Differences In Expression Of Markers Research Articles

PGD: Shared gene linking polycystic ovary syndrome and endometrial cancer, influencing proliferation and migration through glycometabolism.

The relationship among polycystic ovary syndrome (PCOS), endometrial cancer (EC), and glycometabolism remains unclear. We explored shared genes between PCOS and EC, using bioinformatics to unveil their pathogenic connection and influence on EC prognosis. Gene Expression Omnibus datasets GSE226146 (PCOS) and GSE196033 (EC) were used. A protein-protein interaction (PPI) network was constructed to identify the central genes. Candidate markers were screened using dataset GSE54250. Differences in marker expression were confirmed in mouse PCOS and human EC tissues using RT-PCR and immunohistochemistry. The effect of PGD on EC proliferation and migration was explored using Ki-67 and Transwell assays. PGD's impact on the glycometabolic pathway within carbon metabolism was assessed by quantifying glucose content and lactic acid production. R software identified 31 common genes in GSE226146 and GSE196033. Gene Ontology functional classification revealed enrichment in the "purine nucleoside triphosphate metabolism process," with key Kyoto Encyclopedia of Genes and Genomes pathways related to "carbon metabolism." The PPI network identified 15 hub genes. HK2, NDUFS8, PHGDH, PGD, and SMAD3 were confirmed as candidate markers. The RT-PCR analysis validated distinct HK2 and PGD expression patterns in mouse PCOS ovarian tissue and human EC tissue, as well as in normal and EC cells. Transfection experiments with Ishikawa cells further confirmed PGD's influence on cell proliferation and migration. Suppression of PGD expression impeded glycometabolism within the carbon metabolism of EC cells, suggesting PGD as a significant PCOS risk factor impacting EC proliferation and migration through modulation of single carbon metabolism. These findings highlight PGD's pivotal role in EC onset and prognosis.

Multiparameter phenotyping of platelets and characterization of the effects of agonists using machine learning

BackgroundPlatelet function is driven by the expression of specialized surface markers. The concept of distinct circulating subpopulations of platelets has emerged in recent years, but their exact nature remains debatable. ObjectivesTo design a spectral flow cytometry–based phenotyping workflow to provide a more comprehensive characterization, at a global and individual level, of surface markers in resting and activated healthy platelets, and to apply this workflow to investigate how responses differ according to platelet age. MethodsA 14-marker flow cytometry panel was developed and applied to vehicle- or agonist-stimulated platelet-rich plasma and whole blood samples obtained from healthy volunteers, or to platelets sorted according to SYTO-13 (Thermo Fisher Scientific) staining intensity as an indicator of platelet age. Data were analyzed using both user-led and independent approaches incorporating novel machine learning–based algorithms. ResultsThe assay detected differences in marker expression in healthy platelets, at rest and on agonist activation, in both platelet-rich plasma and whole blood samples, that are consistent with the literature. Machine learning identified stimulated populations of platelets with high accuracy (>80%). Similarly, machine learning differentiation between young and old platelet populations achieved 76% accuracy, primarily weighted by forward scatter, cluster of differentiation (CD) 41, side scatter, glycoprotein VI, CD61, and CD42b expression patterns. ConclusionOur approach provides a powerful phenotypic assay coupled with robust bioinformatic and machine learning workflows for deep analysis of platelet subpopulations. Cleavable receptors, glycoprotein VI and CD42b, contribute to defining shared and unique subpopulations. This adoptable, low-volume approach will be valuable in deep characterization of platelets in disease.

Expression of mPGES1 and p16 in feline and human oral squamous cell carcinoma: A comparative oncology approach.

