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Biomedical subjects

W L Wong

Publications and source records attributed to W L Wong.

At least 19 recordsLinked to original sources
PubMed1992

A simple approach to improving sensitivity in a one-step monoclonal antibody-based ELISA for human IIbIIIa using multiple conjugates.

This paper describes an approach which can be used to increase both the speed and the sensitivity of a monoclonal antibody (MAb) based ELISA for human IIbIIIa by using a mixture of enzyme labeled MAbs (conjugates) and a one step incubation format (where analyte and conjugates were incubated simultaneously for 2 h in the MAb coated well). A simple competitive blocking method is described for mapping the relative epitopes of five monoclonal antibodies (MAbs) with minimum preparation of enzyme conjugate. This method permits the selection of three MAbs which recognize distinctly different epitopes, and are suitable for use in a one step ELISA format. Compared to the regular two step ELISA using two MAbs, this simple modification improves the assay sensitivity by three-fold and decreases the incubation time by 75%, while maintaining a high level of precision (2-15%). The assay is very specific and can accurately measure both recombinant and native IIbIIIa.

Antibodies, Monoclonal↗
PubMed1992

Humanization of an anti-p185HER2 antibody for human cancer therapy.

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed. Light- and heavy-chain variable regions were simultaneously humanized in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonucleotides, respectively. The humAb4D5-1 variant does not block the proliferation of human breast carcinoma SK-BR-3 cells, which overexpress p185HER2, despite tight antigen binding (Kd = 25 nM). One of seven additional humanized variants designed by molecular modeling (humAb4D5-8) binds the p185HER2 antigen 250-fold and 3-fold more tightly than humAb4D5-1 and mumAb4D5, respectively. In addition, humAb4D5-8 has potency comparable to the murine antibody in blocking SK-BR-3 cell proliferation. Furthermore, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity against SK-BR-3 cells than mumAb4D5, but it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels.

Adenocarcinoma↗
PubMed1992

HepG2 cells predominantly express the type II interleukin 1 receptor (biochemical and molecular characterization of the IL-1 receptor).

In this study we have characterized the cell surface interleukin 1 (IL-1) receptor in HepG2 hepatoma cells. We found that HepG2 cells bind both IL-1 alpha and beta with high affinity, KDs of 136 and 180 pM and receptor densities of 16,000 and 8500 binding sites/cell respectively. The binding sites appeared to be predominantly type II since phorbol ester treatment of the cells, which selectively downregulates type II IL-1 receptors, reduced binding by 68% while treatment of the cells with an inhibitory monoclonal antibody specific for the type I receptor had no significant effect on IL-1 binding. Competition studies with a modified IL-1 beta analog (Glu4) also revealed binding kinetics more consistent with binding to type II receptors than to type I. Crosslinking and ligand blotting with human 125I-IL-1 demonstrated the presence of two bands, a 78 kDa band typical of crosslinking to type II (p60) receptor, and a 98 kDa band, typical of crosslinking to the type I (p80) receptor. Low level expression of the type I receptor was consistent with molecular biological studies employing polymerase chain reaction (PCR) amplification which indicated that mRNA for the type I receptor was produced by the HepG2 cells. Functional receptors were demonstrated by the induction of IL-8 by IL-1 stimulated cells.

Animals↗
PubMed1992

High level Escherichia coli expression and production of a bivalent humanized antibody fragment.

Many clinical uses of antibodies will require large quantities of fragments which are bivalent and humanized. We therefore attempted to generate humanized F(ab')2 fragments by secretion from E. coli. Titers of 1-2 g l-1 of soluble and functional Fab' fragments have been routinely achieved as judged by antigen-binding ELISA. Surprisingly, this high expression level of Fab' in the periplasmic space of E. coli does not drive dimerization. However, we have developed a protocol to directly and efficiently recover Fab' with the single hinge cysteine in the free thiol state, allowing F(ab')2 formation by chemically-directed coupling in vitro. The E. coli derived humanized F(ab')2 fragment is indistinguishable from F(ab')2 derived from limited proteolysis of intact antibody in its binding affinity for the antigen, p185HER2, and anti-proliferative activity against the human breast tumor cell line, SK-BR-3, which over-expresses p185HER2. This system makes E. coli expression of bivalent antibody fragments for human therapy (or other uses) practical.

Amino Acid Sequence↗
PubMed1992

Modulation of airway inflammation in cystic fibrosis. In vivo suppression of interleukin-8 levels on the respiratory epithelial surface by aerosolization of recombinant secretory leukoprotease inhibitor.

