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J Sadowska

Publications and source records attributed to J Sadowska.

33 records · Page 2Linked to original sources
PubMed1991

Bacterial expression and secretion of various single-chain Fv genes encoding proteins specific for a Salmonella serotype B O-antigen.

Active single-chain Fv molecules encoded by synthetic genes have been expressed and secreted to the periplasm of Escherichia coli using the ompA secretory signal. Four different constructs were developed to investigate the effects of peptide linker design and VL-VH orientation on expression, secretion, and binding to a Salmonella O-polysaccharide antigen. Peptide linker sequences derived from the elbow regions of the Fab molecule were used alone or in combination with the flexible (GGGGS)2 sequence. VL and VH domain order in the single chain molecules had a profound effect on the level of secretion but hardly influenced total expression levels, which were approximately 50 mg/liter, chiefly in the form of inclusion bodies. With VL in the NH2-terminal position, the amount of secreted product obtained was 2.4 mg/liter, but when VH occupied this position the yield was less than 5% of this value. Enzyme immunoassays of the four products showed domain order and linker sequence affected antigen binding by less than an order of magnitude. Attempts to express active Fv from dicistronic DNA were unsuccessful, but active Fv was obtained from single-chain Fv by enzymic cleavage at a site in the elbow linker peptide. The thermodynamic binding parameters of intact and cleaved single-chain Fvs determined by titration microcalorimetry were similar to those of bacterially produced Fab and mouse IgG.

Amino Acid Sequence↗
PubMed1991

Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen.

A 1460-bp DNA encoding the two chains of the antigen-binding fragment (Fab) portion of a monoclonal antibody have been chemically synthesized and expressed in Escherichia coli. The antibody, Se155-4, is specific for a Salmonella serogroup B O-antigen and its crystal structure is under investigation. The genes were synthesized according to a strategy that allows for easy manipulation in genetic engineering studies of the Fab-binding site. Each gene is preceded by the ompA secretory signal and a ribosome-binding site, and has been expressed from the two-cistron DNA under the control of the lac promoter. Active Fab of 50 kDa with an inter-chain disulfide bond has been isolated from the periplasm of E. coli in a one-step affinity purification in high yield (2 micrograms/ml of cells). The bacterially produced Fab is as active as purified mouse Fab in antigen-binding and competitive immunoassays. This is the first example of a completely synthetic Fab gene and provides an ideal system to probe the nature of antigen binding by anti-carbohydrate antibodies.

Amino Acid Sequence↗
PubMed1991

Scanning of key residues in antibody binding sites by two saturation-mutagenesis approaches.

The complementarity-determining region 3 of the heavy chain (CDRH3) generally contributes the most to antibody-antigen binding. His101H in CDRH3 of the antibody Se155-4, which is specific for a trisaccharide epitope of Salmonella serotype B O-antigen, was mutated systematically into all nineteen other amino acids by a double mutation approach. Enzyme immunoassay (EIA) and affinity chromatography showed that the Asn, Gln, Gly and Ser mutants exhibited moderate to strong activity. Some mutants, such as Thr and Pro, had weak binding activity, while the acidic and hydrophobic amino acid substitutions resulted in complete loss of activity. A second mutation approach which randomly changed a selected residue into all other nineteen amino acids, while precluding wild-type transformants, is also described.

Amino Acid Sequence↗
PubMed1990

Interleukin-1 and tumour necrosis factor alpha induced polymorphonuclear leukocyte-endothelial cell adhesion and transendothelial migration in vitro: the effect of apical versus basal monolayer stimulation.

