Khajornsak Tragoolpua

In the Absence of Endogenous Gamma Interferon, Mice Acutely Infected with Neospora caninum Succumb to a Lethal Immune Response Characterized by Inactivation of Peritoneal Macrophages

ABSTRACT Following infection with Neospora caninum, BALB/c mice were shown to be resistant to an acute infection but developed a latent chronic infection. However, BALB/c background gamma interferon (IFN-γ)-deficient mice were sensitive to the acute infection. Since the immune response in IFN-γ-deficient mice is scantly known, we examined the function of macrophages, major histocompatibility complex (MHC) class II expression, T-cell responses, and serum cytokine levels in the mice. All IFN-γ-deficient mice died within 9 days of infection with N. caninum, whereas those treated with exogenous IFN-γ lived longer. Although N. caninum invaded various organs in both types of mice at the early stage of infection, the parasite was not detected in the brains of resistant hosts until 21 days postinfection (dpi). Peritoneal macrophages from IFN-γ-deficient mice were activated by exogenous IFN-γ associated with inhibition of parasite growth and nitric oxide production as were those from BALB/c mice. IFN-γ-deficient mice failed to increase MHC class II expression on macrophages. Moreover, BALB/c mice induced T-cell proliferation while IFN-γ-deficient mice did not. However, in vivo treatment with exogenous IFN-γ induced up-regulated MHC class II expression in IFN-γ-deficient mice. BALB/c mice treated with an antibody to CD4 showed an increase in morbidity and mortality after parasite infection. In serum, significant levels of IFN-γ and interleukin-4 (IL-4) were detected in resistant hosts, whereas IL-10 was detected in IFN-γ-deficient mice. The levels of IL-12 in IFN-γ-deficient mice were higher than those in BALB/c mice at 7 dpi. The present study indicates that early IFN-γ production has a crucial role in the activation of peritoneal macrophages for the induction of protective immune responses againstN. caninum.

Characterization of Neospora caninum Surface Protein NcSRS2 Based on Baculovirus Expression System and Its Application for Serodiagnosis of Neospora Infection

ABSTRACT The baculovirus expression system has proved to be a useful tool for the production of recombinant proteins. Here we have characterized the Neospora caninum surface protein NcSRS2 produced by two types of the recombinant virus and also have developed an enzyme-linked immunosorbent assay (ELISA) using recombinant NcSRS2 for the serologic diagnosis ofNeospora infection. Western blot analysis showed two major protein bands that were detectable in insect cells infected with each recombinant baculovirus, and a lower-molecular-weight protein was detected in culture supernatants from a cell infected with the recombinant virus lacking the hydrophobic C-terminal tail. Analysis of the N-terminal amino acids showed that the secreted NcSRS2 lacked 6 kDa of the N-terminal signal peptide. Moreover, the detergent-soluble protein of insect cells infected with the recombinant baculovirus expressing the full-length NcSRS2 gene was used to develop an ELISA system based on specificity and reactivity to antisera againstToxoplasma gondii, Hammondia heydorni, orN. caninum. Anti-N. caninum mouse, dog, and bovine sera recognized the recombinant NcSRS2 on Western blots. Furthermore, we have shown that the developed ELISA system consistently discriminates indirect fluorescent-antibody test (IFAT)-positive bovine sera against N. caninum from IFAT-negative sera. These results indicate that the ELISA using baculovirus-expressed NcSRS2 can be useful for effective and reliable serodiagnosis of N. caninuminfection.

Antioxidant and Anti-cancer Cell Proliferation Activity of Propolis Extracts from Two Extraction Methods

Antioxidant activity, total phenolic, total flavonoid compounds and cytotoxicity to cancer cell lines of propolis extracts from two extraction methods were investigated in this study. Propolis was collected from Phayao province and extracted with 70% ethanol using maceration and sonication techniques. The antioxidant activity was evaluated by DPPH assay. Total phenolic and flavonoid compounds were also determined. Moreover, the cytotoxicity of propolis was evaluated using MTT assay. The percentage propolis yield after extraction using maceration (18.1%) was higher than using sonication (15.7%). Nevertheless, antioxidant and flavonoid compounds of the sonication propolis extract were significant greater than using maceration. Propolis extract from sonication showed antioxidant activity by 3.30 ± 0.15 mg gallic acid equivalents/g extract. Total phenolic compound was 18.3 ± 3.30 mg gallic acid equivalents/g extract and flavonoid compound was 20.49 ± 0.62 mg quercetin/g extract. Additionally, propolis extracts from two extraction methods demonstrated the inhibitory effect on proliferation of A549 and HeLa cancer cell lines at 24, 48 and 72 hours in a dose-dependent manner. These results are of interest for the selection of the most appropriate method for preparation of propolis extracts as potential antioxidant and anticancer agents.

