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BACKGROUND: IL-5 is produced by the T(H)2 subset of CD4(+) T lymphocytes and is necessary for the eosinophilia typical of allergic conditions. Glucocorticoids such as dexamethasone are highly effective inhibitors of eosinophilic inflammation, and one of their effects is inhibition of IL-5 gene expression. OBJECTIVE: We wished to examine the effect of dexamethasone on the binding of nuclear factors from primary human CD4(+) T lymphocytes to the RE-I and RE-II positively acting regulatory elements of the IL-5 promoter. METHODS: CD4(+) T cells, purified from PBMCs by magnetic bead separation, were activated with anti-CD3 antibody and phorbol myristate acetate. Nuclear extracts were tested in electrophoretic mobility shift assays with probes based on RE-I and RE-II. RESULTS: In extracts from activated cells, the RE-II region of the promoter formed a complex that was shown by supershift assay to contain NFATc. This complex was abolished by treatment of the cells with dexamethasone before activation and was weak or absent in unactivated cells. By contrast, binding to the RE-I region and to the GATA-3 site within RE-I was observed in resting cells and was not affected by activation or treatment with dexamethasone. CONCLUSION: Dexamethasone inhibits the inducible binding of factors to the RE-II region but does not affect the constitutive binding to the RE-I region. Characterization of such molecular effects of glucocorticoids could enable the development of specific inhibitors of IL-5 expression that lack the side effects of glucocorticoids.

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BACKGROUND AND OBJECTIVE: We analyzed the in vivo ocular response to corneal incisions made by Medical Free Electron Laser (MFEL) as a function of scan rate and incision depth. Additionally, we compared biomicroscopy, optical coherence tomography (OCT), and light microscopy as ocular response diagnostic tools. STUDY DESIGN/MATERIALS AND METHODS: Rabbit corneas were incised with pulsed MFEL radiation at 2.94 microm wavelength, scalpel incisions or focal cautery treatment were used as controls. The MFEL beam scan rate ranged from 0.2 to 1.0 mm/second. Ocular effects were monitored for 2 hours postoperatively using OCT and slit lamp examination. Ocular tissue was fixed for light microscopic examination. RESULTS: Anterior chamber fibrin formation correlated with MFEL incision depth. Slower scan rates resulted in deeper incisions and greater fibrin formation. OCT was better than slit lamp biomicroscopy at identifying fibrin attachments. OCT and light microscopy proved to be excellent companion technologies. CONCLUSIONS: Deep corneal incisions in the rabbit produced by the MFEL resulted in fibrin formation in the anterior chamber.

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Treatment of resting murine B lymphocytes with CD40 ligand (CD40L) and IL-4 induces proliferation and a switch in immunoglobulin (Ig) isotype surface expression from IgM and IgD to IgG1 and IgE. Using a fluorescent dye to enable cell sorting according to cell division cycle number, we have examined molecular events associated with B cell differentiation, namely, germ-line transcription and DNA recombination. Digestion-circularisation polymerase chain reaction experiments showed that DNA recombination leading to isotype switching from IgM to IgG1 surface expression is division-dependent and was first detected after B cells had divided three times. Similarly, DNA rearrangement involving the IgE switch region was detectable only after five division cycles. These division cycle numbers correlate with the numbers of divisions required before surface expression of the switched isotype [P.D. Hodgkin, J.-H. Lee, A.B. Lyons, J. Exp. Med. 184 (1996) 277-281]. RT-PCR analyses also revealed that germ-line transcripts for both IgG1 and IgE increased with division number suggesting a threshold expression level may be required for recombination to occur.

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The tetracycline-responsive promoter (TRP) system has been adopted in an attempt to obtain repressible antisense inhibition in a B lymphocyte model in vitro. Levels of secreted IgM protein and mRNA were assessed following the stable transfection of B cell line, HO-2.2, with a series of plasmid constructs containing antisense or sense target sequence DNA (the 3'-untranslated region adjacent to the secreted exon of IgM gene) under the control of the TRP. Significant reduction (approximately 90%) in IgM secretion was observed for clones transfected with antisense plasmids driven by the TRP and containing the IgH enhancer element and the polyadenylation signal sequence from membrane IgM, when compared with untransfected and sense controls. Tetracycline (1 microgram/ml) addition to the culture medium restored the level of IgM secretion in these clones to control values, demonstrating repressibility of antisense inhibition. Transfection of HO-2.2 cells with antisense (or sense) constructs had no detectable effect on membrane IgM protein levels. Hybridisation studies demonstrated that decreased protein production observed in the antisense-transfected clones was most likely attributable to reduced RNA levels. These data show that the TRP can be used for repressible and specific antisense inhibition of gene product expression in B lymphocytes.

