Secondary Abs Research Articles

Detection of Mu‐Opioid Receptors in SH‐SY5Y Human Neuroblastoma Cells by Flow Cytometry

PurposePharmacological actions of morphine are mediated through G‐protein‐coupled receptors (GPCRs), specifically mu‐opioid receptors (MORs). Morphine‐induced activation of GPCRs inhibits adenylyl cyclase and decreases 3’,5’‐cyclic adenosine monophosphate (cAMP). Neuroblastoma SH‐SY5Y cell lines endogenously express MORs. Our aim was to develop a flow cytometry (FC) assay for MORs on the surface of SH‐SY5Y cells. This assay will provide in‐depth understanding of opioid‐induced downstream cAMP signaling.MethodsSH‐SY5Y human neuroblastoma cells (ATCC® CRL2266™) were grown in DMEM/10% FBS and maintained in a humidified incubator at 37ºC/5%CO2. Cells were seeded in 96‐well white, opaque‐bottom plates (5×104cells/well). Transfection conditions were optimized using ViaFect™ (Promega™) and a stably transfected SH‐SY5Y cell line was established using GloSensor™ ‐23F cAMP plasmid (Promega™) containing hygromycin‐resistance gene. Cells were washed in FACS buffer (PBS + 2% FBS) and incubated for 1h at room temperature with monoclonal rabbit anti‐MOR antibody (Ab) (Novus Biologicals). Following the wash, cells were incubated with donkey anti‐rabbit secondary Ab (Abcam). We used monoclonal mouse anti‐muscarinic Ab (Novus Biologicals) and donkey anti‐mouse secondary Ab (Jackson Labs). Both secondary Abs are bound to a fluorophore (Alexa Fluor® 488) which emits light when excited by FITC channel frequency (excitation/emission: 495nm/519nm). A Cytoflex flow cytometer (Beckman Coulter) analyzes cells for FITC fluorescence emission. Multiple primary and secondary concentrations were tested; Human TruStain Fc blocker (BioLegend) was used to prevent nonspecific tagging.ResultsRelative fluorescence emission was measured as percent shift increase from the untagged control group. Because MOR Abs have been tested in a limited capacity, we tested for presence of muscarinic receptors (another endogenously expressed GPCR in these cells) to establish the FC assay. Initial trials with low primary and secondary MOR Ab concentrations showed limited shifts (~1‐2%), however, muscarinic receptor Abs bound to their receptors at lower concentrations compared to MOR Abs. Testing muscarinic receptors showed secondary Ab concentration‐dependent fluorescence shifts indicating that the technique was viable. Studies with increased concentration ratios of primary and secondary MOR Abs showed secondary Ab concentration‐dependent response with maximum % shifts reaching up to 16%. Use of Fc blocker eliminated nonspecific binding, increasing the percent shift.ConclusionCompared to untagged controls, antibody‐tagging on SH‐SY5Y cell surface produced greater immunofluorescence indicating presence of MORs in both non‐transfected and 23F cAMP plasmid transfected cells. Direct binding of receptor modulators leads to altered downstream cAMP signaling as shown in previous in vitro signaling studies and behavioral mouse models. Detection of the MOR is essential to ensure that cAMP signaling is regulated by MOR agonists. In future studies, we plan to investigate the expression of endothelin‐A receptor (ETAR) and explore potential interactions between MOR and ETAR in SH‐SY5Y cells.

PD-L1 Is Regulated By Interferon Gamma and Interleukin 6 through STAT1 and STAT3 Signaling in Cutaneous T-Cell Lymphoma