Neospora caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol anchor in insect cells.

We investigated the terminal location of NcSRS2, a surface antigen of Neospora caninum that has potential use for diagnosis, and demonstrated its importance as a vaccine component against neosporosis, in an insect-baculovirus expression system. To examine the role of the hydrophobic C-terminal tail in NcSRS2, four types of recombinant baculoviruses were constructed. Immunoblotting and N-terminal amino acid analysis revealed cleavage of a 6 kDa of the N-terminal signal peptide in the mature NcSRS2 protein. The recombinant NcSRS2 (rNcSRS2) lacking 25, and 62 amino acids from the termination codon were detected in supernatants from recombinant virus-infected cells, but not in recombinants with truncated 147 amino acids from the termination codon, and intact NcSRS2 gene (401 amino acids). By flow cytometric and confocal laser scanning microscopic analyses, the truncation of the hydrophobic C-terminal tail in NcSRS2 was shown to result in the reduction of protein expression on the cell surface relative to intact rNcSRS2. Except for the recombinant lacking the 147 C-terminal residues, three other rNcSRS2 were detected in the supernatants after treatment with phosphatidylinositol-specific phospholipase C. Our results demonstrate that the N. caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol-anchor molecule in insect cells, and that the hydrophobic C-terminal domain is an essential component for GPI-membrane attachment. We have likewise shown the usefulness of the insect-recombinant baculovirus system in the expression of rNcSRS2.

Generation of functional scFv intrabody to abate the expression of CD147 surface molecule of 293A cells.

BackgroundExpression of intracellular antibodies (intrabodies) has become a broadly applicable technology for generation of phenotypic knockouts in vivo. The method uses surface depletion of cellular membrane proteins to examine their biological function. In this study, we used this strategy to block the transport of cell surface molecule CD147 to the cell membrane. Phage display technology was introduced to generate the functional antibody fragment to CD147, and we subsequently constructed a CD147-specific scFv that was expressed intracellularly and retained in the endoplasmic reticulum by adenoviral gene transfer.ResultsThe recombinant antibody fragments, Fab and scFv, of the murine monoclonal antibody (clone M6-1B9) reacted specifically to CD147 by indirect enzyme-linked immunosorbent assays (ELISA) using a recombinant CD147-BCCP as a target. This indicated that the Fab- and scFv-M6-1B9 displaying on phage surfaces were correctly folded and functionally active. We subsequently constructed a CD147-specific scFv, scFv-M6-1B9-intrabody, in 293A cells. The expression of CD147 on 293A cell surface was monitored at 36 h after transduction by flow cytometry and demonstrated remarkable reduction. Colocalization of scFv-M6-1B9 intrabody with CD147 in the ER network was depicted using a 3D deconvolution microscopy system.ConclusionThe results suggest that our approach can generate antibody fragments suitable for decreasing the expression of CD147 on 293A cells. This study represents a step toward understanding the role of the cell surface protein, CD147.

Intracellular Acidosis Promotes Mitochondrial Apoptosis Pathway: Role of EMMPRIN Down-regulation via Specific Single-chain Fv Intrabody

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a human leukocyte surface molecule that is enriched on the surface of many cancer cells, and it plays an important role in proliferation and metastasis. In this study, we utilized the chimeric adenoviral vector Ad5/F35 carrying gene encoding scFv against EMMPRIN (scFv-M6-1B9) to down-regulate EMMPRIN cell surface expression and investigated programmed cell death response in colorectal cancer (CRC) cell, Caco-2. The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression. This approach led to the inducing of apoptosis, which was relative to the increasing of apoptotic bodies in sub-G1 peak, phosphatidylserine externalization, as well as TUNEL-positive cells. In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3. Moreover, carcinoembryonic antigen (CEA), a tumor marker for CRC, was found to have significantly diminished in both secreted protein and mRNA levels. In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level. Thus, its benefits could be applied to project the future prospects for targeted gene therapy and therapeutic application in monitoring colorectal cancer.