Comparative cancer studies help us determine if discoveries in one species apply to another. Feline and human oral squamous cell carcinoma (FOSCC and HOSCC) are invasive tumours in which inflammation and abnormal p16 expression are reported. Immunohistochemistry was used to determine the expression of p16 and microsomal prostaglandin E2 synthase 1 (mPGES1) in 42 HOSCC and 45 FOSCC samples with known expression of cyclooxygenase 2 (COX2) and cluster of differentiation 147 (CD147). High p16 expression was more common in HOSCC tumour cells compared to adjacent stroma and oral epithelium (p < .05), with a similar but statistically nonsignificant pattern in FOSCC. Interestingly, high mPGES1 expression in FOSCC was more common in the adjacent epithelium compared to the other compartments (p < .05). In HOSCC, mPGES1 was more similar between compartments but was numerically more common in the tumour compartment (p > .05). There were nominal (p > 0.05) differences in marker expression between high and low mPGES1 expressing tumours in both species, including high p16 observed more commonly in high mPGES1 tumours, and COX-2 positive tumours being more common in low mPGES1 tumours. High CD147 HOSCC tumours were more common in the high mPGES1 HOSCC group (p < .05). In the FOSCC cohort, where there was no statistical difference in CD147 expression between high and low mPGES1 tumours, there were numerically higher CD147 cases in the high mPGES1group. Different expression patterns in FOSCC and HOSCC could be related to different risk factors. For example, p16 is a marker of papillomavirus-driven HOSCC, but a causal relationship between papillomaviruses and FOSCC has yet to be definitively demonstrated. The significance of high P16 expression in the absence of papillomavirus infection deserves further study, and the relative contributions of COX2 and mPGES1 to tumour inflammation and progression should be explored. The findings reveal potential similarities in FOSCC and HOSCC biology, while also demonstrating differences that may relate to risk factors and pathogenesis that are unique to each species.

Exploring urinary extracellular vesicles for organ transplant monitoring: A comprehensive study for detection of allograft dysfunction using immune-specific markers

BackgroundAllograft dysfunction (AGD) is a common complication following solid organ transplantation (SOT). This study leverages the potential of urinary extracellular vesicles (UEVs) for the non-invasive detection of AGD. AimWe aimed to assess the diagnostic value of T-cell and B-cell markers characteristic of T-cell-mediated and antibody-mediated rejection in UEV-mRNA using renal transplantation as a model. Materials and methodsUEVs were isolated from 123 participants, spanning healthy controls, functional transplant recipients, and biopsy-proven AGD patients. T-cell and B-cell marker mRNA expressions were evaluated using RT-qPCR. ResultsWe observed significant differences in marker expression between healthy controls and AGD patients. ROC analysis revealed an AUC of 0.80 for T-cell markers, 0.98 for B-cell markers, and 0.94 for combined markers. T-cell markers achieved 81.3 % sensitivity, 80 % specificity, and 80.4 % efficiency. A triad of T-cell markers (PRF1, OX40, and CD3e) increased sensitivity to 87.5 % and efficiency to 82.1 %. B-cell markers (CD20, CXCL3, CD46, and CF3) delivered 100 % sensitivity and 97.5 % specificity. The combined gene signature of T-cell and B-cell markers offered 93.8 % sensitivity and 95 % specificity. ConclusionOur findings underscore the diagnostic potential of UEV-derived mRNA markers for T-cells and B-cells in AGD, suggesting a promising non-invasive strategy for monitoring graft health.

Identification and classification of distinct surface markers of T regulatory cells.

Regulatory T (Treg) cells have emerged as key players in the maintenance of immune homeostasis. Although significant progress has been made in recent years to define the Treg surface markers involved with or identifying their suppressive function, there remains much to be elucidated, and many questions persist. This study determined the expression of surface markers on human peripheral Treg cells and conventional T (Tconv) cells in a steady state and after activation to gain insight into their mechanism of action and more precisely characterize this regulatory population in humans. To screen Treg and Tconv cells, peripheral blood mononuclear cells (PBMCs) were isolated from volunteers, stained with a commercially available lyophilized antibody array comprising 371 surface antigens, and analyzed by flow cytometry. To compare Treg cells with activated Tconv cells, PBMCs were stimulated with PMA and further stained similar to freshly isolated cells. Treg and Tconv cells were positive for 135 and 168 of the 371 antigens, respectively. Based on the frequency distribution, all of the most highly expressed markers identified were shared by both Treg and Tconv cells and participate in T cell activation, act as costimulatory and signaling molecules, or exhibit adhesion and migratory functions. Additionally, we identified several differences in marker expression between Treg and Tconv cells, with most found in the expression of co-stimulatory (ICOS, GITR, 4-1BB) and co-inhibitory (TIGIT, CTLA-4) molecules, as well as chemokine receptors (CXCR4, CXCR5, CCR4, CCR5, CCR7, CCR8, and CXCR7). Furthermore, post-activation expression of surface molecules identified molecules capable of discriminating Treg cells from activated Tconv cells (GITR, 4-1BB, TIGIT, CD120b, and CD39); however, almost all of these markers were also expressed in a small fraction of activated Tconv cells. These results offer insight into the biology of Tregs and contribute to their accurate identification and characterization in variety of immunological diseases as well as physiological processes.

