Quantitative Flow Cytometric Analysis Research Articles

Pioglitazone alleviates lacrimal gland impairments induced by high-fat diet by suppressing M1 polarization

A high-fat diet (HFD) contributes to the pathogenesis of various inflammatory and metabolic diseases. Previous research confirms that under HFD conditions, the extraorbital lacrimal glands (ELGs) can be impaired, with significant infiltration of pro-inflammatory macrophages (Mps). However, the relationship between HFD and Mps polarization in the ELGs remains unexplored. We first identified and validated the differential expression of PPAR-γ in murine ELGs fed ND and HFD through RNA sequencing. Tear secretion was measured using the Schirmer test. Lipid droplet deposition within the ELGs was observed through Oil Red O staining and transmission electron microscopy. Mps phenotypes were determined through quantitative RT-PCR, immunofluorescence, and flow cytometric analysis. An in vitro high-fat culture system for Mps was established using palmitic acid (PA), with supernatants collected for co-culture with lacrimal gland acinar cells. Gene expression was determined through ELISA, immunofluorescence, immunohistochemistry, quantitative RT-PCR, and Western blot analysis. Pioglitazone reduced M1-predominant infiltration induced by HFD by increasing PPAR-γ levels in ELGs, thereby alleviating lipid deposition and enhancing tear secretion. In vitro tests indicated that PPAR-γ agonist shifted Mps from M1-predominant to M2-predominant phenotype in PA-induced Mps, reducing lipid synthesis in LGACs and promoting lipid catabolism, thus alleviating lipid metabolic disorders within ELGs. Conversely, the PPAR-γ antagonist induced opposite effects. In summary, the lacrimal gland is highly sensitive to high-fat and lipid metabolic disorders. Downregulation of PPAR-γ expression in ELGs induces Mps polarization toward predominantly M1 phenotype, leading to lipid metabolic disorder and inflammatory responses via the NF-κb/ERK/JNK/P38 pathway.

Abstract 4559: SMAD3 mediates oxaliplatin resistance in esophageal adenocarcinoma

Abstract Background: Resistance to conventional treatments is a significant barrier to esophageal adenocarcinoma (EAC) therapy. TGF-β signaling appears to be important for EAC tumorigenesis; however, its role in EAC chemoresistance remains understudied. This study investigates the role of SMAD3 in EAC resistance to the frontline chemotherapeutic drug oxaliplatin. Methods: Publicly available datasets were analyzed to determine SMAD3 expression in EAC versus normal samples. Western blot, quantitative real-time polymerase chain reaction, immunofluorescence staining, and flow cytometric analyses were utilized to validate bioinformatic results. Human EAC cell lines (OE33 and OE19) were treated with pharmacologic inhibitors or transfected with small interfering RNA and assessed for DNA damage and apoptotic cell death. Patient-derived organoids and patient-derived xenograft models of EAC were used to confirm our findings in vivo. Results: SMAD3 was significantly upregulated in EAC versus normal tissue samples. Total and phosphorylated SMAD3 protein levels were upregulated in EAC tissues accompanied by increased expression of several genes regulated by SMAD3. Notably, oxaliplatin-resistant EAC cells exhibited overexpression of SMAD3. Chemotherapy non-responding patients showed enrichment of SMAD3 gene expression when compared to responders in the clinical dataset GSE165252. Knockdown or inhibition of SMAD3 hindered DNA repair and sensitized EAC cells to oxaliplatin treatment in vitro and in vivo. Mechanistically, SMAD3 promoted ATM phosphorylation by interacting with protein phosphatase 2A (PP2A) and sequentially inhibiting the binding of PP2A to ATM. Conclusion: Our results reveal a pivotal role for SMAD3 in mediating oxaliplatin resistance in EAC and thus hold a significant potential in guiding future drug development and combination strategies. Citation Format: Farah Ballout, Heng Lu, Nadeem Bhat, Lei Chen, Dunfa Peng, Zheng Chen, Alexander Zaika, Oliver McDonald, Wael El-Rifai. SMAD3 mediates oxaliplatin resistance in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4559.

Panobinostat enhances NK cell cytotoxicity in soft tissue sarcoma.