EMMPRIN reduction via scFv-M6-1B9 intrabody affects α3β1-integrin and MCT1 functions and results in suppression of progressive phenotype in the colorectal cancer cell line Caco-2

Extracellular matrix metalloproteinase inducer (EMMPRIN) exhibits overexpression in various cancers and promotes cancer progression and metastasis via the interaction with its associated molecules. The scFv-M6-1B9 intrabody has a potential ability to reduce EMMPRIN cell surface expression. However, the subsequent effect of scFv-M6-1B9 intrabody-mediated EMMPRIN abatement on its related molecules, α3β1-integrin, MCT1, MMP-2 and MMP-9, is undefined. Our results demonstrated that the scFv-M6-1B9 intrabody efficiently decreased α3β1-integrin cell surface expression levels. In addition, intracellular accumulation of MCT1 and lactate were increased. These results lead to suppression of features characteristic for tumor progression, including cell migration, proliferation and invasion, in a colorectal cancer cell line (Caco-2) although there was no difference in MMP expression. Thus, EMMPRIN represents an attractive target molecule for the disruption of cancer proliferation and metastasis. An scFv-M6-1B9 intrabody-based approach could be relevant for cancer gene therapy.

Propolis extracts from the northern region of Thailand suppress cancer cell growth through induction of apoptosis pathways.

SummaryThe continual increase in mortality rates and number of cancer cases is a matter of serious concern in developing countries. The incorporation of natural products into classical cancer treatment approaches is a promising direction. The mechanisms of A549 and HeLa cancer cell death induction by ethanolic extracts of propolis samples from Phayao, Chiang Mai, and Nan provinces in northern Thailand were investigated in this study. The propolis extract from Chiang Mai showed the highest antioxidant activity and the greatest total phenolic content. The propolis extract from Nan also exhibited the highest total flavonoid content. The proliferation of A549 and HeLa cells grown in the presence of the propolis extracts was suppressed in a dose- and time-dependent manner. Moreover, treatment of both cancer cells with the propolis extracts showed DNA fragmentation and significantly increased the number of the apoptotic cells. On A549 cells, the extrinsic and intrinsic pathways of caspase enzymes were activated by the propolis extracts from Phayao and Chiang Mai. In the case of the propolis extract from Nan, the mechanisms involved apoptosis on the A549 cells were caspase-independent pathway. The extrinsic pathway of the caspase enzyme was triggered by all of the propolis extracts on HeLa cells. Finally, oral administration of the propolis granule produced from the propolis extract from Nan resulted in extended survival of tumour-bearing mice. Therefore, propolis extracts from the northern region of Thailand demonstrated pharmacological properties, both antioxidant and anticancer activities. From these findings, it is evident that propolis extracts can be considered as a naturally obtained agent extremely useful in cancer treatment.

Secretome profile analysis of multidrug-resistant, monodrug-resistant and drug-susceptible Mycobacterium tuberculosis

The emergence of drug-resistant tuberculosis has generated great concern in the control of tuberculosis and HIV/TB patients have established severe complications that are difficult to treat. Although, the gold standard of drug-susceptibility testing is highly accurate and efficient, it is time-consuming. Diagnostic biomarkers are, therefore, necessary in discriminating between infection from drug-resistant and drug-susceptible strains. One strategy that aids to effectively control tuberculosis is understanding the function of secreting proteins that mycobacteria use to manipulate the host cellular defenses. In this study, culture filtrate proteins from Mycobacterium tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains were gathered and profiled by shotgun-proteomics technique. Mass spectrometric analysis of the secreted proteome identified several proteins, of which 837, 892, 838 and 850 were found in M. tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains, respectively. These proteins have been implicated in various cellular processes, including biological adhesion, biological regulation, developmental process, immune system process localization, cellular process, cellular component organization or biogenesis, metabolic process, and response to stimulus. Analysis based on STITCH database predicted the interaction of DNA topoisomerase I, 3-oxoacyl-(acyl-carrier protein) reductase, ESAT-6-like protein, putative prophage phiRv2 integrase, and 3-phosphoshikimate 1-carboxyvinyltransferase with isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin, suggesting putative roles in controlling the anti-tuberculosis ability. However, several proteins with no interaction with all first-line anti-tuberculosis drugs might be used as markers for mycobacterial identification.

Novel potential diagnostic test for Mycobacterium tuberculosis complex using combined immunomagnetic separation (IMS) and PCR‐CTPP

To exploit immunomagnetic separation combined with PCR with confronting two‐pair primers (IMS‐PCR‐CTPP) as a rapid method for detection of Mycobacterium tuberculosis complex (MTC) and identification of Mycobacterium bovis from sputum specimens.