germ layers and showed significantly higher levels of α 2-6 sialic acid (Sia)-specific lectins, known as differentiation potential markers of human MSCs, than ADSCs. LTT-tolerant cells had a normal karyotype and had low telomerase activity, showing little carcinogenetic potency. LTT-tolerant cells also showed significantly increased activity of transmigration in the presence of chemoattractants and had increased expression of migration-related genes compared with ADSCs. In addition, LTT-tolerant cells had stronger suppressive activity against mitogen-stimulated lymphocyte proliferation than ADSCs. Overall, these results indicated that the LTT-tolerant cells in canine ADSCs have similar properties as human Muse cells (although one of the undifferentiated markers is different) and are expected to be a promising tool for regenerative therapy in dogs.

Introduction: Fetal hypoxic events with unclear predictive value are a common indication for placenta examination. We evaluated whether the use of CD15 immunostaining can improve the assessment of severity and duration of fetal hypoxia., Methods: We compared placentas (37-42 gestational weeks) from stillborns/newborns with birth asphyxia (BA) and non-hypoxic newborns. Placental findings were studied in following groups: (1) acute BA (n = 11) due to placental abruption, (2) non-acute BA (n = 121) due to non-acute conditions, (3) non-BA (n = 46) in pregnancies with preeclampsia and gestational diabetes, and (4) controls (n = 30)., Results: A high expression of CD15 in feto-placental resistance vessels (FRVs) was present in non-acute BA (95.9%), but absent in acute BA, non-BA and controls (p < 0.0001). Furthermore, we found no causal relationship of high expression of CD15 in FRVs to coexisting placental conditions, including severity and mechanisms/patterns of placental injury, fetal erythroblastosis, and maternal conditions. According to a multivariate analysis, only a high expression of CD15 in FRVs was independently associated with severe non-acute fetal hypoxia ([OR] = 15.52; 95% [CI] = 5.92-40.67)., Discussion: We have defined a characteristic pattern of CD15 expression in FRVs that allows to interpret various clinical/placental conditions with respect to fetal hypoxia, with an improved predictability compared to conventional analyses. This approach represents a novel diagnostic strategy for placenta examination, which could indirectly assess severity and duration of intrauterine hypoxia in a heterogeneous population of newborns., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), is related to immunological and microbial factors, with the possible implication of enteric viruses. We characterized the interaction between human noroviruses (HuNoVs) and blood group antigens in refractory CD and UC using HuNoV virus-like particles (VLPs) and histological tissues. Immunohistochemistry was conducted on inflammatory tissue samples from the small intestine, colon, and rectum in 15 CD and 9 UC patients. Analysis of the regenerative mucosa of the colon and rectum revealed strong expression of sialylated Lewis a (sLe a ) and Lewis x (sLe x ) antigens and HuNoV VLP binding in the absence of ABO antigen expression in both UC and CD. Competition experiments using sialidase, lectins, and monoclonal antibodies demonstrated that HuNoV attachment mostly involved Le a and, to a lesser extent, Le x moieties on regenerative mucosa in both UC and CD. Further studies will be required to understand the implications of specific HuNoV binding to regenerative mucosa in refractory IBD. IMPORTANCE Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are progressive diseases affecting millions of people each year. Flare-ups during IBD result in severe mucosal alterations of the small intestine (in CD) and in the colon and rectum (in CD and UC). Immunohistochemical analysis of CD and UC samples showed strong expression of known tumoral markers sialyl Lewis a (CA19.9) and sialyl Lewis x (CD15s) antigens on colonic and rectal regenerative mucosa, concurrent with strong human norovirus (HuNov) VLP GII.4 affinity. Sialidase treatment and competition experiments using histo-blood group antigen (HBGA)-specific monoclonal antibodies and lectins clearly demonstrated the implication of the Lewis a moiety and, to a lesser extent, the Lewis x moiety in HuNov recognition in regenerative mucosa of CD and UC tissues. Further studies are required to explore the possible implications of enteric viruses in the impairment of epithelial repair and dysregulation of inflammatory pathways during severe IBD., (Copyright © 2021 Tarris et al.)