Sarcoma is a rare and heterogeneous class of mesenchymal malignancies with poor prognosis. Panobinostat (LBH589) as one of histone deacetylase (HDAC) inhibitors has demonstrated anti-tumor activity in patients with sarcoma, but its mechanisms remains unclear. Here, we found that LBH589 alone inhibited the proliferation and colony formation of soft tissue sarcoma (STS) cell lines. Transcriptome analysis showed that treatment with LBH589 augmented the NK cell-mediated cytotoxicity. Quantitative real-time PCR and flow cytometric analysis (FACS) further confirmed that LBH589 increased the expression of NKG2D ligands MICA/MICB. Mechanistically, LBH589 activated the Wnt/β-catenin pathway by upregulating the histone acetylation in β-catenin promoter. In vitro co-culture experiments and in vivo animal experiments showed that LBH589 increased the cytotoxicity of natural killer (NK) cells while Wnt/β-catenin inhibitor decreased the effects. Our findings suggest that LBH589 facilitates the anti-tumor effect of NK cells, highlights LBH589 an effective assistance drug in NK cell-based immunotherapies.

Efficacy of Different Immunological Approaches Targeting CD22 for the Treatment of Relapsed or Refractory Acute Lymphoblastic Leukemia: A Research Protocol

Introduction: Monoclonal antibodies (mAbs) have emerged as a promising immune-oncological approach to target cancer cells. mAbs have been seen to outperform traditional drug treatments in treating severe cancers despite their low relative cytotoxicity due to their high selectivity. CD22 is expressed in 60-90% of individuals with B-cell Acute Lymphoblastic Leukemia (B-ALL), and is rapidly internalized when bound to an antibody, making it an effective point of entry for cytotoxic agents. Epratuzumab is an anti-CD22 mAb, effective against B-ALL. Epratuzumab-SN-38 (Emab-SN-38) and Inotuzumab ozogamicin (InO) are promising anti-CD22 Antibody-Drug Conjugates (ADCs). Methods: Epratuzumab, Inotuzumab, and Emab-SN38 treatments will be evaluated in vitro and in vivo. B lymphocytes collected from a 30-35-year-old R/R ALL patient will be purified and expanded. A cell culture assay will evaluate the treatments. Cells will be engrafted into humanized mice. Mice will be assorted into four treatment groups: saline (control), Epratuzumab, Inotuzumab, and Emab-SN-38. Quantitative flow cytometric analysis will be used to assess treatment effectiveness. Complete Response will be determined as ≅ zero human leukemic cells, Partial Response as ≤5% cells, and Remission as >5% cells or with identifiable clinical signs. Mice will be followed for 6 months after the last dose of treatment to assess for relapse and survival rate. Results: It is expected that all three treatments will result in more significant results regarding tumour shrinkage and rate of cancer growth than saline. The ADCs are expected to perform better than unconjugated Epratuzumab. Relapse and Adverse Event rates are expected to be lowest in Epratuzumab-SN-38. Discussion: The comparison of the effectiveness of these treatments are expected to establish Emab-SN-38 as a potential treatment option and propel research into other cytotoxic agents which could be used in conjugation with Epratuzumab and other mAbs. Conclusion: ADCs combine the cytotoxicity of chemotherapy and the specificity of mAbs to treat R/R ALL. The ADCs are expected to outperform Epratuzumab in decreasing leukemic cell load given their potent targeted cytotoxicity. Emab-SN-38 is expected to be less toxic but as effective as Inotuzumab. These results could inform research on safer and more potent ADCs in treating R/R ALL via CD22.

A Novel Function of Mitochondrial Phosphoenolpyruvate Carboxykinase as a Regulator of Inflammatory Response in Kupffer Cells.

Background: There has been a recent appreciation that some metabolic enzymes can profoundly influence the nature of the immune response produced in macrophages. However, the role of mitochondrial phosphoenolpyruvate carboxykinase (PCK2) in immune response remains unknown. This study aims to investigate the role of PCK2 in lipopolysaccharides (LPS)-induced activation in Kupffer cells. Methods: Inflammatory cytokines were determined by real-time quantitative reverse transcription-polymerase chain action (qRT-PCR) and flow cytometric analysis using a cytometric bead array. Western blotting and immunofluorescence staining were used to determine PCK2 expression and subcellular distribution under confocal laser microscopy. qRT-PCR, flow cytometry, and high-performance liquid chromatography (HPLC) were used to determine mitochondrial function. Pharmacological inhibition, knockdown, and overexpression of PCK2 were used to confirm its function. Co-immunoprecipitation (Co-IP) was performed to determine MAPK/NF-κB phosphorylation. Results: Inflammatory response was significantly increased in LPS-treated mice and Kupffer cells. During the inflammatory process, the protein level of PCK2 was significantly upregulated in Kupffer cells. Interestingly, the localization of PCK2 was mainly in cytosol rather than mitochondria after LPS stimulation. Gain-of-function and loss-of-function analyses found that PCK2 overexpression significantly upregulated the levels of inflammation markers, whereas PCK2 knockdown or inhibition significantly mitigated LPS-induced inflammatory response in Kupffer cells. Furthermore, PCK2 promoted protein phosphorylation of NF-κB and AKT/MAPK, the major signaling pathways, controlling inflammatory cascade activation. Conclusion: We identified a novel function of PCK2 in mediating LPS-induced inflammation and provided mechanistic insights into the regulation of inflammatory response in Kupffer cells. Therefore, PCK2 may serve as a novel therapeutic target for the regulation of Kupffer cells-mediated inflammatory responses.

