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Watchara Kasinrerk

Publications and source records attributed to Watchara Kasinrerk.

17 recordsLinked to original sources
PubMed2006

A novel approach using streptavidin magnetic bead-sorted in vivo biotinylated survivin for monoclonal antibody production.

One major obstacle in antibody production is the lack of highly purified immunogen. In this study, we describe an alternative strategy to circumvent this problem. A nucleotide sequence encoding a full-length of human survivin was cloned into pAK400cb. After transforming into an E. coli Origami B strain, survivin-biotin carboxyl carrier protein (BCCP) fusion protein was generated in the cytoplasm, where the BCCP domain serves as a target for in vivo biotinylation. The biotinylated heterologous protein was subsequently immobilized on streptavidin-coated magnetic particles and separated from other proteins in a magnetic field. The survivin-coated beads were used to raise immune responses in BALB/c mice for hybridoma production. A number of hybrid clones were found to secrete anti-survivin antibodies. Three established clones were selected for single cell cloning. All generated monoclonal antibodies specifically reacted with the standard human recombinant survivin. Two out of three monoclonal antibodies recognized survivin in tumor extracts. The present method has advantages in facilitating monoclonal antibody production by making antigen purification steps unnecessary.

Animals↗
PubMed2006

Binding of multivalent CD147 phage induces apoptosis of U937 cells.

CD147 is a broadly expressed cell-surface molecule and serves as a signaling receptor for extracellular cyclophilins. CD147 also appears to interact with immune cells, but its counter-receptor on these cells has not been clearly described. In the present report, we displayed multiple copies of the CD147 extracellular domain (CD147Ex) on VCSM13 phage to study the interaction of CD147 with its ligand. Recognition of phage containing fusion protein of CD147Ex and gpVIII (CD147Ex phage) by four different anti-CD147 mAbs indicated that at least parts of the CD147 are properly folded. Specific binding of CD147Ex phage to various cell types was demonstrated by flow cytometry. Morphological changes, however, were observed only in U937, a monocytic cell line, after 24 h incubation with multivalent CD147Ex phage. After 48 h, U937 cell propagation ceased. Staining with annexin V and the presence of cleaved caspase-3 indicated that many of the CD147Ex phage-treated cells had lost viability through apoptotic cell death. The above results suggest that CD147 induces apoptosis in U973 cells and that at least a portion of this cell death program involves a caspase-dependent pathway.

Annexin A5↗
PubMed2006

Vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein NS1 and complement.

BACKGROUND: Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thirty years ago, complement activation was proposed to be a key underlying event, but the cause of complement activation has remained unknown. METHODS: The major nonstructural dengue virus (DV) protein NS1 was tested for its capacity to activate human complement in its membrane-associated and soluble forms. Plasma samples from 163 patients with DV infection and from 19 patients with other febrile illnesses were prospectively analyzed for viral load and for levels of NS1 and complement-activation products. Blood and pleural fluids from 9 patients with DSS were also analyzed. RESULTS: Soluble NS1 activated complement to completion, and activation was enhanced by polyclonal and monoclonal antibodies against NS1. Complement was also activated by cell-associated NS1 in the presence of specific antibodies. Plasma levels of NS1 and terminal SC5b-9 complexes correlated with disease severity. Large amounts of NS1, complement anaphylatoxin C5a, and the terminal complement complex SC5b-9 were present in pleural fluids from patients with DSS. CONCLUSIONS: Complement activation mediated by NS1 leads to local and systemic generation of anaphylatoxins and SC5b-9, which may contribute to the pathogenesis of the vascular leakage that occurs in patients with DHF/DSS.

Adolescent↗
PubMed2006

CD147 contains different bioactive epitopes involving the regulation of cell adhesion and lymphocyte activation.

CD147 is a leukocyte surface molecule which belongs to the immunoglobulin superfamily. It is broadly expressed on various cell types and is a lymphocyte activation-associated molecule. In order to study the function of CD147, five CD147 monoclonal antibodies (mAbs) were generated: M6-2F9; M6-1D4; M6-1F3; M6-1B9; and M6-1E9. Biochemical characterizations and cross-blocking experiments indicated that M6-1B9 and M6-1E9 recognize the same or contiguous epitopes on CD147. By employing COS transfectants expressing CD147 membrane-distal domain (domain 1) and membrane-proximal domain (domain 2), mAbs M6-2F9, M6-1D4, M6-1B9, and M6-1E9 were shown to recognize epitopes located on domain 1 of the molecule. Functional studies indicated that engagement of CD147 by mAbs M6-1B9 and M6-1E9 strongly inhibited lymphocyte proliferation induced by a CD3 mAb. In contrast, mAbs M6-2F9, M6-1D4, and M6-1F3 induced U937 homotypic cell aggregation. The results indicate that CD147 contains at least two bioactive domains. Epitopes responsible for induction of cell aggregation are different from those regulating lymphocyte activation.