The success of investigations on the structure and function of the genome (genomics) has been paralleled by an equally awesome progress in the analysis of protein structure and function (proteomics). We propose that the investigation of carbohydrate structures that go beyond a cell's metabolism is a rapidly developing frontier in our expanding knowledge on the structure and function of carbohydrates (glycomics). No other functional system appears to be suited as well as the nervous system to study the functions of glycans, which had been originally characterized outside the nervous system. In this review, we describe the multiple studies on the functions of Lewis X , the human natural killer cell antigen-1 (HNK-1), as well as oligomannosidic and sialic (neuraminic) acids. We attempt to show the sophistication of these structures in ontogenetic development, synaptic function and plasticity, and recovery from trauma, with a view on neurodegeneration and possibilities to ameliorate deterioration. In view of clinical applications, we emphasize the need for glycomimetic small organic compounds which surpass the usefulness of natural glycans in that they are metabolically more stable, more parsimonious to synthesize or isolate, and more advantageous for therapy, since many of them pass the blood brain barrier and are drug-approved for treatments other than those in the nervous system, thus allowing a more ready access for application in neurological diseases. We describe the isolation of such mimetic compounds using not only Western NIH, but also traditional Chinese medical libraries. With this review, we hope to deepen the interests in this exciting field.

Alopecia areata (AA), which is defined as an autoimmune hair loss disease, has a serious impact on the quality of life for patients with AA worldwide. In this study, to our knowledge, a previously unreported method of AA induction in C3H mice has been established and validated. Using this method, we showed that dermal injection of 1-3 million of a mixture of skin cells freshly isolated from AA-affected skin induces AA in more than 80% of healthy mice. Contrary to the previous protocol, the induction of AA by this approach does not need any surgical AA skin grafting, cell manipulation, or high number of activated T cells. We also showed that dermal injection of adherent myeloid cells (mainly CD11b+) in healthy mice is as potent as a mixture of none adherent CD3+ T cells and CD19+ B cells in the induction of AA. Interestingly, most of the mice (7 out of 8) that received non-adherent cells developed AA universalis, whereas most of the mice (5 out of 7) that received adherent cells developed patchy AA. Finally, we found a high number of stage-specific embryonic antigen-expressing cells whose expression in monocytes in an inflammatory disease causes the release of inflammatory cytokines, TNF-α and IL-1β, from these cells in AA-affected skin., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Giant cell arteritis (GCA) is a common form of primary systemic vasculitis in adults, with no reliable indicators of prognosis or treatment responses. We used single cell technologies to comprehensively map immune cell populations in the blood of patients with GCA and identified the CD66b+CD15+CD10lo/-CD64- band neutrophils and CD66bhiCD15+CD10lo/-CD64+/bright myelocytes/metamyelocytes to be unequivocally associated with both the clinical phenotype and response to treatment. Immature neutrophils were resistant to apoptosis, remained in the vasculature for a prolonged period of time, interacted with platelets, and extravasated into the tissue surrounding the temporal arteries of patients with GCA. We discovered that immature neutrophils generated high levels of extracellular reactive oxygen species, leading to enhanced protein oxidation and permeability of endothelial barrier in an in vitro coculture system. The same populations were also detected in other systemic vasculitides. These findings link functions of immature neutrophils to disease pathogenesis, establishing a clinical cellular signature of GCA and suggesting different therapeutic approaches in systemic vascular inflammation.

Background: The flow cytometry analysis of GPI-linked proteins on red blood cells and leukocytes is crucial for paroxysmal nocturnal hemoglobinuria (PNH) diagnostics. However, the commonly used multicolor panels cannot be implemented in low-resourced hematology laboratories. In order to develop a simple prediagnostic test for PNH screening, we analyzed the diagnostic accuracy of the two-color (FLAER/CD15) detection of GPI-deficient neutrophils., Methods: We reanalyzed multicolor data set of 1594 peripheral blood samples of patients screened for PNH applying only two markers (FLAER/CD15). The quantitative positivity/negativity was reported. Then, these results were compared in a blinded manner with previously obtained multicolor data from the same samples., Results: Among the 1594 samples included in the study, 507 samples were PNH-positive by the multicolor assay. The two-color method revealed 510 PNH-positive samples. The detailed examination of this discrepancy revealed 12 false-positives and 9 false-negatives. Therefore, FLAER/CD15 screening method displayed 98.90% of the diagnostic specificity and 98.22% of the sensitivity., Conclusion: This simple two-color evaluation of FLAER-negative neutrophils is a highly effective screening test for PNH. Although this approach is not intended to replace the multicolor diagnostic procedure, it could minimize the number of patients requiring a conventional multicolor flow cytometric assay., (© 2020 John Wiley & Sons Ltd.)