Background: Signaling interactions within the cutaneous T cell lymphoma (CTCL) microenvironment often create a permissive environment for CTCL growth. In response to oncogenic signals from proliferating tumor cells, the tumor microenvironment changes throughout disease progression. Studies have shown that dendritic cells and macrophages are critical tumorigenic regulators of the microenvironment in hematologic malignancies. Tumor cells escape immune surveillance by manipulating immune checkpoint receptors like PD1, and activation of the PD1 receptor by PD-L1 transduces a signal that leads to the inhibition of T-cell functions. Tumor cells, resting dendritic cells, and macrophages in the tumor microenvironment express PD-L1; and proinflammatory cytokines like IFN-γ and IL 6 upregulate PD-L1 on tumor cells. However, the mechanisms of PD-L1 upregulation in CTCL remain unknown.Methods: Immunoblots: Primary patient peripheral blood mononuclear cells (PBMCs), CTCL cell lines (Mac1 and Mac2a) and mature monocyte-derived dendritic cells (MoDCs) from healthy patients were treated with IFNγ, IL-6, a a pan JAK/STAT inhibitor (tofacitinib), and a PD-L1 inhibitor for 72 hrs. Cell lysates were collected and subjected to immunoblot analysis with antibodies against PD-L1, pSTAT1, and pSTAT3. Immunohistochemistry: Paraffin sections of patients' skin biopsies were incubated with mAbs against CD163 PD-L1, and pSTAT3 at 4°C overnight. Sections were incubated with appropriate secondary Abs for 2 hrs at room temperature. The images were taken with a Zeiss 700 confocal microscope. Flow Cytometry: Migrated skin cells from skin explant cultures taken from CTCL patients were incubated with fluorochrome-conjugated mAbs and analyzed on an LSR Fortessa (BD) flow cytometer. The gating parameters were as follows: epidermal LCs (CD1a+, CD14neg, HLA-DRbright), dermal CD1a+ DCs (CD1a+, CD14neg, HLA-DRbright, CD11b+, CD11c+), and dermal CD14+ DCs (CD1aneg, CD14+, HLA-DRbright, CD11b+, CD11c+). Gates were set for collection and analysis of at least 10,000 live events, and the data were analyzed with FlowJo 9.7.6 software (TreeStar).Results: Our idata indicate that T cells in CTCL have an exhausted phenotype, presumably due to chronic Ag stimulation by cutaneous DCs. Macrophages and dendritic cell subsets in lesional skin from mycosis fungoides patients as well as PBMCs from Sézary syndrome (SS) patients express PD-L1. To elucidate the mechanism of PD-L1 regulation, we treated PBMCs from SS patients and CTCL cell lines with IFN-γ, IL-6, a pan JAK/STAT inhibitor (tofacitinib), and a PD-L1 inhibitor (durvalumab). We found upregulation of PD-L1 protein expression by PBMCs when treated with IFN-γ and IL-6, through upregulated STAT1 and STAT3 signaling detected by Western blots. A pan JAK/STAT inhibitor abrogated the increased expression of PD-L1. Monocyte-derived dendritic cells from healthy donors exhibit upregulated PD-L1 expression by IFN-γ, but not with IL-6. Durvalumab treatment reduced pSTAT3 and PD-L1 expression. We have therefore initiated a clinical trial with a PD-L1 inhibitor (durvalumab) in patients with mycosis fungoides (MF)/Sézary syndrome (SS) and observed promising initial responses.Conclusions: PBMCs from Sezary syndrome patients and moDCs from healthy donor upregulate PD-L1 through STAT signaling initiated by proinflammatory cytokines IFN-γ and IL-6. Durvalumab also reduced PD-L1 and pSTAT3 expression in patient PBMCs. PD-L1 expression may result from the proinflammatory background of the tumor microenvironment and activation of the PD1 receptor by PD-L1 transduces a signal that leads to the inhibition of T-cell functions in CTCL. DisclosuresKadin:Allergan, Inc: Honoraria. Querfeld:Soligenix: Research Funding; Actelion: Honoraria, Research Funding; Medivir: Honoraria; Mallinckrodt: Honoraria; Kyowa: Research Funding; Elorac: Research Funding; Celgene: Honoraria, Research Funding; MiRagen: Honoraria, Research Funding; Trillium Therapeutics: Research Funding; City of Hope: Employment; Mindera: Consultancy.

Diagnosis of leptomeningeal metastasis (LM) through identification of circulating tumor cells (CTCs) in cerebrospinal fluid (CSF).

