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Biomedical subjects

A S Kang

Publications and source records attributed to A S Kang.

At least 19 recordsLinked to original sources
PubMed2005

The detection of protective antigen (PA) associated with spores of Bacillus anthracis and the effects of anti-PA antibodies on spore germination and macrophage interactions.

The protective antigen (PA) component of the anthrax toxins is an essential virulence factor of Bacillus anthracis and is the major protective immunogen. The kinetics of PA production during growth of B. anthracis, and the roles of anti-PA antibody in host immunity are not clearly defined. Production of PA by the vegetative organisms peaks during the shift from exponential to stationary phase of growth. Recently, PA was also found to be associated with spores. In our study, PA-specific mRNA was detected in spores by RT-PCR within 15-min of exposure to germinant. PA protein was detected by immunomagnetic electrochemiluminescence (ECL) on spores within 1 h of exposure to a germination medium and was rapidly released into the supernatant. PA was not demonstrated on ungerminated spores by RNA analysis, ECL, or spore-based anti-PA ELISA; however, it was detected on ungerminated spores by immunoelectron microscopy (immunoem). In rabbits, PA induces polyclonal antibodies (Abs) that, in addition to their anti-toxin neutralizing activities, exhibit anti-spore activities. In this study, the anti-spore effects of a human monoclonal Ab specific for PA (AVP-hPA mAb, Avanir Pharmaceuticals) were characterized. AVP-hPA mAb retarded germination in vitro, and enhanced the phagocytic and sporicidal activities of macrophages. The activities were comparable to those of the polyclonal rabbit anti-rPA Ab. Assays to detect germination inhibitory activity (GIA) in serum from vaccinated mice and guinea pigs suggested a possible role for anti-PA Abs in protection. Thus, anti-PA Ab-mediated, anti-spore activities may play a role in protection during the early stages of an anthrax infection.

Animals↗
PubMed2001

Stage-dependent localization of a novel gene product of the malaria parasite, Plasmodium falciparum.

A novel Plasmodium falciparum gene, MB2, was identified by screening a sporozoite cDNA library with the serum of a human volunteer protected experimentally by the bites of P. falciparum-infected and irradiated mosquitoes. The single-exon, single-copy MB2 gene is predicted to encode a protein with an M(r) of 187,000. The MB2 protein has an amino-terminal basic domain, a central acidic domain, and a carboxyl-terminal domain with similarity to the GTP-binding domain of the prokaryotic translation initiation factor 2. MB2 is expressed in sporozoites, the liver, and blood-stage parasites and gametocytes. The MB2 protein is distributed as a approximately 120-kDa moiety on the surface of sporozoites and is imported into the nucleus of blood-stage parasites as a approximately 66-kDa species. Proteolytic processing is favored as the mechanism regulating the distinct subcellular localization of the MB2 protein. This differential localization provides multiple opportunities to exploit the MB2 gene product as a vaccine or therapeutic target.

Amino Acid Sequence↗
PubMed2000

The I-Ag7 MHC class II molecule linked to murine diabetes is a promiscuous peptide binder.

Susceptibility to insulin-dependent diabetes mellitus is linked to MHC class II genes. The only MHC class II molecule expressed by nonobese diabetic (NOD) mice, I-Ag7, shares a common alpha-chain with I-Ad but has a peculiar beta-chain. As with most beta-chain alleles linked to diabetes susceptibility, I-Ag7 contains a nonaspartic residue at position beta57. We have produced large amounts of empty I-Ag7 molecules using a fly expression system to characterize its biochemical properties and peptide binding by phage-displayed peptide libraries. The identification of a specific binding peptide derived from glutamic acid decarboxylase (GAD65) has allowed us to crystallize and obtain the three-dimensional structure of I-Ag7. Structural information was critical in evaluating the binding studies. I-Ag7, like I-Ad, appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate and contain small and/or small hydrophobic residues at P4 and P6 of the peptide, a motif frequently found in most globular proteins. The degree of promiscuity is increased for I-Ag7 over I-Ad as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains. So, although I-Ad and I-Ag7 are structurally closely related, stable molecules and good peptide binders, they differ functionally in their ability to bind significantly different peptide repertoires that are heavily influenced by the presence or the absence of a negatively charged residue at position 57 of the beta-chain. These characteristics link I-Ag7 with autoimmune diseases, such as insulin-dependent diabetes mellitus.