Animals↗
PubMed2006

Chicken egg yolk antibodies specific for the gamma chain of human hemoglobin for diagnosis of thalassemia.

Immunoglobulin Y (IgY) technology was used to generate anti-hemoglobin Bart's (Hb Bart's) IgY antibodies (Abs) for development into an enzyme-linked immunosorbent assay (ELISA) test for thalassemia diagnosis. Hb Bart's purified from the hemolysate of a patient with Hb Bart's hydrops fetalis (homozygous alpha-thalassemia) was used to immunize a chicken via the pectoralis muscle. After water dilution and sodium sulfate precipitation, 40 to 70 mg of IgY could be extracted from an egg. IgY, first detected in sera 2 weeks after immunization, reached the highest titer at week 4, and the titer remained stable for at least 2 weeks before declining. The pattern of Ab response in the yolk was the same as in the serum but was somewhat delayed. The IgY Abs produced reacted with gamma globin, Hb Bart's, Hb F, normal cord hemolysate (Hbs F plus A), and Hb Bart's hydrops fetalis (Hbs Bart's plus Portland) and to a lesser degree with beta globin, Hb A, Hb A2 and adult hemolysate (Hbs A plus A2), but the Abs did not react with alpha globin. Immunoaffinity purification with Hb A coupled to Sepharose was used to isolate an unbound IgY that reacted with Hb F, Hb Bart's, and gamma globin, and this IgY was used to develop an ELISA test for thalassemia diagnosis. The results of direct ELISA analyses of 336 hemolysate samples from individuals with various known thalassemia genotypes and phenotypes and from healthy individuals confirmed the specificity of the polyclonal Abs for Hbs containing Hb F and Hb Bart's. This specificity, which was due to the Abs' strong reactivity in cases of pathologic thalassemic diseases and weak reactivity in cases of nonpathologic thalassemic diseases, depended on the levels of Hb Bart's and Hb F.

Animals↗
PubMed2005

Twin-arginine signal peptide attributes effective display of CD147 to filamentous phage.

A novel phagemid (pTat8) was constructed in this study to improve the quality of a molecule displayed on filamentous phage. The twin-arginine translocation (Tat) pathway was chosen for transporting and integrating a CD147 molecule into a phage particle via gpVIII. The parent vector pComb8-CD147Ex was modified by substituting a Sec signal sequence (PelB) with a twin-arginine signal sequence from trimethylamine N-oxide reductase (TorA). The characteristics of the CD147 displayed on the phage particle were evaluated by Sandwich ELISA and Western immunoblotting. A Tat-dependent leader was found to be superior to the Sec leader for the phage display of CD147. Our findings further support the involvement of an Escherichia coli Tat translocase in mediating the integration of a hydrophobic transmembrane protein into the inner membrane. This modified phagemid will be useful in phage display technique when the correctly folded structure is required (i.e., antibody libraries and ligand-receptor tracing).

Basigin↗
PubMed2004

Alterations of pr-M cleavage and virus export in pr-M junction chimeric dengue viruses.