3567 Background: Diagnosis of LM from solid tumors can be challenging. The TargetSelector (TS) CTC detection assay has demonstrated highly specific and sensitive CTC capture both for epithelial (CK+) and non-epithelial (CK-) subsets. The assay utilizes a ten-antibody (ab) capture cocktail followed by biotinylated secondary abs that bind to CTCs, enriched in a microfluidic device. TS targeted next-generation sequencing (NGS) assay detects somatic mutations in 12 breast cancer-related genes. The aim was to determine whether TS can improve sensitivity in the diagnosis of LM compared to CSF cytology by lumbar puncture (LP). Methods: CSF was collected prospectively from patients (pts) with a prior solid tumor diagnosis and suspicion of LM. CTCs were isolated from CSF using the TS platform. Cells were stained with cytokeratin (CK), CD45, streptavidin and DAPI. CTCs captured in a microchannel were classified as CK + or -. Peripheral blood samples obtained at time of LP underwent similar CTC analysis. Cell-free total nucleic acids (cfTNA) were extracted from plasma and CSF followed by NGS. Data analysis used the Ion Torrent Suite with annotation and report curation by Ion Reporter and Oncomine Knowledgebase Reporter software respectively. Results: There were 14 pts (13 women and 1 man), median age 56 years (range 32-75) with cancers of the breast (10), lung (1), colon (1), CNS lymphoma (1) or glioma (1). Pts had received a median of 2.5 lines of systemic metastatic therapy (range 0-8). CSF cytology was not sent for 1 pt and TS was not performed for 1 pt. TS and standard cytology had 89% agreement in pts with metastatic breast cancer (MBC, 8/9). Of the 6 pts for whom CTCs were detected in CSF by TS, 3 pts had + cytology (all MBC), 2 pts had - cytology and 1 pt with MBC was not tested by cytology. Of the 3 pts with + CSF by cytology (all MBC), all were detected by TS (Table). Among 5 MBC pts with CTCs present in CSF, ER status was concordant in 2 of 5 (40%). HER2 status was concordant in 3 of 4 (75%) evaluable pts and not determined in 1 pt. Analysis of cfDNA from CSF identified somatic mutations in 3 pts (TP53, PIK3CA, CCND1, respectively). In 1 of 3 pts, the mutation identified in the CSF (PIK3CA) in HR+/HER2- MBC was also identified in the blood. Conclusions: TargetSelector is a viable platform for the detection of breast cancer CTCs in the CSF. NGS performed on CSF samples can identify potentially actionable mutations. [Table: see text]

Tantalum Oxide Nanoparticle-Based Mass Tag for Mass Cytometry.

Mass cytometry (MC) is a bioanalytical technique that uses metal-tagged antibodies (Abs) for high-dimensional single-cell immunoassays. Currently, this technology can measure over 40 parameters simultaneously on individual cells using metal-chelating polymer (MCP) based reagents. However, MC can in principle detect up to 135 parameters with the development of new elemental mass tags. Here we report the development of a tantalum oxide nanoparticle (NP)-based mass tag for MC immunoassays. Uniform-sized amine-functionalized tantalum oxide NPs (d ∼ 5.7 nm) were synthesized via a one-pot two-step reverse microemulsion method. These amine-functionalized NPs were further modified with azide groups by reacting with azide-PEG2k succinimidyl carboxymethyl ester (NHS-PEG2k-N3) cross-linkers. The Ab-NP conjugates were prepared by reacting azide-functionalized NPs with dibenzocyclooctyne (DBCO)-functionalized primary or secondary Abs (DBCO-Ab) followed by fast protein size exclusion liquid chromatography (FPLC) purification. Three Ab-NP conjugates (TaO2-PEG2k-goat antimouse, TaO2-PEG2k-CD25, TaO2-PEG2k-CD196) were fabricated and tested in MC immunoassays. For the TaO2-PEG2k-goat antimouse conjugate, we showed that it can effectively detect abundant CD20 biomarkers on Ramos cells. For TaO2-PEG2k-CD25 and TaO2-PEG2k-CD196 conjugates, we demonstrated that these Ab-NP conjugates could be integrated into the commercial Ab staining panels for high-dimensional single-cell immune profiling of human peripheral blood mononuclear cells.

Lanthanide nanoparticles for high sensitivity multiparameter single cell analysis.