Decellularized adipose tissue scaffolds guide hematopoietic differentiation and stimulate vascular regeneration in a hindlimb ischemia model

Cellular therapies to stimulate therapeutic angiogenesis in individuals with critical limb ischemia (CLI) remain under intense investigation. In this context, the efficacy of cell therapy is dependent on the survival, biodistribution, and pro-angiogenic paracrine signaling of the cells transplanted. Hematopoietic progenitor cells (HPC) purified from human umbilical cord blood using high aldehyde dehydrogenase-activity (ALDHhi cells) and expanded ex vivo, represent a heterogeneous mixture of progenitor cells previously shown to support limb revascularization in mouse models of CLI. The objectives of this study were to investigate the utility of bioscaffolds derived from human decellularized adipose tissue (DAT) to guide the differentiation of seeded HPC in vitro and harness the pro-angiogenic capacity of HPC at the site of ischemia after implantation in vivo. Probing whether the DAT scaffolds altered HPC differentiation, label-free quantitative mass spectrometry and flow cytometric phenotype analyses indicated that culturing the HPC on the DAT scaffolds supported their differentiation towards the pro-angiogenic monocyte/macrophage lineage at the expense of megakaryopoiesis. Moreover, implantation of HPC in DAT scaffolds within a unilateral hindlimb ischemia model in NOD/SCID mice increased cell retention at the site of ischemia relative to intramuscular injection, and accelerated the recovery of limb perfusion, improved functional limb use and augmented CD31+ capillary density when compared to DAT implantation alone or saline-injected controls. Collectively, these data indicate that cell-instructive DAT scaffolds can direct therapeutic HPC differentiation towards the monocyte/macrophage lineage and represent a promising delivery platform for improving the efficacy of cell therapies for CLI.

Interleukin-dependent modulation of the expression of MHC class I and MHC class II genes in chicken HD11 cells

Interleukins (ILs) regulate cell surface antigens known as activation markers, which have distinct functional roles. However, the regulation of major histocompatibility complex (MHC) class I, MHC class II, and related genes by cytokines in chickens is not well understood. In the present study, we evaluated the influence of certain recently discovered chicken interleukins—i.e., IL-11, IL-12B, IL-17A, IL-17B, IL-26, and IL-34—on the expression and regulation of genes related to MHC class I, MHC class II, and the associated proteins in an HD11 chicken macrophage cell line. We used quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunocytochemical, and flow cytometric analyses to assess dose- and time-dependent expression in the HD11 cell line and found that the ILs induced MHC class I, MHC class II, and associated protein. As NF-κB is actively involved in cell activation and is constitutively activated in many immune cells, we also determined whether NF-κB regulates MHC class I, MHC class II, and related gene expression in the HD11 cell line. The NF-κB inhibitor sulfasalazine (Sz) dose-dependently inhibited MHC class I and MHC class II in the HD11 cell line. Sz also downregulated the expression of MHC class I, MHC class II, and the associated proteins in the IL-induced HD11 cell line. The expression of MHC class I, MHC class II, and associated genes was accompanied by the Sz-sensitive degradation of the p65 (RelA) and p50 subunits of NF-κB and IκBα. Our results indicate that the different effects of each IL on the expression of genes related to MHC class I, MHC class II, and the associated proteins are involved with the regulation of the dose and duration of antigenic peptide presentation and, thus, also influence Th1, Th2, and Th17 production.

Downregulation of PD-L1 via amide analogues of brefelamide: Alternatives to antibody-based cancer immunotherapy.