Amino Acid Motifs↗
PubMed2000

A structural framework for deciphering the link between I-Ag7 and autoimmune diabetes.

Susceptibility to murine and human insulin-dependent diabetes mellitus correlates strongly with major histocompatibility complex (MHC) class II I-A or HLA-DQ alleles that lack an aspartic acid at position beta57. I-Ag7 lacks this aspartate and is the only class II allele expressed by the nonobese diabetic mouse. The crystal structure of I-Ag7 was determined at 2.6 angstrom resolution as a complex with a high-affinity peptide from the autoantigen glutamic acid decarboxylase (GAD) 65. I-Ag7 has a substantially wider peptide-binding groove around beta57, which accounts for distinct peptide preferences compared with other MHC class II alleles. Loss of Asp(beta57) leads to an oxyanion hole in I-Ag7 that can be filled by peptide carboxyl residues or, perhaps, through interaction with the T cell receptor.

Alleles↗
PubMed2000

Is calciphylaxis best treated surgically or medically?

BACKGROUND: Calciphylaxis is a rare, painful, life-threatening problem of cutaneous necrosis and refractory healing in patients with uremia and secondary hyperparathyroidism. The pathogenesis involves abnormalities in calcium and phosphorus metabolism and acute deposition of calcium in tissues. METHOD: The clinical course of 16 patients who were diagnosed with calciphylaxis at our institution from 1994 through 1998 was reviewed. RESULTS: Fourteen female patients and 2 male patients had chronic renal disease, secondary hyperparathyroidism, and characteristic skin necrosis (mean age, 56 years; range, 39-70 years). All patients underwent intensive medical therapy, including ongoing hemodialysis (n = 16 patients), parathyroidectomy (n = 7 patients), and debridement of cutaneous lesions (n = 8 patients). Mean serum values in surgical and nonsurgical patients were significantly different for phosphorus, calcium-phosphorus product, and parathormone levels. Median survival was 9.4 months; 15 patients (93%) have died. The median survival time for parathyroidectomy versus nonparathyroidectomy was 14.8 and 6.3 months (P =.22), for skin debridement versus nondebridement was 14.1 and 6.1 months (P =.08), and for diabetic versus nondiabetic patients was 6.5 and 13.9 months (P =.11). CONCLUSIONS: Calciphylaxis has a female preponderance, with a dismal prognosis. A multidisciplinary approach that uses frequent hemodialysis to normalize calcium and phosphorus levels and local debridement of skin lesions seems prudent. Parathyroidectomy cannot be recommended routinely in all patients, unless severe hyperparathyroidism mandates intervention.

Adult↗
PubMed1999

Expression of a functional antibody fragment in the gut of Rhodnius prolixus via transgenic bacterial symbiont Rhodococcus rhodnii.

Expression within insects of foreign antiparasitic gene products via microbial symbionts could be used to prevent transmission of vector-borne pathogens to vertebrate hosts. Genetically transformed symbiotic bacteria Rhodococcus rhodnii expressed functional antibody fragments (rDB3 encoding murine V(H)/K which binds progesterone) that were exported into the gut lumen of the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae), a vector of Chagas disease. Transgenic symbionts were maintained in successive nymphal instars and adults of Rhodnius prolixus despite competition with native untransformed Rhodococcus rhodnii. This is the first description of a functional mammalian antibody fragment expressed in an insect. Our system is a model for constructing paratransgenic insects (insects carrying transformed symbionts) with compromised ability to transmit pathogens.

Animals↗
PubMed1998

Modulation of antibody display on M13 filamentous phage.

Here we describe a phage vector for the display of single chain antibodies and polypeptides on the surface of filamentous M13 phage which permits facile manipulation of the valency of display. The gene encoding the polypeptide is fused to a synthetic copy of the major coat protein VIII gene (gpVIII) which permits incorporation into the phage during assembly of the filament. Here we examine the growth parameters of phage propagation on the subsequent selection of an anti-progesterone antibody fragment from a mixture of display phage. Our results suggest that the density of the polypeptides displayed on phage may be modulated by altering growth conditions. This ability to influence polypeptide display density on filamentous phage may provide a versatile approach for accessing complex libraries and the capture of weaker ligand receptor interactions by avidity, whilst the potential to access and discriminate between higher affinity interactions is not negated.