Changes in Expression of the Vascular Endothelial Growth Factor in the Kidneys of Diabetic Rats During Aging

Background. Diabetic nephropathy is the leading cause of end-stage renal disease (ESRD) in most developed countries. A number of specific factors may contribute to the onset and progression of diabetic nephropathy. One of those specific factors is vascular endothelial growth factor (VEGF). We studied the expression of VEGF in the kidneys of diabetic rats during aging. Methods. Male Sprague-Dawley rats were injected with 55mg/kg streptozotocin (STZ) (DM group) or with citrate buffer (control group). Kidneys were collected after 2 weeks, 2 months, 6 months and 12 months, and were analyzed in three different kidney structures: glomeruli, proximal (PCT) and distal convoluted tubules (DCT). Sections were stained immunohistochemically, using VEGF.Results. Significant differences in marker expression were observed after 2 months, with higher VEGF expression in diabetic rats. Positive VEGF cells were detected exclusively in the glomeruli of diabetic rats after 2 months. After 2 weeks of DM onset, high VEGF expression was demonstrated in the distal tubules of diabetic rats, as well as a complete absence of VEGF expression in the distal tubules of the control group. Conclusions. The major change in expression of VEGF occurs after 2 months of DM onset, particularly in the DTC, implying an early onset of pathophysiological changes in diabetic kidneys, which would usually occur with aging. These findings help to contribute to our understanding of changes associated with DN and guide towards potentially appropriate treatment modalities.Key words: diabetes mellitus, kidneys, nephropathy, VEGF

An Immune Atlas of Nephrolithiasis: Single-Cell Mass Cytometry on SIRT3 Knockout and Calcium Oxalate-Induced Renal Injury.

Background As a common urological disease with a high recurrence rate, nephrolithiasis caused by CaOx may elicit a strong immunologic response. We present a CyTOF-based atlas of the immune landscape in nephrolithiasis models to understand how the immune system contributes to, and is affected by, the underlying response caused by SIRT3 knockout and CaOx inducement. Materials and Methods We performed a large-scale CyTOF analysis of immune cell abundance profiles in nephrolithiasis. The immunophenotyping data were collected from four different mouse models, including the SIRT3 wild-type or knockout, including and excluding CaOx inducement. Unsupervised analysis strategies, such as SPADE and viSNE, revealed the intrarenal resident immune components and the immune alterations caused by SIRT3 knockout and CaOx-induced renal injury. Results An overview analysis of the immune landscape identified T cells and macrophages as the main immune cell population in nephrolithiasis models. Highly similar phenotypes were observed among CD4+ and CD8+ T cell subsets, including cells expressing Ki67, Ly6C, Siglec-F, and TCRβ. Macrophages expressed a characteristic panel of markers with varied expression levels including MHC II, SIRPα, CD11c, Siglec-F, F4/80, CD64, and CD11b, indicating more subtle differences in marker expression than T cells. The SIRT3KO/CaOx and SIRT3WT/CaOx groups exhibited global differences in the intrarenal immune landscape, whereas only small differences existed between the SIRT3KO/CaOx and SIRT3KO/Ctrl groups. Among the major immune lineages, the response of CD4+ T cells, NK cells, monocytes, and M1 to CaOx inducement was regulated by SIRT3 expression in contrast to the expression changes of B cells, DCs, and granulocytes caused by CaOx inducement. The panel of immune markers influenced by CaOx inducement significantly varied with and without SIRT3 knockout. Conclusion In a CaOx-induced nephrolithiasis model, SIRT3 has a critical role in regulating the immune system, especially in reducing inflammatory injury. The characteristic panel of altered immune clusters and markers provides novel insights leading to improved prediction and management of nephrolithiasis.

Characterizing the tumor microenvironment in rare renal cancer histological types.

