Biomedical subjects

B S Prabhakar

Publications and source records attributed to B S Prabhakar.

At least 19 recordsLinked to original sources

PubMed2006

MADD/DENN splice variant of the IG20 gene is necessary and sufficient for cancer cell survival.

The IG20 gene is overexpressed in human tumors and cancer cell lines, and encodes at least four splice variants (SVs) namely, IG20pa, MADD, IG20-SV2 and DENN-SV. Earlier, gain-of-function studies showed that IG20-SVs can exhibit diverse functions and play a critical role in cell proliferation and apoptosis. Expression of exogenous IG20pa or DENN-SV rendered cells either susceptible or resistant to induced apoptosis, respectively, whereas MADD and IG20-SV2 had no apparent effect. In order to understand the contrasting effects of the IG20-SVs in a physiologically more relevant system, we expressed exon-specific small hairpin RNAs (shRNAs) to selectively knockdown specific IG20-SVs. Consistent with an earlier study, knockdown of all IG20-SVs resulted in spontaneous apoptosis of HeLa and PA-1 cells. In addition, we unambiguously demonstrated that knockdown of MADD can render cells susceptible to spontaneous apoptosis but had no discernible effect on cell proliferation, colony size or cell cycle progression. Moreover, expression of MADD alone, and not DENN-SV, in the absence of endogenous IG20-SVs was sufficient to prevent spontaneous apoptosis. Our results show the utility of shRNAs for selective knockdown of particular IG20-SVs and their potential therapeutic value in cancer. Further, they demonstrate that MADD alone is sufficient and necessary for cancer cell survival.

Alternative Splicing↗

PubMed2005

Correlation of disease evolution with progressive inflammatory cell activation and migration in the IL-4 transgenic mouse model of atopic dermatitis.

Atopic dermatitis is a chronic inflammatory skin disease characterized by inflammatory cell infiltration in the skin. In order to assess the roles of inflammatory cells in this disease, we analysed the activation status and surface markers of various leucocytes in the IL-4 transgenic mouse model of atopic dermatitis, by flow cytometry, immuofluorescence microscopy, and T cell proliferation assays. The studies were performed with a nontransgenic mouse control and transgenic mice at three disease stages: before disease onset, early skin disease, and late skin disease, so that we can delineate the immunological sequence of events. As the skin disease evolves, the skin draining lymph node cells from IL-4-Tg mice show a spontaneous proliferation and a progressively enhanced proliferative response to stimulants including anti-CD3, Con A, PHA, and Staphylococcus enterotoxins A and B. As the disease evolves, the percent of lymphoid organ T cells expressing activation molecules (CD44 and CD69) and costimulatory molecules (ICOS and PD-1) are progressively increased; the percent and total number of T cells are reduced in an incremental manner in the secondary lymphoid organs while the number of T cells infiltrating the skin increases in an incremental fashion; the total number of dendritic antigen presenting cells, macrophages, and NK cells gradually increases in the lymphoid organs. Collectively, our results suggest that there is a continued and progressive migration of activated inflammatory cells from the secondary lymphoid organs into the skin where they participate in immune responses resulting in the pathology associated with inflammation.

Animals↗

PubMed2004

Early up-regulation of Th2 cytokines and late surge of Th1 cytokines in an atopic dermatitis model.

We investigated cytokine profiles in interleukin (IL)-4 transgenic (Tg) mice with a skin inflammatory disease resembling human atopic dermatitis. cDNA microarray revealed that the mRNAs encoding IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-13, tumour necrosis factor (TNF)-alpha, TNF-beta and interferon (IFN)-gamma were up-regulated in the skin of late lesion Tg mice and to a lesser degree in non-lesion Tg mice when compared to those of non-Tg mice. Real time reverse transcription-polymerase chain reaction (RT-PCR) analyses indicated that the cDNA copy numbers of IL-1beta, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma from the skin of late, early and non-lesions increased significantly compared to non-Tg mice. IL-2 and IL-12p40 cDNA copy numbers were increased significantly in early, but not late, lesions. Interestingly, IL-1beta, IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, TNF-alpha, and IFN-gamma cDNAs were increased significantly the skin of before-onset and/or non-lesion mice. Flow cytometry analyses demonstrated an increased percentage of keratinocytes producing IL-4 as the disease progressed. The percentage of IL-2, IL-4, IL-10 and IFN-gamma-producing T cells and IL-12-producing antigen-presenting cells in skin-draining lymph nodes and inflammatory skin also increased, particularly in mice with late lesion. These results suggest that disease induction is primarily triggered by Th2 cytokines and that Th1, Th2 and non-Th proinflammatory cytokines are all involved in the disease process.

