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P Chong

Publications and source records attributed to P Chong.

At least 37 records · Page 2Linked to original sources
PubMed1997

A strategy for rational design of fully synthetic glycopeptide conjugate vaccines.

The present study describes a strategy to rationally design fully synthetic glycopeptide conjugate vaccines. Glycopeptide immunogens were constructed by coupling synthetic oligosaccharides comprising repeating units of synthetic 3-beta-D-ribose-(1-1)-D-ribitol-5-phosphate (sPRP) to synthetic peptides containing potent T-helper cell determinants and B-cell epitopes of the Haemophilus influenzae type b (Hib) outer membrane proteins (OMPs) P1, P2, and P6. Rabbit immunogenicity studies revealed that some of these fully synthetic glycoconjugates were capable of eliciting high titers of both anti-PRP and anti-OMP immunoglobulin G antibodies. In addition, we systematically investigated the factors which could influence their immunogenicity. We observed that the magnitude of the anti-PRP antibody response markedly depended on the relative spatial orientation of sPRP and T-cell epitopes, the anti-PRP antibody response was enhanced when a multiple antigenic peptide was used as a carrier, the anti-PRP antibody response was optimal for three PRP repeating units, and lipidation of peptide-PRP conjugates had a minimal effect on the magnitude of the anti-PRP antibody response. The results of this study clearly demonstrate that coupling a carbohydrate hapten to a peptide can provide T-cell help and convert it into a T-cell-dependent antigen. The antisera raised against these conjugates were also found to be protective against Hib infection in the infant rat model of bacteremia.

Amino Acid Sequence↗
PubMed1996

Effects of interferon-alpha on cytokine profile, T cell receptor repertoire and peptide reactivity of human allergen-specific T cells.

A large panel of T cell clones (TCC) specific for the recombinant form of Poa pratensis allergen (rKBG7.2 or Poa p9) were established from the peripheral blood of grass pollen-sensitive donor in the absence or presence of recombinant interferon-alpha (IFN-alpha) in bulk culture and their pattern of cytokine secretion, peptide reactivity and TCR V beta repertoire was examined. The majority of allergen-specific TCC derived in absence of IFN-alpha produced high amounts of interleukin-4 (IL-4) and IL-5 but not IFN-gamma (Th2 cells), while most of TCC derived in presence of IFN-alpha produced IFN-gamma but not, or limited amounts of, IL-4 and IL-5 (Th1 or Th0 cells). Of 24 TCC established in the presence of IFN-alpha, 22 were able to recognize a single allergen peptide, p26, while none of the clones established in the absence of IFN-alpha showed a similar specificity. The majority of both clones expressed the V beta 2 element regardless of whether they were established in the presence of INF-alpha, but the presence of IFN-alpha favored the expansion of V beta 2+, V beta 17+ and V beta 22+ Poa p9-specific T cells, whereas in the absence of IFN-alpha, other TCR V beta-bearing T cells (V beta 5, and V beta 6.7, and V beta 14) were expanded in addition to V beta 2+ T cells. None of V beta 2+ clones established in the absence of IFN-alpha reacted with p26, whereas all the V beta 2+ clones established in its presence in the absence of interferon-alpha reacted with p26, whereas all the V beta 2+ clones established in its presence reacted to this peptide. IFN-alpha also shifted the TCR V beta repertoire of both Poa p9- and Lolium perenne group 1 (Lol p1)-specific T cell lines generated from the same patient and from a different grass-sensitive individual. These data demonstrate that IFN-alpha modulates the development of allergen-specific T cells in vitro, and suggest that IFN-alpha may represent a useful tool for novel immunotherapeutic approaches in allergic disorders.

Allergens↗
PubMed1996

Multiple B- and T-cell epitopes on a major allergen of Kentucky Bluegrass pollen.

The B- and T-cell epitopes of a recombinant grass allergen, rKBG60, were delineated using a set of overlapping synthetic peptides. Direct binding by enzyme-linked immunosorbent assay (ELISA) utilizing serum pools led to the identification of 13 murine immunoglobulin-, and nine to 13 human IgG- and five to seven human IgE-reactive overlapping peptides. Of the peptides which bound to human IgE antibodies, all but three peptides bound to human and/or murine IgG antibodies. Furthermore, eight out of 12 synthetic peptides induced antigen-specific antibodies in mice, suggesting that these peptides contained epitopes that recognized and/or induced T cells. These results, in conjunction with cross-recognition of different peptides at the C-terminus of rKBG60 by antibodies to neighbouring or non-overlapping peptides suggest that the C-terminus of this antigen represents a dominant antigenic and allergenic site. Peripheral blood mononuclear cell (PBMC) proliferation studies using these synthetic peptides for 13 grass allergic individuals indicated that seven potential human T-cell epitopes exist on this allergen. Taken together, the results demonstrate that multiple B- and T-cell epitopes exist on this major group of grass allergens, the majority of which are localized at the C-terminus of this antigen.