Background: The Sd a antigen and its biosynthetic enzyme B4GALNT2 are highly expressed in healthy colon but undergo a variable down-regulation in colon cancer. The biosynthesis of the malignancy-associated sialyl Lewis x (sLe x ) antigen in normal and cancerous colon is mediated by fucosyltransferase 6 (FUT6) and is mutually exclusive from that of Sd a . It is thought that the reduced malignancy associated with high B4GALNT2 was due to sLe x inhibition., Methods: We transfected the cell lines SW480 and SW620, derived respectively from a primary tumor and a metastasis of the same patient, with the cDNAs of FUT6 or B4GALNT2, generating cell variants expressing either the sLe x or the Sd a antigens. Transfectants were analyzed for growth in poor adherence, wound healing, stemness and gene expression profile., Results: B4GALNT2/Sd a expression down-regulated all malignancy-associated phenotypes in SW620 but only those associated with stemness in SW480. FUT6/sLe x enhanced some malignancy-associated phenotypes in SW620, but had little effect in SW480. The impact on the transcriptome was stronger for FUT6 than for B4GALNT2 and only partially overlapping between SW480 and SW620., Conclusions: B4GALNT2/Sd a inhibits the stemness-associated malignant phenotype, independently of sLe x inhibition. The impact of glycosyltransferases on the phenotype and the transcriptome is highly cell-line specific.

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called "targeting by timing." Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation, and N - and O -glycans present in each compartment were analyzed by LC-MS. We found abundant paucimannosidic N -glycans and lack of O -glycans in the early-formed azurophil granules, whereas the later-formed specific and gelatinase granules and secretory vesicles contained complex N- and O- glycans with remarkably elongated N -acetyllactosamine repeats with Lewis epitopes. Immunoblotting and histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils, and their complexity increased progressively from azurophil granules to specific granules and then to gelatinase granules, suggesting temporal changes in the glycosylation machinery indicative of "glycosylation by timing" during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step toward a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Venkatakrishnan et al.)