Animals↗
PubMed1998

Gene therapy with a single chain interleukin 12 fusion protein induces T cell-dependent protective immunity in a syngeneic model of murine neuroblastoma.

A major goal of tumor immunotherapy is the effective eradication of established metastases associated with the induction of a T cell-mediated protective immunity. We achieved this in a poorly immunogenic murine neuroblastoma model by gene therapy with a single chain interleukin 12 (scIL-12) fusion protein that assures equal expression of its p35 and p40 subunits. Thus, NXS2 hybrid neuroblastoma cells (C1300 x dorsal root ganglion cells), which form experimental bone marrow and liver metastases in syngeneic A/J mice, were transduced with a gene encoding murine interleukin 12, monomerized by introduction of a protein linker between the p35 and p40 protein chains of this heterodimeric cytokine. We demonstrate for the first time that subcutaneous vaccination with these transduced cells induces a protective immunity, as indicated by the complete absence of liver and bone marrow metastasis after challenge with NXS2 wild-type tumor cells. Furthermore, vaccination of animals with established liver and bone marrow metastases completely eradicated liver metastases and suppressed bone marrow metastases. The local and systemic immune response against scIL-12-transduced NXS2 cells is largely dependent on CD8(+) T cells. This was demonstrated in vivo by depletion of immunocompetent A/J mice with monoclonal anti-CD4 and anti-CD8 antibodies and in vitro by specific major histocompatibility complex, class I-restricted CD8(+) T cell-mediated killing of NXS2 and their parental C1300 neuroblastoma cells. In conclusion, we demonstrate successful anti-tumor immunotherapy with an scIL-12 fusion protein that could facilitate clinical application of interleukin 12 gene therapy.

Amino Acid Sequence↗
PubMed1997

Bacterial expression and purification of recombinant Plasmodium yoelii circumsporozoite protein.

We report the expression and purification of recombinant rodent malarial Plasmodium yoelii circumsporozoite surface protein (PyCSP) in Escherichia coli. To facilitate purification of the recombinant protein, the PyCSP was expressed as an amino-terminal fusion protein to glutathione S-transferase and as a carboxy-terminal fusion protein to a hexahistidyl tag. The expression of the fusion protein was controlled by the inducible tac promoter. Under optimal conditions the immunoreactive PyCSP represented approximately 0.04% of the total cell lysate. Western blot analysis probing with an anti-PyCSP antibody revealed a wide array of immunoreactive bands. Material isolated by affinity purification on glutathione-Sepharose 4B resin also contained multiple bands indicative of premature termination or carboxyl-terminal degradation. Analysis of protein retained on a nickel nitrilotriacetic acid resin revealed evidence of amino-terminal deleted material. Combining the two mild affinity purifications resulted in isolation of a single immunoreactive protein of approximate molecular weight of 96 kDa. We anticipate that the approach described in this study will facilitate the production of highly purified recombinant proteins.

Animals↗
PubMed1995

Characterization of a progesterone-binding, three-domain antibody fragment (VH/K) expressed in Escherichia coli.

The heavy chain variable region (VH) and the kappa light chain of the anti-progesterone monoclonal antibody (mAb) DB3, have been expressed as a single-chain three-domain polypeptide, designated VH/K, and secreted into the periplasmic space of Escherichia coli (E. coli). The linker sequence was derived from the VH-CH1 elbow region. The C kappa domain provides a sensitive detection tail for Western blotting and enzyme-linked immunosorbent assay (ELISA). Periplasmic extracts of transformed E. coli contained material that bound progesterone and related steroids with similar specificity and affinity to DB3, and displayed the DB3 idiotype and kappa chain epitopes. Reference to the crystal structure of DB3 suggests that all the characteristics of the combining site interaction with steroids are retained in the bacterially expressed material. Western blotting demonstrated material with a molecular weight equivalent to three domains after reduction, but six domains in the unreduced state, suggesting that the VH/K polypeptide is assembled in the periplasm as a disulphide-bridged dimer. The VH/K construct provides a novel route to expression of antibody combining sites in E. coli for antibody engineering.