The tumor microenvironment (TME), including immune cells, cancer‐associated fibroblasts, endothelial cells, adjacent normal cells, and others, plays a crucial role in influencing tumor behavior and progression. Here, we characterized the TME in 83 primary renal tumors and matched metastatic or recurrence tissue samples (n = 15) from papillary renal cell carcinoma (pRCC) types 1 (n = 20) and 2 (n = 49), collecting duct carcinomas (CDC; n = 14), and high‐grade urothelial carcinomas (HGUC; n = 5). We investigated 10 different markers of immune infiltration, vasculature, cell proliferation, and epithelial‐to‐mesenchymal transition by using machine learning image analysis in conjunction with immunohistochemistry. Marker expression was compared by Mann–Whitney and Kruskal–Wallis tests and correlations across markers using Spearman's rank correlation coefficient. Multivariable Poisson regression analysis was used to compare marker expression between histological types, while accounting for variation in tissue size. Several immune markers showed different rates of expression across histological types of renal carcinoma. Using pRCC1 as reference, the incidence rate ratio (IRR) of CD3+ T cells (IRR [95% confidence interval, CI] = 2.48 [1.53–4.01]) and CD20+ B cells (IRR [95% CI] = 4.38 [1.22–5.58]) was statistically significantly higher in CDC. In contrast, CD68+ macrophages predominated in pRCC1 (IRR [95% CI] = 2.35 [1.42–3.9]). Spatial analysis revealed CD3+ T‐cell and CD20+ B‐cell expressions in CDC to be higher at the proximal (p < 0.0001) and distal (p < 0.0001) tumor periphery than within the central tumor core. In contrast, expression of CD68+ macrophages in pRCC2 was higher in the tumor center compared to the proximal (p = 0.0451) tumor periphery and pRCC1 showed a distance‐dependent reduction, from the central tumor, in CD68+ macrophages with the lowest expression of CD68 marker at the distal tumor periphery (p = 0.004). This study provides novel insights into the TME of rare kidney cancer types, which are often understudied. Our findings of differences in marker expression and localization by histological subtype could have implications for tumor progression and response to immunotherapies or other targeted therapies.

Central human B cell tolerance manifests with a distinctive cell phenotype and is enforced via CXCR4 signaling in hu-mice

Central B cell tolerance, the process restricting the development of many newly generated autoreactive B cells, has been intensely investigated in mouse cells while studies in humans have been hampered by the inability to phenotypically distinguish autoreactive and nonautoreactive immature B cell clones and the difficulty in accessing fresh human bone marrow samples. Using a human immune system mouse model in which all human Igκ+ B cells undergo central tolerance, we discovered that human autoreactive immature B cells exhibit a distinctive phenotype that includes lower activation of ERK and differential expression of CD69, CD81, CXCR4, and other glycoproteins. Human B cells exhibiting these characteristics were observed in fresh human bone marrow tissue biopsy specimens, although differences in marker expression were smaller than in the humanized mouse model. Furthermore, the expression of these markers was slightly altered in autoreactive B cells of humanized mice engrafted with some human immune systems genetically predisposed to autoimmunity. Finally, by treating mice and human immune system mice with a pharmacologic antagonist, we show that signaling by CXCR4 is necessary to prevent both human and mouse autoreactive B cell clones from egressing the bone marrow, indicating that CXCR4 functionally contributes to central B cell tolerance.

Phenotypical Characterization and Neurogenic Differentiation of Rabbit Adipose Tissue-Derived Mesenchymal Stem Cells.

Although the rabbit is a frequently used biological model, the phenotype of rabbit adipose-derived mesenchymal stem cells (rAT-MSCs) is not well characterized. One of the reasons is the absence of specific anti-rabbit antibodies. The study aimed to characterize rAT-MSCs using flow cytometry and PCR methods, especially digital droplet PCR, which confirmed the expression of selected markers at the mRNA level. A combination of these methods validated the expression of MSCs markers (CD29, CD44, CD73, CD90 and CD105). In addition, cells were also positive for CD49f, vimentin, desmin, α-SMA, ALDH and also for the pluripotent markers: NANOG, OCT4 and SOX2. Moreover, the present study proved the ability of rAT-MSCs to differentiate into a neurogenic lineage based on the confirmed expression of neuronal markers ENO2 and MAP2. Obtained results suggest that rAT-MSCs have, despite the slight differences in marker expression, the similar phenotype as human AT-MSCs and possess the neurodifferentiation ability. Accordingly, rAT-MSCs should be subjected to further studies with potential application in veterinary medicine but also, in case of their cryopreservation, as a source of genetic information of endangered species stored in the gene bank.