The cytokines interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF alpha) enhance polymorphonuclear leukocyte (PMNL) adhesion to vascular endothelium by an endothelial cell dependent mechanism in vitro and induce PMNL infiltration in vivo In this study, we employed human umbilical vein endothelium (HUVE) cultured on microporous membrane filters to form a monolayer, a system in which PMNL adherence and PMNL transendothelial migration could be measured using 51Cr-labelled human PMNL. In this system, it was found that PMNL adhesion and migration were dependent on prior treatment of the HUVE monolayer with IL-1 or TNF alpha for at least 2 h and that cytokine could be removed prior to the addition of PMNL without any effect on the response. PMNL adherence to the HUVE was maximal by 30 min and was followed by progressive migration of PMNL across the monolayer and the membrane filter into the lower chamber. The effect of apical surface versus basal surface exposure of the HUVE monolayer to IL-1 alpha and TNF alpha on subsequent PMNL interaction with the HUVE monolayer in the absence of cytokine was examined. Apical or basal stimulation induced comparable PMNL adherence at 30 min following addition of PMNL (35.5% and 43.1%). However, basal (i.e., abluminal) exposure to IL-1 or TNF alpha of the HUVE induced significantly greater PMNL transendothelial migration (e.g., 27.8% vs. 15.4%; P less than 0.01). The expression of endothelial-leukocyte adhesion molecules ELAM-1 and ICAM-1 following apical versus basal stimulation was determined by ELISA on viable cells. These adhesion molecules were upregulated to a similar extent under both conditions. These observations suggest that spacial localization or orientation of adhesion molecules may be influenced by basal versus apical cytokine stimulation or that other mechanisms are responsible for the preferential PMNL migration with basal stimulation. These findings may have implications for the in vivo interactions of PMNL with vascular endothelium, depending on whether the endothelium is exposed to IL-1 of TNF alpha via the blood on the luminal (apical) surface or via the extravascular space on the abluminal (basal) surface.

Cell Adhesion↗
PubMed1990

Endotoxin-stimulated human macrophages produce a factor that induces polymorphonuclear leucocyte infiltration and is distinct from interleukin-1, tumour necrosis factor alpha and chemotactic factors.

Previously we reported that rabbit macrophages (M phi) in the presence of nanogram quantities of endotoxin (LPS) release factors that induce polymorphonuclear leucocyte (PMNL) infiltration into the skin of rabbits following i.d. injection. The predominant factor was a de novo synthesized protein of 45,000 MW on gel filtration that was distinguishable from IL-1 but not from TNF alpha. Here we examined human monocytes, in vitro monocyte-derived M phi and peritoneal M phi for the production of an analogous protein. Upon stimulation with LPS, they all rapidly (6 hr) produced a factor(s) that caused PMNL accumulation in the skin of rabbits when injected i.d. This activity, referred to as PMNL-recruiting activity (PRA), was heat labile and its production was blocked by cycloheximide. By Sephadex-G100 chromatography the major PRA of cultured M phi or peritoneal M phi had a molecular weight (MW) of 45,000-60,000. The active fractions were free of IL-1 (less than 0.2 U/ml) and Superose-12 FPLC chromatography separated the peak of PRA, which eluted at 45,000 MW, from TNF alpha, eluting at 20,000 MW. The peak PRA was not neutralized by antisera to IL-1 alpha, IL-1 beta, TNF alpha, IL-6 or GM-CSF, indicating that it was distinct from these cytokines. The major PRA did not induce the migration of PMNL in vitro in a filter chemotaxis assay. In contrast to the M phi, the major PRA produced by LPS-stimulated monocytes eluted at 15,000-20,000 MW, contained IL-1 activity and was neutralized by antisera to IL-1 alpha and IL-1 beta. Monocytes from a few donors also produced the 45,000-60,000 MW PRA simultaneously. We conclude that human peritoneal M phi and in vitro monocyte-derived M phi exposed to LPS secrete a protein of 45,000-60,000 MW, which is a potent inducer of PMNL infiltration but is distinct from IL-1, TNF alpha, IL-6, GM-CSF and PMNL chemotactic factors.

Animals↗
PubMed1987

[Not Available].

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History, Modern 1601-↗
PubMedDate not supplied

Rabbit alveolar macrophages stimulated with endotoxin and lung fragments from endotoxemic rabbits produce a leukocyte infiltration-inducing factor that lacks IL-1, TNF alpha, or chemotactic activity.