Animals↗

PubMed2003

Relevance of differential immunogenicity of human and mouse recombinant desmoglein-3 for the induction of acantholytic autoantibodies in mice.

Pemphigus vulgaris (PV) is an antibody-mediated autoimmune disease of the skin and mucous membranes. Desmoglein-3 (dsg-3) expressed in the suprabasal layer of the skin serves as an autoantigen in PV. Passive transfer of sera, either from patients with PV or from experimental animals immunized with a recombinant human dsg3 (hdsg3) into neonatal BALB/c mice results in blister formation, suggesting strongly that there is significant cross-reactivity between the mouse dsg3 (mdsg3) and the hdsg3. However, efforts to induce disease in adult mice through active immunization using hdsg-3 have not been successful, suggesting that the epitopes required for the induction of pathogenic antibodies in adult mice might not be present in hdsg3. Therefore, in this study, we expressed a full-length mdsg3 in insect cells and compared its serological reactivity with that of the hdsg3 using species specific polyclonal sera and a panel of seven monoclonal antibodies (MoAbs) with unique binding specificities to hdsg3. Studies using sera demonstrated a considerable cross-reactivity, while studies using MoAbs exhibited specific epitope differences between the two proteins. Because of these differences, we reasoned that immunization with mdsg3 might induce disease in adult mice. Immunization of four strains of mice (i.e. BALB/c, DBA/1, HRS/J and SJL/J) with mdsg3 resulted in considerable antibody response, but failed to induce lesions. However, sera from immunized BALB/c mice induced acantholysis of neonatal mouse skin in vitro. These studies indicated that our inability to induce lesions in adult mice through active immunization is not due to differences in the ability of mouse and human dsg3 to induce acantholytic antibodies, but due probably to structural differences between adult and neonatal mouse skin. Alternatively, immunization with a combination of dsg3 protein along with other proteins might be necessary to induce pemphigus disease in adult mice. Nevertheless, our current studies show that molecular mechanisms leading to the production of acantholytic antibodies in mice can now be studied using homologous mdsg3.

Animals↗

PubMed2001

Contrasting effects of IG20 and its splice isoforms, MADD and DENN-SV, on tumor necrosis factor alpha-induced apoptosis and activation of caspase-8 and -3.

We identified a novel cDNA (IG20) that is homologous to cDNAs encoding a protein differentially expressed in normal and neoplastic cells (DENN-SV) and human MADD (MAPK-activating death domain-containing protein). Furthermore, we show that the above variants most likely result from alternative splicing of a single gene. Functional analyses of these variants in permanently transfected HeLa cells revealed that IG20 and DENN-SV render them more susceptible or resistant to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis, respectively. All variants tested could interact with TNF receptor 1 and activate ERK and nuclear factor kappaB. However, relative to control cells, only cells expressing IG20 showed enhanced TNF-alpha-induced activation of caspase-8 and -3, whereas cells expressing DENN-SV showed either reduced or no caspase activation. Transfection of these cells with a cDNA encoding CrmA maximally inhibited apoptosis in HeLa-IG20 cells. Our results show that IG20 can promote TNF-alpha-induced apoptosis and activation of caspase-8 and -3 and suggest that it may play a novel role in the regulation of the pleiotropic effects of TNF-alpha through alternative splicing.

Alternative Splicing↗

PubMed2001

Targeted delivery of anti-CTLA-4 antibody downregulates T cell function in vitro and in vivo.

