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1.  Validation of a Microsphere Immunoassay for Serological Leptospirosis Diagnosis in Human Serum by Comparison to the Current Gold Standard 
PLoS Neglected Tropical Diseases  2015;9(3):e0003636.
A microsphere immunoassay (MIA) utilising Luminex xMap technology that is capable of determining leptospirosis IgG and IgM independently was developed. The MIA was validated using 200 human samples submitted for routine leptospirosis serology testing. The traditional microscopic agglutination (MAT) method (now 100 years old) suffers from a significant range of technical problems including a dependence on antisera which is difficult to source and produce, false positive reactions due to auto-agglutination and an inability to differentiate between IgG and IgM antibodies. A comparative validation method of the MIA against the MAT was performed and used to determine the ability of the MIA to detect leptospiral antibodies when compared with the MAT. The assay was able to determine samples in the reactive, equivocal and non-reactive ranges when compared to the MAT and was able to differentiate leptospiral IgG antibodies from leptospiral IgM antibodies. The MIA is more sensitive than the MAT and in true infections was able to detect low levels of antibody in the later stages of the acute phase as well as detect higher levels of IgM antibody earlier in the immune phase of the infection. The relatively low cost, high throughput platform and significantly reduced dependency on large volumes of rabbit antisera make this assay worthy of consideration for any microbiological assay that currently uses agglutination assays.
Author Summary
Leptospirosis is a zoonotic disease caused by spirochaetes of the genus Leptospira and affects millions of people, worldwide, each year. Laboratory diagnosis of leptospirosis currently relies on methods that are flawed in many areas. Current methods are outdated, time consuming and expensive. They rely on a continuous supply of animal products (rabbit anti-sera) and require specialist expertise and equipment. The current gold standard diagnostic assay for leptospirosis (MAT) cannot determine IgG from IgM antibodies and relies on live cultures, which presents problems in the way of maintenance and attenuation. Development of a new diagnostic assay for serological diagnosis of leptospirosis that is specific, sensitive and able to discriminate between IgG and IgM classes of antibodies—as well as being more cost effective—will significantly improve the capabilities for detecting leptospirosis infections. It will provide medical professionals with more valuable diagnostic information and public health professionals with improved epidemiological information.
PMCID: PMC4373873  PMID: 25807009
2.  Effect of Serum Heat-Inactivation and Dilution on Detection of Anti-WNV Antibodies in Mice by West Nile Virus E-protein Microsphere Immunoassay 
PLoS ONE  2012;7(9):e45851.
Immunopathogenesis studies employing West Nile virus (WNV) mice model are important for the development of antivirals and vaccines against WNV. Since antibodies produced in mice early during WNV infection are essential for clearing virus from the periphery, it is important to detect early and persistent anti-WNV antibodies. ELISA and plaque reduction neutralization tests are traditionally used for detection of anti-WNV antibodies and WNV-neutralizing antibodies, respectively. Although these assays are sensitive and specific, they are expensive and time consuming. Microsphere immunoassays (MIA) are sensitive, specific, allow for high throughput, are cost effective, require less time to perform than other methods, and require low serum volumes. Several assay parameters such as serum heat-inactivation (HI) and dilution can alter WNV MIA sensitivity. We examined the effect of these parameters on WNV E-protein MIA (WNV E-MIA) for the enhanced detection of anti-WNV IgM and IgG antibodies. WNV E-MIA was conducted using serial dilutions of HI and non-HI (NHI) serum collected at various time points from mice inoculated with WNV. HI significantly enhanced detection of IgM and IgG antibodies as compared to NHI serum. WNV IgM and IgG antibodies in HI sera were detected earlier at day 3 and IgM antibodies persisted up to day 24 after infection. HI serum at 1∶20 dilution was found to be optimal for detection of both IgM and IgG antibodies as compared to higher-serum dilutions. Further, addition of exogenous complement to the HI serum decreased the WNV E-MIA sensitivity. These results suggest that serum-HI and optimal dilution enhance WNV E-MIA sensitivity by eliminating the complement interference, thereby detecting low-titer anti-WNV antibodies during early and late phases of infection. This improved MIA can also be readily employed for detection of low-titer antibodies for detection of other infectious agents and host proteins.
PMCID: PMC3457982  PMID: 23049879
3.  Development of a Fluorescent-Microsphere Immunoassay for Detection of Antibodies Specific to Equine Arteritis Virus and Comparison with the Virus Neutralization Test▿  
The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP51-255], M1-162, and N1-110), as well as partial sequences of these structural proteins (GP51-116, GP575-112, GP555-98, M88-162, and N1-69) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP555-98 protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP555-98 MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP555-98 MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.
PMCID: PMC2223870  PMID: 18032597
4.  Insulin-like growth factor-I receptor in proliferation and motility of pancreatic cancer 
AIM: To develop a molecular therapy for pancreatic cancer, the insulin-like growth factor-I (IGF-I) signaling pathway was analyzed.
METHODS: Pancreatic cancer cell lines (MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4) were cultured in media with 10 mL/L fetal bovine serum. Western blotting analysis was performed to clarify the expression of IGF-I receptor (IGF-IR). Picropodophyllin (PPP), a specific inhibitor of IGF-IR, LY294002, a specific inhibitor of phosphatidylinositol 3 kinase (PI3K), and PD98059, a specific inhibitor of mitogen-activated protein kinase, were added to the media. After 72 h, a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay was performed to analyze cell proliferation. A wound assay was performed to analyze cell motility with hematoxylin and eosin (HE) staining 48 h after addition of each inhibitor.
RESULTS: All cell lines clearly expressed not only IGF-IR but also phosphorylated IGF-IR. PPP significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 36.9% ± 2.4% (mean ± SD), 30.9% ± 5.5%, 23.8% ± 3.9%, 37.1% ± 5.3%, 10.4% ± 4.5%, 52.5% ± 4.5% and 22.6% ± 0.4%, at 2 μmol/L, respectively (P < 0.05). LY294002 significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 44.4% ± 7.6%, 32.9% ± 8.2%, 53.9% ± 8.0%, 52.8% ± 4.0%, 32.3% ± 4.2%, 51.8% ± 4.5%, and 30.6% ± 9.4%, at 50 μmol/L, respectively (P < 0.05). PD98059 did not significantly suppress cell proliferation. PPP at 2 μmol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 3.0% ± 0.2%, 0%, 0%, 2.0% ± 0.1%, 5.0% ± 0.2%, 3.0% ± 0.1%, and 5.0% ± 0.2%, respectively (P < 0.05). LY294002 at 50 μmol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 to 3.0% ± 0.2%, 0%, 3.0% ± 0.2%, 0%, 0%, 0% and 3% ± 0.1%, respectively (P < 0.05). PD980509 at 20 μmol/L did not suppress motility. Cells were observed by microscopy to analyze the morphological changes induced by the inhibitors. Cells in medium treated with 2 μmol/L PPP or 50 μmol/L LY294002 had pyknotic nuclei, whereas those in medium with 20 μmol/L PD98059 did not show apoptosis.