Based on the knowledge that neutrophil elastase (NE) in cystic fibrosis (CF) epithelial lining fluid (ELF) can induce human bronchial epithelial cells to express the gene for interleukin 8 (IL-8), an 8.5-kD neutrophil chemoattractant, we have evaluated CF ELF for the presence of IL-8, and investigated the ability of aerosolized recombinant secretory leukoprotease inhibitor (rSLPI) to suppress NE, and hence IL-8, levels on the respiratory epithelial surface in CF. Enzyme-linked immunoassay revealed 21.9 +/- 4.8 nM IL-8 in CF ELF compared with none in normals. Active NE was detectable in ELF of all individuals with CF and was significantly decreased (P < 0.03) after aerosolization of rSLPI. Human bronchial epithelial cells exposed to CF ELF recovered before rSLPI therapy expressed IL-8 mRNA transcripts, but ELF recovered after rSLPI therapy induced far less bronchial epithelial cell IL-8 gene expression. Consistent with this, rSLPI aerosol therapy caused a marked reduction in CF ELF IL-8 levels (P < 0.05) and neutrophil number (P < 0.02). There was also a clear association between CF ELF active NE and IL-8 levels (r = 0.94). These data suggest that rSLPI therapy not only suppresses respiratory epithelial NE levels, but also breaks a cycle of inflammation on the CF epithelial surface.

Adult↗
PubMed1992

Thyroid follicular cells produce interleukin-8.

Interleukin-8 (IL-8) is a potent proinflammatory cytokine known to be produced by several cell types. To elucidate whether endocrine cells can also make IL-8, we have tested supernatants from eleven thyroid follicular cell primary cultures. IL-8 was readily detected under basal conditions (range 3.4-32.1 ng/ml from 1 x 10(5) cells in 3 days) and was increased 4-20 fold by stimulation with IL-1. TSH and tumor necrosis factor had an inconsistent effect, while gamma-interferon reduced basal and IL-1-stimulated IL-8 production. Since IL-8 can act as a chemoattractant for lymphocytes, these observations may explain in part the accumulation of lymphocytes within the gland in autoimmune thyroiditis.

Cells, Cultured↗
PubMed1992

The musculoskeletal manifestations of epidermolysis bullosa: an analysis of 19 cases with a review of the literature.

Epidermolysis bullosa encompasses a group of rare disorders characterized by marked skin fragility and blister formation. In patients with dystrophic epidermolysis bullosa, skeletal and soft-tissue abnormalities are an important feature. An analysis of the musculoskeletal manifestations in 19 patients is presented. In the hands and feet, features included generalized osteoporosis, wedge-shaped thinning and hooking of distal phalanges, overconstricted bones, acro-osteolysis, flexion contractures, metatarsal and metacarpal subluxation, distal trophic changes, webbing of digits, encasement of the whole extremity in a pouch of skin, soft-tissue calcification and retarded skeletal maturity. Previously undescribed findings in the hands and feet are bony ankylosis of the proximal interphalangeal joints, resorption of the metatarsal and metacarpal heads, shortened metatarsal bones, carpal and tarsal fusion and destruction, and cystic changes of the distal radius and ulna. In the remainder of the skeleton, hip dysplasia with premature osteoarthritis, knee joint bony ankylosis and thoracic and thoraco-lumbar scoliosis are other undescribed findings.

Adolescent↗
PubMed1991

Increase in glia-derived nerve growth factor following destruction of hippocampal neurons.

It is currently believed that under normal conditions hippocampal neurons synthesize nerve growth factor (NGF) which may provide trophic support for cholinergic neurons projecting from the basal forebrain. The concept that glial cells are mobilized to increase the production of NGF following destruction of hippocampal neurons was examined. Excitotoxin-induced destruction of the dorsal hippocampal neurons resulted in a massive and prolonged increase in NGF-like immunoreactivity (LI). Immunostaining for NGF-LI and the glial marker, glial fibrillary acidic protein (GFAP), revealed that the source of increased NGF-LI production following the lesion were reactive astrocytes. Thus, glial cells assume the role of providing trophic support following loss of target neurons.

Animals↗
PubMed1991

p185HER2 signal transduction in breast cancer cells.