CTLA-4 is a T cell surface molecule that binds to the costimulatory molecules CD80 and CD86 on antigen-presenting cells and downregulates T cell function. Therefore, we wanted to test whether antigen-specific activated T cells could be inhibited through directed CTLA-4 signaling using a bispecific antibody (BiAb) capable of simultaneously binding to CTLA-4 and a tissue-specific antigen. The BiAb was prepared by linking two separate monoclonal antibodies against CTLA-4 and the thyroid-stimulating hormone receptor (TSHR). The mouse B cell lymphoma line M12 (H2(d)) was used to induce alloreactive T cells in CBA/J mice (H2(k)); M12 cells stably transfected with the cDNA encoding murine TSHR (mM12) were used to restimulate the alloresponse in vitro. Results of assays for in vitro T cell proliferation, IL-2 production, and cytotoxicity in the presence of BiAb demonstrated that the BiAb could inhibit the T cell alloresponse when stimulated with mM12 cells but not with M12 cells. This effect was dependent on binding of TSHR-bound BiAb to CTLA-4, since the addition of soluble CTLA-4-Ig blocked the inhibitory effect. Injection of mM12 cells, along with the BiAb, not with antibodies against TSHR or CTLA-4 either separately or together, into CBA/J mice (H2(k)) downregulated alloreactive T cell responses. Our study demonstrated that the presence of CTLA-4 signaling molecules on the surface of target cells can protect those cells from immune attack by antigen-specific T cells and suggested that a similar approach could have potential therapeutic value in transplant rejection and tissue-specific autoimmune diseases.

Abatacept↗

PubMed2001

Studies using recombinant fragments of human TSH receptor reveal apparent diversity in the binding specificities of antibodies that block TSH binding to its receptor or stimulate thyroid hormone production.

Patients with Graves' disease have autoantibodies that bind to the TSH receptor and stimulate the thyroid, leading to hyperthyroidism. Earlier studies have shown that the ectodomain of the glycosylated human TSH receptor contains epitopes that could adsorb these pathogenic antibodies. Further studies with mutated cDNAs, chimeric proteins, peptides, and antipeptide antibodies suggested that alterations in the conformation of the protein could lead to loss of reactivity, and that thyroid-stimulating antibodies interact with the N-terminal region of the TSH receptor. Although many of these studies provided valuable insights, they were somewhat inconclusive due to limitations inherent to each of the approaches. In an attempt to further define regions within the TSH receptor with which thyroid-stimulating antibodies interact, we expressed seven recombinant TSH receptor fragments in insect cells and tested them for their ability to neutralize TSH binding inhibitory Igs and thyroid-stimulating antibody activity in the sera of patients with Graves' disease. The fragments containing amino acids 22-305 were able to neutralize the TSH binding inhibitory Ig activity, whereas a fragment containing amino acids 54-254 was able to neutralize the thyroid-stimulating antibodies. Fragments containing additional amino acids, flanking residues 54-254, failed to neutralize the thyroid-stimulating antibody activity, suggesting that thyroid-stimulating antibody epitopes are masked. Our studies show that thyroid autoantibodies, with different functional properties, bind to distinct conformational epitopes on the TSH receptor.

Animals↗

PubMed2000

Adjuvant effects of cholera toxin b subunit on immune response to recombinant thyrotropin receptor in mice.

We had previously shown that BALB/c mice immunized with the extracellular domain of human thyrotropin receptor (ETSHR) developed moderate hyperthyroxinemia. The antibody responses in these mice were predominantly of the IgG1 subclass. Since cholera toxin B subunit (CT-B) has direct effects on the thyroid, and is known to activate B lymphocytes and cause enhanced IgG1 production, we tested the ability of CT-B to modulate the antibody response to ETSHR. CT-B is unique in that it not only elicits a strong immune response to itself, but more importantly, when given with other antigens acts as a potent adjuvant. In the present study, BALB/c mice given ETSHR with CFA or CT-B via ip route showed higher titers of antibodies to ETSHR when compared to mice similarly immunized with ETSHR alone, or with IFA. Antibodies in ETSHR+CT-B immunized mice were mostly of the IgG1 subclass and reacted predominantly with ETSHR peptides 1 (aa 22-41), 21 (aa 322-341), and 23 (352-371). In contrast, animals immunized with ETSHR+CFA showed IgG1, IgG2a and IgG2b responses and reacted with peptides 1 and 21. Furthermore, mice immunized with ETSHR along with CT-B showed significantly higher levels of thyrotropin (TSH) binding inhibitory immunoglobulins (TBII) compared to those that did not receive CT-B. None of the mice immunized with a control antigen showed antibody response to ETSHR. These results suggested that CT-B could enhance and modulate immune response to ETSHR.

Adjuvants, Immunologic↗

PubMed2000

Clinical and environmental isolates of Burkholderia cepacia exhibit differential cytotoxicity towards macrophages and mast cells.