Quality control for human induced pluripotent stem cells (hiPSCs) is important for efficient and stable production of hiPSC-derived cell therapy products to be used for transplantation. During cell culture, hiPSCs spontaneously undergo morphological changes and lose pluripotent properties. Such cells are termed deviated cells, which are altered from the undifferentiated state of hiPSCs, and express the early differentiation marker stage-specific embryonic antigen 1 (SSEA-1). In this study, we searched for soluble SSEA-1 + glycoproteins secreted from deviated cells generated by culturing hiPSCs in cell culture medium containing heat-inactivated supplements. Glycoproteins obtained from cell culture supernatants of SSEA-1 + deviated cells were enriched by an O-glycan binding lectin and blotted with anti-SSEA-1 antibody. A single protein band at >250 kDa specifically detected by anti-SSEA-1 antibody was identified as fibronectin (FN) by LC-MS/MS analysis and immunoprecipitation combined with western blotting, indicating that FN is a carrier protein of SSEA-1. We then constructed a sandwich enzyme-linked immunosorbent assay to detect SSEA-1 + FN secreted from deviated cells. This FN-SSEA-1 test proved to be both sensitive and specific, allowing for non-destructive detection of SSEA-1 + deviated cells within mixed cell population, with a lower limit of detection of 100 cells/mL. The developed assay may provide a standard technology for quality control of hiPSCs used for regenerative medicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The finding of corpse parts poses several challenges for the forensic pathologist presenting implications for identification, diagnosis of death and determination of wounds vitality. Further interpretative difficulties in cases of cadaveric dismemberment derive from the scarcity of tanatochronological parameters useful to estimate the post-mortem interval (PMI) and the absence of uniform investigative protocols in the different centres of forensic pathology. The present study proposes an investigation protocol for the cadaveric dismemberment through the discussion of a case series. The study group consisted of cases in which the dismemberment was performed after the murder. For all cases, a study protocol based on crime scene investigation, post-mortem computed tomography (PMCT), autopsy, toxicological, histological, immunohistochemical and genetic investigations was implemented. In particular, the standardised use of radiographic study before the autopsy allows all to have information that can guide the forensic pathologist during the autopsy. The use of immunohistochemistry allows an assessment of the vitality of the lesions possibly involved in the determinism of death, as well as of the surfaces of dismemberment, representing a tool of considerable utility for forensic purposes. The genetic investigations allow the identification of the victims, while the toxicological ones highlight the possible abuse of substances. The implemented protocol presents a demonstrated usefulness in improving diagnostic accuracy in corpse dismemberment cases., (Copyright © 2020 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Myeloid-derived suppressor cells with polymorphonuclear morphology (PMN-MDSCs) contribute to the progression and immune evasion of prostate cancer. However, the spatial distribution of tumor-infiltrating PMN-MDSCs in primary and metastatic prostate cancer, especially in the context of comparison between the epithelial and stromal compartments of the tumor, has not been characterized. Here, we describe a multicolor immunofluorescence staining study of 90 primary tumors, 37 lymph node metastases (all with matched primary tumors) and 35 bone metastases using archived samples. CD11b + CD15 + cells were identified as PMN-MDSCs and pan-cytokeratin + cells were identified as prostate epithelial cells. We found that, in both primary tumor and metastases, PMN-MDSCs infiltrate much more readily in the stromal area compared with the epithelial area of the tumor regions. In comparison to the stromal area of primary tumors, the stromal area of either lymph node metastases or bone metastases was infiltrated with more PMN-MDSCs. In primary tumors, stromal PMN-MDSCs were associated with vascularization, segmented neutrophils, patient age and close juxtaposition to neoplastic epithelial cells. These results reveal the stroma rather than the epithelia of prostate cancer as the major hotbed for PMN-MDSCs and support the role of PMN-MDSCs in the metastatic progression of prostate cancer., (© 2020 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and pore formation have been described, little is known about receptor binding of TcAs. Here, we identify heparins/heparan sulfates and Lewis antigens as receptors for different TcAs from insect and human pathogens. Glycan array screening reveals that all tested TcAs bind negatively charged heparins. Cryo-EM structures of Morganella morganii TcdA4 and Xenorhabdus nematophila XptA1 reveal that heparins/heparan sulfates unexpectedly bind to different regions of the shell domain, including receptor-binding domains. In addition, Photorhabdus luminescens TcdA1 binds to Lewis antigens with micromolar affinity. Here, the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a glycan dependent association mechanism of Tc toxins on the host cell surface.

Background: The phenotypical identification of diagnostic cells is crucial for the diagnosis of Hodgkin's lymphoma (HL) on fine-needle aspiration cytology (FNAC). The aim of this study is to evaluate the immunocytochemical (ICC) expression of CD30, CD15, and PAX5 in Hodgkin's cells (HC) and Reed-Sternberg cells (RSC) on smears and cell-blocks (CB) of HL and to compare the performance of each antibody on smears and CB., Methods: In 21 FNAC cases of histologically confirmed classical HL, ICC identification of HC and RSC was performed using CD15, CD30, and PAX5 on smears and CB, respectively., Results: CD30 was positive in 19/21 cases (90.5%; 11/11 smears and 8/10 CB), CD15 was positive in 14/21 cases (66.7%; 5/11 smears and 9/10 CB), and PAX5 was positive in 13/21 cases (61.9%; 9/11 smears and 4/10CB)., Conclusions: CD15, CD30, and PAX5 are useful to the FNAC identification of HC and RSC. CD30 is the most sensitive, followed by CD15 and PAX5, which are more effective on CB and smears, respectively., (© 2019 Wiley Periodicals, Inc.)

Prostate-specific antigen (PSA) is the most frequently used biomarker for the screening of prostate cancer. Understanding the structure of cancer-specific glycans can help us improve PSA assay. In the present study, we analysed the glycans of PSA obtained from culture medium containing cancer tissue-originated spheroids (CTOS) which have similar characteristics as that of the parent tumour to explore the new candidates for cancer-related glycoforms of PSA. The glycan profile of PSA from CTOS was determined by comparing with PSA from normal seminal plasma and cancer cell lines (LNCaP and 22Rv1) using lectin chromatography and mass spectrometry. PSA from CTOS was mostly sialylated and the content of Wisteria floribunda agglutinin reactive glycan (LacdiNAc) was similar to that of PSA derived from seminal plasma and 22Rv1. Conversely, concanavalin A (Con A)-unbound PSA was definitely detected from the three cancer origins but was almost negligible in seminal PSA. Two novel types of PSA were elucidated in the Con A-unbound fraction: one is a high molecular weight PSA with highly branched N-glycans, and the other is a low molecular weight PSA without N-glycans. Furthermore, the existence of Lewis X antigen group on PSA was indicated. These PSAs will be candidates for new cancer-related markers.

