Potential prognostic and diagnostic application of a novel monoclonal antibody against keratinocyte growth factor receptor.
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ABSTRACT:
SUBMITTER: Ceccarelli S
PROVIDER: S-EPMC4155171 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Three different genotypes of chikungunya virus (CHIKV) have been classified: East/Central/South African (ECSA), West African (WA), and Asian. Previously, a rapid immunochromatographic (IC) test detecting CHIKV E1-antigen showed high sensitivity for certain ECSA-genotype viruses, but this test showed poor performance against the Asian-genotype virus that is spreading in the American continents. We found that the reactivity of one monoclonal antibody (MAb) used in the IC rapid diagnostic test (RDT) is affected by a single amino acid substitution in E1. Therefore, we developed new MAbs that exhibited specific recognition of all three genotypes of CHIKV. METHODS:Using a combination of the newly generated MAbs, we developed a novel version of the IC RDT with improved sensitivity to Asian-genotype CHIKV. To evaluate the sensitivity, specificity, and cross-reactivity of the new version of the IC RDT, we first used CHIKV isolates and E1-pseudotyped lentiviral vectors. We then used clinical specimens obtained in Aruba in 2015 and in Bangladesh in 2017 for further evaluation of RDT sensitivity and specificity. Another alphavirus, sindbis virus (SINV), was used to test RDT cross-reactivity. RESULTS:The new version of the RDT detected Asian-genotype CHIKV at titers as low as 10^4 plaque-forming units per mL, a concentration that was below the limit of detection of the old version. The new RDT had sensitivity to the ECSA genotype that was comparable with that of the old version, yielding 92% (92 out of 100) sensitivity (95% confidence interval 85.0-95.9) and 100% (100 out of 100) specificity against a panel of 100 CHIKV-positive and 100 CHIKV-negative patient sera obtained in the 2017 outbreak in Bangladesh. CONCLUSIONS:Our newly developed CHIKV antigen-detecting RDT demonstrated high levels of sensitivity and lacked cross-reactivity against SINV. These results suggested that our new version of the CHIKV E1-antigen RDT is promising for use in areas in which the Asian and ECSA genotypes of CHIKV circulate. Further validation with large numbers of CHIKV-positive and -negative clinical samples is warranted. (323 words).
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Project description:The insulin-like growth factors (IGFs) signaling system has been shown to play important roles in neoplasia. The IGF receptor type 1 (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and there is substantial experimental and clinical evidence that targeting IGF-IR is a promising therapeutic strategy against cancer. It has been previously reported that a mouse monoclonal antibody (mAb), 4G11, blocked IGF-I binding to IGF-IR and downregulated the IGF-IR in MCF-7 cells. We cloned this antibody, constructed a human-mouse chimeric antibody, designated m590, and characterized it. The chimeric IgG1 m590 bound to cell-associated IGF-IR on NWT c43 stably transfected cells and MCF-7 breast cancer cells as efficiently as the parental murine antibody. Using purified IGF-IR extracellular domains, we found that both the chimeric m590 and the parental 4G11 antibodies bind to conformational epitopes on IGF-IR. Neither of these antibodies bound to the insulin receptor (IR) ectodomain. Furthermore, IgG1 m590 blocked the binding of IGF-I and IGF-II to IGF-IR, and inhibited both IGF-I and IGF-II induced phosphorylation of IGF-IR in MCF-7 cells. These results suggest that m590 could be an useful antibody in diagnosis and treatment of cancer, as well as a research tool.
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Project description:Fibroblast growth factor receptor-2 (FGFR-2) plays an important role in tumorigenesis. In thyroid cancer it has been observed a FGFR-2 down-modulation, but the role of this receptor has not been yet clarified. Therefore, we decided to examine the expression of both FGFR-2 isoform, FGFR-2-IIIb and FGFR-2-IIIc, in different histological thyroid variants such as hyperplasia, follicular adenoma and papillary carcinoma. Immunohistochemistry and quantitative Real-Time PCR analyses were performed on samples of hyperplasia, follicular adenoma and papillary carcinoma, compared with normal thyroid tissue. Thyroid hyperplasia did not show statistically significant reduction in FGFR-2 protein and mRNA levels. Interestingly, in both follicular adenoma and papillary carcinoma samples we observed a strongly reduced expression of both FGFR-2 isoforms. We speculate that FGFR-2 down-modulation might be an early event in thyroid carcinogenesis. Furthermore, we suggest the potential use of FGFR-2 as an early marker for thyroid cancer diagnosis.