CONCLUSION: IGF-IR and PI3K are good candidates for molecular therapy of pancreatic cancer.
PMCID: PMC2856825  PMID: 20397262
Insulin-like growth factor-I receptor; Phosphatidylinositol 3 kinase; Pancreatic neoplasms
5.  Effects of lorglumide on growth and invasion of human pancreatic cancer cell line Mia PaCa-2 in vitro through the cholecystokinin-cholecystokinin-1 receptor pathway 
Background: Cholecystokinin (CCK) has been found to be a growth stimulant through its special receptor pathway, especially for gastrointestinal malignancies. Although the CCK-1 receptor has been shown to be highly expressed in resected human pancreatic cancer samples, its role is less clear.
Objective: The aim of this in vitro study was to investigate the CCK-1 receptor expression and the function of the CCK-CCK-1 receptor pathway in the human pancreatic adenocarcinoma cell line, Mia PaCa-2.
Methods: The expression of the CCK-1 receptor in Mia PaCa-2 cells was detected by reverse-transcriptase polymerase chain reaction and flow cytometry. CCK-1 receptor agonist CCK-8S (the major transmitter form of CCK) and antagonist lorglumide were cultured respectively with Mia PaCa-2. Three groups were created for this study: CCK-8S group (Mia PaCa-2 cells treated with CCK-8S), lorglumide group (Mia PaCa-2 cells treated with lorglumide), and the control group (Mia PaCa-2 cells alone). Investigators were blinded to group designation. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry were used to detect the cell growth, cell cycle, and apoptosis. Apoptosis index rate was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. Cell invasion ability was observed by invasion assay. Expression of matrix metalloproteinase-2 (MMP-2) was measured by Western blotting.
Results: Mia PaCa-2 cells were found to express the CCK-1 receptor. Compared with the control group (70.2% [1.5%]), CCK-8S was associated with significant mean (SD) cell proliferation (85.1% [1.7%]; P = 0.039), and the ratio in the S stage of the cell cycle increased significantly (50.5% [1.7%] vs 42.2% [1.4%]; P = 0.021). CCK-8S was also associated with increased Mia PaCa-2 cell invasion ability (123.8 [1.7] vs 102.1 [5.8]; P = 0.005 vs control). Compared with the control group, lorglumide was associated with significantly inhibited cell growth (52.1% [1.8%]; P = 0.002) and cell invasion (77.6% [1.2%]; P = 0.003). Lorglumide also induced G0/G1 cell cycle arrest and apoptosis (27.1% [3–5%] vs 3–7% [0.6%]; P = 0.003 vs control). The change of invasion ability appeared to be mediated by MMP-2 expression, which was upregulated by CCK-8S and downregulated by lorglumide.
Conclusion: The findings of this in vitro study suggest that CCK may exert a trophic action on the Mia PaCa-2 cell line, while lorglumide inhibited the cell growth and invasion.
PMCID: PMC3969616  PMID: 24688146
cholecystokinin; CCK-1 receptor; pancreatic cancer; apoptosis; MMP-2
6.  Validation of a Microsphere-Based Immunoassay for Detection of Anti-West Nile Virus and Anti-St. Louis Encephalitis Virus Immunoglobulin M Antibodies▿  
A microsphere-based immunoassay (MIA) was previously developed that is capable of determining the presence of anti-West Nile (WN) virus or anti-St. Louis encephalitis (SLE) virus immunoglobulin M (IgM) antibodies in human serum or cerebrospinal fluid. The original data set on which the classification rules were based comprised 491 serum specimens obtained from the serum bank at the Division of Vector-Borne Infectious Diseases of the Centers for Disease Control and Prevention (DVBID). The classification rules were used to provide a result and to determine whether confirmatory testing was necessary for a given sample. A validation study was coordinated between the DVBID and five state health laboratories to determine (i) the reproducibility of the test between different laboratories, (ii) the correlation between the IgM-enzyme-linked immunosorbent assay (MAC-ELISA) and the MIA, and (iii) whether the initial nonspecific parameters could be refined to reduce the volume of confirmatory testing. Laboratorians were trained in the method, and reagents and data analysis software developed at the DVBID were shipped to each validating laboratory. Validating laboratories performed tests on approximately 200 samples obtained from their individual states, the collections of which comprised approximately equal numbers of WN virus-positive and -negative samples, as determined by MAC-ELISA. In addition, 377 samples submitted to the DVBID for arbovirus testing were analyzed using the MIA and MAC-ELISA at the DVBID only. For the specimens tested at both the state and the DVBID laboratories, a correlation of results indicated that the technology is readily transferable between laboratories. The detection of IgM antibodies to WN virus was more consistent than detection of IgM antibodies to SLE virus. Some changes were made to the analysis software that resulted in an improved accuracy of diagnosis.
PMCID: PMC2043310  PMID: 17609393
7.  Mesothelin confers pancreatic cancer cell resistance to TNF-α-induced apoptosis through Akt/PI3K/NF-κB activation and IL-6/Mcl-1 overexpression 
Molecular Cancer  2011;10:106.
Previous studies showed that mesothelin (MSLN) plays important roles in survival of pancreatic cancer (PC) cells under anchorage dependent/independent conditions as well as resistance to chemotherapy. The recent success of intratumorally-injected adeno-encoded, chemo/radiation-inducible-promoter driven hTNF-α, (TNFerade) + gemcitabine in pre-clinical models of PC have renewed interest in use of TNF-α as a therapeutic component. To help find additional factors which might affect the therapy, we examined the resistance of MSLN-overexpressing pancreatic cancer cell lines to TNF-α-induced growth inhibition/apoptosis.