Markers of steroid receptor, kinase signalling pathways and Ki-67 expression in relation to tamoxifen sensitivity and resistance

Background: It remains clinically important to identify ER positive breast cancers likely to respond to tamoxifen (TAM) and so we aimed to select a group of biomarkers able to predict response. We also assessed whether data from different sample types [tumor microarrays (TMAs) and core biopsies] or tumor sites could be combined for biomarker studies. Methods: A total of 123 endocrine treatment naive patients with known ER and HER2 status treated with TAM had paraffin-embedded tumor tissue available either as TMAs (n=102) or core biopsies (n=21). TMA cores were collected from three different tumor sites, two central and one peripheral. Ten biomarkers were evaluated by immunohistochemistry, for % positivity and/or H-Score, comprising: ER, HER2, Ki67, phosphorylated forms of ER (Ser118), IGF1R, PRAS40, Akt & MAPK (ERK1/2), and PTEN & androgen receptor expression (AR). Each tumor was analysed for Akt1 E17K somatic mutation using BEAMing technology. Patient outcome was assessed by clinical benefit (CB) rate & survival analyses [time to progression (TTP) and time to death (TTD)]. Results: There was no significant difference in % positivity or H-Score between central & peripheral tumor sites for all biomarkers examined. After False Discovery Rate (FDR) correction differences (P<0.05) were observed between the two central samples only for HER2 & pER118 and pPRAS40. However, differences in biomarker expression were common between core biopsies and TMAs. Only 2/123 (1.6%) tumors had Akt1 E17K mutations. Univariate and multivariate analyses identified that lower levels of PTEN and higher levels of Ki-67 (% positivity) were predictive of poor outcome (TTP & TTD) following TAM. Higher ER. lower Ki-67 and AR/ER ratio <2 predicted increased CB rate. Conclusions: There were few differences in marker expression between TMAs from different intratumoral sites. More marked differences between TMAs and core biopsies suggest caution if combining such datasets. Loss of PTEN, a key regulator of the PI3K/Akt pathway, was the only RTK/kinase signaling biomarker related to poorer clinical outcome. PTEN along with ER & lower Ki-67 proved the most predictive markers for better outcome (TTP & TTD and/or CBR) following TAM treatment. Keywords: ER+ breast cancer; Akt pathway; tamoxifen

Use of dual-marker staining to differentiate between lung squamous cell carcinoma and adenocarcinoma

ObjectiveTo assess the value of dual-marker immunostaining for detecting p40 and napsin A, and cytokeratin 5/6 (CK5/6) and thyroid transcription factor 1 (TTF1) in single sections of lung cancer tissue, for differentiating between lung squamous cell carcinoma and adenocarcinoma.MethodsLung cancer tissue sections from 58 patients were stained by dual-marker immunostaining using a mixtures of anti-p40 and anti-napsin A, and anti-CK5/6 and anti-TTF1 primary antibodies. Sections stained with single markers were used as controls. Nuclear or cytoplasmic staining was considered as indicating positive p40 or napsin A expression, respectively, and cytoplasmic or nuclear staining was considered as indicating positive CK5/6 or TTF1 expression, respectively.Resultsp40/napsin A and CK5/6/TTF1 dual-marker staining showed high sensitivity, specificity, positive predictive value, and negative predictive value for the diagnosis of squamous cell carcinoma and adenocarcinoma respectively. There were no differences in marker expression between dual-marker and single-marker staining.ConclusionsDual-marker immunostaining is a relatively easy, time- and cost-conserving staining method for detecting two markers in a single section using one procedure and one chromogen. p40 and napsin A, and CK5/6 and TTF1 dual-marker staining were suitable for the differential diagnosis of lung squamous cell carcinoma and adenocarcinoma.

Establishment and characterization of proliferating primary cultures of equine epidermal keratinocytes

Skin-derived tissue cultures are a useful model to study molecular mechanisms of skin renewal and pathogenesis of dermal diseases. Horses often suffer from skin diseases, skin trauma and problems with proper wound healing, which could be improved by in vitro grown keratinocyte grafts. Herein we describe establishment and characterization of equine skin-derived primary cell cultures, using enzymatic and explant methods. The established cell lines of primary equine keratinocytes (peK) maintained high proliferative capacity for over five passages and expressed different epithelial/keratinocyte-specific markers. Characterization of the primary culture was performed in parallel with localization studies of the markers in the skin histological sections, using commercially available antibodies. Relative expression of typical differentiation stage-specific markers was determined in the established cell lines, using RT-qPCR. Basal (proliferating) keratinocytes were the predominant cell type in the established cell lines, but low expression of post-mitotic keratinocyte markers was also detected. Differences in marker expression were observed neither between the peK originating from two different animals nor between the peK established with two different methods (enzymatically or by explanting). The described methods in combination with the suggested characterization and differentiation markers are suitable for establishment of proliferating peK and evaluation of their differentiation status.