The therapeutic blockade of immune checkpoint has emerged as an effective treatment option for a broad range of tumors. However, the objective tumor response is still limited to a small number of cases and tumor types. The full utility of monoclonal antibody (mAb)-based treatment is hindered by several inherent limitations. Thus, there is an urgent requirement to explore alternative modalities targeting the same pathways. In the present study, two amide analogues of brefelamide, TPFS-201 and TPFS-202, were identified as small molecular immune checkpoint inhibitors, as they downregulated PD-L1 expression in tumor cells. PD-L1 was suppressed in cancer cells treated with TPFD compounds at both mRNA and protein levels, as detected by reverse transcription quantitative PCR and flow cytometric analysis, respectively. Reporter assays using a PD-L1 promoter luciferase construct confirmed the transcriptional inhibition of PD-L1 by TPFS compunds. TPFS compound-mediated PD-L1 downregulation in cancer cells consequently restored T cell activity, as identified by the reduction of apoptosis and an increase in interleukin-2 promoter activity in Jurkat T cells, which were co-cultured with TPFS compound-treated A549 cells. TPFS compound-mediated PD-L1 inhibition was partially abolished by the disruption of the putative transcriptional co-activator with PDZ (TAZ)/TEA domain (TEAD)-binding motif in the PD-L1 promoter. The inhibitory effect of TPFS compounds on PD-L1 was markedly inhibited in mouse cell lines, which is consistent with previous research demonstrating that PD-L1 regulation by TAZ is not conserved in mice due to distinct promoter sequences flanking the TAZ/TEAD-binding motif. Together, the data of the current study indicated the potential utility of the brefelamide amide analogues as small molecule immune checkpoint inhibitors, thereby providing therapeutic alternatives, which could be used as monotherapy or in combination with mAbs-based treatment.

Quercetin promotes the survival of granulocytic myeloid-derived suppressor cells via the ESR2/STAT3 signaling pathway

Quercetin is a natural product that has been shown to induce tumor apoptosis and necrosis through multiple mechanisms. Tumor-induced myeloid-derived suppressor cell (MDSC) expansion negatively regulates the immune response by inhibiting T cell function through signal transducer and activator of transcription 3 (STAT3) activation, thereby facilitating tumor escape from host immune surveillance. Thus MDSC is an attractive target for cancer immunotherapy to enhance cytotoxic T cell responses. However, the effects of quercetin on MDSC are poorly understood. Here, we demonstrate that quercetin treatment enhanced mouse- and human- derived granulocytic-myeloid-derived suppressor cells (G-MDSC) survival and promoted the secretion of T cell-suppressive factors in vitro. Bioinformatics analysis further showed that quercetin was highly correlated with the estrogen receptor signaling pathway, which was confirmed by quantitative reverse transcription-polymerase chain reaction and flow cytometric analysis. These findings highlight the potential advantages and feasibility of quercetin in reinforcing the suppressive property of G-MDSC. Thus impact of G-MDSC should be taken into consideration when quercetin is applied to tumor therapy.

Inhibition of TIM-4 protects against cerebral ischaemia-reperfusion injury.

TIM‐4 plays an important role in ischaemia‐reperfusion injury of liver and kidney; however, the effects of TIM‐4 on cerebral ischaemia‐reperfusion injury (IRI) are unknown. The purpose of the present study was to investigate the potential role of TIM‐4 in experimental brain ischaemia‐reperfusion injury. In this study, cerebral ischaemia reperfusion was induced by transient middle cerebral artery occlusion (MCAO) for 1 hour in C57/BL6 mice. The TIM‐4 expression was detected in vivo or vitro by real‐time quantitative polymerase chain reaction, Western blot and flow cytometric analysis. In vivo, the administration of anti‐TIM‐4 antibodies significantly suppressed apoptosis, inhibited inflammatory cells and enhanced anti‐inflammatory responses. In vitro, activated microglia exhibited reduced cellular proliferation and induced IRI injury when co‐cultured with neurons; these effects were inhibited by anti‐TIM‐4 antibody treatment. Similarly, microglia transfected with TIM‐4 siRNA and stimulated by LPS + IFN‐γ alleviated the TIM‐4‐mediated damage to neurons. Collectively, our data indicate that the inhibition of TIM‐4 can improve the inflammatory response and exerts a protective effect in cerebral ischaemia‐reperfusion injury.

Skeletal unloading reduces cluster of differentiation (CD) 38 expression in the bone marrow and osteoblasts of mice

BackgroundMechanical unloading induces bone loss in human weight-loaded bones. The findings of recent studies have revealed that cluster of differentiation 38 knockout mice display bone loss similar to that observed in osteoporosis. This study aimed to determine whether the expression of cluster of differentiation 38 is implicated in skeletal unloading and reloading. MethodsEight-week-old male C57BL/6J mice were assigned to control, tail-suspension, or reloading after tail-suspension groups. In the tail-suspension group, tail suspension elevated the hind limbs for 1 week. The bilateral femurs and tibias from the groups were evaluated for cluster of differentiation 38 immunocytochemistry, and the cluster of differentiation 38 messenger ribonucleic acid levels and the expression of cluster of differentiation 38 and other cell-surface antigens were evaluated using quantitative real-time polymerase chain reaction and flow cytometric analyses. ResultsIn the tail-suspension group, the alkaline phosphatase reactivity, cluster of differentiation 38 immunoreactivity in the bone marrow and osteoblasts, and the expression of cluster of differentiation 38 messenger ribonucleic acid and that of other cell-surface antigens were significantly lower than those in the control group. In the reloading after tail-suspension group, the level of cluster of differentiation 38 expression was restored to the same level as that in the control group. ConclusionsCluster of differentiation 38 expression declined after skeletal unloading and recovered to normal levels after reloading. In the bone marrow, cluster of differentiation 38 expression plays a crucial role in bone formation in response to mechanical stress.