Stable MSLN overexpressing MIA PaCa-2 cells (MIA-MSLN), stable MSLN-silenced AsPC-1 cells (AsPC-shMSLN) and other pancreatic cells (MIA-PaCa2, Panc 28, Capan-1, BxPC3, PL 45, Hs 766T, AsPC-1, Capan-2, Panc 48) were used. NF-κB activation was examined by western blots and luciferase reporter assay. TNF-α induced growth inhibition/apoptosis was measured by MTT, TUNEL assay and caspase activation. IL-6 was measured using luminex based assay.
Compared to low endogenous MSLN-expressing MIA PaCa-2 and Panc 28 cells, high endogenous MSLN-expressing Capan-1, BxPC3, PL 45, Hs 766T, AsPC-1, Capan-2, Panc 48 cells were resistant to TNF-α induced growth inhibition. Stable MSLN overexpressing MIA-PaCa2 cells (MIA-MSLN) were resistant to TNF-α-induced apoptosis while stable MSLN-silenced AsPC1 cells (AsPC-shMSLN) were sensitive. Interestingly, TNF-α-treated MIA-MSLN cells showed increased cell cycle progression and cyclin A induction, both of which were reversed by caspase inhibition. We further found that MIA-MSLN cells showed increased expression of anti-apoptotic Bcl-XL and Mcl-1; deactivated (p-Ser75) BAD, and activated (p-Ser70) Bcl-2. Constitutively activated NF-κB and Akt were evident in MIA-MSLN cells that could be suppressed by MSLN siRNA with a resultant increase in sensitivity of TNF-α induced apoptosis. Blocking NF-κB using IKK inhibitor wedelolactone also increased sensitivity to TNF-α-mediated cytotoxicity with concomitant decrease in Mcl-1. Blocking Akt using PI3K inhibitor also had a likewise effect presumably affecting cell cycle. MIA-MSLN cells produced increased IL-6 and were increased furthermore by TNF-α treatment. SiRNA-silencing of IL-6 increased TNF-α sensitivity of MIA-MSLN cells.
Our study delineates a MSLN-Akt-NF-κB-IL-6-Mcl-1 survival axis that may be operative in PC cells, and might help cancer cells' survival in the highly inflammatory milieu evident in PC. Further, for the success of TNFerade + gemcitabine to be successful, we feel the simultaneous inhibition of components of this axis is also essential.
PMCID: PMC3175472  PMID: 21880146
Pancreatic cancer; Mesothelin; TNF-α; Apoptosis
8.  The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines 
The anti-tumor activity of glucose analogs 2-deoxy-glucose (2-DG) and D-allose was investigated alone or in combination with p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 or platinum analogs as a strategy to pharmacologically target glycolytic tumor phenotypes.
Hypoxia inducible factor-1 alpha (HIF-1α) protein accumulation in pancreatic cell lines treated with SB202190 alone and in combination with glucose analogs was analyzed by Western blot. HIF-1α transcriptional activity was measured in MIA PaCa-2 cells stably transfected with a hypoxia response element luciferase reporter following treatment with glucose analogs alone, and in combination with SB202190. Induction of cleaved poly(ADP-ribose) polymerase (PARP) was measured by Western blot in the MIA PaCa-2 cells. In vitro anti-proliferative activity of 2-DG and D-allose alone, or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines), or with SB202190 were investigated using the MTT assay.
SB202190 decreased HIF-1α protein accumulation and transcriptional activity. 2-DG demonstrated greater anti-proliferative activity than D-allose. Pre-treatment with SB202190 enhanced activity of both 2-DG and D-allose in MIA PaCa-2, BxPC-3, ASPC-1, and SK-OV-3 cells. The combination of D-allose and platinum agents was additive to moderately synergistic in all but the OVCAR-3 and HEY cells. SB202190 pre-treatment further enhanced activity of D-allose and 2-DG with platinum agents in most cell lines investigated.
SB202190 induced sensitization of tumor cells to 2-DG and D-allose may be partially mediated by inhibition of HIF-1α activity. Combining glucose analogs and p38 MAPK inhibitors with chemotherapy may be an effective approach to target glycolytic tumor phenotypes.
PMCID: PMC4391305  PMID: 25888489
2-deoxy-glucose; D-allose; Hypoxia inducible factor 1α; Pancreatic cancer; Ovarian cancer; p38 mitogen activated protein kinase
9.  Targeting cMET with INC280 impairs tumour growth and improves efficacy of gemcitabine in a pancreatic cancer model 
BMC Cancer  2015;15:71.
Expression and activation of the cMET receptor have been implicated in tumor progression and resistance to chemotherapy in human pancreatic cancer. In this regard we assessed the effects of targeting cMET in pancreatic cancer models in vitro and in vivo.
Human (L3.6pl, BxP3, HPAF-II, MiaPaCa2) and murine (Panc02) pancreatic cancer cell lines, endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) were used for the experiments. Furthermore, the human pancreatic cancer cell line MiaPaCa2 with acquired resistance to gemcitabine was employed (MiaPaCa2(G250)). For targeting the cMET receptor, the oral available, ATP-competitive inhibitor INC280 was used. Effects of cMET inhibition on cancer and stromal cells were determined by growth assays, western blotting, motility assays and ELISA. Moreover, orthotopic xenogeneic and syngeneic mouse (BALB-C nu/nu; C57BL/6) models were used to assess in vivo efficacy of targeting cMET alone and in combination with gemcitabine.
Treatment with INC280 impairs activation of signaling intermediates in pancreatic cancer cells and ECs, particularly when cells were stimulated with hepatocyte growth factor (HGF). Moreover, motility of cancer cells and ECs in response to HGF was reduced upon treatment with INC280. Only minor effects on VSMCs were detected. Interestingly, MiaPaCa2(G250) showed an increase in cMET expression and cMET inhibition abrogated HGF-induced effects on growth, motility and signaling as well as DFX-hypoxia HIF-1alpha and MDR-1 expression in vitro. In vivo, therapy with INC280 alone led to inhibition of orthotopic tumor growth in xenogeneic and syngeneic models. Similar to in vitro results, cMET expression was increased upon treatment with gemcitabine, and combination of the cMET inhibitor with gemcitabine improved anti-neoplastic capacity in an orthotopic syngeneic model. Immunohistochemical analysis revealed a significant inhibition of tumor cell proliferation (Ki67) and tumor vascularization (CD31). Finally, combination of gemcitabine with INC280 significantly prolonged survival in the orthotopic syngeneic tumor model even when treatment with the cMET inhibitor was initiated at an advanced stage of disease.