During the export of flavivirus particles through the secretory pathway, a viral envelope glycoprotein, prM, is cleaved by the proprotein convertase furin; this cleavage is required for the subsequent rearrangement of receptor-binding E glycoprotein and for virus infectivity. Similar to many furin substrates, prM in vector-borne flaviviruses contains basic residues at positions P1, P2, and P4 proximal to the cleavage site; in addition, a number of charged residues are found at position P3 and between positions P5 and P13 that are conserved for each flavivirus antigenic complex. The influence of additional charged residues on pr-M cleavage and virus replication was investigated by replacing the 13-amino-acid, cleavage-proximal region of a dengue virus (strain 16681) with those of tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV) and by comparing the resultant chimeric viruses generated from RNA-transfected mosquito cells. Among the three chimeric viruses, cleavage of prM was enhanced to a larger extent in JEVpr/16681 than in YFVpr/16681 but was slightly reduced in TBEVpr/16681. Unexpectedly, JEVpr/16681 exhibited decreased focus size, reduced peak titer, and depressed replication in C6/36, PS, and Vero cell lines. The reduction of JEVpr/16681 multiplication correlated with delayed export of infectious virions out of infected cells but not with changes in specific infectivity. Binding of JEVpr/16681 to immobilized heparin and the heparin-inhibitable infection of cells were not altered. Thus, diverse pr-M junction-proximal sequences of flaviviruses differentially influence pr-M cleavage when tested in a dengue virus prM background. More importantly, greatly enhanced prM cleavability adversely affects dengue virus export while exerting a minimal effect on infectivity. Because extensive changes of charged residues at the pr-M junction, as in JEVpr/16681, were not observed among a large number of dengue virus isolates, these results provide a possible mechanism by which the sequence conservation of the pr-M junction of dengue virus is maintained in nature.

Amino Acid Sequence↗
PubMed2004

Production of IgY anti-mouse IgG antibodies from chicken eggs.

IgY technology offers several advantages over antibody production in mammals. In this study, we applied IgY technology for the production of anti-mouse IgG polyclonal antibodies and developed a FITC conjugate reagent. Two hens were immunized 3 times with mouse IgG, one via the pectoralis and the other via the calf muscles. Specific antibodies could be detected in the sera two weeks after the immunization, and maximum levels were reached at week 10. The hen which was immunized via the pectoralis muscle produced a much higher antibody response than the hen immunized via the calf muscle. In egg yolk, specific antibodies appeared 2 weeks after the first immunization, reached a plateau after week 11 and remained high until week 20. IgY were extracted from egg yolk by sodium sulfate precipitation. Approximately 40 mg of IgY could be extracted from a single egg. The extracted IgY was labeled with FITC. The so-produced antibody-FITC conjugate reacted to all mouse IgG isotypes and could be used to determine leukocyte sub-populations in blood samples by flow cytometry.

Animals↗
PubMed2003

Displaying and epitope mapping of CD147 on VCSM13 phages: influence of Escherichia coli strains.

The external domain of a human leukocyte surface molecule, CD147 was displayed on the surface of phage. Two Escherichia coli laboratory strains, XL-1 Blue and TG-1, were chosen to separately propagate the recombinant phages. By sandwich enzyme linked immunosorbent assay (ELISA), CD147 on phage particles were individually captured by six CD147 mAbs and subsequently detected by anti-M13 conjugated HRP. All mAbs specifically bound the CD147 on phage particles derived from TG-1. On the contrary, only four of them could recognize the CD147 on phages produced by XL-1 Blue. The results indicate that the environment in the TG-1 periplasm is more appropriate than that of XL-1 Blue for promoting the suitable folding of CD147. This finding emphasizes the importance of selecting the appropriate E. coli host for display of a complex protein. The epitopes of CD147 displayed on the phage were further mapped by competitive inhibition ELISA, which is a reliable and economical method. Certain clusters of mAb recognition areas were identified and will provide valuable information for the discovery of the ligand for CD147.

Antigens, CD↗
PubMed2003

A flow cytometric method for enumeration of lymphocyte sub-populations in sample containing lysis-resistant red blood cells.

Determination of lymphocyte sub-populations is usually carried out by flow cytometry using two-color immunophenotyping reagent. By this technique, however, the combination of FSC and SSC with CD45-FITC/CD14-PE is unable to identify the lymphocyte population in a sample containing lysis-resistant red blood cells (RBC). The actual values of lymphocyte sub-populations, therefore, cannot be determined in these RBC contaminated samples. To overcome this problem, we describe here the use of 7-aminoactinomycin D (7-AAD) to exclude lysis-resistant RBC from white blood cells (WBC). By adding 7-AAD, lymphocytes of samples containing RBC could be identified by using FL3/SSC, therefore, the actual number of lymphocyte sub-populations of the stained cells was obtained. We have proved that 7-AAD can be used to exclude contaminated RBC and has no effect on the measurement of lymphocyte sub-populations by using two-color immunophenotyping reagent. In routine blood samples that contain lysis-resistant RBC, 7-AAD markedly increased the purity of lymphocytes in the lymphocyte gate to >95% and the lymphocyte sub-populations therefore could be correctly determined. The described method is inexpensive, simple and gives successful analysis of lymphocyte sub-populations in a sample containing lysis-resistant RBC.