Burkholderia cepacia is an emerging opportunistic pathogen that causes fatal infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Various environmental isolates of B. cepacia are, however, capable of degrading environmental pollutants, such as trichloroethylene, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), etc., and are also highly effective in controlling plant diseases caused by nematodes and fungi. Such strains have therefore been proposed for environmental release to clean up toxic dump sites or as biopesticides. Various efforts to distinguish between clinical and environmental isolates of B. cepacia with regard to their virulence characteristics have produced ambiguous results, suggesting that newer methods are needed to test for the presence or absence of pathogenic potential in B. cepacia strains proposed for environmental release. We now report that several clinical strains of B. cepacia secrete cytotoxic factors that allow macrophage and mast cell death in the presence of external ATP. Several environmental strains had reduced activity in this regard. We also demonstrate that, while all the strains secrete enzymes that have nucleoside diphosphate kinase (Ndk), adenylate kinase (Ak) and 5'-nucleotidase activity, the level of secretion of the 5'-nucleotidase (and/or ATPase/phosphatase) appears to be lower in the environmental strains than in the clinical strains. The secretion of these enzymes is specifically activated in the presence of eukaryotic proteins such as alpha2-macroglobulin. As macrophage-or mast cell surface-associated P2Z receptors promote their cell death in the presence of mM concentrations of ATP, and as the secreted ATP-using enzymes generate various phosphorylated or non-phosphorylated adenine nucleotides that may even be better agonists than ATP in activating the P2Z receptors or may act through the activation of additional purinergic receptors, such enzymes may play an important role in allowing B. cepacia to evade host defence.

Adenosine Diphosphate↗

PubMed1999

BALB/c mice produce blister-causing antibodies upon immunization with a recombinant human desmoglein 3.

Pemphigus vulgaris (PV) is an Ab-mediated autoimmune blistering disease of mucotaneous surfaces. Over 95% of the patients with PV express DR4 or DRw6, and the disease is characterized by the presence of autoantibodies directed against desmoglein 3 (Dsg 3), a protein expressed on keratinocytes. An appropriate animal model is required to understand immunoregulation and to address the role of immunogenetic components in the production of pathogenic Abs that are characteristic of PV. Therefore, we turned to the development of a mouse model. Four strains of female mice (BALB/c, DBA/1, SJL/J, and HRS/J) were screened for their ability to produce pathogenic anti-Dsg 3 Abs. We demonstrated that only BALB/c mice immunized with a full-length Dsg 3 can produce pathogenic Abs capable of causing acantholysis of human foreskin in culture and blistering in neonatal mice. This observation suggested that either H-2d or the BALB background contains the immunogenetic makeup necessary for the production of pathogenic anti-Dsg 3 Abs. No correlation was noted between a given isotype and the pathogenic potential of autoantibodies from different strains of mice. Similarly, the pattern of reactivity of Abs with a panel of 46 synthetic peptides that span the entire Dsg 3 failed to reveal any association between binding specificity and the pathogenic potential, and suggested that pathogenic Abs might recognize conformational epitopes. Moreover, our studies showed that the epitopes recognized by pathogenic Abs are contained within the extracellular Dsg 3.

Animals↗

PubMed1999

Induction of experimental autoimmune Graves' disease in BALB/c mice.

We immunized BALB/c mice with M12 cells (H-2d) expressing either mouse (mM12 cells) or human thyrotropin receptor (TSHR) (hM12 cells). Immunized mice developed autoantibodies to native TSHR by day 90 and, by day 180, showed considerable stimulatory Ab activity as measured by their ability to enhance cAMP production (ranging from 6. 52 to 20.83 pmol/ml in different treatment groups relative to 1.83 pmol/ml for controls) by TSHR-expressing Chinese hamster ovary cells. These mice developed severe hyperthyroidism with significant elevations in both tetraiodothyronine and triiodothyronine hormones. Tetraiodothyronine levels in different experimental groups ranged from a mean of 8.66-12.4 microg/dl, relative to 4.8 microg/dl in controls. Similarly, mean triiodothyronine values ranged from 156.18 to 195.13 ng/dl, relative to 34.99 ng/dl for controls. Next, we immunized BALB/c mice with a soluble extracellular domain of human TSHR (TBP), or TBP expressed on human embryonic kidney cells (293 cells) (293-TBP cells). These mice showed severe hyperthyroidism in a manner very similar to that described above for mice immunized with the mouse TSHR or human TSHR, and exhibited significant weight loss, with average weight for treatment groups ranging from 20.6 to 21.67 g, while controls weighed 24.2 g. Early after onset of the disease, histopathological examination of thyroids showed enlargement of colloids and thinning of epithelial cells without inflammation. However, later during disease, focal necrosis and lymphocytic infiltration were apparent. Our results showed that conformationally intact ectodomain of TSHR is sufficient for disease induction. Availability of a reproducible model in which 100% of the animals develop disease should facilitate studies aimed at understanding the molecular pathogenesis of Graves' disease.