We reported previously that rabbit pleural and peritoneal macrophages (Møs) and human Mø stimulated with endotoxin (LPS) release a protein factor of 45 to 60 kd that induces local polymorphonuclear leukocyte (PMNL) infiltration upon intradermal injection in rabbits. In the case of the human Mø product, it was shown to be distinct from interleukin-1 (IL-1), tumor necrosis factors (TNF alpha), granulocyte macrophage colony stimulating factor (GMCSF), IL-6, and lower molecular weight PMNL chemotactic factors. Here, we examined resident rabbit alveolar Møs to determine if they produce a similar factor following in vitro or in vivo exposure to LPS. Following LPS exposure (0.3 to 30 ng/ml), alveolar Møs obtained from normal rabbits by bronchoalveolar lavage released PMNL recruiting activity within 3 h, as measured by the accumulation of 51Cr labeled blood PMNL at injected skin sites. Production of this activity was blocked by cycloheximide; it was heat labile and not affected by polymyxin B, which neutralized the LPS. On gel filtration chromatography, a major peak of activity was eluted at 45 to 60 kd and was free of IL-1 but partially overlapped with rabbit TNF alpha. Although active in vivo in PMNL recruitment into the tissues, these fractions did not induce PMNL migration in vitro in a filter chemotaxis assay. After sodium dodecyl sulfate (SDS), polyacrylamide gel electrophoresis (PAGE) the predominant PMNL recruiting factor (PRF) eluted from gel slices corresponded to 22 to 32 kd, suggesting that this protein is a dimer under gel filtration conditions. These gel eluates did not contain TNF alpha activity. Following iv administration of sublethal doses of LPS (3 micrograms/kg) or of antibiotic killed Escherichi coli (10(9)/kg), peripheral lung fragments from perfused lungs spontaneously produced this PRF during ex vivo culture without further LPS stimulation. Lung tissue from normal rabbits did not release PRF spontaneously. We conclude that resident alveolar Møs produce a PRF protein in response to LPS that is distinct from IL-1, TNF alpha, and chemotactic factors and that the production of a similar protein by lung cells (probably Møs) is probably induced in vivo during endotoxemia or bacteremia. This factor may contribute to PMNL accumulation in the lung during pathologic processes.

Animals↗
PubMedDate not supplied

The expression of aromatase, estrogen receptor alpha and estrogen receptor beta in mouse Leydig cells in vitro that derived from cryptorchid males.

A broad expression of aromatase and estrogen receptors (ERs) in the testis suggests an important role for estrogens in regulating testicular cell function and reproductive events. The aim of the present study was to show whether Leydig cells in vitro isolated from cryptorchid testes of two inbred strains of mice, KE and CBA, are a site of estrogen synthesis. Using immunocytochemistry, aromatase, estrogen receptor alpha(ERalpha), and estrogen receptor beta(ERbeta) were localized in cultured Leydig cells. Immunoreactive aromatase was found in the cytoplasm of control Leydig cells and those isolated from cryptorchid males, however the intensity of immunostaining was different, being stronger in Leydig cells deriving from cryptorchid mice. The strongest aromatase immunostaining was found in cryptorchid-KE Leydig cells. Strong immunoexpression of ERalpha was detected in the nuclei of both KE-and CBA-Leydig cells. The intensity of ERalpha immunostaining was stronger in cultured cells deriving from cryptorchid testes. ERbeta immunoexpression was detected predominantly in KE-Leydig cells. Control CBA-Leydig cells were negative for ERbeta or the result was inconclusive, whereas in cryptorchid CBA-Leydig cells a weak immunostaining was present in their nuclei. Western blot analysis confirmed the results obtained by immunocytochemistry. In KE- and CBA-Leydig cells aromatase as a band of 55 kDa protein was present, whereas ERalpha molecular weight was 67 kDa on Western blots. No band was detected for ERbeta. Radioimmunological analysis revealed that androgen and estrogen levels secreted by Leydig cells in vitro were strain-dependent. Additionally, in KE-Leydig cells that derived from cryptorchid mice estrogen level was distinctly higher in comparison with that of the respective control.

Animals↗