Mass cytometry (MC) is a high throughput multiparameter analytical technique for determining biomarker expression in cells. In MC, antibodies (Abs) are tagged with heavy metal isotopes via conjugation to metal chelating polymers (MCPs). To improve the sensitivity of MC towards low abundance biomarkers, we are developing nanoparticle (NP)-based reagents as mass tags for Abs. We examine the use of silica-coated NaHoF4 NPs (d ∼ 12 nm) decorated with PEG5k conjugated to thiol-modified primary or secondary Abs for MC assays. We compare the sensitivity of NP-Ab conjugates to MCP-Ab conjugates towards seven biomarkers with varying expression levels across six cell lines. We also perform a multi-parameter assay using a cocktail of both NP- and MCP-based reagents to detect seven cellular markers in peripheral blood mononuclear cells (PBMCs). In the case of highly abundant markers, signal enhancements from NP-Ab conjugates offer minimal advantages over MCP-Ab conjugates, which already give strong signals. In the case of biomarkers with lower abundance, the level of signal enhancements depended on the nature of the biomarker being detected, or on the type of detection method used. When comparing the indirect detection of CD14 on THP-1 cells using NPs or MCPs conjugated to secondary Abs, the NP reagents offered little signal enhancements compared to the MCP reagents. However, in the case of direct CD14 detection on THP-1 or U937 cells using NPs or MCPs conjugated to primary Abs, a 30- or 450-fold signal enhancement was seen from the NP-based reagent. In the experiments where both NP-Ab and MCP-Ab conjugates were used together to stain PBMCs, we found that the presence of the NP-Ab conjugates did not affect the function of MCP-Ab conjugates, and the NP-Ab conjugates showed minimal non-specific interaction with cells without the target biomarker (CD14). Furthermore, these NP-Ab conjugates could be used to identify rare CD14+ monocytes from the PBMC mixture with a 20-fold signal increase when compared to the use of only MCP-Ab conjugates. Collectively, the strong signal amplification obtained from NP reagents demonstrate the potential of these reagents to be used in conjunction with MCP-reagents to detect rare cellular markers or cell types that may otherwise be overlooked when using MCP-reagents alone.

High-affinity IgM+ memory B cells are defective in differentiation into IgM antibody-secreting cells by re-stimulation with a T cell-dependent antigen

IgM antibodies (Abs) are thought to play a major role in humoral immunity but only at the early stage of the primary immune response. However, two subsets of IgM+ memory B cells (MBCs), one with high affinity gained by means of multiple somatic hypermutation (SHM) and the other with low affinity and no SHMs, are generated through the germinal center (GC)-dependent and GC-independent (non-GC) pathway, respectively, after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP)-chicken γ-globulin. Surprisingly, an analysis of antibody-secreting cells reveals that a large amount of anti-NP IgM Ab with few SHMs is secreted during the recall response, indicating that only non-GC MBCs have terminal differentiation potential. Since secondary IgM Abs are capable of binding to dinitrophenyl ligands, they likely provide broad cross-reactivity in defense against microbial infection.

OR14 Next-generation HLA antibody reagents

Aim A humanized monoclonal antibody (Ab) pan-reactive for HLA Class I is useful for many HLA testing applications, including validation and confirmation of HLA Ab assays and platforms. By engineering the Ab so it is also available in subtype-specific (IgG1-4) and allotype-specific forms, it becomes useful for validating and characterizing Ab assays dependent upon complement-fixation, and validating the specificity of secondary Abs used as detection reagents. Methods The IgG1-4 and allotype-specific forms of this Ab were tested with the detection reagents for our flow cytometric crossmatch (FCXM) assay and single-antigen bead (SAB) assay. The IgG1-4 Abs were also tested in the CDC assay, with and without AHG. The IgG1 Ab was tested at dilutions against cells in the FCXM. The IgG1-4 Abs were tested at multiple dilutions in the SAB and C1q assays, and with and without heat-inactivation and EDTA treatment of sera. Results All lots of AHG used as detection reagents in the FCXM and SAB assays were able to detect the IgG1-4 Abs, and the FCXM reagents detected all 29 isoallotypes. No false-reactivity was seen in the Class II SAB assay. In the CDC assay, IgG1-3 were positive both with and w/out AHG, but IgG4 was only positive when AHG was added. When tested against cells from 15 different individuals, the IgG1 Ab provided consistent reactivity, regardless of the HLA type of the donor cells. When serial dilutions of the IgG1-4 Abs were tested in the SAB and C1q assays, a distinct hierarchy of sensitivity in the C1q assay was apparent, with IgG3 > IgG1 > IgG2. IgG4 was negative in the C1q assay. Conclusions These IgG1-4 monoclonal Abs, directed against a monomorphic Class I epitope, are valuable reagents for validating and characterizing the multiple HLA antibody assays typically employed in a modern HLA laboratory. In Red Cell serology, it has been demonstrated that some IgG isoallotypes are NOT detected by the secondary AHG detection reagents, making these Abs useful to confirm detection with different lots of AHG. These antibodies are also useful as controls in antibody assays that rely on complement-fixation (i.e. CDC and C1q). Finally, when titrated appropriately, these Abs can give consistent low, medium, or strongly-positive reactivity in the SAB and FCXM assays, making them useful as a reagent to standardize and/or normalize results.