FGF8, FGF10 and FGF receptor 2 in foreskin of children with hypospadias: an analysis of immunohistochemical expression patterns and gene transcription

Fibroblast growth factors (FGFs) play a crucial role in early embryogenesis of the genital tubercle and are involved in the development of hypospadias, affecting both endo- and ectodermally derived tissues. It was hypothesized that expression of FGFs could be qualitatively or quantitatively altered in skin of children with hypospadias. The objective of the study was to investigate expression patterns and transcription levels of FGF8, FGF10, and FGF Receptor 2 (FGFR2) in patients with hypospadias compared to normal controls. Skin samples from the ventro-lateral aspect of the foreskin of 32 patients with hypospadias (17 distal and 15 proximal, mean age 25 months) and 10 normal foreskin samples (mean age 77 months) were analyzed by immunohistochemistry. Staining, localization, and distribution of positive cells in epidermis and dermis were categorized independently by two researchers. Complementary DNA (cDNA) samples prepared from messenger RNA (mRNA) isolates of the same samples were analyzed by quantitative polymerase chain reaction (qPCR), comparing expressions of FGF8, FGF10, and FGFR2 with loading controls. Patients with hypospadias consistently showed aberrant immunohistochemical staining patterns for FGF8/FGF10/FGFR2 in epidermis and dermis compared to patients without penile malformation (p<0.01 for all markers). qPCR displayed no difference in expression levels on mRNA level (FGFR2 p=0.44, FGF8 p=0.77, and FGF10 p=0.17) comparing normal foreskin with foreskin from patients with hypospadias. Figure. Theresults point at an impact of FGF signaling during embryological development of hypospadias on skin, as an ectodermally derived tissue. Similar to the urethral development, this might be a result of mesothelial-epithelial interactions. The differing expression patterns in immunohistochemistry are not matched by a quantitative difference in marker expression on the mRNA level, putatively caused by post-translational modifications or alterations of the downstream pathway. FGFs, particularly FGF10 and FGFR2, are critically involved in wound healing. There are significant differences in localization and distribution of FGF8, FGF10, and FGFR2 in comparisons of normal foreskin to foreskin of patients with hypospadias, whereas there is no difference in the quantitative expression of these markers on the mRNA level. This confirms the notion that penile skin is affected as well by the embryological aberrations during the embryogenesis of hypospadias.

Distinct release strategies are required to modulate macrophage phenotype in young versus aged animals

The role of innate immunity and macrophages in the host response to biomaterials has received renewed attention. A context-dependent spectrum of macrophage phenotypes are shown to affect tissue integration and performance of implanted biomaterials and medical devices. Recent studies by our group demonstrated that the host response in aged animals was characterized by delayed macrophage recruitment, differences in marker expression and a shifted pro-inflammatory (M1) response, associated with an unresolved host response in the long-term. The present work sought to study the effects of single and sequential cytokine delivery regimens in aged mice to restore delayed recruitment of macrophages and shift the inflammatory host response towards an M2-like phenotype, using MCP-1 (macrophage chemotactic protein-1) and IL-4 (interleukin-4), respectively. Implantation of cytokine-eluting implants showed a preserved response to MCP-1 in both young and aged animals, restoring delayed macrophage recruitment in aged mice. However, the response elicited by IL-4, sequential delivery of MCP-1/IL-4 and coating components was distinct in young versus aged mice. While single delivery of IL-4 did not counteract the high inflammatory response observed in aged mice, the sequential delivery of MCP-1/IL-4 was capable of restoring both recruitment and shifting the macrophage response towards an M2-like phenotype, associated with decreased implant scarring in the long-term. In young mice, sequential delivery was not as effective as IL-4 alone at promoting an M2-like response, but did result in a reduction of M1 macrophages and capsule deposition downstream. These results demonstrate that a proper understanding of patient/context-dependent biological responses are needed to design biomaterial-based therapies with improved outcomes in the setting of aging.

Fibronectin-conjugated thermoresponsive nanobridges generate three dimensional human pluripotent stem cell cultures for differentiation towards the neural lineages

Production of 3-dimensional neural progenitor cultures from human pluripotent stem cells offers the potential to generate large numbers of cells. We utilised our nanobridge system to generate 3D hPSC aggregates for differentiation towards the neural lineage, and investigate the ability to passage aggregates while maintaining cells at a stem/progenitor stage. Over 38 days, aggregate cultures exhibited upregulation and maintenance of neural-associated markers and demonstrated up to 10 fold increase in cell number. Aggregates undergoing neural induction in the presence or absence of nanobridges demonstrated no differences in marker expression, proliferation or viability. However, aggregates formed without nanobridges were statistically significantly fewer and smaller by passage 3. Organoids, cultured from aggregates, and treated with retinoic acid or rock inhibitor demonstrated terminal differentiation as assessed by immunohistochemistry. These data demonstrate that nanobridge 3D hPSC can differentiate to neural stem/progenitor cells, and be maintained at this stage through serial passaging and expansion.