Allergens↗
PubMed1996

Identification of domains in rubella virus genomic RNA and capsid protein necessary for specific interaction.

In rubella virus-infected cells, genomic 40S and subgenomic 24S RNAs are present in the cytoplasm of infected cells. However, encapsidation by rubella virus capsid protein is specific for 40S genomic RNA. As a first step toward understanding the assembly of rubella virus nucleocapsid at the molecular level, the interaction between capsid protein and genomic RNA was studied by Northwestern (RNA-protein) blot analysis. RNA probes prepared by in vitro transcription were used to localize the RNA sequence that participates in binding to the capsid protein. We have identified a 29-nucleotide RNA sequence (nucleotides 347 to 375) that is essential for the binding. By using overlapping synthetic peptides of capsid protein, a peptide domain (residues 28 to 56) that displays specific RNA-binding activity of capsid protein has been located. This result suggests that the specific recognition of viral RNA during rubella virus assembly involves, at least in part, the nucleocapsid protein.

Amino Acid Sequence↗
PubMed1996

Host genetic and adjuvant factors influence epitope specificity to a major recombinant grass allergen.

The role of host genetic and adjuvant factors in the induction of immune responses to a major recombinant Kentucky bluegrass allergen was examined utilizing five strains of mice and two different adjuvants. Analysis of the recombinant allergen-specific antibodies induced in these strains indicated that the antibodies of various isotypes were differentially regulated. In terms of IgE antibody response, BDF1 and DBA/2 were characterized as high responder, whereas BALB/C, CBA/J and C57BL/6 were intermediate and SJL was a low responder. In different strains, both dextran sulfate (DS) and complete Freund's adjuvant (CFA), as adjuvants, induced recombinant allergen-specific IgE antibodies of similar titer, however, CFA induced higher IgG2a and lower IgM antibodies compared to DS. Further, analysis of T cell proliferative responses of the splenocytes of different strains demonstrated that these strains varied also in their capacity to respond to synthetic peptides. Furthermore, utilizing a panel of synthetic peptides corresponding to the recombinant allergen, we demonstrated that the antibodies induced by the recombinant allergen with CFA in different strains vary with respect to their epitope specificity. In the BDF1 strain, compared to DS, CFA as adjuvant induced recombinant allergen-specific antibodies of additional peptide specificity. Taken together, these results suggest that both host genetic background and adjuvants govern the fine specificity of antibodies produced against this recombinant Kentucky bluegrass allergen.

Adjuvants, Immunologic↗
PubMed1996

The insulin-mimetic agent vanadate promotes receptor endocytosis and inhibits intracellular ligand-receptor degradation by a mechanism distinct from the lysosomotropic agents.

Vanadate (sodium orthovanadate) is an insulin-mimetic agent and phosphotyrosine phosphatase inhibitor that has been proposed as a potential therapeutic agent for diabetes. We previously reported that vanadate decreased the number of cell-surface insulin receptors but inhibited receptor degradation in cultured lymphocytes (IM-9) (1). To determine whether vanadate affected receptors without intrinsic tyrosine kinase activity, its effects on LDL and transferrin receptors and their ligands were examined. Vanadate exposure resulted in a dose- and time-dependent decrease in LDL binding to cultured human fibroblasts associated with a decrease in cell surface receptor number while total solubilized cell LDL receptors increased. Vanadate also inhibited the LDL-mediated downregulation of total cellular LDL receptors in the absence and presence of cycloheximide consistent with an inhibition of LDL receptor degradation. In the case of the ligand, vanadate augmented the accumulation of intact 125I-LDL associated with an inhibition of up to 80% of the ability of LDL to decrease cholesterol synthesis. Since these actions were similar to the effects of lysosomotropic agents, we examined the effect of vanadate on intraendosomal pH using the fluorescent probe acridine orange. In contrast with chloroquine and NH4Cl, vanadate did not neutralize the pH of the acidic intracellular compartment. Furthermore, after a transient insulin-like effect, chronic exposure to vanadate diminished 125I-diferric transferrin binding to rat adipocytes. In contrast with the inhibitory action of NH4Cl, intracellular 59Fe uptake remained unaffected and was proportional to cell-surface binding capacity in the presence of vanadate. These data demonstrate a chronic effect of vanadate to promote the accumulation of intracellular receptors and to inhibit ligand and receptor degradation. The latter effect is not mediated by pH changes, appears to be localized to a late endosomal/lysosomal compartment, and suggests a possible role for tyrosine dephosphorylation in the regulation of receptor-ligand degradation.