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Project description:Airway epithelial cells are exposed to environmental toxicants that result in airway injury. Naphthalene (NA) causes site-selective damage to Clara cells in mouse distal airways. N-terminally truncated recombinant human keratinocyte growth factor (DeltaN23-KGF) protects against acute lung injury. The present study investigated whether or not DeltaN23-KGF protects against NA-induced acute Clara cell damage by measuring airway responses specifically and in order to identify underlying molecular mechanisms. Mice were treated with DeltaN23-KGF or PBS 33 h prior to injection of 200 mg.kg body weight(-1) NA. Lung function was analysed by head-out body plethysmography. Distal airways isolated by microdissection were assessed for cell permeability using ethidium homodimer-1. Immunohistochemistry of Clara cell-specific protein in conjunction with a physical dissector was used to quantify Clara cell numbers. RNA was isolated from frozen airways in order to analyse gene expression using quantitative RT-PCR. DeltaN23-KGF prevented NA-induced airflow limitation and Clara cell permeability, and resulted in twice as many Clara cells compared with PBS pre-treatment. DeltaN23-KGF-pre-treated mice exhibited increased expression of proliferating cell nuclear antigen mRNA. Cytochrome P(450) isoform 2F2, which converts NA into its toxic metabolite, was reduced by approximately 50%. The present results demonstrate that pre-treatment with N-terminally truncated recombinant human keratinocyte growth factor protects against naphthalene-induced injury. This suggests that N-terminally truncated recombinant human keratinocyte growth factor exerts its beneficial effect through a decrease in the expression of cytochrome P(450) isoform 2F2.
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Project description:Monoclonal antibodies (mAbs) have long provided powerful research tools for virologists to understand the mechanisms of virus entry into host cells and of antiviral immunity. Even so, commercial development of human (or humanized) mAbs for the prophylaxis, preemptive and acute treatment of viral infections has been slow. This is surprising, as new antibody discovery tools have increased the speed and precision with which potent neutralizing human antiviral mAbs can be identified. As longstanding barriers to antiviral mAb development, such as antigenic variability of circulating viral strains and the ability of viruses to undergo neutralization escape, are being overcome, deeper insight into the mechanisms of mAb action and engineering of effector functions are also improving the efficacy of antiviral mAbs. These successes, in both industrial and academic laboratories, coupled with ongoing changes in the biomedical and regulatory environments, herald an era when the commercial development of human antiviral mAb therapies will likely surge.
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Project description:IntroductionDuck circovirus (DuCV) infection is currently recognized as an important immunosuppressive disease in commercial duck flocks in China. Specific antibodies against DuCV viral proteins are required to improve diagnostic assays and understand the pathogenesis of DuCV infection.Methods and resultsTo generate DuCV-specific monoclonal antibodies (mAbs), a recombinant DuCV capsid protein without the first 36 N-terminal amino acids was produced in Escherichia coli. Using the recombinant protein as an immunogen, a mAb was developed that reacted specifically with the DuCV capsid protein, expressed in E. coli and baculovirus systems. Using homology modeling and recombinant truncated capsid proteins, the antibody-binding epitope was mapped within the region of 144IDKDGQIV151, which is exposed to solvent in the virion capsid model structure. To assess the applicability of the mAb to probe the native virus antigen, the murine macrophage cell line RAW267.4 was tested for DuCV replicative permissiveness. Immunofluorescence and Western blot analysis revealed that the mAb recognized the virus in infected cells and the viral antigen in tissue samples collected from clinically infected ducks.DiscussionThis mAb, combined with the in vitro culturing method, would have widespread applications in diagnosing and investigating DuCV pathogenesis.
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Project description:Human cytomegalovirus glycoprotein B (gB) represents a target for diagnosis and treatment in view of the role it plays in virus entry and spread. Nevertheless, to our knowledge, rare detection of a gB antigen has been reported in transplant patients and limited information is available about diagnostic gB monoclonal antibodies (mAbs). Our aim was to develop gB mAbs with diagnostic potential. Hydrophilic gB peptides (ST: amino acids 27-40, SH: amino acids 81-94) of favorable immunogenicity were synthesized and used to immunize BALB/c mice. Two mAbs, named ZJU-FH6 and ZJU-FE6, were generated by the hybridoma technique and limited serial dilution and then characterized by indirect ELISA, Western blotting, immunoprecipitation, and immunohistochemical staining. The mAbs displayed high titers of specific binding affinities for the ST and SH synthetic peptides at an mAb dilution of 1:60,000 and 1:240,000, respectively. Western blotting and immunoprecipitation indicated that these mAbs recognized both denatured and native gB of the Towne and AD169 strains. The mAbs, when used as the primary antibody, showed positive staining in cells infected with both Towne and AD169 strains. The mAbs were then tested on patients submitted to allogeneic hematopoietic stem cell transplantation. The gB antigen positivity rates of the patients tested using ZJU-FH6 and ZJU-FE6 were 62.0 and 63.0%, respectively. The gB antigen showed a significant correlation with the level of pp65 antigen in peripheral blood leukocytes. In conclusion, two potential diagnostic gB mAbs were developed and were shown to be capable of recognizing gB in peripheral blood leukocytes in a reliable manner.
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