Adoptive Transfer↗

PubMed1999

Neuroendocrine-induced synthesis of bone marrow-derived cytokines with inflammatory immunomodulating properties.

Although cytokines and other soluble regulators of immunity are known to be involved in hematopoiesis, little is known about the signals that induce the synthesis of those mediators locally. Based on recent studies linking the neuroendocrine hormone thyrotropin [thyroid-stimulating hormone (TSH)] to immune cell function in other tissues, we investigated the capacity of TSH to activate cytokine responses from bone marrow cells. These studies reveal that stimulation of the TSH receptor on bone marrow cells-using highly purified or recombinant TSH or by direct stimulation with anti-TSH receptor antibodies-rapidly induces the synthesis of cytokines from bone marrow cells that are classically used in the regulation of inflammatory responses. Of 13 cytokines screened for activity by ELISA or by RNase protection assays for gene expression, IL-6, IFN-beta, TNFalpha, TNFbeta, TGFbeta2, and lymphotoxin-beta responses were reproducibly induced by TSH within 2-3 h of stimulation. Intracellularly, TSH stimulation of bone marrow cells caused rapid increases in cAMP levels and induced the phosphorylation of the Jak2 protein kinase, thereby defining a novel G-protein-coupled receptor/cytokine synthesis pathway. These findings demonstrate that TSH can serve as a primary inductive signal of cytokine production by bone marrow cells.

Animals↗

PubMed1999

Comparison of immune responses to extracellular domains of mouse and human thyrotropin receptor.

The mouse and human thyrotropin receptors show greater than 87% homology in their amino acid sequences. However, glycosylated extracellular domains of mouse (mET-gp) and human (hET-gp) thyrotropin receptors showed differences in their ability to react with patient autoantibodies to thyrotropin receptor (TSHR). To test for potential differences in their immunogenicity, we immunized BALB/c mice with either gel pure non-glycosylated ectodomain of human TSHR (ETSHR II), or hET-gp (hET-gp III), or mET-gp (mET-gp III). Alternatively, mice were primed with gel pure hET-gp or mET-gp and subsequently immunized with insect cells expressing hET-gp (hET-gp II) or mET-gp (mET-gp II) respectively. All groups of mice immunized with TSHR developed high titers of antibodies against the respective immunogens. As shown earlier, sera obtained from mice immunized with ETSHR showed strong reactivity to peptide 1 (aa 22-41) and weak reactivity to peptides 23 (aa 352-371), 24 (aa 367-386), 25 (aa 382-401), and 26 (aa 397-415). Mice immunized with hET-gp or mET-gp showed comparable titers to peptides 1 and 23 and lower reactivity to other peptides. Mice immunized with hET-gp showed higher TBII reactivity (52.2%) compared to mice immunized with either ETSHR (20.9%) or mET-gp (34.5%). Peptides from the C-terminal region of ETSHR could neutralize the TBII activities of sera from mice immunized with ETSHR or hET-gp but not mET-gp. Compared to corresponding control mice, T4 levels in mET-gp II mice were only marginally higher. These data suggested that outcome of immunization with mouse ETSHR is comparable to that seen after immunization with human ETSHR.

Amino Acid Sequence↗

PubMed1999

Selective binding of thyrotropin receptor autoantibodies to recombinant extracellular domain of thyrotropin/lutropin-chorionic gonadotropin receptor chimeric proteins.