A partially agonistic monoclonal antibody, 4D5, known to bind to the extracellular domain of p185HER2 and shown to inhibit long term growth of p185HER2-overexpressing breast cancer cells, was used to study signal transduction and phosphotyrosyl protein substrates associated with this receptor. Normal breast epithelial cells and breast carcinoma cells expressing low levels of p185HER2 were not affected by 4D5. HER2/neu-overexpressing breast cancer cells (BT-474 and SK-Br-3) exposed to 4D5 exhibited rapid phosphorylation of both p185HER2 and an associated 56-kDa phosphotyrosyl protein (ptyr56). Paralleling the 4D5- stimulated phosphorylation of p185HER2 and ptyr56 was a 5-10-fold induction of c-fos mRNA and phosphatidylinositol 4-kinase activity and a 2-fold induction of inositol 1,4,5-trisphosphate 3'-kinase activity. The increased phosphatidylinositol 4-kinase activity immunoprecipitated with p185HER2 and also co-eluted with ptyr56 from an antiphosphotyrosine immunoaffinity column. These results indicate that short term (less than 6 h) 4D5 activation of p185HER2 in overexpressing breast cancer cells produces agonistic-like signaling typical of homologous tyrosine kinase growth factor receptors such as epidermal growth factor receptor. The data also suggest that ptyr56 represents a novel phosphorylated substrate associated with 4D5-stimulated p185HER2.

Blotting, Western↗
PubMed1991

Increase in nerve growth factor-like immunoreactivity and decrease in choline acetyltransferase following contusive spinal cord injury.

We have previously described a graded spinal cord injury model in the rat. Mild contusive injury results in an initially severe functional deficit that is attenuated over time to reveal the mild chronic deficits that characterize this injury. In this study, we have shown that mild contusive injury also results in a significant decrease in choline acetyltransferase (ChAT) activity during the first week after injury. At 1 week ChAT activity is maximally reduced at the site of the contusion and is also significantly lowered throughout the spinal cord. ChAT activity then rebounds during the following 3 weeks, partially at the injury site where there is considerable loss of gray and white matter, and completely in rostral and caudal cord segments. The rebound in ChAT activity is temporally associated with the partial recovery of function. Further, the changes in ChAT activity after injury are mirrored by changes in nerve growth factor-like immunoreactivity (NGF-LI) as determined by a specific two-site ELISA. NGF-LI increases significantly after injury, reaching a maximum at 7 days after contusion and at the injury site. However, levels of NGF-LI are also significantly increased throughout the spinal cord. NGF-LI then decreases at 2 and 4 weeks as ChAT activity rebounds. Further experiments will be needed to examine the possibility of a role for NGF in promoting the recovery of function after spinal cord injury.

Animals↗
PubMed1991

Inhibition of translational initiation by metalloendoprotease antagonists. Evidence for involvement of sequestered Ca2+ stores.

Synthetic oligopeptide inhibitors of metalloendoprotease activity have been shown to block membrane fusion events, to slow transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi, and to perturb Ca2+ homeostasis. Effects of such agents on translational activity, which requires Ca2+ sequestered putatively within the ER, were examined in this study. Cbz-Gly-Phe-NH2 (where Cbz is benzyloxycarbonyl) provoked rapid inhibition of amino acid incorporation into a broad spectrum of proteins in GH3 pituitary, C6 glial, and Neuro-2a cells but not in reticulocytes, which lack ER. Polysome accumulation and incorporation were reduced concurrently, indicating that the dipeptide acted to slow translational initiation. Inhibitions were largest at low extracellular Ca2+, were reversed by increasing extracellular Ca2+, were comparable to those achieved in the presence of EGTA or Ca2+ ionophores, and were observed with assorted metalloendoprotease antagonists but not with leupeptin. At concentrations inhibitory to protein synthesis Cbz-Gly-Phe-NH2 mobilized cell-associated 45Ca, lowered cytosolic free Ca2+, and did not generate inositol phosphates. Cells treated for 3-4 h with Cbz-Gly-Phe-NH2 reacquired the ability to synthesize proteins at nearly normal rates; a phorbol ester or cAMP-elevating agent was necessary for such recovery in GH3, but not C6 or Neuro-2a, cells. GRP78, which may function in the folding and assembly of secretory proteins and in translational accommodation to agents that deplete sequestered Ca2+ stores, was induced during such treatments. Accumulation of GRP78 mRNA in treated preparations was reduced as extracellular Ca2+ was increased. Extended exposure to dipeptide followed by brief recovery in its absence rendered protein synthesis resistant to inhibition by Ca2+ ionophore. It is concluded that metalloendoprotease antagonists suppress translational initiation as a consequence of their capacity to mobilize sequestered Ca2+ stores.