Adipocytes↗
PubMed1995

Murine granulocytic cell adhesion to bone marrow hemonectin is mediated by mannose and galactose.

Hemonectin (HN) is a bone marrow (BM) protein that promotes specific attachment of immature granulocytes and their precursors within the BM. We report that HN is a glycoprotein containing both mannose and galactose residues, and provide evidence that these carbohydrates mediate granulocytic cell adhesion to HN. Carbohydrate structure was determined by digoxigenin-conjugated lectin binding to HN and indicated the presence of mannose, galactose, sialic acid, and the absence of fucose-linked oligosaccharides. The role of carbohydrates in mediating cell adhesion was examined by chemical and enzymatic deglycosylation. Deglycosylation of HN with trifluoromethanesulfonic acid, which cleaves N- and O-linked oligosaccharides, inhibits 66% of cell attachment to HN, and results in an apparent decrease in molecular weight from 60 to 50 kD. Enzymatic deglycosylation with endo-B-N-acetylglucosaminidase H, which hydrolyzes specific N-linked mannose residues, inhibits 30% of cell adhesion to HN. Finally, the role of these specific sugars in hemonectin-mediated cell adhesion was confirmed with neoglycoprotein blocking. Preincubation of BM cells with mannosyl- and galactosyl-BSA probes produces a dose-dependent inhibition of cell attachment to HN, whereas fucosyl-BSA does not inhibit cell adhesion to HN. These results show that mannose and galactose partially mediate adhesion of BM granulocytes to HN.

Animals↗
PubMed1995

The sequencing of the 80-kDa D15 protective surface antigen of Haemophilus influenzae.

The 80-kDa D15 antigen (D-15-Ag) has previously been shown to be a target for protective immunity and conserved amongst typeable and nontypeable Haemophilus influenzae. Here, the gene encoding D-15-Ag is shown to encode a 797-aa polypeptide which, after cleavage of the predicted signal peptide, would have a molecular mass of 85,632 Da.

Amino Acid Sequence↗
PubMed1995

Removal of superglue from the external ear using acetone: case report and literature review.

The ability of superglue preparations (cyanoacrylate) to bond instantly to skin as well as inanimate objects presents a special problem when instilled into the external ear canal. We present two cases of patients who had it successfully removed under the microscope with the help of acetone BP to debond it from the skin, without any damage to the meatus or the tympanic membrane. The third patient had the superglue removed without the help of acetone but sustained damage to the tympanic membrane. A literature review of similar cases is included.

Acetone↗
PubMed1995

HIV-1 recombinant poxvirus vaccine induces cross-protection against HIV-2 challenge in rhesus macaques.

Rhesus macaques were immunized with attenuated vaccinia or canarypox human immunodeficiency virus type 1 (HIV-1) recombinants and boosted with HIV-1 protein subunits formulated in alum. Following challenge with HIV-2SBL6669, three out of eight immunized macaques resisted infection for six months and another exhibited significantly delayed infection, whereas all three naive controls became infected. Immunizations elicited both humoral and cellular immune responses; however, no clear correlates of protection were discerned. Although more extensive studies are now called for, this first demonstration of cross-protection between HIV-1 and -2 suggests that viral variability may not be an insurmountable problem in the design of a global AIDS vaccine.

AIDS Vaccines↗
PubMed1995

A comparison of flow cytometry and microcytotoxicity for the evaluation of alloimmune therapy in patients with recurrent spontaneous abortions.

PROBLEM: There is substantial data that support the efficacy of paternal leukocyte immunization (PLI) for the treatment of alloimmune mediated miscarriage; however, there is confusion regarding the laboratory test that should be performed to determine levels of maternal anti-paternal leukocyte antibodies (MAPLA). METHOD: Popular methodologies employed include: 1) microcytotoxicity (MCX), 2) mixed lymphocyte culture (MLC), and 3) cell flow cytometry crossmatch (FCXM). Cell flow cytometry crossmatch correlates well with the more difficult MLC assay although the former proves the more sensitive study. This work compares the MCX assays with FCXM. The study group consisted of ten women who had a history of three or more spontaneous abortions (SABs). All ten had very low levels (< 10%) of MAPLA as measured by FCXM. Following PLI all subjects demonstrated elevated levels (> 50%) of MAPLA by FCXM. At 12 weeks gestation, sera were simultaneously measured for MAPLA by MCX and FCXM. RESULTS: Although all ten patients had very high levels of MAPLA by FCXM during pregnancy, five of ten had antibodies to HLA Class I and two of ten had antibodies to HLA Class II paternal antigens by MCX. Furthermore, all patients who were positive by MCX to paternal Class I antigens were also positive to Class I antigens not seen in either parent. Both patients who were positive by MCX to paternal Class II antigens were also positive to maternal Class II antigens. Notable is that all ten women eventually delivered healthy infants. CONCLUSION: Based on this preliminary study, the MCX assay is neither sensitive or reliable enough to determine the need and/or to monitor the effectiveness of PLI. Flow cytometry should be the modality of choice when determining the need for alloimmunotherapy and to monitor the effectiveness of treatment.