Animals↗
PubMed2003

Construction of high-density display of CD147 ectodomain on VCSM13 phage via gpVIII: effects of temperature, IPTG, and helper phage infection-period.

Production of VCSM13 phage displaying a high density of CD147 ectodomain (CD147Ex) was achieved when culturing conditions were modulated. A phagemid expressing CD147Ex was constructed and used to produce phage display CD147Ex gpVIII fusion protein in TG1 Escherichia coli. Displaying of CD147Ex via gpVIII was successfully increased when growing the transformed TG1 at 25 degrees C with IPTG-stimulation. In addition to temperature and IPTG-stimulation, the VCSM13 helper phage infection-period particularly affected the insertion of CD147Ex into phage progeny. By sandwich ELISA, incorporation of the CD147Ex into phage particle was confirmed. The correct size of the CD147Ex-gpVIII fusion protein at 28kDa was demonstrated by Western immunoblotting. Multivalent display of CD147Ex on phage particles will be valuable in discovering its ligand partner.

Antigens, CD↗
PubMed2003

Production of anti-dengue NS1 monoclonal antibodies by DNA immunization.

Monoclonal antibodies against dengue NS1 protein were generated following immunization of mice with plasmid DNA encoding the transmembrane form of NS1 from dengue serotype 2 virus. A mammalian expression vector, pDisplay, was engineered to direct cell surface expression of dengue NS1 and tested for transient expression in COS cells. Two mice were immunized intramuscularly with six doses of 100 microg of plasmid at 2-week intervals; one mouse received a booster of live virus prior to the last plasmid injection. Both mice showed antibody responses against dengue antigens in dot enzyme immunoassay. Following fusion, hybridomas were screened with dot enzyme immunoassay against all four dengue serotypes. Specificity to the NS1 protein was confirmed by western blot analysis. Among five anti-dengue NS1 monoclonal antibodies generated, two clones were serotype 2 specific, two clones reacted with all four serotypes and the last also reacted with Japanese encephalitis virus. Reactivity against native or denatured forms of NS1 revealed three clones with reactivity to linear epitopes and two clones recognizing conformational epitopes. Such diverse specificity of anti-dengue NS1 monoclonal antibodies indicates that DNA immunization, especially with the combination of virus boosting, is an efficient way of producing monoclonal antibodies against viral protein. This has opened up a possibility of producing monoclonal antibodies to rare viral proteins that are difficult to isolate or purify.

Animals↗
PubMed2003

Effect of bitter melon (Momordica charantia Linn) on level and function of natural killer cells in cervical cancer patients with radiotherapy.

Cervical cancer patients have a defective immune system. There is a decrease of total white blood cell count including lymphocytes and natural killer (NK) cells. NK cells, one type of lymphocytes, play a role to eliminate cancer cells by antibody dependent cell mediated cytotoxicity (ADCC) mechanism. Previous studies have shown that P-glycoprotein (170 kDa, transmembrane protein) may be a transporter for cytokine releasing in ADCC mechanism. This study proposed to explore the role of bitter melon intake in cervical cancer patients undergoing normal treatment (radiotherapy). Subjects were divided into three groups: 1) normal control (women 35-55 years, n = 35), 2) patient control (n = 30) and 3) patient treatment (n = 30) groups. Patient control and patient treatment groups were cervical cancer patients (stage II or III) treated with radiotherapy (without or with bitter melon ingestion). Blood samples of patient control and patient treatment groups were analyzed for NK cells percentage and P-glycoprotein level. Bitter melon is a Thai herb. Previous studies have shown that bitter melon can stimulate lymphocyte activity in vitro and in vivo (mouse). The authors hope that bitter melon could stimulate the increase of NK cells percentage and P-glycoprotein level on the membrane in blood samples from cervical cancer patients who ingest bitter melon. The results showed an increased percentage of NK cells in patient control and patient treatment groups. The increase in each group is significant (p < 0.05) when compared with the percentage of NK cells from second and third blood sampling time (after radiation with of without bitter melon intake for 45 and 90 days) with first blood sampling time (before treatment). The results also show a significant decrease of P-glycoprotein level (p < 0.05) in second and third blood sampling times when compared with first blood sampling time of the patient treatment group. There was no significant difference of P-glycoprotein (P-gp) level from first, second and third blood sampling times in patient control group. Bitter melon ingestion did not affect NK cell level but it affected the decrease of P-gp level on NK cell membrane.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Production of antibodies by single DNA immunization: comparison of various immunization routes.