Abstract P5-12-07: Expression of adipocyte fatty acid binding protein promotes obesity-associated mammary tumor growth

Abstract Background: The underlying mechanisms that drive obesity-related breast cancer remain unclear. Adipocyte/macrophage fatty acid binding protein (A-FABP) is linked to obesity and breast cancer, while transforming growth factor (TGF)β appears to play a pleiotropic role in breast cancer, suppressing its development but promoting its progression. Whether the proteins work together to drive breast cancer progression is not known. We evaluated the expression of these two markers in matched serum from healthy women and women with breast cancer. Hypothesis: A-FABP and TGFβ drive breast cancer development and progression. Methods: Serum was collected under an institutional review board approved protocol. A-FABP was measured in serum collected from 275 women (92 with breast cancer and 183 without) and TGFβ from 245 matched women (92 with breast cancer and 153 without). A-FABP levels were measured using a human A-FABP4 ELISA kit while TGFβ was measured using human TGFβ ELISA kit. The Kruskal-Wallis test was used to determine if a difference in marker expression existed between women with and without breast cancer, as well as in women with early vs. more advanced breast cancer. Linear mixed effect models were used to examine the relationship between A-FABP and BMI, as well as between TGFβ and BMI, controlling for age, menopause status and a diagnosis of breast cancer. Results:A-FABP expression was higher in obese than lean postmenopausal women, both those with breast cancer (mean : 44.9 vs. 25.1 ng/mL, p=0.002) and without breast cancer (39.4 vs. 26.9 ng/mL, p=0.003). A-FABP expression was also higher in premenopausal obese vs. lean women with cancer (28.9 vs. 12.7 ng/mL, p=0.027), but not in premenopausal healthy women. A-FABP expression was higher in postmenopausal obese vs. lean women with early stage (0-2A) breast cancer (45.6 vs. 21.9 ng/mL, p=0.013) and was inversely associated with HER-2 expression, though being of borderline significance (p=0.060). This was most notable among triple negative vs. ER/PR negative HER2 positive breast cancers. Considering both early and advanced breast cancer, TGFβ expression trended higher in post- than in pre-menopausal obese women with breast cancer (138.9 vs. 68.7 pg/mL, p=0.061), however among premenopausal women with advanced (Stages 2B-3C) disease, TGFβ expression was 5 fold higher in lean than obese individuals (251.7 vs. 48.2 pg/mL, p=0.029) but trended higher in obese vs lean postmenopausal women. TGFβ and A-FABP were found to be significantly associated (ρ=0.14, p=0.024). Impact: Both A-FABP and TGFβ expression are associated with postmenopausal breast cancer among obese women, and their expression in matched samples is significantly associated.. TGFβ is associated with premenopausal advanced breast cancer in premenopausal women. TGFβ is known to induce epithelial mesenchymal transition and may play a role in pregnancy associated breast cancer. Further studies are needed to determine if A-FABP and TGFβ work together in postmenopausal breast cancer. Citation Format: Sauter E, Hao J, Yan X, Kong M, Li B. Expression of adipocyte fatty acid binding protein promotes obesity-associated mammary tumor growth [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-07.

Cell death in the human infant central nervous system and in sudden infant death syndrome (SIDS).

The brainstem has been a focus of sudden infant death syndrome (SIDS) research with amassing evidence of increased neuronal apoptosis. The present study extends the scope of brain regions examined and determines associations with known SIDS risk factors. Immunohistochemical expression of cell death markers, active caspase-3 and TUNEL, was studied in 37 defined brain regions in infants (aged 1-12 months) who died suddenly and unexpectedly (SUDI). A semi-quantitative mean score of marker expression was derived for each region and scores compared between three SUDI subgroups: explained SUDI (eSUDI; n = 7), SIDS I (n = 8) and SIDS II (n = 13). In eSUDI, active caspase-3 scores were highest in several nuclei of the rostral medulla, and lowest in the hypothalamus and cerebellar grey matter (GM). TUNEL was highest in regions of the hippocampus and basal ganglia, and lowest in the thalamus and cerebellar GM. TUNEL scores were higher in SIDS II compared to eSUDI in the amygdala (p = 0.03) and 5/9 nuclei in the rostral medulla (p = 0.04 - 0.01), and higher in SIDS II compared to SIDS I in the amygdala (p < 0.01), putamen (p = 0.01), lentiform nucleus (p = 0.03) and parietal (p = 0.03) and posterior frontal (p = 0.02) cortex. Active caspase-3 was greater in the hypoglossal nucleus (p = 0.03) of SIDS I compared to eSUDI infants. Co-sleeping, cigarette smoke exposure and the presence of an upper respiratory tract infection in SIDS infants was associated with differences in marker expression. This study affirms the sensitivity of the brainstem medulla to cell death in SIDS, and highlights the amygdala as a new region of interest.