These data provide evidence that targeting cMET in combination with gemcitabine may be effective in human pancreatic cancer and warrants further clinical evaluation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1064-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4340491  PMID: 25884642
Pancreatic cancer; cMET; Resistance; Survival
10.  Antagonistic analogs of growth hormone-releasing hormone increase the efficacy of treatment of triple negative breast cancer in nude mice with doxorubicin; A preclinical study 
Oncoscience  2014;1(10):665-673.
This study evaluated the effects of an antagonistic analog of growth hormone-releasing hormone, MIA-602, on tumor growth, response to doxorubicin, expression of drug resistance genes, and efflux pump function in human triple negative breast cancers.
HCC1806 (doxorubicin-sensitive) and MX-1 (doxorubicin-resistant), cell lines were xenografted into nude mice and treated with MIA-602, doxorubicin, or their combination. Tumors were evaluated for changes in volume and the expression of the drug resistance genes MDR1 and NANOG. In-vitro cell culture assays were used to analyze the effect of MIA-602 on efflux pump function.
Therapy with MIA-602 significantly reduced tumor growth and enhanced the efficacy of doxorubicin in both cell lines. Control HCC1806 tumors grew by 435%, while the volume of tumors treated with MIA-602 enlarged by 172.2% and with doxorubicin by 201.6%. Treatment with the combination of MIA-602 and doxorubicin resulted in an increase in volume of only 76.2%. Control MX-1 tumors grew by 907%, while tumors treated with MIA-602 enlarged by 434.8% and with doxorubicin by 815%. The combination of MIA-602 and doxorubicin reduced the increase in tumor volume to 256%. Treatment with MIA-602 lowered the level of growth hormone-releasing hormone and growth hormone-releasing hormone receptors and significantly reduced the expression of multidrug resistance (MDR1) gene and the drug resistance regulator NANOG. MIA-602 also suppressed efflux pump function in both cell lines.
We conclude that treatment of triple negative breast cancers with growth hormone-releasing hormone antagonists reduces tumor growth and potentiates the effects of cytotoxic therapy by nullifying drug resistance.
PMCID: PMC4278278  PMID: 25593995
triple negative breast cancer; drug resistance; combination therapy; growth-hormone-releasing hormone; antagonist; GHRH analogs
11.  Role of intracellular and extracellular annexin A1 in migration and invasion of human pancreatic carcinoma cells 
BMC Cancer  2014;14:961.
Annexin A1 (ANXA1), a 37 kDa multifunctional protein, is over-expressed in tissues from patients of pancreatic carcinoma (PC) where the protein seems to be associated with malignant transformation and poor prognosis.
The expression and localization of ANXA1 in MIA PaCa-2, PANC-1, BxPC-3 and CAPAN-2 cells were detected by Western Blotting and Immunofluorescence assay. Expression and activation of Formyl Peptide Receptors (FPRs) were shown through flow cytometry/PCR and FURA assay, respectively. To investigate the role of ANXA1 in PC cell migration and invasion, we performed in vitro wound-healing and matrigel invasion assays.
In all the analyzed PC cell lines, a huge expression and a variable localization of ANXA1 in sub-cellular compartments were observed. We confirmed the less aggressive phenotype of BxPC-3 and CAPAN-2 compared with PANC-1 and MIA PaCa-2 cells, through the evaluation of Epithelial-Mesenchymal Transition (EMT) markers. Then, we tested MIA PaCa-2 and PANC-1 cell migration and invasiveness rate which was inhibited by specific ANXA1 siRNAs. Both the cell lines expressed FPR-1 and -2. Ac2-26, an ANXA1 mimetic peptide, induced intracellular calcium release, consistent with FPR activation, and significantly increased cell migration/invasion rate. Interestingly, in MIA PaCa-2 cells we found a cleaved form of ANXA1 (33 kDa) that localizes at cellular membranes and is secreted outside the cells, as confirmed by MS analysis. The importance of the secreted form of ANXA1 in cellular motility was confirmed by the administration of ANXA1 blocking antibody that inhibited migration and invasion rate in MIA PaCa-2 but not in PANC-1 cells that lack the 33 kDa ANXA1 form and show a lower degree of invasiveness. Finally, the treatment of PANC-1 cells with MIA PaCa-2 supernatants significantly increased the migration rate of these cells.
This study provides new insights on the role of ANXA1 protein in PC progression. Our findings suggest that ANXA1 protein could regulate metastasis by favouring cell migration/invasion intracellularly, as cytoskeleton remodelling factor, and extracellularly like FPR ligand.
PMCID: PMC4301448  PMID: 25510623
Annexin A1; Pancreatic cancer; Formyl peptide receptors; Cell migration; Cell invasion
12.  Targeting microspheres and cells to polyethylene glycol-modified biological surfaces 
It has previously been demonstrated that damaged arterial tissue can be acutely modified with protein-reactive polyethylene glycol (PEG) to block undesirable platelet deposition. This concept might be expanded by employing PEG-biotin and its strong interaction with avidin for site-specific targeted delivery. Toward this end, cultured endothelial cells (ECs) were surface modified with PEG-biotin and the available biotin was quantified with flow cytometry. NeutrAvidin-coated microspheres and PEG-biotin modified ECs with NeutrAvidin as a bridging molecule were delivered under arterial shear stress to PEG-biotin modified ECs on a coverslip as well as scrape-damaged bovine carotid arteries. After incubation with a 10 mM solution for 1 min, 8 × 107 PEG-biotin molecules/EC were found and persisted for up to 120 h. Perfused microspheres adhered to NHS-PEG-biotin treated bovine carotid arteries with 60 ± 16 microspheres/mm2 versus 11 ± 4 microspheres/mm2 for control arteries (p < 0.015). Similarly, 22 ± 5 targeted ECs/mm2 adhered to NHS-PEG-biotin treated bovine carotid arteries versus 6 ± 2 ECs/mm2 for control arteries (p < 0.01). The targeting strategy demonstrated here might ultimately find application for drug delivery, gene therapy, or cell therapy where localization to specific labeled vascular regions is desired following catheter-based or surgical procedures.
PMCID: PMC2873022  PMID: 17177289
polyethylene glycol; targeted delivery; surface modification; biotin; avidin
13.  Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system 
The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR) that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG) phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR.