Abortion, Habitual↗
PubMed1995

Immunogenicity of synthetic peptides of Haemophilus influenzae type b outer membrane protein P1.

To identify the B- and T-cell epitopes of P1 of Haemophilus influenzae type b, 13 peptides covering 90% of the protein were chemically synthesized. Mouse, guinea pig, and rabbit antisera raised against purified native P1 were tested for their reactivities against the peptides in peptide-specific enzyme-linked immunosorbent assays (ELISAs). Six immunodominant linear B-cell epitopes were mapped to residues 103 to 137, 189 to 218, 248 to 283, 307 to 331, 384 to 412, and 400 to 437 of the mature P1 protein. When P1 peptides were screened for their reactivities with three human convalescent-phase serum specimens, peptides corresponding to residues 39 to 64, 226 to 253, and 400 to 437 reacted strongly with the antisera. Four regions (residues 39 to 64, 226 to 253, 339 to 370, and 400 to 437) contained murine T-cell epitopes. Rabbit antipeptide antisera were tested for their reactivities with the immunizing peptides and P1 protein by ELISA and immunoblots. All anti-P1 peptide antisera except those raised against peptide HIBP1-8 (residues 279 to 312) or HIBP1-8-keyhole limpet hemocyanin conjugate were shown to be specific for their respective immunizing peptides by ELISA. In addition, rabbit antisera raised against the synthetic peptides corresponding to residues 1 to 29, 39 to 64, 103 to 137, 189 to 218, 226 to 253, 248 to 283, 307 to 331, and 400 to 437 of the mature P1 protein recognized the P1 protein from both typeable and nontypeable isolates. These results suggest that these peptides contain epitopes highly conserved among typeable and nontypeable strains of H. influenzae. However, none of the antipeptide antisera have bactericidal activity, nor were they protective against H. influenzae type b in the infant rat model of bacteremia.

Amino Acid Sequence↗
PubMed1994

Roles of disulfide bonds in recombinant human interleukin 6 conformation.

Human IL-6 has two disulfide bonds linking Cys45 to Cys51 and Cys74 to Cys84, respectively. Previous site-directed mutagenesis studies have demonstrated that the Cys74-Cys84 bond is essential for full biological and receptor binding activities. To address the structural importance of these disulfide bonds in the formation and stabilization of IL-6 secondary and tertiary structures, we have generated a panel of disulfide bond-deficient rIL-6 analogs both by chemical reduction and alkylation as well as by site-directed mutagenesis. Conformational changes affecting these rIL-6 analogs were probed by circular dichroism spectroscopy, as well as reactivity with monoclonal antibodies, and correlated with changes in biological activities. We have shown that the first disulfide bridge (Cys45-Cys51) is highly sensitive to reduction and, therefore, more solvent-exposed or less thermodynamically stable. Contrary to previous reports, this bridge contributes, although minimally, to the full biological activity of the cytokine. However, no significant changes in secondary or tertiary structures were observed upon removal of this bond. In marked contrast, analogs lacking the disulfide bridge between Cys74 and Cys84 exhibited as little as 0.5% and 0.05% wild-type biological and receptor binding activities, respectively. These dramatic changes correlated with a slight reduction in alpha-helical content and a decreased reactivity with the neutralizing monoclonal antibody mAb8 which recognizes a conformational epitope associated with the active site. Our results suggest that the second disulfide bridge plays a critical role in maintaining the spatial relationship between the putative IL-6 A and D helices.

Alkylation↗
PubMed1994

Immunogenicity study of a synthetic T-cell epitope of rubella virus capsid protein recognized by human T cells in different strains of mice.