The extracellular domain of the glycosylated human thyrotropin receptor (ET-gp) contains epitopes that can adsorb pathogenic antibodies from sera of patients with Graves' disease (GD). In an attempt to define the regions within the ETSHR with which autoantibodies interact, we expressed extracellular domains of eight thyrotropin receptor/chorionic gonadotropin receptor (TSHR/LH-CGR) chimeric proteins in insect cells. The levels of expression were high and chimeric proteins were glycosylated. Chimeric proteins designated as EMc2+4 and EMc2+3+4, in which amino acids (aa) 90-165 and 261-370, and aa 90-370, respectively, of TSHR were replaced with corresponding aa of LH-CGR, partially reversed the thyrotropin binding inhibitory immunoglobulin (TBII) activity of experimental anti-TSHR antisera (anti-ET-gp). The other six chimeras almost completely reversed the TBII activity of these anti-ET-GP antisera. Next, we tested the ability of these chimeric proteins to reverse the TBII activity of GD patients' sera. Similar to our earlier study, ET-gp protein reversed the TBII activity of all eight GD patients' sera tested. Chimera EMc2, in which aa 90-165 of TSHR has been replaced with corresponding aa of LH-CGR, and EMc2+4 partially reversed the TBII activity of only three of the eight GD patients' sera. However, the other six chimeric proteins failed to neutralize the TBII activity of any of GD patients' sera. These data showed the following: (1) There is considerable heterogeneity amongst autoantibodies in GD patients' sera, (2) The TBII activity of some, but not others, is dependent on aa 90-165 and 261-370, and (3) Most Graves' sera, with TBII activity, failed to react with chimeric proteins in which either N-terminal or C-terminal regions of the extra cellular domain of the TSHR were replaced with corresponding regions of LH-CGR. These results suggest that the TBII activity of GD patients' sera is dependent on conformational epitopes and replacement of certain regions of TSHR with homologous regions of LH-CGR results in sufficient alteration in the conformation of the protein leading to loss of reactivity.

Animals↗

PubMed1999

P2Z-Independent and P2Z receptor-mediated macrophage killing by Pseudomonas aeruginosa isolated from cystic fibrosis patients.

We demonstrate that a mucoid, alginate-producing strain of Pseudomonas aeruginosa isolated from the lungs of a cystic fibrosis (CF) patient secretes multiple enzymes with nucleoside diphosphate kinase (Ndk), ATPase, adenylate kinase, 5'-nucleotidase, and ATP-modifying enzymatic activities. The secretion is triggered at high cell density and in complex media but is greatly reduced when the mucoid cells are grown in mineral salts media or in presence of 5.0 mM Ca2+ or Mg2+. Interestingly, the secretion is triggered primarily in the mucoid CF isolate of strain 8821M (or in strain FRD1) but not in a nonmucoid laboratory strain, PAO1. The purified secreted Ndk shows 100% match in its N-terminal amino acid sequence with that of purified intracellular Ndk and demonstrates similar enzymatic properties. The N-terminal sequence of the purified ATPase isolated from an ndk knockout mutant shows its identity with that of the heat shock chaperonin Hsp60. During fractionation, the flowthrough fraction from a Mono Q column demonstrates the presence of 5'-nucleotidase, adenylate kinase, and a putative ATP reductase activity. These fractions demonstrate high cytotoxic activities for murine peritoneal primary macrophages which can be further stimulated in the presence of ATP or inhibited by pretreatment of macrophages with oxidized ATP (oATP). The cytotoxicity associated with ATP-induced stimulation is believed to be due to activation of macrophage surface-associated P2Z (P2X7) receptors, which are one of the purinergic receptors responsible for pore formation on macrophage membrane. Blocking of these receptors by pretreatment with oATP blocks ATP-induced macrophage cell death. Thus mucoid P. aeruginosa cells elaborate enzymes that modulate the external ATP levels of macrophages, thereby modulating macrophage cell death through P2Z receptor activation. Evidence for the presence of secreted cytotoxic agents that act independently of P2Z receptor activation is also presented.

Adenosine Diphosphate↗

PubMed1999

Direct detection of Sabin poliovirus vaccine strains in stool specimens of first-dose vaccinees by a sensitive reverse transcription-PCR method.