A liposome detection reagent was prepared, which consisted of a population of liposomes ~120 nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA) in human serum. This ILPCR assay exhibited a linear dose–response curve from 10-10 M to 10-16 M CEA. Within this range the assay coefficient of variance was <6 % for repeatability and <2 % for reproducibility. The assay detection limit was 13 fg/mL, which is 1,500-times more sensitive than current clinical assays for CEA. An ILPCR assay to quantify HIV-1 p24 core protein in buffer was also developed.
The ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the liposomes allows nonspecific DNA in the assay medium to be degraded with DNase I prior to quantification of the encapsulated reporter by PCR, which reduces false-positive results and improves quantitative accuracy. The ability to encapsulate multiple reporters per liposome also helps overcome the effect of polymerase inhibitors present in biological specimens. Finally, the biotin-labeled liposome detection reagent can be coupled through a NeutrAvidin bridge to a multitude of biotin-labeled probes, making ILPCR a highly generic assay system.
PMCID: PMC3466442  PMID: 22726242
14.  Serum Markers to Detect Metastatic Uveal Melanoma 
Anticancer research  2007;27(4A):1897-1900.
Osteopontin is over expressed in metastatic uveal melanoma (UM). S-100β and melanoma-inhibitory activity (MIA) serum levels are elevated in metastatic cutaneous melanoma. The ability of osteopontin, S-100β and MIA serum levels to be used as non-invasive markers for detecting metastatic UM was tested.
Materials and Methods
Osteopontin, S-100β, and MIA levels were measured by ELISA assays in 18 patients with metastatic UM and in 38 patients who were disease-free (DF) for at least 10 years after treatment of the primary tumor. Paired serum samples from 8 patients before and after development of metastasis were analyzed. Forty-four healthy controls (C) were compared to the other two groups.
Serum osteopontin, MIA, and S-100β levels were significantly higher in patients with metastatic UM as compared to patients who were DF for at least 10 years after treatment (p=0.0001) or with age matched controls. Serum osteopontin, MIA, and S-100β levels were significantly higher (p<0.005) after metastasis formation than before the clinical detection of metastasis in the 8 patients. Receiver operator characteristic analysis was performed for metastatic patients vs. DF and vs. C and the area under the curve was calculated for each marker and for the combination of the 3 markers which was 91%.
Elevated serum osteopontin, MIA and S-100β levels correlate with metastatic UM to the liver. When used in combination, these markers provide a highly sensitive and specific method to detect hepatic metastases and therefore provide for earlier therapeutic intervention that can prolong survival.
PMCID: PMC1986737  PMID: 17649791
Uveal melanoma; metastasis; markers; osteopontin; S-100; MIA
15.  MIA is a potential biomarker for tumour load in neurofibromatosis type 1 
BMC Medicine  2011;9:82.
Neurofibromatosis type 1 (NF1) is a frequent genetic disease characterized by multiple benign tumours with increased risk for malignancy. There is currently no biomarker for tumour load in NF1 patients.
In situ hybridization and quantitative real-time polymerase reaction were applied to investigate expression of cartilage-specific genes in mice bearing conditional inactivation of NF1 in the developing limbs. These mice do not develop tumours but recapitulate aspects of NF1 bone dysplasia, including deregulation of cartilage differentiation. It has been recently shown that NF1 tumours require for their growth the master regulator of cartilage differentiation SOX9. We thus hypothesized that some of the cartilage-specific genes deregulated in an Nf1Prx1 mouse model might prove to be relevant biomarkers of NF1 tumours. We tested this hypothesis by analyzing expression of the SOX9 target gene product melanoma-inhibitory activity/cd-rap (MIA) in tumour and serum samples of NF1 patients.
Increased expression of Mia was found in Nf1-deficient cartilage in mice. In humans, MIA was expressed in all NF1-related tumours and its serum levels were significantly higher in NF1 patients than in healthy controls. Among NF1 patients, MIA serum levels were significantly higher in those with plexiform neurofibromas and in those with large number of cutaneous (> 1,000) or subcutaneous (> 100) neurofibromas than in patients without such tumours. Most notably, MIA serum levels correlated significantly with internal tumour burden.
MIA is a potential serum biomarker of tumour load in NF1 patients which could be useful in following the disease course and monitoring the efficacy of therapies.
PMCID: PMC3224593  PMID: 21726432
16.  Maternal Immune Activation Alters Fetal Brain Development through Interleukin-6 
Schizophrenia and autism are thought to result from the interaction between a susceptibility genotype and environmental risk factors. The offspring of women who experience infection while pregnant have an increased risk for these disorders. Maternal immune activation (MIA) in pregnant rodents produces offspring with abnormalities in behavior, histology, and gene expression that are reminiscent of schizophrenia and autism, making MIA a useful model of the disorders. However, the mechanism by which MIA causes long-term behavioral deficits in the offspring is unknown. Here we show that the cytokine interleukin-6 (IL-6) is critical for mediating the behavioral and transcriptional changes in the offspring. A single maternal injection of IL-6 on day 12.5 of mouse pregnancy causes prepulse inhibition (PPI) and latent inhibition (LI) deficits in the adult offspring. Moreover, coadministration of an anti-IL-6 antibody in the poly(I:C) model of MIA prevents the PPI, LI, and exploratory and social deficits caused by poly(I:C) and normalizes the associated changes in gene expression in the brains of adult offspring. Finally, MIA in IL-6 knock-out mice does not result in several of the behavioral changes seen in the offspring of wild-type mice after MIA. The identification of IL-6 as a key intermediary should aid in the molecular dissection of the pathways whereby MIA alters fetal brain development, which can shed new light on the pathophysiological mechanisms that predispose to schizophrenia and autism.
PMCID: PMC2387067  PMID: 17913903
schizophrenia; autism; cytokine; poly(I:C); maternal immune activation; IL-6; influenza
17.  Mamma Mia: A Feasibility Study of a Web-Based Intervention to Reduce the Risk of Postpartum Depression and Enhance Subjective Well-Being 
JMIR Research Protocols  2013;2(2):e29.
Currently, 10-15% of women giving birth suffer from symptoms of postpartum depression. Due to a lack of knowledge of this condition and the stigma associated with it, as well as few treatment options, a large proportion of postpartum women with depression remain untreated. Internet-based interventions have been found effective in treating depression, anxiety, phobias, and addictions. Hence, we developed such program (“Mamma Mia”) with the aim of reducing the risk for postpartum depression and enhance subjective well-being. Mamma Mia is based on positive psychology, metacognitive therapy, and couples therapy. It starts in gestational week 22, and lasts until 6 months after birth. During pregnancy, Mamma Mia is delivered weekly (every Monday). After birth, Mamma Mia is delivered three times per week for six weeks. The remaining weeks, the program is delivered more sporadically. In total, Mamma Mia consists of 44 sessions. The program is individualized, interactive, and tunneled (ie, the user is guided through the program in a pre-determined manner).