The Therapeutic CD38 Monoclonal Antibody Daratumumab Induces Programmed Cell Death via Fcγ Receptor-Mediated Cross-Linking.

Emerging evidence suggests that FcγR-mediated cross-linking of tumor-bound mAbs may induce signaling in tumor cells that contributes to their therapeutic activity. In this study, we show that daratumumab (DARA), a therapeutic human CD38 mAb with a broad-spectrum killing activity, is able to induce programmed cell death (PCD) of CD38(+) multiple myeloma tumor cell lines when cross-linked in vitro by secondary Abs or via an FcγR. By comparing DARA efficacy in a syngeneic in vivo tumor model using FcRγ-chain knockout or NOTAM mice carrying a signaling-inactive FcRγ-chain, we found that the inhibitory FcγRIIb as well as activating FcγRs induce DARA cross-linking-mediated PCD. In conclusion, our in vitro and in vivo data show that FcγR-mediated cross-linking of DARA induces PCD of CD38-expressing multiple myeloma tumor cells, which potentially contributes to the depth of response observed in DARA-treated patients and the drug's multifaceted mechanisms of action.

Topical fentanyl stimulates healing of ischemic wounds in diabetic rats.

Topically applied opioids promote angiogenesis and healing of ischemic wounds in rats. We examined if topical fentanyl stimulates wound healing in diabetic rats by stimulating growth-promoting signaling, angiogenesis, lymphangiogenesis and nerve regeneration. We used Zucker diabetic fatty rats that develop obesity and diabetes on a high fat diet due to a mutation in the Leptin receptor. Fentanyl blended with hydrocream was applied topically on ischemic wounds twice daily, and wound closure was analyzed regularly. Wound histology was analyzed by hematoxylin and eosin staining. Angiogenesis, lymphangiogenesis, nerve fibers and phospho-platelet derived growth factor receptor-β (PDGFR-β) were visualized by CD31-, lymphatic vessel endothelium-1, protein gene product 9.5- and anti-phospho PDGFR-β-immunoreactivity, respectively. Nitric oxide synthase (NOS) and PDGFR-β signaling were analyzed using Western immunoblotting. Fentanyl significantly promoted wound closure as compared to phosphate-buffered saline (PBS). Histology scores were significantly higher in fentanyl-treated wounds, indicative of increased granulation tissue formation, reduced edema and inflammation, and increased matrix deposition. Fentanyl treatment resulted in increased wound angiogenesis, lymphatic vasculature, nerve fibers, nitric oxide, NOS and PDGFR-β signaling as compared to PBS. Phospho-PDGFR-β co-localized with CD31 co-staining for vasculature. Topically applied fentanyl promotes closure of ischemic wounds in diabetic rats. Increased angiogenesis, lymphangiogenesis, peripheral nerve regeneration, NO and PDGFR-β signaling are associated with fentanyl-induced tissue remodeling and wound healing.

Fine Specificity of B-Cell Tolerance to Blood Group Antigens Following ABO-Incompatible (Aboi) Heart Transplantation

Introduction: Histo-blood group ABH(O) ags are oligosaccharides present as glycolipids or glycoproteins. A, B or H epitopes are carried by 6 different internal carbohydrate backbones (type I-VI), forming antigenically distinct variants, each type differentially expressed in human tissues. In heart, only type II structures are expressed (on vascular endothelium) whereas red cells express type I-IV. Identifying natural antibody (ab) clones specific to these ABH epitopes may be important in the setting of ABOi transplantation. Our objectives were to 1) develop a diagnostic tool to detect abs specific to ABH epitopes on all 6 carbohydrate backbones and 2) characterize abs in volunteers and patients who received ABOi heart transplants as infants. Methods: Chemically synthesized A type I-VI, B type I-VI and H type I-VI were printed onto microarray slides; spot morphology and ags were confirmed by monoclonal abs to ABH ags. After initial optimization, plasma from different blood group individuals and patients were hybridized and bound IgM, IgG and IgA detected with fluorescently-labelled secondary abs. Slides were scanned with a microarray scanner and Mean Fluorescent Intensities (MFI) determined using Imagene software. Results: Subtype-specific IgM, IgG and IgA abs varied substantially amongst individuals with different blood groups as well as the same blood group. In infants who received ABOi transplants, abs to donor blood group ags were not detected against type II subtype (fig 1) compared to same blood type controls (fig 2). Immunohistochemistry studies on cardiac biopsies confirmed that only type II carbohydrates were expressed on endothelium.[Figure 1][Figure 2]Conclusions: This analysis demonstrates that infant recipients of ABOi transplants remain deficient in production of antibodies to A-ag expressed in the cardiac graft, despite detection of antibodies to irrelevant A-ag detected by agglutination assay. Thus the ABO-microarray is valuable for subtype-specific blood group abs relevant to the graft. This tool will allow enhanced safety of ABO-incompatible organ transplantation and improve study of mechanisms of tolerance and/or accommodation in the clinical setting. Microarray technology provides analysis of tiny sample volumes (< 5 ml plasma), and simultaneous characterization of all ab isotypes, invaluable when testing infants.