Amino Acid Sequence↗
PubMed1991

Platelet factor 4 selectively inhibits binding of TGF-beta 1 to the type I TGF-beta 1 receptor.

A low molecular weight inhibitor of TGF-beta 1 binding was detected in partially purified human platelet extracts by using Hep 3B hepatoma cells in the binding assays. The inhibitory protein was purified to homogeneity and was identified as platelet factor 4 on the basis of its amino acid sequence. TGF-beta 1 binding to Hep 3B cells was almost completely inhibited by 100 nM concentrations of platelet factor 4, but TGF-beta 1 binding to NRK 49F fibroblasts was inhibited only slightly. Affinity cross-linking experiments revealed that these differences in the inhibition of TGF-beta 1 binding by platelet factor 4 were due to differences in the complements of TGF-beta 1 binding proteins present on these two cell types. In Hep 3B cells the majority of bound TGF-beta 1 was cross-linked to a complex which had an apparent molecular weight of 70 kDa. TGF-beta 1 binding to this protein was the most sensitive to inhibition by platelet factor 4. Based on its size and TGF-beta 1 binding properties, we believe this protein is the type I TGF-beta 1 receptor. Hep 3B cells also had a high-affinity TGF-beta 1 binding protein which appeared as an 80 kDa complex, and which we believe to be the type II TGF-beta 1 receptor. TGF-beta 1 binding to this protein was not inhibited by platelet factor 4. TGF-beta 1 was also cross-linked to complexes of higher molecular weights in Hep 3B cells, but it was not clear whether any of them represented the type III TGF-beta 1 receptor. In NRK 49F cells, the majority of bound TGF-beta 1 was cross-linked to a high molecular weight complex which probably represented the type III TGF-beta 1 receptor. NRK 49F cells also had type I TGF-beta 1 receptors and platelet factor 4 inhibited binding to these receptors in the NRK cells. Since the type I receptor contributed only a small percentage of total TGF-beta 1 binding, however, the overall effects of platelet factor 4 on TGF-beta 1 binding to NRK 49F cells were negligible. We were unable to demonstrate specific or saturable binding of platelet factor 4 to Hep 3B cells using either direct binding or affinity cross-linking assays. Thus, it is not clear whether platelet factor 4 inhibits TGF-beta 1 binding by competition for binding to the type I receptor. Modest concentrations of TGF-beta 1 reduced the adherence of Hep 3B cells to tissue culture dishes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Effects of Ca2+ and ionophore A23187 on protein synthesis in intact rabbit reticulocytes.

1. Amino acid incorporation in intact rabbit reticulocytes was unaffected by depletion of Ca2+ with EGTA. 2. The Ca2+ ionophore A23187 strongly inhibited incorporation in reticulocytes incubated in 1 mM Ca2+ but not in EGTA. Polysomal profiles and average ribosomal transit times of cells treated with Ca2+ ionophore at 1 mM Ca2+ were characteristic of translational elongation block. 3. The behavior of reticulocytes with respect to Ca2+ and A23187 contrasts with that of nucleated cells possessing endoplasmic reticulum in which protein synthesis is inhibited at translational initiation by either Ca2+ depletion or by exposure to Ca2+ ionophore.

Animals↗
PubMed1991

Generation of polyclonal antibodies against recombinant human activin A.

A goat antiserum to purified recombinant human activin A (rhAct-A), a dimer formed by two beta A-subunits of inhibin, has been produced. The immunoreactivity of the antiserum has been evaluated in an antigen coated enzyme-linked immunosorbent assay, in a radioimmunoassay using iodinated rhAct-A, and by Western blot analysis. The antiserum demonstrated some cross reactivity to inhibin A, a structurally related heterodimer which contains an identical beta A-subunit coupled to a distinct, though similar, alpha subunit. A simple radioimmunoassay for rhAct-A in tissue culture supernatant has been developed with rhAct-A affinity column purified polyclonal antiserum. The assay is precise and sensitive with a range of 0.31-40 ng/ml. The cross reactivity of inhibin A in the RIA is about 4.3%. Despite its cross-reactivity this antiserum will facilitate studies of the physiology of activin A and inhibin A which includes a Western blot analysis where a molecular size distinction is accomplished.

Activins↗