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An in vitro model has been developed in an attempt to optimise antisense inhibition in B cells as a prelude to transgenic studies. The hypotheses tested were that i) the 3'-untranslated region would be an appropriate target for antisense inhibition; 2) the immunoglobulin heavy chain intronic enhancer could be used to enhance antisense inhibition via increased production of antisense transcripts; and 3) the mouse metallothionein-1 promoter would allow induction of antisense inhibition in B cells. Secreted IgM protein and mRNA were monitored following the stable transfection of a B cell line, HO-2.2, with a series of plasmid constructs containing antisense or sense target sequence DNA under the control of either the mouse metallothionein-1 promoter or homologous (ie same promoter as target sequence) immunoglobulin heavy chain promoter. The 3'-untranslated region proved to be an appropriate target resulting in 70% inhibition of IgM secretion. Compared with untransfected and sense controls, significant decreases in IgM secretion (and RNA levels) were detected in clones transfected with antisense constructs utilising the mouse metallothionein-1 promoter and the immunoglobulin heavy chain intronic enhancer elements. These clones exhibited a further significant reduction in secreted IgM production upon zinc induction. Hybridisation studies demonstrated that decreased protein production was most likely attributable to reduction in RNA levels. In contrast, transfection with antisense constructs had no effect on membrane IgM protein levels which not only confirmed the specificity of antisense action but meant that the B cell remained sensitive to receptor ligation. We conclude that reasonable antisense inhibition of gene product expression can be achieved in B cells by targeting the 3'-untranslated region and using both an inducible promoter (mouse metallothionein-1) and the IgH enhancer to aid antisense RNA production.

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We have prepared purified cytotrophoblasts from human term placentas and examined the sensitivity of fura-2 loaded cells to the nucleotides ATP and UTP and to changes in extracellular Ca2+ concentration ([Ca2+]o). Purified cytotrophoblasts were obtained by collagenase digestion and separation according to density using self-generated Percoll gradients. The cytotrophoblast fraction was free of red cell and largely free of white cell contamination (as assessed by uniformly negative staining for vimentin and the failure of > 90% of fura-2 loaded cells to respond to the chemotactic peptide fMet-Leu-Phe). Purified cells secreted progesterone in a linear fashion over several hours in the presence of 25-hydroxycholesterol. The cells ranged in size from approximately 7.5 to 50 microns in diameter as described previously for purified cytotrophoblasts, and an analysis of cells for sensitivity to [Ca2+]o or nucleotides suggested functional heterogeneity within the cytotrophoblast population. Small cells (7.5-10 microns) were negative for cytokeratin-8 and, after loading with fura-2, were insensitive to extracellular nucleotides but sensitive to elevations in [Ca2+]o. Medium-sized cells (12-20 microns) were largely cytokeratin-positive (70% of cells) and sensitive to both ATP and UTP but largely insensitive to [Ca2+]o. Large cells (25-50 microns) were uniformly cytokeratin-positive (100% of cells) and, after fura-2 loading, sensitive to both [Ca2+]o and extracellular ATP or UTP. We examined the likely origin of small, medium and large cytotrophoblasts using an immunomagnetic cell sorting procedure that separates villous cytotrophoblasts (which do not express major histocompatibility class I antigens) from extravillous cytotrophoblasts. This procedure resulted in the selective sedimentation of almost all medium and large cells, leading to the conclusion that the small cells were villous cytotrophoblasts whereas medium and large cells were predominantly extravillous in origin. The data suggest that small, medium and large cytotrophoblasts have distinct roles in the function of the term placenta.

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The CD45 or leucocyte-common antigens are encoded by a single gene but can be found in various forms due to alternative splicing of three exons near the 5' end of the gene. The CD45 antigens are major glycoproteins of all types of leucocytes. Monoclonal antibodies recognizing restricted epitopes of CD45 have been used to distinguish phenotypic and functional subsets of lymphocytes. To facilitate epitope mapping and biochemical studies, we have expressed the extracellular portions for four different isoforms of rat CD45 in Chinese hamster ovary cells. Constructs were prepared to give four soluble CD45 isoforms, with sequence incorporating either all three alternative exons (sCD45.ABC), the B exon (sCD45.B), the C exon (sCD45.C), or no alternative exons (sCD45.O). These were expressed at approximately 5 mg/l of spent tissue culture supernatant and were antigenically active with monoclonal antibodies (mAb) that recognize all CD45 isoforms. The MRC OX22 and OX32 mAb have been used to split rat CD4+ T cells into functionally distinct subpopulations and the epitopes for these were mapped to the product of exon C. The epitope for MRC OX33, a marker for B cells, requires expression of either the A exon or the A/B exon junction. Electron microscopy showed that the extra segments contributed to an extended structure as has been predicted from the sequence. The shape of the molecule is discussed with regard to other molecules at the leucocyte cell surface.