Fine Specificity of B-Cell Tolerance to Blood Group Antigens Following ABO-Incompatible (Aboi) Heart Transplantation

Introduction: Histo-blood group ABH(O) ags are oligosaccharides present as glycolipids or glycoproteins. A, B or H epitopes are carried by 6 different internal carbohydrate backbones (type I-VI), forming antigenically distinct variants, each type differentially expressed in human tissues. In heart, only type II structures are expressed (on vascular endothelium) whereas red cells express type I-IV. Identifying natural antibody (ab) clones specific to these ABH epitopes may be important in the setting of ABOi transplantation. Our objectives were to 1) develop a diagnostic tool to detect abs specific to ABH epitopes on all 6 carbohydrate backbones and 2) characterize abs in volunteers and patients who received ABOi heart transplants as infants. Methods: Chemically synthesized A type I-VI, B type I-VI and H type I-VI were printed onto microarray slides; spot morphology and ags were confirmed by monoclonal abs to ABH ags. After initial optimization, plasma from different blood group individuals and patients were hybridized and bound IgM, IgG and IgA detected with fluorescently-labelled secondary abs. Slides were scanned with a microarray scanner and Mean Fluorescent Intensities (MFI) determined using Imagene software. Results: Subtype-specific IgM, IgG and IgA abs varied substantially amongst individuals with different blood groups as well as the same blood group. In infants who received ABOi transplants, abs to donor blood group ags were not detected against type II subtype (fig 1) compared to same blood type controls (fig 2). Immunohistochemistry studies on cardiac biopsies confirmed that only type II carbohydrates were expressed on endothelium.[Figure 1][Figure 2]Conclusions: This analysis demonstrates that infant recipients of ABOi transplants remain deficient in production of antibodies to A-ag expressed in the cardiac graft, despite detection of antibodies to irrelevant A-ag detected by agglutination assay. Thus the ABO-microarray is valuable for subtype-specific blood group abs relevant to the graft. This tool will allow enhanced safety of ABO-incompatible organ transplantation and improve study of mechanisms of tolerance and/or accommodation in the clinical setting. Microarray technology provides analysis of tiny sample volumes (< 5 ml plasma), and simultaneous characterization of all ab isotypes, invaluable when testing infants.

Dual Inhibition of Met Kinase and Angiogenesis to Overcome HGF-Induced EGFR-TKI Resistance in EGFR Mutant Lung Cancer

Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a serious problem in the management of EGFR mutant lung cancer. We recently reported that hepatocyte growth factor (HGF) induces resistance to EGFR-TKIs by activating the Met/PI3K pathway. HGF is also known to induce angiogenesis in cooperation with vascular endothelial growth factor (VEGF), which is an important therapeutic target in lung cancer. Therefore, we hypothesized that dual inhibition of HGF and VEGF may be therapeutically useful for controlling HGF-induced EGFR-TKI-resistant lung cancer. We found that a dual Met/VEGF receptor 2 kinase inhibitor, E7050, circumvented HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer cell lines by inhibiting the Met/Gab1/PI3K/Akt pathway in vitro. HGF stimulated VEGF production by activation of the Met/Gab1 signaling pathway in EGFR mutant lung cancer cell lines, and E7050 showed an inhibitory effect. In a xenograft model, tumors produced by HGF-transfected Ma-1 (Ma-1/HGF) cells were more angiogenic than vector control tumors and showed resistance to gefitinib. E7050 alone inhibited angiogenesis and retarded growth of Ma-1/HGF tumors. E7050 combined with gefitinib induced marked regression of tumor growth. Moreover, dual inhibition of HGF and VEGF by neutralizing antibodies combined with gefitinib also markedly regressed tumor growth. These results indicate the therapeutic rationale of dual targeting of HGF-Met and VEGF-VEGF receptor 2 for overcoming HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer.