DNA immunization is a recent vaccination method that induces humoral and cellular immune responses in a range of hosts. Different immunization routes induce a different degree of the immune response. In the present report, we demonstrate that multiple intramuscular immunizations of plasmid DNA encoding various leukocyte surface molecules induced a specific antibody response. In contrast, a single intramuscular immunization could not induce antibody production. To study the induction of antibody response after a single immunization of plasmid DNA, mice were single-dose intramuscularly, intraperitoneally, intravenously and intrasplenically immunized, simultaneously, with the same preparation of plasmid DNA encoding CD147 membrane protein. We observed that only the intrasplenic route induced specific antibody production. The induction of antibody by intrasplenic immunization was confirmed by using plasmid DNA encoding CD54 molecule. By this single-dose DNA intrasplenic immunization, the generated antibodies could be detected in mice up to 6 months. These results suggest that the injected DNA is expressing the relevant protein antigen in the spleen for several months after injection. Our results demonstrate that direct immunization of antigen-encoding DNA into the spleen is a more effective method for induction of antibody production. This finding may support future investigations of DNA vaccination strategies that specifically promote the uptake of plasmid by splenocytes. Intrasplenic immunization may also be helpful in the understanding of the early events of the immune response to DNA vaccine and be useful as an effective route for the induction of immune responses.

Animals↗
PubMed2002

Engagement of Na,K-ATPase beta3 subunit by a specific mAb suppresses T and B lymphocyte activation.

In order to identify new molecules involved in regulation of T cell proliferation, we generated various mAb by immunization of mice with the T cell line Molt4. We found one mAb (termed P-3E10) that down-regulated the in vitro T cell proliferation induced by CD3-specific OKT3 mAb. The P-3E10 mAb was also able to inhibit IFN-gamma, IL-2, IL-4 and IL-10 production of OKT3-activated T cells. The antigen recognized by P-3E10 mAb is broadly expressed on all hematopoietic as well as on all non-hematopoietic cell lines tested so far. Within peripheral blood leukocytes, the P-3E10 antigen was detected on lymphocytes, monocytes and granulocytes. Human umbilical vein endothelial cells (HUVEC) also scored positively. By evaluating the effect of P-3E10 mAb on these cell types we found that it also inhibited anti-IgM-induced B cell proliferation. However, it did not block growth factor-mediated proliferation of HUVEC, and spontaneous proliferation of SupT-1, Jurkat, Molt4 and U937 cell lines. Moreover, it did not influence phagocytosis of human blood monocytes and granulocytes. Biochemical analysis revealed that the P-3E10 antigen is a protein with a mol. wt of 45-50 kDa under non-reducing and 50-55 kDa under reducing conditions. By using a retroviral cloning system, the P-3E10 antigen was cloned. Sequence analysis revealed the P-3E10 antigen to be identical to the beta3 subunit of the Na,K-ATPase.

Amino Acid Sequence↗
PubMedDate not supplied

Immunomodulatory activities of Centella asiatica and Rhinacanthus nasutus extracts.

Centella asiatica (CA) and Rhinacanthus nasutus (RN )have been used for treatment of various illnesses, but the mechanisms of action remain largely unknown. This study focused on the influence of CA and RN extracts on cell-mediated and humoral immune responses. In human peripheral blood mononuclear cells (PBMCs), CA (water extract) and RN (water and ethanol extracts) significantly increased proliferation and the production of IL-2 and TNF-alpha. In contrast, an ethanol extract of CA inhibited human PBMC mitogenesis and the production of IL-2 and TNF-alpha. BALB/c mice treated with CA extracts (100 mg/kg bw) showed higher responses to both primary and secondary antibodies against BSA when compared with non-treated group. Only the secondary antibody response was increased in RN extract-treated mice. The present study revealed immunomodulating activity of CA and RN extracts with regard to both non-specific cellular and humoral immune responses. The data available to date suggest that they may have chemopreventive or anticancer potential.

Acanthaceae↗