Lately, stem cell approaches have provided new information on reproductive organ function and additionally recommended novel treatment possibilities. The type(s) and differentiation potential of stem cells present in the mammalian ovary are largely unknown; while oogonial stem cells have been reported, we explored the possibility that multipotent stem cells may reside in the ovary and have wide differentiation potential. In this experimental study, homogenates of whole mouse ovaries were sorted using the stem cell surface markers stem cell antigen-1 and stage specific embryonic antigen-1/CD15. Viable double-positive cells 3-10 μm in diameter were evaluated immediately after sorting and after culture using differentiation conditions. Ovarian-derived stem cells were differentiated into the three main cell types: adipocytes, chondrocytes, or osteocytes. The subsequent culture was performed in media containing bone morphogenetic protein 4 (BMP-4) and/or retinoic acid (RA). RA, BMP-4 or the two agents in combination, consistently stimulated germ cell gene expression. RA treatment strongly stimulated germline gene expression and also the development of cells that were morphologically reminiscent of oocytes. The germ cell genes Dazl, Ddx4, Figla, Gdf-9, Nobox, Prdm9, and Sycp-1 were all detected at low levels. Remarkably, treatment with BMP-4 alone significantly increased protein expression of the granulosa cell product anti-Müllerian hormone (AMH). We have shown that an inclusive isolation protocol results in the consistent derivation of multipotent stem cells from the adult ovary; these cells can be differentiated towards the germ cell fate (RA alone), somatic ovarian cell fate as indicated by AMH production (BMP-4 alone), or classical mesenchymal cell types. Taken together, these data suggest the presence of multipotent mesenchymal stem cells in the murine ovary.

The differentiation of primordial germ cells (PGCs) is a fundamental step in development. PR domain-containing protein 14 (PRDM14) and B lymphocyte-induced maturation protein 1 (BLIMP1) play pivotal roles in mouse PGC specification. In the present study, we assessed the roles of chicken orthologs of PRDM14 and BLIMP1 in PGC development. PRDM14 and BLIMP1 were expressed in blastodermal cells and PGCs. The in vivo knockdown of PRDM14 or BLIMP1 by introducing a replication-competent retroviral vector expressing shRNAs to the blastodermal stage of embryos reduced the number of SSEA-1 or chicken vasa homologue-positive PGCs on day 5.5-6.5. Since the inhibition of Activin receptor-like kinase 4/5/7 in cultured PGCs reduced the expression of PRDM14, BLIMP1, and NANOG, and that of MEK inhibited PRDM14 expression, the expression of these genes seems to be controlled by Activin A and FGF2 signaling. Overall, PRDM14, BLIMP1, and NANOG seem to be involved in the self-renewal of PGCs in cultured PGCs and embryos., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Domestic cats suffer from a range of inherited genetic diseases, many of which display similarities with equivalent human conditions. Developing cellular models for these inherited diseases would enable drug discovery, benefiting feline health and welfare as well as enhancing the potential of cats as relevant animal models for translation to human medicine. Advances in our understanding of these diseases at the cellular level have come from the use of induced pluripotent stem cells (iPSCs). iPSCs can differentiate into virtually any cell type and can be derived from adult somatic cells, therefore overcoming the ethical implications of destroying embryos to obtain embryonic stem cells. No studies, however, report the generation of iPSCs from domestic cats [feline iPSCs (fiPSCs)]. Feline adipose-derived fibroblasts were infected with amphotropic retrovirus containing the coding sequences for human Oct4 , Sox2 , Klf4 , cMyc , and Nanog . Isolated iPSC clones were expanded on inactivated mouse embryonic fibroblasts in the presence of feline leukemia inhibitory factor (fLIF). Retroviral delivery of human pluripotent genes gave rise to putative fiPSC colonies within 5-7 days. These iPS-like cells required fetal bovine serum and fLIF for maintenance. Colonies were domed with refractile edges, similar to mouse iPSCs. Immunocytochemistry demonstrated positive staining for stem cell markers: alkaline phosphatase, Oct4 , Sox2 , Nanog , and SSEA1. Cells were negative for SSEA4. Expression of endogenous feline Nanog was confirmed by quantitative polymerase chain reaction. The cells were able to differentiate in vitro into cells representative of the three germ layers. These results confirm the first generation of induced pluripotent stem cells from domestic cats. These cells will provide valuable models to study genetic diseases and explore novel therapeutic strategies.

