Cytochrome P450 2S1 depletion enhances cell proliferation and migration in bronchial epithelial cells, in part, through modulation of prostaglandin E(2) synthesis.

Cytochromes P450 (P450s) contribute to the metabolic activation and inactivation of various endogenous substrates. Despite years of research, the physiological role of CYP2S1 remains unknown. CYP2S1 has demonstrated NADPH P450-reductase-independent metabolism of cyclooxygenase (COX)-derived prostaglandins [e.g., prostaglandin G(2) (PGG(2))] at nanomolar concentrations. Arachidonic acid is converted to prostaglandin precursors [PGG(2) and prostaglandin H(2) (PGH(2))] through COX. These precursors are used to synthesize numerous prostanoids, including PGE(2). Prostaglandin E(2) (PGE(2)) promotes cell proliferation and cell migration and inhibits apoptosis. CYP2S1 metabolism of PGG(2) presumably sequesters PGG(2) and PGH(2), making them unavailable for synthesis of prostanoids such as PGE(2). Whether CYP2S1 contributes to prostaglandin metabolism and influences cell physiological remains to be determined. The purpose of this study was to evaluate the physiological role of CYP2S1, if any, in human bronchial epithelial cells [SV40-derived bronchial epithelial cell line (BEAS-2B)]. To do this, we used small interfering RNA to deplete CYP2S1 mRNA and protein by approximately 75% and evaluated the impact of CYP2S1 depletion on cell proliferation and migration. CYP2S1 depletion enhanced both cell proliferation and migration in BEAS-2B cells. Consistent with the proposed role of CYP2S1 in PGE(2) synthesis, the reduction in CYP2S1 expression doubled intracellular PGE(2) levels. Pharmacological administration of PGE(2) enhanced cell proliferation in BEAS-2B cells but failed to promote migration. Our data reveal an important role for CYP2S1 in the regulation of cell proliferation and migration, occurring in part through modulation of prostaglandin synthesis.

The use of vitamin A-deficient diets and jugular vein ligation to increase intracranial pressure in chickens (Gallus gallus).

A technique was developed to record intracranial cerebrospinal fluid pressure (iCSFp) in chicks and mature chickens. Using that procedure, 2 methods were found to effect a significant elevation in intracranial pressure: 1) feeding a purified diet to roosters for 40 d containing 25% of the bird's requirement for vitamin A, and 2) ligating both jugular veins in birds. The purified diet significantly reduced plasma retinol levels from 1.080 to 0.046 microg/mL, and iCSFp was significantly elevated from 63.0 to 106.0 mmH2O (P < or = 0.05). Two limitations for using hypovitaminosis A diets were capillary fragility and a cisterna magna that did not develop to the size of that structure in birds of the same age fed control diets with adequate vitamin A content. The second procedure, a reversible surgical technique, showed that within 2.5 h from jugular vein ligation, intracranial pressure rose to 109.7 mmH2O, comparable with levels attained following feeding a vitamin A deficient diet to roosters. Bilateral clamping of the jugular veins overnight resulted in an elevation of iCSFp to 127 +/- 8.86 mmH2O. Results suggest that the chicken may be a useful animal model to investigate intracranial hypertension and its accompanying headaches known to occur in humans.

Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms.

The vascular endothelial growth factor (VEGF) family encompasses four polypeptides that result from alternative splicing of mRNA. We have previously demonstrated differences in the secretion pattern of these polypeptides. Stable cell lines expressing VEGFs were established in human embryonic kidney CEN4 cells. VEGF121, the shortest form, was secreted and freely soluble in tissue culture medium. VEGF189 was secreted, but was almost entirely bound to the cell surface or extracellular matrix. VEGF165 displayed an intermediary behavior. Suramin induced the release of VEGF189, permitting its characterization as a more basic protein with higher affinity for heparin than VEGF165 or VEGF121, but with similar endothelial cell mitogenic activity. Heparin, heparan sulfate, and heparinase all induced the release of VEGF165 and VEGF189, suggesting heparin-containing proteoglycans as candidate VEGF-binding sites. Finally, VEGF165 and VEGF189 were released from their bound states by treatment with plasmin. The released 34-kDa dimeric species are active as endothelial cell mitogens and as vascular permeability agents. We conclude that the bioavailability of VEGF may be regulated at the genetic level by alternative splicing that determines whether VEGF will be soluble or incorporated into a biological reservoir and also through proteolysis following plasminogen activation.

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.

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.

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.

Ligand-mediated immunofunctional assay for quantitation of growth hormone-binding protein in human blood.

Human serum contains a high affinity GH-binding protein (GHBP) whose amino-terminal sequence is identical to the extracellular domain of the GH receptor. Current methods that measure GHBP are laborious, require size or charcoal separation of the GH/GHBP complex, and may be influenced by ambient GH concentrations. We have developed a novel assay method that allows quantitation of the total amount of functional GHBP in serum or plasma. The assay can also be used to measure the concentration of the circulating GH/GHBP complex. An anti-GHBP monoclonal antibody, which recognizes both free GHBP and GH-bound GHBP, is used to capture the GHBP on a microtiter plate. Recombinant human GH is added to saturate all binding sites, and an anti-GH antibody conjugated with horseradish peroxidase is used to detect the amount of GH (endogenous and exogenous) bound to the GHBP. The same procedure, but without incubation with GH, allows measurement of the endogenous GH/GHBP complex. The assay is sensitive (detection range, 31-2000 pmol/L), with average inter- and intraassay precisions of 11.3% and 7.3%, respectively. Measurements in random blood samples from 16 healthy adults showed that all subjects had clearly detectable GHBP concentrations (range, 65.8-305.6 pmol/L). In contrast, GHBP levels were undetectable in samples from 2 patients with Laron-type dwarfism. We believe that this ligand-mediated immunofunctional assay, which combines the simplicity and specificity of an enzyme-linked immunosorbent assay with the ability to detect only biochemically active binding protein, will be useful for studies of the role of the GHBP in health and disease.