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Treatment of covalently cross-linked or heat-aggregated oligomers of human IgG with 4 mM-tetranitromethane abrogated their C1q-binding activity. In contrast, tetranitromethane modification of rabbit IgG oligomers, under identical conditions, had no effect upon their C1q-binding activity. The tetranitromethane treatment led to nitration of about ten tyrosine residues per IgG molecule in both species, and the modification was specific for tyrosine residues. Reduction of the nitrated protein with Na2S2O4 did not lead to recovery of C1q-binding activity in human IgG oligomers or to loss of activity in rabbit IgG oligomers. Tryptic peptides from the nitrated proteins were isolated and a peptide containing nitrotyrosine-319 was recovered from human IgG, as well as peptides from both species corresponding to the region around nitrotyrosine-278. These data are consistent with the inactivation of C1q-binding activity in human IgG being the result of nitration of tyrosine-319; the rabbit IgG is unaffected by nitration because position 319 is phenylalanine. The evidence supports the C1q-receptor site proposed by Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek [(1980) Nature (London) 288, 338-344]: residues 316-338.

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The neutral leukocyte proteases involved in the release of free amino acids in incubation mixtures of blood cell lysates and of human serum albumin with leukocyte lysates were characterized by several techniques including 1H nuclear magnetic resonance spectroscopy, electrophoresis, high performance liquid chromatography, gel filtration, amino acid analysis, and NH2-terminal analysis. The data suggested that the enzymes which contributed significantly to the extensive protein hydrolysis observed were two endopeptidases and three exopeptidases. Identification and analysis of the products obtained in incubation mixtures of human serum albumin with elastase and leucine aminopeptidase were compared with the products obtained in incubation mixtures of the protein with leukocyte lysates.

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The origin of low frequency methyl resonances which appear in the spin-echo 1H nuclear magnetic resonance spectra of incubated blood cell lysates was investigated by several techniques including 1H and 13C nuclear magnetic resonance spectroscopy, electrophoresis, high performance liquid chromatography, gel filtration, and amino acid analysis. These resonances were identified as arising from methyl moieties of leucine and valine. Other peaks which also appeared in the spectra of incubated blood cell lysates were assigned to methyl groups of alanine and threonine. The free amino acids are products of neutral proteases located on the leukocyte membrane or able to act on the extracellular medium. Since more than one enzyme appears to be implicated, it is possible that both membrane and granule proteases take part in the hydrolysis. Comparison of rates of product formation in white cell lysates incubated with human serum albumin, and with red cell lysate, suggests that erythrocyte peptidases also contribute to proteolysis in the latter case.

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A new HPLC-based assay for the direct determination of the extent of maleylation of proteins is described. The rationale is founded on the HPLC detection of maleic acid that results from cleavage of maleyl lysyls by low pH treatment. The method, employing a C-18 reverse-phase column eluted isocratically with 0.01 M acetic acid, pH 5.4, and absorbance monitored at 210 nm, is sensitive (lower limit 1 pmol) and linear in the range 1 pmol-0.5 mumol and allows recovery of the native protein.

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Treatment of covalently crosslinked rabbit IgG oligomers with diethylpyrocarbonate resulted in the loss of their C1q binding activity. The inactivation was a first-order process with respect to time in the range 0-8 min, and modifier concentration from 0 to 2.39 mM. Hydroxylamine treatment of diethylpyrocarbonate-treated IgG oligomers led to 80% recovery of their C1q binding activity. Diethylpyrocarbonate treatment of IgG oligomers had little effect on their absorbance at 278 nm, but led to an increase in their absorbance at 242 nm. The apparent pKa of the modified residues was 6.91 +/- 0.12. These data are consistent with diethylpyrocarbonate modification of histidine residues leading to loss of C1q binding activity in rabbit IgG oligomers. Modification of four histidine residues per IgG molecule was associated with the loss of C1q binding activity. Thus, there may be two histidine residues at or near the C1q binding sites in the CH2 domains of rabbit IgG.

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