A faster immunofluorescence assay for tracking infection progress of human cytomegalovirus

Immunofluorescence assay (IFA) is one of the most frequently used methods in the biological sciences and clinic diagnosis, but it is expensive and time-consuming. To overcome these limitations, we developed a faster and more cost-effective IFA (f-IFA) by modifying the standard IFA, and applied this method to track the progression of human cytomegalovirus (HCMV) infection in different cells. The f-IFA that we developed not only saves time, but also dramatically reduces the quantity of antibody (Ab), which will facilitate the application of IFA in clinic diagnosis. f-IFA requires only 15 min for blocking, 10 min incubation for each primary and secondary Abs, followed by 1 min extensive wash after each incubation. Only 25 μl of diluted Ab solution was needed for each coverslip at the primary and secondary Ab incubation steps. In addition, all steps were performed at room temperature. This f-IFA has been applied successfully to follow virion entry (pp65) and expression of viral genes (IE1, UL44, and pp65) in order to track the details of HCMV infection process. We found that ∼0.5% HCMV-infected T98G cells formed multiple-micronuclei (IE1 and nucleus staining) and had virus shedding (pp65 staining) by f-IFA, which could not be detected by the traditional IFA. Our results indicated that f-IFA is a sensitive, convenient, fast, and cost-effective method for investigating the details of virus infection progress, especially HCMV infection. The faster and cost-effective feature with higher sensitivity and specificity implies that f-IFA has potential applications in clinical diagnosis.

A Highly Conserved Cytoplasmic Cysteine Residue in the α4 Nicotinic Acetylcholine Receptor Is Palmitoylated and Regulates Protein Expression

Nicotinic acetylcholine receptor (nAChR) cell surface expression levels are modulated during nicotine dependence and multiple disorders of the nervous system, but the mechanisms underlying nAChR trafficking remain unclear. To determine the role of cysteine residues, including their palmitoylation, on neuronal α4 nAChR subunit maturation and cell surface trafficking, the cysteines in the two intracellular regions of the receptor were replaced with serines using site-directed mutagenesis. Palmitoylation is a post-translational modification that regulates membrane receptor trafficking and function. Metabolic labeling with [(3)H]palmitate determined that the cysteine in the cytoplasmic loop between transmembrane domains 1 and 2 (M1-M2) is palmitoylated. When this cysteine is mutated to a serine, producing a depalmitoylated α4 nAChR, total protein expression decreases, but surface expression increases compared with wild-type α4 levels, as determined by Western blotting and enzyme-linked immunoassays, respectively. The cysteines in the M3-M4 cytoplasmic loop do not appear to be palmitoylated, but replacing all of the cysteines in the loop with serines increases total and cell surface expression. When all of the intracellular cysteines in both loops are mutated to serines, there is no change in total expression, but there is an increase in surface expression. Calcium accumulation assays and high affinity binding for [(3)H]epibatidine determined that all mutants retain functional activity. Thus, our results identify a novel palmitoylation site on cysteine 273 in the M1-M2 loop of the α4 nAChR and determine that cysteines in both intracellular loops are regulatory factors in total and cell surface protein expression of the α4β2 nAChR.

Identification of Key Amino Acid Residues That Determine the Ability of High Risk HPV16-E7 to Dysregulate Major Histocompatibility Complex Class I Expression

High risk human Papillomavirus (HPV) types are the major causative agents of cervical cancer. Reduced expression of major histocompatibility complex class I (MHC I) on HPV-infected cells might be responsible for insufficient T cell response and contribute to HPV-associated malignancy. The viral gene product required for subversion of MHC I synthesis is the E7 oncoprotein. Although it has been suggested that high and low risk HPVs diverge in their ability to dysregulate MHC I expression, it is not known what sequence determinants of HPV-E7 are responsible for this important functional difference. To investigate this, we analyzed the capability to affect MHC I of a set of chimeric E7 variants containing sequence elements from either high risk HPV16 or low risk HPV11. HPV16-E7, but not HPV11-E7, causes significant diminution of mRNA synthesis and surface presentation of MHC I, which depend on histone deacetylase activity. Our experiments demonstrate that the C-terminal region within the zinc finger domain of HPV-E7 is responsible for the contrasting effects of HPV11- and HPV16-E7 on MHC I. By using different loss- and gain-of-function mutants of HPV11- and HPV16-E7, we identify for the first time a residue variation at position 88 that is highly critical for HPV16-E7-mediated suppression of MHC I. Furthermore, our studies suggest that residues at position 78, 80, and 88 build a minimal functional unit within HPV16-E7 required for binding and histone deacetylase recruitment to the MHC I promoter. Taken together, our data provide new insights into how high risk HPV16-E7 dysregulates MHC I for immune evasion.