Relationship Between EGFR Expression in Oral Cancer Cell Lines and Cetuximab Antibody-dependent Cell-mediated Cytotoxicity.

Cetuximab treatment targets the epidermal growth factor receptor (EGFR) overexpressed in oral cancer. This study aimed to investigate the anti-tumour activity of cetuximab against oral cancer cell lines with respect to antibody-dependent cell-mediated cytotoxicity (ADCC), and determine the correlation between ADCC and EGFR expression. EGFR expression in oral cancer cells was measured by quantitative flow cytometric analysis and immunohistochemistry. ADCC activity was measured by 4-h calcein release assays. Cetuximab-mediated ADCC against oral cancer cells was detectable at a concentration of 0.1 μg/ml. A high correlation was observed between the number of EGFR molecules on the surface of oral cancer cells and ADCC (correlation coefficient: 0.847; p=0.032). ADCC is an important mechanism underlying the therapeutic effect of cetuximab, and EGFR expression in tumour cells might serve as a predictive marker to evaluate the effect of cetuximab treatment.

A Long-Acting Pharmacological Grade Interleukin-7 Molecule Logarithmically Accelerates Ucart Proliferation, Differentiation, and Tumor Killing

Background: Chimeric antigen receptor T cell (CART) therapy is revolutionizing modern cancer therapy, with two anti-CD19 CARTs FDA-approved for relapsed/refractory B cell lymphoma/leukemia and many other CARTs for solid and liquid tumors currently undergoing clinical trials. Our group recently demonstrated multiplexed CRISPR/Cas9 gene-editing of anti-CD7 CARTs to produce CD7 and T cell receptor alpha constant (TRAC)-deleted “off-the-shelf” universal (U)CART7s that effectively kill CD7+ T cell lymphoma in vivo without causing GVHD or fratricide (Cooper et al, Leukemia, 2018). However, in current clinical practice, suboptimal CART persistence and tumor killing permit tumor cell escape and, ultimately, disease relapse. Reasoning that a pro-lymphoid growth factor could promote CART efficacy, we supplemented UCART infusion with subcutaneous injections of the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc, NT-I7) in vivo using a CD19+ lymphoma xenograft model.Methods: To create anti-CD19 universal CARTs (UCART19), we activated human T cells on CD3/CD28 beads, electroporated the T cells with Cas9 mRNA and a TRAC-targeted gRNA, and virally transduced an anti-CD19 scFv 3rd generation CAR containing a peptidase 2A-cleaved human CD34 construct for both purification and tracking in vivo. Residual TRAC+ cells were depleted using magnetic selection. For xenograft tumor modeling in vivo, we injected NOD-scid IL2Rgammanull (NSG) mice with 5x105 RamosCBR-GFP cells four days prior to UCART19 (2x106 cells). Mice were treated with NT-I7 (10mg/kg SC) on days +1, +15 and +29 post UCART19 infusion.Results:RamosCBR-GFP mice receiving NT-I7 without UCART19 (NT-I7 only group) survived marginally longer (24 day med survival) than mice receiving RamosCBR-GFP cells alone (No tx group) (21 day medium survival, p=0.018, NT-I7 only vs. No Tx). While RamosCBR-GFP mice treated with UCART19 alone (UCART19 group) survived 33 days, 100% of RamosCBR-GFP mice treated with UCART19 and NT-I7 (UCART19+NT-I7 group) were alive at 80 days (Fig 1a), with no mouse showing signs of xenogeneic GVHD (p<0.0001, UCART19+NT-I7 vs. UCART19). At three weeks post UCART19 infusion, bioluminescent imaging (BLI) revealed minimal tumor signal in UCART19+NT-I7 treated mice (108 vs. 1010 photon flux/s, p<0.05, UCART19+NT-I7 vs. UCART19) and near-undetectable photon flux/s at four weeks (107 vs 1011 photon flux/s, p<0.0001, UCART19+NT-I7 vs. UCART19). Quantitative 17-parameter flow cytometric analyses of the blood, bone marrow, and spleens revealed an up to ~8000-fold increase in UCART19 cells in NT-I7-treated mice four weeks post UCART19 infusion (Fig 1a). These UCART19 cells demonstrated a predominantly effector and effector memory phenotype.Discussion: CARTs engineered to express interleukin-7 and CCL19 showed increased migration to and killing of solid tumors (Adachi et al, Nature Biotechnology, 2018). However, genetically engineered potentiation strategies lack “off-switches” and may preclude additional genetic enhancements required for universal “off-the-shelf” CART development. Here, we demonstrate that a pharmacological grade long-acting interleukin-7 agonist can potentiate adoptive cellular therapies. Specifically, NT-I7 can dramatically enhance gene modified T cell proliferation, persistence and tumor killing in vivo, resulting in enhanced survival, providing a tunable clinic-ready adjuvant for reversing suboptimal CART activity in vivo. [Display omitted] DisclosuresCooper:WUGEN: Consultancy, Equity Ownership. Lee:NeoImmuneTech: Employment. Park:NeoImmuneTech: Employment.