A multiplex reverse transcription-PCR method was optimized to monitor the duration of excretion of Sabin poliovirus strains in stools of vaccinees following administration of the first dose of the trivalent oral vaccine. The assay detected approximately 1 50% tissue culture infective dose of each poliovirus serotype spiked into cell culture media. Although PCR inhibitors were frequently encountered in the stool specimens, a 1:20 dilution of the extracted RNA was sufficient to obtain a positive PCR result. Analysis of 195 stool specimens collected from 26 vaccinees showed that poliovirus types 1, 2, and 3 were identified more frequently by PCR than by tissue culture isolation. The percentages of specimens positive by PCR for poliovirus types 1, 2, and 3 were 67.2, 82.6, and 53.8, respectively. In contrast, the culture method identified types 1, 2, and 3 virus in 55.4, 64.1, and 27.7% of the samples, respectively. Poliovirus type 2 excretion was detected by PCR in practically all of the oral poliovirus vaccine recipients for 4 to 8 weeks following vaccination. In contrast, excretion of type 1 and 3 viruses was more variable, with a range of 1 to 8 weeks. Shedding of type 3 virus ceased in approximately 70% of vaccinees within a week after immunization. In addition to an enhanced sensitivity for the detection of poliovirus, this PCR method permits the direct characterization of virus in stool specimens without further passage in culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen.

Animals↗

PubMed1999

Regulation of thyrotropin receptor protein expression in insect cells.

Expression of large quantities of conformationally intact thyrotropin receptor (TSHR) is essential to understand the structure-function relationship of the receptor. We expressed three different constructs of full-length human TSHR in insect cells: (a) a TSHR cDNA lacking signal sequence (TSHR-ns), (b) a TSHR cDNA containing human TSHR signal sequence (TSHR-hs) and (c) a TSHR cDNA with baculovirus envelope protein encoded signal sequence gp-67 (TSHR-gp). No unique protein band, corresponding to any of these recombinant proteins, was visible upon Coomassie Blue staining after SDS-PAGE. However, Western blot using TSHR specific monoclonal antibody showed unique bands around 80, 100 and 100 kDa in TSHR-ns, TSHR-hs and TSHR-gp virus infected insect cells respectively. All three full-length TSHR proteins could neutralize the TSH binding inhibitory immunoglobulin (TBII) activity from sera of experimental animals. However, only glycosylated proteins (TSHR-hs and TSHR-gp) neutralized the TBII activity of sera from autoimmune thyroid patients, confirming the importance of glycosylation for patient autoantibody reactivity. Expression levels of full-length TSHR proteins were much lower than the levels of similarly produced corresponding ectodomains of TSHR proteins. Southern blot and Northern blot analyses showed that DNA and RNA levels in full-length TSHR virus infected insect cells were comparable to the levels found in cells infected with viruses encoding only the ectodomain of TSHR. These data suggest that full-length TSHR expression is very low and is regulated at the translational level.

Animals↗

PubMed1999

Glycosylated ectodomain of the human thyrotropin receptor induces antibodies capable of reacting with multiple blocking antibody epitopes.

Recently, we showed that the glycosylated ectodomain of the human thyrotropin receptor (hET-gp) reacts with autoantibodies from autoimmune thyroid disease (AITD) patients' sera. To better understand the effects of glycosylation of thyrotropin receptor (TSHR) in antibody induction, we immunized rabbits with hET-gp protein. The rabbits developed relatively high titers of antibodies with highly potent TSH binding inhibitory immunoglobulin (TBII) and thyroid stimulatory blocking antibody (TSBAb) activities. Both the hET-gp and a nonglycosylated ectodomain of the human TSHR (hETSHR) protein significantly reversed the TBII as well as TSBAb activity. Based on the ability of synthetic peptides to significantly reverse the functional activity of these rabbit antisera, we identified three discrete regions of the TSH R, represented by amino acids 202-221, 292-311 and 367-386, as TBII epitopes and four regions represented by amino acids 352-371, 367-386, 382-401 and 392-415 as TSBAb epitopes. These data demonstrate that rabbit antibodies that bind to amino acids 367-386 mediate their TSBAb activity by inhibiting the binding of TSH to TSHR; whereas, antibodies to regions 352-415, excluding aa 367-386, exert their TSBAb activity by affecting a step subsequent to TSH binding. Coincident with the elevation of TBII and TSBAb activity, serum total T4 levels declined and thus suggested that the antibodies exerted functional effects on thyroid in vivo. Together, these data demonstrate that glycosylated hET-gp protein is a more potent immunogen and it can induce a broader antibody response directed against multiple TBII and TSBAb epitopes.

Animals↗