Differential expression of junctional adhesion molecules in systemic sclerosis (SSc) skin

ObjectiveJunctional adhesion molecule (JAM)-A and JAM-C regulate leucocyte-endothelial cell (EC) interactions. JAMs also interact at inter-EC junctions regulating vascular permeability and are implicated in pathophysiological processes involving leucocyte transmigration, tight...

Involvement of junctional adhesion molecules in lymphatic vascular remodelling in rheumatoid arthritis

Background and objectiveRheumatoid arthritis (RA) is a chronic systemic autoimmune disease, in which neovascularisation of the synovium is regarded as a crucial step in its development and progression. Lymphatic neovascularisation...

Localization in the cell of the protein forming ATP-dependent potassium-selective channels in a bilayer lipid membrane. An ultrastructural study

The localization in the cell of the protein forming the ATP-dependent potassium-selective channels in the bilayer lipid membrane has been studied. The electron microscope investigation of rat liver and heart tissue sections after their incubation with Abs against this protein and the visualization of the protein with secondary Abs conjugated with colloid gold were carried out. Colloid gold particles were observed both in mitochondrial membranes and in membranes of endoplasmic and sarcoplasmic reticulum. In heart mitochondria, these particles were significantly greater than in liver mitochondria. The localization of the channel protein both in mitochondria and reticulum, as well as the structural similarity between the mitochondrial channel and the precursor of calreticulin suggest that the channel protein belongs to the family of calreticulins. The possible function of the protein as a channel subunit of the mitochondrial ATP-dependent potassium channel is discussed.

Claudin Association with CD81 Defines Hepatitis C Virus Entry

Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.

Human Prostatic Acid Phosphatase, an Authentic Tyrosine Phosphatase, Dephosphorylates ErbB-2 and Regulates Prostate Cancer Cell Growth

Cellular prostatic acid phosphatase (cPAcP), an authentic tyrosine phosphatase, is proposed to function as a negative growth regulator of prostate cancer (PCa) cells in part through its dephosphorylation of ErbB-2. Nevertheless, the direct interaction between cPAcP and ErbB-2 has not been shown nor the specific dephosphorylation site of ErbB-2 by cPAcP. In this report, our data show that the phosphorylation level of ErbB-2 primarily at Tyr(1221/2) correlates with the growth rate of both LNCaP and MDA PCa2b human PCa cells. Further, cPAcP reciprocally co-immunoprecipitated with ErbB-2 in a non-permissive growth condition. Expression of wild type cPAcP, but not inactive mutant, by cDNA in cPAcP-null LNCaP C-81 cells results in decreased tyrosine phosphorylation of ErbB-2 including Tyr(1221/2). Concurrently, Tyr(317) phosphorylation of p52(Shc), proliferating cell nuclear antigen expression, and cell growth are decreased in these cells. Conversely, decreased cPAcP expression by short hairpin RNA in LNCaP C-33 cells was associated with elevated phosphorylation of ErbB-2 initially at Tyr(1221/2). Its downstream p52(Shc), ERK1/2, Akt, Src, STAT-3, and STAT-5 were activated, and cell proliferation, proliferating cell nuclear antigen, and cyclin D1 expression were increased. Stable subclones of C-33 cells by small interfering PAcP had elevated Tyr(1221/2) phosphorylation of ErbB-2 and exhibited androgen-independent growth and increased tumorigenicity in xenograft female animals. In summary, our data together indicate that in prostate epithelia, cPAcP interacts with and dephosphorylates ErbB-2 primarily at Tyr(1221/2) and hence blocks downstream signaling, leading to reduced cell growth. In PCa cells, decreased cPAcP expression is associated with androgen-independent cell proliferation and tumorigenicity as seen in advanced hormone-refractory prostate carcinomas.