Skeletal Muscle Gene Expression Study of Monozygous Twins with 35 Years of Divergent Exercise History

Variations in physical ability and capacities among individuals depend on both genetic inheritance and lifestyle. Previous research has yet to utilize a human model of monozygous (MZ) twins with substantial and long-term differences in exercise backgrounds to identify molecular and cellular mechanisms underlying exercise-mediated adaptations in muscle and whole-body phenotype. PURPOSE: In particular, this study examined skeletal muscle expression of genes related to muscle growth, inflammation, metabolism, and fiber type distribution in MZ twins with 35 years of substantially differing exercise history. METHODS: Muscle biopsies were collected from the vastus lateralis of male, 52-year-old MZ twins. Reverse transcriptase-polymerase chain reaction was used for the quantification of mRNAs associated with the following gene markers of various adaptive responses: a) skeletal muscle fiber type shift: MyHC-1, MyHC-2a, and MyHC-2x; b) adaptations in oxidative capacity: transcription factor A of the mitochondria (TFAM), and citrate synthase; c) angiogenesis: endothelial nitric oxide synthase, and vascular endothelial growth factor; d) muscular growth and satellite cell activation: myostatin, Pax7 (PAX7), mechano-growth factor (IGF1Ec), insulin-like growth factor a (IGF1Ea), and MyoD; and e) the inflammatory response: TWEAK (TNFSF12), the FN14 TWEAK receptor (TNFRSF12A) and tumor necrosis factor-α (TNF). RESULTS: MyHC-2x was expressed at a lower level in the trained subject relative to the untrained subject. No differences were observed for other markers of fiber type or metabolic or angiogenesis gene products. Some differences were observed in the expression of genes related to muscle growth including elevations in PAX7, MGF and IGF1Ea, and a reduction in MyoD. Finally, the only difference in expression of markers for the inflammatory response was an elevation in TNFRSF12A in the trained twin. CONCLUSIONS: At the mRNA-level, differences in expression of some key markers related to muscle fiber type, muscle growth, and the inflammatory response were observed in the trained vs the untrained twin. These data highlight the adaptability of skeletal muscle at the molecular level with decades of divergent physical activity patterns.

Changes in expression of special AT-rich sequence binding protein 1 and phosphatase and tensin homologue in kidneys of diabetic rats during ageing.

Background Diabetic nephropathy (DN) is a common complication of diabetes mellitus (DM). We studied the expression of special AT-rich sequence binding protein 1 (SATB1) and phosphatase and tensin homologue (PTEN) in the kidneys of diabetic rats during ageing. Methods Male Sprague Dawley rats were injected with 55 mg/kg streptozotocin (STZ) (DM group) or with citrate buffer (control group). Kidneys were collected after 2 weeks, 6 months and 12 months, and were analysed in three different kidney structures: glomeruli, proximal (PCT) and distal convoluted tubules (DCT). Sections were stained immunohistochemically, using SATB1 and PTEN. Results Significant differences in marker expression were observed after 2 weeks, with higher SATB1 expression and lower PTEN expression in diabetic rats. PTEN was more highly expressed in controls after 6 and 12 months. After 12 months, there was higher SATB1 expression in diabetic rats. In the glomeruli, control rats had higher PTEN expression, whereas diabetic rats had higher SATB1 expression, after 12 months. PTEN expression increased from 2 weeks to 12 months in both the PCT and DCT of control rats. SATB1 was expressed exclusively in the PCT of diabetic rats after 2 weeks, and its expression in the DCT was higher in controls. After 6 months, both the PCT and DCT showed higher SATB1 expression in diabetic rats. Conclusions The major changes in expression of SATB1 and PTEN occur after 2 weeks of DM onset, particularly in the PCT, implying an early onset of pathophysiological changes in diabetic kidneys, which would normally occur with ageing. These findings help to contribute to our understanding of changes associated with DN and guide towards possible appropriate treatment modalities.