A single-tube two-color flow cytometric method for distinguishing between febrile bacterial and viral infections

The aim of this study was to develop a rapid single-tube two-color flow cytometric method to distinguish between febrile bacterial and viral infections.In this prospective comparative study, the quantitative flow cytometric analysis of CD35 and CD64 on isolated human leukocytes was obtained from 286 hospitalized febrile patients, of which 197 patients were found to have either a bacterial (n = 136) or viral (n = 61) infection. The data from infected patients was compared to 49 healthy controls and 23 patients in an inflammatory state.We developed a flow cytometric marker for bacterial infections, defined as the two-color bacterial infection index (TC-BI-index), by incorporating the quantitative analysis of CD35 and CD64 on isolated neutrophils, monocytes, and B-lymphocytes, which displayed 90% sensitivity and specificity in distinguishing between microbiologically confirmed bacterial (n = 77) and viral infections (n = 61) within 45 min.We propose that TC-BI-index test will be useful in assisting physicians to ascertain whether antibiotic treatment is required, thus limiting unnecessary antimicrobial usage.

Genetic analysis in a compound heterozygote family with familial hypercholesterolemia.

Homozygous familial hypercholesterolemia (FH) is rare, with an incidence of ~one in a million and commonly presents with a genetic mutation. The genetic variations of families with FH were clinically analyzed to investigate the association between the phenotype and genotype of patients. Direct sequencing was conducted for the proband and her parents to detect mutations in the fragment of 18 exons of the low‑density lipoprotein receptor (LDLR) and apolipoprotein B100 Q3500R in the peripheral blood genomic DNA. The gene sequences were compared with normal ones to find mutations using GenBank. The QX200 Droplet Digital PCR system was used to detect target DNA copy number variations of the proband and her parents. The functional alterations resulting from the novel mutations were verified by quantitative polymerase chain reaction, western blotting and flow cytometric analyses. The lipid levels of the proband and her parents were all elevated. Genetic testing results indicated that the proband and her mother had a novel heterozygous missense mutation (C377G, 28893T>G) in exon 8 of the LDLR gene, whereas the proband and her father had LDLR gene DNA fragment deletions in exon 18. Clinically, the proband was of a compound heterozygous genotype and her parents were of the simple heterozygous genotype. Furthermore, both mutations led to impaired expression and LDL binding and internalization function of LDLR invitro. The proband's genotype was confirmed to be compound heterozygous FH, leading to clinical manifestations in line with the homozygous FH phenotype. The phenotype is highly associated with the genotype in this type of compound heterozygous FH.

Linkage disequilibrium and functional analysis of PRE1 insertion together with SNPs in the promoter region of IGFBP7 gene in different pig breeds.

Polymorphisms in regions upstream of transcription initiation site may modify the transcriptional activity of target genes by changing promoter activity. This study aims to determine whether or not polymorphisms at porcine IGFBP7 promoter region affect gene expression. In this study, eight SNPs and one PRE1 insertion in this region were first confirmed. The PRE1 insertion was widespread in 20 Chinese indigenous breeds, but was not observed in three commercial breeds. A perfect linkage disequilibrium, consisting of six of those SNPs and a PRE1, was observed with two haplotypes (h1 and h2) in five pig breeds. The h1 haplotype had an overwhelming superiority distribution in Large White, Landrace, and Bama mini-pig; in turn, the h2 only existed in the PRE1 presence breeds. As the haplotypes and PRE1 were located at gene promoter regions, we further investigated the transfection of plasmids with three different fragments of IGFBP-7 promoter region (H1, H2, RF). The CMV promoter of the pEGFP-N1 was substituted by these three different fragments, respectively. Different transcriptional and translational activities of EGFP in PK-15 cells were observed in these three recombinant plasmids by quantitative real-time PCR and flow cytometric analysis. The results indicated that H1 had the higher transcriptional and translational activities of EGFP as compared to the H2 (P < 0.05, P < 0.05). As compared to the RF group, EGFP mRNA expression level was significantly higher in H1 groups (P < 0.05). The IGFBP-7 promoter polymorphisms detected in this study may be important functional variants and potential genetic markers for pig population genetic study.