Animals↗
PubMed1994

Crystallization, sequence and preliminary crystallographic data for transmission-blocking anti-malaria Fab 4B7 with cyclic peptides from the Pfs25 protein of P. falciparum.

X-ray quality crystals of an Fab fragment from a transmission-blocking monoclonal antibody 4B7 (MAb 4B7) against a sexual stage protein Pfs25 of Plasmodium falciparum were grown as uncomplexed and peptide-complexed forms. Initially, the intact immunoglobulin was crystallized because cleavage with pepsin or papain did not produce a homogeneous product. Further proteolytic trials with elastase produced a suitable Fab fragment from which crystals have been obtained, both for the free Fab and in complex with cyclic peptides and in the presence of linear peptides. While linear peptides bind to MAb 4B7, cyclic peptides modeled on a predicted beta-hairpin loop of the third EGF-like domain of Pfs25 bind better and readily co-crystallize with the Fab. The genes for the variable domain of the Fab have been cloned, sequenced and the primary amino-acid sequence for the complete Fab deduced. This work explores the use of glycerol as an additive and the modification of the peptide sequence outside the epitope for improving in the crystallization. Data sets have been collected from crystals of several Fab-peptide complexes and from uncomplexed Fab to resolutions ranging from 2.4 to 3.3 A. The packing arrangements of several crystal forms have been determined by molecular replacement, and refinement of their three-dimensional structures is in progress. The three-dimensional structure of this Fab complexed with the various peptides will aid in an understanding of the mode by which this antibody recognizes and prevents transmission of the malaria parasite.

Journal Article↗
PubMed1992

In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library.

We have used a combinatorial immunoglobulin library approach to obtain monoclonal antibodies from nonimmune adult mice, thereby establishing the principles of (i) accessing naive combinatorial antibody libraries for predetermined specificities and (ii) increasing the affinity of the selected antibody binding sites by random mutagenesis. A combinatorial Fab library expressing immunoglobulin mu and kappa light-chain fragments on the surface of filamentous phage was prepared from bone marrow of nonimmunized, adult BALB/c mice with the multivalent display vector pComb8. Phage displaying low affinity Fabs (binding constants, 10(4)-10(5) M-1) binding to a progesterone-bovine serum albumin conjugate were isolated from the library. Random mutagenesis of the heavy- and light-chain variable regions expressed in the mono-valent phage display vector pComb3 was performed by error-prone PCR, and subsequently clones with improved affinity for the hapten conjugate were selected. We demonstrate that antibodies with desirable characteristics from a nonimmune source may be selected and affinity maturation may be achieved by using the twin vectors pComb8 and pComb3, thus opening the route to obtaining specific antibodies from a generic library and bypassing immunization.

Amino Acid Sequence↗
PubMed1991

Antibody redesign by chain shuffling from random combinatorial immunoglobulin libraries.

A number of experiments on the shuffling of heavy and light chains from antibodies of defined specificity for the transition-state analogue hapten nitrophenyl phosphonamidate are described. The experiments report on the promiscuity of heavy and light chains in binding antigen and the feasibility of antibody redesign by this shuffling process. The concepts of incestuous and extraclonal promiscuous association are described. Shuffling opens the possibility of generating panels of antibodies with related specificity but of distinct idiotypic composition that may have significance in the use of human monoclonal antibodies in therapy.

Animals↗
PubMed1991

Assembly of combinatorial antibody libraries on phage surfaces: the gene III site.

A phagemid system was developed for the monovalent display of combinatorial antibody Fab libraries on the surface of filamentous phage M13. Fab fragments were fused to the carboxyl-terminal domain of the gene III protein. Phage displaying Fab fragments on their surface, or Phabs, were enriched by 10(3)- to 10(5)-fold on antigen-coated surfaces over nonspecific phage. The method may replace current antibody cloning techniques.

Antibodies, Bacterial↗
PubMed1991

Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces.

We describe a method based on a phagemid vector with helper phage rescue for the construction and rapid analysis of combinatorial antibody Fab libraries. This approach should allow the generation and selection of many monoclonal antibodies. Antibody genes are expressed in concert with phage morphogenesis, thereby allowing incorporation of functional Fab molecules along the surface of filamentous phage. The power of the method depends upon the linkage of recognition and replication functions and is not limited to antibody molecules.

Antibodies, Monoclonal↗