The purpose of the present study was to pilot test the intervention in order to assess the feasibility and acceptance among program users.
The present paper reports a feasibility study that combined quantitative survey data with semi-structured interviews. Participants (N=103) were recruited via hospitals, well-baby clinics, and Facebook. Due to time constraint in completing the current study, our results were based on participation in one of the two phases: pregnancy or maternity. Participants in the pregnancy phase were surveyed 4 and 8 weeks after intervention enrollment, and participants in the postnatal phase were surveyed 2 and 4 weeks after intervention enrollment. The survey assessed perceived usefulness, ease-of-use, credibility, and unobtrusiveness. All measures were filled in by participants at both measurement occasions. Data were analyzed by running descriptives and frequencies with corresponding percentages. Binomial tests were carried out to investigate whether demographics differed significantly from a 50/50 distribution. Paired sample t tests were used to examine differences between time 1 and 2. Four participants were interviewed in the qualitative follow-up study, where they were given the opportunity to address and elaborate on similar aspects as assessed in the survey.
More than two-thirds of users found Mamma Mia to be of high quality and would recommend Mamma Mia to others. By far, most also found the amount of information and frequency of the intervention schedule to be appropriate. Mamma Mia was perceived as a user-friendly and credible intervention.
Overall, the user acceptance of Mamma Mia was good and our findings add to the feasibility of the program. The effect of Mamma Mia on depression and subjective well-being will be evaluated in a large randomized controlled trial, and if found to be effective, Mamma Mia could serve as a low-threshold prevention program.
PMCID: PMC3742405  PMID: 23939459
pilot project; Internet; early intervention; depression postpartum; health promotion; well-being; eHealth
18.  Excess glucose induces hypoxia-inducible factor-1α in pancreatic cancer cells and stimulates glucose metabolism and cell migration 
Cancer Biology & Therapy  2013;14(5):428-435.
Pancreatic cancer patients frequently show hyperglycemia, but it is uncertain whether hyperglycemia stimulates pancreatic cancer cells. We have investigated whether excess glucose induces hypoxia-inducible factor-1α (HIF-1α) and stimulates glucose metabolism and cell migration in pancreatic cancer cells. We studied wild-type (wt) MiaPaCa2 pancreatic cancer cells and a MiaPaCa2 subline (namely si-MiaPaCa2) that had HIF-1α-specific small interfering RNA. Wt-MiaPaCa2 cells are known to be HIF-1α-positive in hypoxia and HIF-1α-negative in normoxia, whereas si-MiaPaCa2 cells are devoid of HIF-1α in both normoxia and hypoxia. We incubated these cells with different amounts of glucose and determined HIF-1α mRNA and protein by real-time polymerase chain reaction and western blotting. We determined glucose consumption, lactate production and intracellular hexokinase-II and ATP to assess glucose metabolisms and determined pyruvate dehydrogenase kinase-1, reactive oxygen species and fumarate to assess mitochondrial activities. Further, we studied cell migration using a Boyden chamber. Excess glucose (16.7−22.2mM) increased HIF-1α in hypoxic wt-MiaPaCa2 cells. HIF-1α expression increased ATP contents and inhibited mitochondrial activities. Extracellular glucose and hypoxia stimulated glucose metabolisms independent of HIF-1α. Excess glucose stimulated the migration of wt- and si-MiaPaCa2 cells in both normoxia and hypoxia. Thus, glucose stimulated cell migration independent of HIF-1α. Nevertheless, hypoxic wt-MiaPaCa2 cells showed greater migrating ability than their si-MiaPaCa2 counterparts. We conclude that (1) excess glucose increases HIF-1α and ATP in hypoxic wt-MiaPaCa2 cells, (2) extracellular glucose and hypoxia regulate glucose metabolisms independent of HIF-1α and (3) glucose stimulates cell migration by mechanisms that are both dependent on HIF-1α and independent of it.
PMCID: PMC3672187  PMID: 23377827
pancreatic cancer; hypoxia-inducible factor-1; glucose; glycolysis; cell migration; hexokinase-II; reactive oxygen species
19.  Hypoxia-inducible factor-1 (HIF-1) is involved in the regulation of hypoxia-stimulated expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and MCP-5 (Ccl12) in astrocytes 
Neuroinflammation has been implicated in various brain pathologies characterized by hypoxia and ischemia. Astroglia play an important role in the initiation and propagation of hypoxia/ischemia-induced inflammation by secreting inflammatory chemokines that attract neutrophils and monocytes into the brain. However, triggers of chemokine up-regulation by hypoxia/ischemia in these cells are poorly understood. Hypoxia-inducible factor-1 (HIF-1) is a dimeric transcriptional factor consisting of HIF-1α and HIF-1β subunits. HIF-1 binds to HIF-1-binding sites in the target genes and activates their transcription. We have recently shown that hypoxia-induced expression of IL-1β in astrocytes is mediated by HIF-1α. In this study, we demonstrate the role of HIF-1α in hypoxia-induced up-regulation of inflammatory chemokines, human monocyte chemoattractant protein-1 (MCP-1/CCL2) and mouse MCP-5 (Ccl12), in human and mouse astrocytes, respectively.
Primary fetal human astrocytes or mouse astrocytes generated from HIF-1α+/+ and HIF-1α+/- mice were subjected to hypoxia (<2% oxygen) or 125 μM CoCl2 for 4 h and 6 h, respectively. The expression of HIF-1α, MCP-1 and MCP-5 was determined by semi-quantitative RT-PCR, western blot or ELISA. The interaction of HIF-1α with a HIF-1-binding DNA sequence was examined by EMSA and supershift assay. HIF-1-binding sequence in the promoter of MCP-1 gene was cloned and transcriptional activation of MCP-1 by HIF-1α was analyzed by reporter gene assay.