Resident cell lineages are preserved in pulmonary vascular remodeling.

Pulmonary vascular remodeling is the main pathological hallmark of pulmonary hypertension disease. We undertook a comprehensive and multilevel approach to investigate the origin of smooth muscle actin‐expressing cells in remodeled vessels. Transgenic mice that allow for specific, inducible, and permanent labeling of endothelial (Cdh5‐tdTomato), smooth muscle (Acta2‐, Myh11‐tdTomato), pericyte (Cspg4‐tdTomato), and fibroblast (Pdgfra‐tdTomato) lineages were used to delineate the cellular origins of pulmonary vascular remodeling. Mapping the fate of major lung resident cell types revealed smooth muscle cells (SMCs) as the predominant source of cells that populate remodeled pulmonary vessels in chronic hypoxia and allergen‐induced murine models. Combining in vivo cell type‐specific, time‐controlled labeling of proliferating cells with a pulmonary artery phenotypic explant assay, we identified proliferation of SMCs as an underlying remodeling pathomechanism. Multicolor immunofluorescence analysis showed a preserved pattern of cell type marker localization in murine and human pulmonary arteries, in both donors and idiopathic pulmonary arterial hypertension (IPAH) patients. Whilst neural glial antigen 2 (chondroitin sulfate proteoglycan 4) labeled mostly vascular supportive cells with partial overlap with SMC markers, PDGFRα‐expressing cells were observed in the perivascular compartment. The luminal vessel side was lined by a single cell layer expressing endothelial markers followed by an adjacent and distinct layer defined by SMC marker expression and pronounced thickening in remodeled vessels. Quantitative flow cytometric analysis of single cell digests of diverse pulmonary artery layers showed the preserved separation into two discrete cell populations expressing either endothelial cell (EC) or SMC markers in human remodeled vessels. Additionally, we found no evidence of overlap between EC and SMC ultrastructural characteristics using electron microscopy in either donor or IPAH arteries. Lineage‐specific marker expression profiles are retained during pulmonary vascular remodeling without any indication of cell type conversion. The expansion of resident SMCs is the major underlying and evolutionarily conserved paradigm of pulmonary vascular disease pathogenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Myc binding protein 2 suppresses M2-like phenotypes in macrophages during zymosan-induced inflammation in mice.

MYCBP2 is an E3 ubiquitin ligase, which is well characterized as a key element in the inhibition of neuronal growth, synapse formation and synaptic strength by regulating several signaling pathways. Although MYCBP2 was suspected to be expressed also in immune cells, to date nothing is known about its role in inflammation. We used Multi-epitope ligand cartography (MELC), a method for multiple sequential immunohistology, to show that MYCBP2 is strongly expressed in monocyte-derived macrophages during zymosan-induced inflammation. We generated a myeloid-specific knockout mouse and found that loss of MYCBP2 in myeloid cells reduced nociceptive (painful) behavior during the resolution phase (1-3 days after zymosan injection). Quantitative MELC analyses and flow cytometric analysis showed an increased number of CD206-expressing macrophages in the inflamed paw tissue. Fittingly, CD206 and arginase 1 expression was upregulated in MYCBP2-deficient bone marrow-derived macrophages after polarization with IL10 or IL4. The regulation of protein expression in these macrophages by MYCBP2 varied depending on the polarization signal. The increased IL10-induced CD206 expression in MYCBP2-deficient macrophages was mediated by p38 MAPK, while IL4-induced CD206 expression in MYCBP2-deficient macrophages was mediated by protein kinase A.

