Peripheral T-cell lymphomas expressing CD30 and CD15 expand the spectrum of anaplastic large cell lymphoma, ALK-negative.

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.

Androgen drives melanoma invasiveness and metastatic spread by inducing tumorigenic fucosylation.

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.

Targeting myeloid chemotaxis to reverse prostate cancer therapy resistance.

Inflammation is a hallmark of cancer1. 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 modalities2-5. 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-DRloCD15+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.

Spatial predictors of immunotherapy response in triple-negative breast cancer.

Immune checkpoint blockade (ICB) benefits some patients with triple-negative breast cancer, but what distinguishes responders from non-responders is unclear1. Because ICB targets cell-cell interactions2, 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 cytometry3 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.

Establishment of Bactrian Camel Induced Pluripotent Stem Cells and Prediction of Their Unique Pluripotency Genes.

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.

New insight on the role of Helicobacter pylori cagA in the expression of cell surface antigens with important biological functions in gastric carcinogenesis.

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.

Phenotyping clonal populations of glioma stem cell reveals a high degree of plasticity in response to changes of microenvironment.

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.

An Introduction to the Relationship Between Lewis x and Malignancy Mainly Related to Breast Cancer and Head Neck Squamous Cell Carcinoma (HNSCC).

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.

Glioblastoma Contains Topologically Distinct Proliferative and Metabolically Defined Subpopulations of Nestin- and Glut1-Expressing Cells.

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.

The Frequency and Effect of Granulocytic Myeloid-Derived Suppressor Cells on Mycobacterial Survival in Patients With Tuberculosis: A Preliminary Report.

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 H37Rv-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 H37Rv 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.

Expression of the Carbohydrate Lewis Antigen, Sialyl Lewis A, Sialyl Lewis X, Lewis X, and Lewis Y in the Placental Villi of Patients With Unexplained Miscarriages.

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.

Differential expression of stem cell markers in proliferating cells in glioma.

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.

Long-Term Trypsin Treatment Promotes Stem Cell Potency of Canine Adipose-Derived Mesenchymal Stem Cells.

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.

Specific Norovirus Interaction with Lewis x and Lewis a on Human Intestinal Inflammatory Mucosa during Refractory Inflammatory Bowel Disease.

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 (sLea) and Lewis x (sLex) 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 Lea and, to a lesser extent, Lex 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.

α1,3-Fucosyltransferase-IX, an enzyme of pulmonary endogenous lung stem cell marker SSEA-1, alleviates experimental bronchopulmonary dysplasia.

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.

Myeloid Adherent Cells Are Involved in Hair Loss in the Alopecia Areata Mouse Model.

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-1ß, from these cells in AA-affected skin.

The Sda Synthase B4GALNT2 Reduces Malignancy and Stemness in Colon Cancer Cell Lines Independently of Sialyl Lewis X Inhibition.

BACKGROUND: The Sda 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 (sLex) antigen in normal and cancerous colon is mediated by fucosyltransferase 6 (FUT6) and is mutually exclusive from that of Sda. It is thought that the reduced malignancy associated with high B4GALNT2 was due to sLex 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 sLex or the Sda antigens. Transfectants were analyzed for growth in poor adherence, wound healing, stemness and gene expression profile. RESULTS: B4GALNT2/Sda expression down-regulated all malignancy-associated phenotypes in SW620 but only those associated with stemness in SW480. FUT6/sLex 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/Sda inhibits the stemness-associated malignant phenotype, independently of sLex inhibition. The impact of glycosyltransferases on the phenotype and the transcriptome is highly cell-line specific.

ROS-producing immature neutrophils in giant cell arteritis are linked to vascular pathologies.

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.

SSEA-1-positive fibronectin is secreted by cells deviated from the undifferentiated state of human induced pluripotent stem cells.

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.

Polymorphonuclear MDSCs are enriched in the stroma and expanded in metastases of prostate cancer.

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.