Sequence analyses identified HIF-1-binding sites in the promoters of MCP-1 and MCP-5 genes. Both hypoxia and HIF-1α inducer, CoCl2, strongly up-regulated HIF-1α expression in astrocytes. Mouse HIF-1α+/- astrocytes had lower basal levels of HIF-1α and MCP-5 expression. The up-regulation of MCP-5 by hypoxia or CoCl2 in HIF-1α+/+ and HIF-1α+/- astrocytes was correlated with the levels of HIF-1α in cells. Both hypoxia and CoCl2 also up-regulated HIF-1α and MCP-1 expression in human astrocytes. EMSA assay demonstrated that HIF-1 activated by either hypoxia or CoCl2 binds to wild-type HIF-1-binding DNA sequence, but not the mutant sequence. Furthermore, reporter gene assay demonstrated that hypoxia markedly activated MCP-1 transcription but not the mutated MCP-1 promoter in transfected astrocytes.
These findings suggest that both MCP-1 and MCP-5 are HIF-1 target genes and that HIF-1α is involved in transcriptional induction of these two chemokines in astrocytes by hypoxia.
PMCID: PMC1872020  PMID: 17474992
20.  Effects of intratracheal administration of nuclear factor-kappaB decoy oligodeoxynucleotides on long-term cigarette smoke-induced lung inflammation and pathology in mice 
Respiratory Research  2009;10(1):79.
To determine if nuclear factor-κB (NF-κB) activation may be a key factor in lung inflammation and respiratory dysfunction, we investigated whether NF-κB can be blocked by intratracheal administration of NF-κB decoy oligodeoxynucleotides (ODNs), and whether decoy ODN-mediated NF-κB inhibition can prevent smoke-induced lung inflammation, respiratory dysfunction, and improve pathological alteration in the small airways and lung parenchyma in the long-term smoke-induced mouse model system. We also detected changes in transcriptional factors. In vivo, the transfection efficiency of NF-κB decoy ODNs to alveolar macrophages in BALF was measured by fluorescein isothiocyanate (FITC)-labeled NF-κB decoy ODNs and flow cytometry post intratracheal ODN administration. Pulmonary function was measured by pressure sensors, and pathological changes were assessed using histology and the pathological Mias software. NF-κB and activator protein 1(AP-1) activity was detected by the electrophoretic motility shift assay (EMSA). Mouse cytokine and chemokine pulmonary expression profiles were investigated by enzyme-linked immunosorbent assay (ELISA) in bronchoalveolar lavage fluid (BALF) and lung tissue homogenates, respectively, after repeated exposure to cigarette smoke. After 24 h, the percentage of transfected alveolar macrophages was 30.00 ± 3.30%. Analysis of respiratory function indicated that transfection of NF-κB decoy ODNs significantly impacted peak expiratory flow (PEF), and bronchoalveolar lavage cytology displayed evidence of decreased macrophage infiltration in airways compared to normal saline-treated or scramble NF-κB decoy ODNs smoke exposed mice. NF-κB decoy ODNs inhibited significantly level of macrophage inflammatory protein (MIP) 1α and monocyte chemoattractant protein 1(MCP-1) in lung homogenates compared to normal saline-treated smoke exposed mice. In contrast, these NF-κB decoy ODNs-treated mice showed significant increase in the level of tumor necrosis factor-α(TNF-α) and pro-MMP-9(pro-matrix metalloproteinase-9) in mice BALF. Further measurement revealed administration of NF-κB decoy ODNs did not prevent pathological changes. These findings indicate that NF-κB activation play an important role on the recruitment of macrophages and pulmonary dysfunction in smoke-induced chronic lung inflammation, and with the exception of NF-κB pathway, there might be complex mechanism governing molecular dynamics of pro-inflammatory cytokines expression and structural changes in small airways and pulmonary parenchyma in vivo.
PMCID: PMC2751757  PMID: 19706153
21.  Development of Robust and Standardized Cantilever Sensors Based on Biotin/Neutravidin Coupling for Antibody Detection 
Sensors (Basel, Switzerland)  2013;13(4):5273-5285.
A cantilever-based protein biosensor has been developed providing a customizable multilayer platform for the detection of antibodies. It consists of a biotin-terminated PEG layer pre-functionalized on the gold-coated cantilever surface, onto which NeutrAvidin is adsorbed through biotin/NeutrAvidin specific binding. NeutrAvidin is used as a bridge layer between the biotin-coated surface and the biotinylated biomolecules, such as biotinylated bovine serum albumin (biotinylated BSA), forming a multilayer sensor for direct antibody capture. The cantilever biosensor has been successfully applied to the detection of mouse anti-BSA (m-IgG) and sheep anti-BSA(s-IgG) antibodies. As expected, the average differential surface stress signals of about 5.7 ± 0.8 × 10−3 N/m are very similar for BSA/m-IgG and BSA/s-IgG binding, i.e., they are independent of the origin of the antibody. A statistic evaluation of 112 response curves confirms that the multilayer protein cantilever biosensor shows high reproducibility. As a control test, a biotinylated maltose binding protein was used for detecting specificity of IgG, the result shows a signal of bBSA layer in response to antibody is 5.8 × 10−3 N/m compared to bMBP. The pre-functionalized biotin/PEG cantilever surface is found to show a long shelf-life of at least 40 days and retains its responsivity of above 70% of the signal when stored in PBS buffer at 4 °C. The protein cantilever biosensor represents a rapid, label-free, sensitive and reliable detection technique for a real-time protein assay.
PMCID: PMC3673136  PMID: 23604028
cantilever; biosensor; protein; multilayer; NeutrAvidin; biotin
22.  Fast, Antigen-Saving Multiplex Immunoassay To Determine Levels and Avidity of Mouse Serum Antibodies to Pertussis, Diphtheria, and Tetanus Antigens ▿ † 
To enhance preclinical evaluation of serological immune responses to the individual diphtheria, tetanus, and pertussis (DTP) components of DTP combination vaccines, a fast hexavalent bead-based method was developed. This multiplex immunoassay (MIA) can simultaneously determine levels of specific mouse serum IgG antibodies to P antigens P.69 pertactin (P.69 Prn), filamentous hemagglutinin (FHA), pertussis toxin (Ptx), and combined fimbria type 2 and 3 antigens (Fim2/3) and to diphtheria toxin (Dtx) and tetanus toxin (TT) in a single well. The mouse DTP MIA was shown to be specific and sensitive and to correlate with the six single in-house enzyme-linked immunosorbent assays (ELISAs) for all antigens. Moreover, the MIA was expanded to include avidity measurements of DTP antigens in a multivalent manner. The sensitivities of the mouse DTP avidity MIA per antigen were comparable to those of the six individual in-house avidity ELISAs, and good correlations between IgG concentrations obtained by both methods for all antigens tested were shown. The regular and avidity mouse DTP MIAs were reproducible, with good intra- and interassay coefficients of variability (CV) for all antigens. Finally, the usefulness of the assay was demonstrated in a longitudinal study of the development and avidity maturation of specific IgG antibodies in mice having received different DTP vaccines. We conclude that the hexaplex mouse DTP MIA is a specific, sensitive, and high-throughput alternative for ELISA to investigate the quantity and quality of serological responses to DTP antigens in preclinical vaccine studies.