Purpose: Metastatic non-small cell lung (NSCLC) cancer represents one of the most common types of brain metastasis. The mechanisms involved in how circulating cancer cells transmigrate into brain parenchyma are not fully understood. The aim of this work was to investigate the role of fucosylated carbohydrate epitopes CD15 and sialyated CD15s in cancer adhesion to brain-derived endothelial cells and determine their influence in blood-brain barrier (BBB) disruption METHODS: Three distinct, independent methods were used to measure brain endothelial integrity and include voltohmmeter (EVOM™), impedance spectroscopy (CellZscope®) and electric cell-substrate impedance sensing system (ECIS™). Two fucosyltransferases (FUT4 and 7) responsible for CD15 and CD15s synthesis were modulated in four human cancer cell lines (three lung cancer and one glioma)., Results: Overexpression of CD15 or CD15s epitopes led to increase in adhesion of cancer cells to cerebral endothelial cells compared with wild-type and cells with silenced CD15 or CD15s (p < 0.01). This overexpression led to the disruption of cerebral endothelial cell monolayers (p < 0.01). Knockdown of FUT4 and FUT7 in metastatic cancer cells prevented disruption of an in vitro BBB model. Surprisingly, although the cells characterised as 'non-metastatic', they became 'metastatic' -like when cells were forced to over-express either FUT4 or FUT7., Conclusions: Results from these studies suggest that overexpression of CD15 and CD15s could potentiate the transmigration of circulating NSCLC cells into the brain. The clinical significance of these studies includes the possible use of these epitopes as biomarkers for metastasis.

To evaluate the early absolute CD64/CD15/CD45 neutrophil count as a marker of prognosis of sepsis outcome the absolute CD64/CD15/CD45 count was measured by flow cytometry in 65 patients with confirmed or suspected Gram-negative sepsis and organ dysfunction. Serum interleukin(IL)-8 and interferon-gamma (IFNγ) were measured by an enzyme immunoassay. An absolute count lower than 2500 cells/mm 3 could early discriminate non-survivors with sensitivity 82.9% (OR 3.46, 95%CIs 1.10-10.95, p 0.042). After forward step-wise Cox- regression analysis, it was found that acute coagulopathy, acute renal injury, and an early absolute CD64/CD15/CD45 count lower than 2500/mm 3 were independently associated with unfavorable outcome. The OR for death among patients with an absolute CD64/CD15/CD45 neutrophil count greater than 2500/mm 3 and circulating IL-8 greater than 95 pg/ml was 0.44; this was significantly increased to 7.44 among patients with an absolute CD64/CD15/CD45 neutrophil count lower than 2500/mm 3 (p 0.045 by the Breslow-Day's test; p 0.046 by the Tarone's test). An absolute CD64/CD15/CD45 count below 2500/mm 3 can be a useful prognosticator of sepsis outcome and a probable indicator of sepsis immunosuppression.

Background: Structural or functional defects in the placenta, are the primary cause of growth restriction of the fetus. Morphological examination of such placentas from intrauterine growth restricted (IUGR) fetuses often appears deceptively normal. Evaluation of angiogenesis and fetoplacental vasculature is critical to understand the underlying pathogenesis of fetal growth restriction in both idiopathic as well as cases where it is thought to be secondary to complications like preeclampsia (PE). We analyzed the immaturity of fetoplacental vasculature using CD15, which is a stage specific embryonic antigen known to be expressed in immature endothelium., Material and Methods: One hundred and twelve placentas (81 from IUGR and 31 from gestationally appropriate samples (appropriate for gestational age (AGA)) were collected based on stringent inclusion criteria, and subjected to detailed examination of morphology and microscopy along with immunostaining for CD15. IUGR placentas known to have villous immaturity such as those associated with gestational diabetes, Rh negative pregnancies and anemia were excluded. The time of clinical onset of IUGR, associated complications like PE and oligohydramnios along with clinical variables were recorded. CD15 expression was scored in both distal and proximal vasculature and the values in IUGR and AGA pregnancies were compared and correlated with clinical variables., Results: The mean CD 15 scores in both proximal vasculature (PV) as well as distal (DV) vasculature were significantly higher in the IUGR group compared to AGA (17.7 versus 5.16 in PV and 50.8 versus 23.7 in distal vasculature (DV)). Gestational age had no influence on CD15 staining in PV or DV in IUGR group, whereas preterm AGAs expressed higher CD15 only in the distal vessels. PE, oligohydramnios and the time of onset of IUGR did not influence the fetal vascular immaturity, as measured by CD15 scores. Although none of the clinical or obstetric factors influenced CD15 staining in AGA, fetal vessel immaturity in the IUGR group remained high even after adjusting for confounding variables like maternal age, gestational age and birth weight. Histological features suggestive of chronic hypoxia were significantly higher in IUGR placentas, compared to AGA and correlated positively with CD15 expression., Conclusion: Fetoplacental endothelium in both PV and DV is immature in IUGR irrespective of the gestational age or any other associated factors and CD15 immunodetection is a valuable marker for assessment of immaturity.