The immunogenicity of a human immunodominant T-cell epitope C9 (residues 205-233) of rubella virus capsid protein was studied in three strains of mice using C9 lipopeptide. This peptide induced strong T-cell responses in all three strains of mice. The minimal T-cell epitope C9B (residues 205-216) recognized by human T cells with HLA-DR4 phenotype did not specifically stimulate proliferation of T cells from mice in vitro. T cells specific for C9 from immunized mice were shown to be CD4+, in agreement with results of similar studies in RV-seropositive humans.

Amino Acid Sequence↗
PubMed1994

High fecundity rates following in-vitro fertilization and embryo transfer in antiphospholipid antibody seropositive women treated with heparin and aspirin.

This study was undertaken to explore whether intervention with heparin and aspirin (H/A) in selected patients undergoing in-vitro fertilization (IVF) and embryo transfer could improve fecundity rates. Specifically, it explored the possibility that women diagnosed with organic pelvic disease who demonstrated antiphospholipid antibodies (APA) could benefit from H/A administration in a similar manner to that used in patients with recurrent pregnancy loss. We used an enzyme-linked immunosorbent assay for six different phospholipids to identify patients who expressed APA before they underwent IVF/embryo transfer. This study was confined to the first IVF/embryo transfer cycle that followed assessment of APA status and accordingly, the number of IVF/embryo transfer cycles corresponds with the number of patients treated. APA seropositive patients were treated with aspirin, 81 mg orally q.d., and heparin 5000 IU s.c. b.i.d., beginning on day 1 of controlled ovarian stimulation. The endpoint for success was a live birth or an ultrasound confirming fetal cardiac activity (a viable pregnancy). The prevalence of APA in patients diagnosed with organic pelvic disease (53%) was much higher than in those without female pathology (14%). The administration of H/A to APA seropositive patients significantly (P < 0.05) improved the viable pregnancy rate (49%) compared to the untreated APA seropositive group (16%). The viable pregnancy rate for APA seropositive women treated with H/A was also significantly (P < 0.001) higher than for untreated APA seronegative patients (27%). We conclude that all women undergoing IVF/embryo transfer should be tested for APA prior to initiating ovarian stimulation and those with APA seropositivity should be treated with H/A.

Adult↗
PubMed1994

Characterization of antiphospholipid antibodies in women with recurrent spontaneous abortions.

Antiphospholipid antibodies are important in the etiology of recurrent pregnancy loss. To date, most studies have concentrated on antibodies to cardiolipin specifically. In this study, the serum of 352 women with recurrent pregnancy loss was studied by enzyme-linked immunosorbent assay for antibodies to six phospholipid epitopes: cardiolipin, phosphoserine, phosphoglycerol, phosphoethanolamine, phosphatidic acid and phosphoinositol. Of these women, 59.1% had either an IgG or IgM antibody to one of the six phospholipids. This compared to only 4.6% in the control group. Approximately 75% of the isotypes were IgM. The most common phospholipid epitope was phosphoserine. However, in patients with antibodies to only one phospholipid, phosphoethanolamine was the most common. These findings support recent evidence that antiphospholipid antibodies may interfere with the formation of syncytiotrophoblasts in the placenta. In addition, antiphospholipid antibodies occur more frequently in patients who suffer recurrent miscarriages than was previously thought.

Abortion, Habitual↗
PubMed1993

Structure-function analysis of the C-terminal segment of human interleukin-6.

It has been hypothesized that interleukin-6 (IL-6) and granulocyte-colony-stimulating factor (G-CSF) may fold as four-alpha-helix bundle proteins. To probe the functional role of the putative fourth helical segment of IL-6 (D-helix), a chimeric IL-6/G-CSF analog containing the predicted D-helix of G-CSF as well as a panel of IL-6 D-helix point mutants were analyzed for their respective secondary structure, antigenicity, and receptor binding and biological activities. The putative D-helix of IL-6 could not be replaced by its G-CSF counterpart in spite of their high degree of similarity and thus is indispensable for the antigenic and functional integrity of the IL-6 receptor binding site. Conversely, the grafting of the G-CSF D-helix did not confer any G-CSF activity to IL-6. A synthetic helical peptide containing the IL-6 D-helix was inactive, even when mixed with or linked to a peptide from the A-helix known to be involved in the active site. However, the conserved residues F173, R179, and R182 found in the D-helices of both IL-6 and G-CSF critically contribute to the architecture of the IL-6 active site. Indeed, mutation of F173 or R179 markedly affected IL-6 receptor binding and biological activities, but not the conformation of a major neutralization epitope. Furthermore, substitution of R182 resulted in a significant unfolding of the D-helix accompanied by a drastic loss in IL-6 antigenicity and functional activities. Nevertheless, residues other than F173, R179, and R182 also contribute to IL-6 specificity.

Amino Acid Sequence↗