PMCID: PMC3122557  PMID: 21325488
23.  HOXB7 mRNA is overexpressed in pancreatic ductal adenocarcinomas and its knockdown induces cell cycle arrest and apoptosis 
BMC Cancer  2013;13:451.
Human homeobox genes encode nuclear proteins that act as transcription factors involved in the control of differentiation and proliferation. Currently, the role of these genes in development and tumor progression has been extensively studied. Recently, increased expression of HOXB7 homeobox gene (HOXB7) in pancreatic ductal adenocarcinomas (PDAC) was shown to correlate with an invasive phenotype, lymph node metastasis and worse survival outcomes, but no influence on cell proliferation or viability was detected. In the present study, the effects arising from the knockdown of HOXB7 in PDAC cell lines was investigated.
Real time quantitative PCR (qRT-PCR) (Taqman) was employed to assess HOXB7 mRNA expression in 29 PDAC, 6 metastatic tissues, 24 peritumoral tissues and two PDAC cell lines. siRNA was used to knockdown HOXB7 mRNA in the cell lines and its consequences on apoptosis rate and cell proliferation were measured by flow cytometry and MTT assay respectively.
Overexpression of HOXB7 mRNA was observed in the tumoral tissues and in the cell lines MIA PaCa-2 and Capan-1. HOXB7 knockdown elicited (1) an increase in the expression of the pro-apoptotic proteins BAX and BAD in both cell lines; (2) a decrease in the expression of the anti-apoptotic protein BCL-2 and in cyclin D1 and an increase in the number of apoptotic cells in the MIA PaCa-2 cell line; (3) accumulation of cell in sub-G1 phase in both cell lines; (4) the modulation of several biological processes, especially in MIA PaCa-2, such as proteasomal ubiquitin-dependent catabolic process and cell cycle.
The present study confirms the overexpression of HOXB7 mRNA expression in PDAC and demonstrates that decreasing its protein level by siRNA could significantly increase apoptosis and modulate several biological processes. HOXB7 might be a promising target for future therapies.
PMCID: PMC3851693  PMID: 24088503
Pancreatic ductal adenocarcinoma; Homeobox; HOXB7; siRNA; Gene expression
24.  Melanoma-inhibiting activity (MIA) mRNA is not exclusively transcribed in melanoma cells: low levels of MIA mRNA are present in various cell types and in peripheral blood 
British Journal of Cancer  1999;81(6):1066-1070.
The detection of minimal amounts of melanoma cells by tyrosinase reverse transcription polymerase chain reaction (RT-PCR) is seriously hampered by false negative reports in blood of melanoma patients with disseminated melanoma. Therefore, additional assays which make use of multiple melanoma markers are needed. It has been shown that introduction of multiple markers increases the sensitivity of detection. Melanoma inhibitory activity (MIA) is one such melanoma-specific candidate gene. To test the specificity of MIA PCR, we performed 30 and 60 cycles of PCR with two different sets of MIA specific primers on 19 melanoma and 16 non-melanoma cell lines. MIA mRNA was detected in 16 out of 19 melanoma cell lines and in seven out of 16 non-melanoma cell lines after 30 cycles of PCR. However, MIA mRNA could be detected in all cell lines after 60 cycles of PCR. Also, in 14 out of 14 blood samples of melanoma patients, five out of six blood samples of non-melanoma patients and in seven out of seven blood samples of healthy volunteers, MIA mRNA was detected after 60 cycles of PCR, whereas no MIA PCR product could be detected in any of the blood samples after 30 cycles of PCR. We conclude that low levels of MIA transcripts are present in various normal and neoplastic cell types. Therefore, MIA is not a suitable marker gene to facilitate the detection of minimal amounts of melanoma cells in blood or in target organs of the metastatic process. © 1999 Cancer Research Campaign
PMCID: PMC2362958  PMID: 10576666
MIA; melanoma; circulating cancer cells; RT-PCR
25.  Whole Exome Sequencing Identifies TTC7A Mutations for Combined Immunodeficiency with Intestinal Atresias 
Combined Immunodeficiency with Multiple Intestinal Atresias (CID-MIA) is a rare hereditary disease characterized by intestinal obstructions and profound immune defects.
We sought to determine the underlying genetic causes of CID-MIA by analyzing the exomic sequence of 5 patients and their healthy direct relatives from 5 unrelated families.
We performed whole exome sequencing on 5 CID-MIA patients and 10 healthy direct family members belonging to 5 unrelated families with CID-MIA. We also performed targeted Sanger sequencing for the candidate gene TTC7A on 3 additional CID-MIA patients.
Through analysis and comparison of the exomic sequence of the individuals from these 5 families, we identified biallelic damaging mutations in the TTC7A gene, for a total of 7 distinct mutations. Targeted TTC7A gene sequencing in 3 additional unrelated patients with CID-MIA revealed biallelic deleterious mutations in two of them, as well as an aberrant splice product in the third patient. Staining of normal thymus showed that the TTC7A protein is expressed in thymic epithelial cells as well as in thymocytes. Moreover, severe lymphoid depletion was observed in the thymus and peripheral lymphoid tissues from two patients with CID-MIA.
We identified deleterious mutations of the TTC7A gene in 8 unrelated patients with CID-MIA and demonstrated that the TTC7A protein is expressed in the thymus. Our results strongly suggest that TTC7A gene defects cause CID-MIA.
Clinical Implications
Damaging mutations in the gene TTC7A should be scrutinized in patients with CID-MIA. Characterization of the role of this protein in the immune system and intestinal development, as well as in thymic epithelial cells may have important therapeutic implications.
PMCID: PMC3759618  PMID: 23830146
Combined Immunodeficiency with Multiple Intestinal Atresias; Tetracopeptide Repeat Domain 7A; Whole Exome Sequencing; Thymus

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