Stage-specific embryonic antigen 1 (SSEA-1) is an antigenic epitope (also called CD15 antigen) defined as a Lewis X carbohydrate structure and known to be expressed in murine embryonal carcinoma cells, mouse embryonic stem cells (ESCs), and murine and human germ cells, but not human ESCs/induced pluripotent stem cells (iPSCs). It is produced by α1,3-fucosyltransferase IX gene ( FUT9 ), and F9 ECCs having a disrupted FUT9 locus by gene targeting are reported to exhibit loss of SSEA-1 expression on their cell surface. Mouse ESCs are pluripotent cells and therefore known as "naïve stem cells (NSCs)." In contrast, human ESCs/iPSCs are thought to be epiblast stem cells (EpiSCs) that are slightly more differentiated than NSCs. Recently, it has been demonstrated that treatment of EpiSCs with several reprograming-related drugs can convert EpiSCs to cells similar to NSCs, which led us to speculate that SSEA-1 may have been expressed in these NSC-like EpiSCs. Immunocytochemical staining of these cells with anti-SSEA-1 revealed increased expression of this epitope. RT-PCR analysis also confirmed increased expression of FUT9 transcripts as well as other stemness-related transcripts such as REX-1 ( ZFP42 ). These results suggest that SSEA-1 can be an excellent marker for human NSCs.

Impact Statement: Neural stem cells (NCSs) are integral to establishing in vitro models and regenerative medicine. To this day, there is an unmet need to enrich these cells from a heterogeneous cell population for clinical applications without irreversible manipulation. We identified a method to propagate human NCSs via computational analysis of their mechanical signature. In this study, we report a novel analytical method for mechanical forces in three-dimensional cultures. Further, our results revealed that stemness may, in part, be mediated by physical properties of the extracellular matrix. In conclusion, our findings have potential implications in understanding stem cell mechanobiology for enrichment or differentiation.

Unveiling the mechanism of the relapse of acute lymphoblastic leukemia (ALL) is the key to improve the prognosis of ALL and remains a huge challenge. Glycan-based interactions play a vital role in immune surveillance, cell-cell adhesion and cell-matrix interaction, contributing to treatment failure in tumor. However, the glycan essential for leukemia development and its upstream regulatory mechanism by oncogenic drivers were rarely reported. Here, we demonstrated that Le X , a well-characterized cancer-related glycan epitope, strengthened the cell-matrix interaction via glycosylating α5β1 integrin under the control of the driver oncogenic Ikaros isoform (IK6) in ALL. By analyzing the expression profile of Ikaros and the level of FUT4/Le X in clinical samples, we found that FUT4/Le X was positively correlated with dysfunctional Ikaros isoforms. IK1 (Full length Ikaros) regulates the level of FUT4 as a transcription repressor, while IK6 abolished the wild-type Ikaros mediated transcriptional repression and resulted in higher level of FUT4 expression. Moreover, we demonstrated that FUT4 could activate α5β1-mediated sequential signal transduction and accelerate adhesion and invasion between integrin α5β1 in leukemia cells and fibronectin in extracellular matrix (ECM) via increasing glycosylation. Together, our study provides a new insight into the mechanisms by which Ikaros mutation induced ALL cells invasion and a potential strategy for drug-resistance ALL by blocking Le X in combination with common chemotherapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)