Vildagliptin ameliorates pulmonary fibrosis in lipopolysaccharide-induced lung injury by inhibiting endothelial-to-mesenchymal transition

BackgroundPulmonary fibrosis is a late manifestation of acute respiratory distress syndrome (ARDS). Sepsis is a major cause of ARDS, and its pathogenesis includes endotoxin-induced vascular injury. Recently, endothelial-to-mesenchymal transition (EndMT) was shown to play an important role in pulmonary fibrosis. On the other hand, dipeptidyl peptidase (DPP)-4 was reported to improve vascular dysfunction in an experimental sepsis model, although whether DPP-4 affects EndMT and fibrosis initiation during lipopolysaccharide (LPS)-induced lung injury is unclear. The aim of this study was to investigate the anti-EndMT effects of the DPP-4 inhibitor vildagliptin in pulmonary fibrosis after systemic endotoxemic injury.MethodsA septic lung injury model was established by intraperitoneal injection of lipopolysaccharide (LPS) in eight-week-old male mice (5 mg/kg for five consecutive days). The mice were then treated with vehicle or vildagliptin (intraperitoneally, 10 mg/kg, once daily for 14 consecutive days from 1 day before the first administration of LPS.). Flow cytometry, immunohistochemical staining, and quantitative polymerase chain reaction (qPCR) analysis was used to assess cell dynamics and EndMT function in lung samples from the mice.ResultsLung tissue samples from treated mice revealed obvious inflammatory reactions and typical interstitial fibrosis 2 days and 28 days after LPS challenge. Quantitative flow cytometric analysis showed that the number of pulmonary vascular endothelial cells (PVECs) expressing alpha-smooth muscle actin (α-SMA) or S100 calcium-binding protein A4 (S100A4) increased 28 days after LPS challenge. Similar increases in expression were also confirmed by qPCR of mRNA from isolated PVECs. EndMT cells had higher proliferative activity and migration activity than mesenchymal cells. All of these changes were alleviated by intraperitoneal injection of vildagliptin. Interestingly, vildagliptin and linagliptin significantly attenuated EndMT in the absence of immune cells or GLP-1.ConclusionsInhibiting DPP-4 signaling by vildagliptin could ameliorate pulmonary fibrosis by downregulating EndMT in systemic LPS-induced lung injury.

Abstract C26: Analysis of stem cell number &amp; potency in African-American breast tissue

Abstract Breast cancer (BC) survival among African-American (AA) women 5 years after initial diagnosis is only 74%, significantly lower than the 88% survival observed in women of European white (EW) ancestry. The differences in outcomes have been attributed to the greater prevalence of Triple Negative Breast Cancer (TNBC) in AA women and, indeed, stage for stage, TNBC is more lethal than luminal type BCs. Why AA women are more likely to develop TNBC remains a major question. In the past, disparities in BC outcomes were frequently attributed to socioeconomic factors, but increasing data suggests that it is based on intrinsic differences in AA breast tissue compared with EW. Risk factors for BC have been extensively investigated and include elements impacting breast differentiation and development, including timing of thelarche and menarche, as well as timing and number of pregnancies. Breast formation (thelarche) occurs in AA girls 2 years earlier than EW girls in the US. The purpose of this study was to better understand the etiology of premature thelarche and, by extension, BC etiology in AA women. We utilized a unique tissue engineering system that our laboratory has developed for culture of human breast epithelial cells, both normal and malignant. This system allows for long-term (&amp;gt;3 months) growth of normal primary epithelial cell cultures. These cultures form 3-dimensional “epispheres,” made up of 40-100 epithelial cells, which can then differentiate into organotypic branching ducts and lobules. We have established explant cultures of normal breast epithelial cells from 48/48 non-diseased women undergoing breast reduction mammoplasty. Twelve of these (25%) were AA women socioeconomically matched with the EW women. Analysis of these cultures resulted in a significant multivariate model for the timing of ductal formation (P = 0.005) involving only the ancestry of the donor and her height. Thus, intrinsic biological differences exist between AA and EW breast tissue that might impact upon the incidence or etiology of BC. We hypothesized that stem cells from AA women would be present in higher proportions or alternatively demonstrate higher potency than EW. Breast epithelial stem cells were defined by the absence of CD24 antigen and the presence of CD44 and CD49f antigens on the cell surface using quantitative flow cytometric analysis. Cloning efficiency, defined as sorted single cells that grew into colonies, was quantified as a percentage of the total cells sorted and verified microscopically. Potency, defined as colonies that exhibited multiple cell types, was quantified as a percentage of the total cloning efficiency. Our results indicate that AA samples, on average, contained 48.4% breast stem cells, while EW samples manifested a significantly smaller proportion, only 7.2% (P = 0.018). Cloning efficiency and potency analyses are currently ongoing. In summary AA women display higher proportions of stem cells in normal breast tissues relative to EW, which may be pertinent to premature thelarche and by extension, triple negative breast cancer etiology. Citation Format: Omar Ibrahim, Stephen G. Grant, Nicole T. Myers, Amie B. Courtney, Nancy A. lalanne, Jean J. Latimer. Analysis of stem cell number &amp; potency in African-American breast tissue. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr C26.