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T Keler

Publications and source records attributed to T Keler.

34 records · Page 2Linked to original sources
PubMed1995

Construction and characterization of a humanized anti-gamma-Ig receptor type I (Fc gamma RI) monoclonal antibody.

The murine mAb 22 (M22) binds to the human high affinity receptor for the Fc portion of IgG (Fc gamma RI). This mAb recognizes an epitope on Fc gamma RI that is distinct from the natural ligand (Fc) binding site, and, therefore, can bind to Fc gamma RI in the presence of saturating levels of IgG found in vivo. Fc gamma RI is expressed only on cytotoxic effector cells and it acts as an efficient mediator (trigger molecule) of effector functions. Directing tumor cells or other pathogens to Fc gamma RI utilizing M22 conjugated to an anti-tumor- or to an anti-pathogen-specific mAb may represent an effective method to enhance their removal and destruction under physiologic conditions. Humanization of M22 may improve the therapeutic potential of the mAb since murine mAbs normally elicit a human anti-mouse Ab response. In this report we describe the humanization of M22 by CDR-grafting onto human V region frameworks based on KOL VH and REI V kappa attached to human IgG1 and kappa constant domains. This humanized mAb, H22, displays equivalent binding specificity and affinity as its murine counterpart. In addition, H22 is able to trigger superoxide release from Fc gamma RI-bearing cells, a finding that demonstrates that H22 triggers Fc gamma RI functions. The successful humanization of mAb 22 is an important step in the development of Fc gamma RI-directed immunotherapy.

Amino Acid Sequence↗
PubMed1993

Growth promotion of transfected hepatoma cells by liver fatty acid binding protein.

Former studies have linked hepatocyte growth with liver fatty acid binding protein (L-FABP) of rat liver cytosol. In search for the roles of L-FABP in hepatocytes, we previously stably transfected rat L-FABP sense and antisense cDNAs into rat hepatoma HTC cells that do not contain L-FABP RNA or protein, thereby providing a zero-background, homologous cell model of L-FABP-expression suitable for controlled studies of its intracellular functions in hepatocyte-derived cells. The present study demonstrates the abilities of L-FABP to promote DNA synthesis and cell growth, preserve cell morphology, extend survival, and act cooperatively with unsaturated fatty acids in the transfected hepatoma cells in the absence of serum. Following removal of serum, the three control L-FABP-nonexpressing cell lines increased in cell lines increased in cell number for 24 hr and thereafter declined, whereas the three L-FABP-expressing cell lines exhibited a 39% higher rate of DNA synthesis per cell at 24 hr and grew in cell number for 48 hr. As a result, at 72 hr there were 2.5-fold (avg.) as many L-FABP-expressing cells than L-FABP-nonexpressing cells. In addition, the L-FABP-expressing cells retained their original polygonal morphology at 48 hr, when in contrast most of the control nonexpressing cells were spherical in shape with membrane blebs. In an effort to identify the agonists that collaborate with L-FABP in the growth promotion and preservation of cell morphology, various free fatty acids were examined at 48 hr for their ability to eliminate the differences in behavior of the two cell types in the serum-free medium. The unsaturated fatty acids, oleic acid (18:1 omega 9), linoleic acid (18:2 omega 6), alpha-linolenic acid (18:3 omega 3), and arachidonic acid (20:4 omega 6), at 1 microM markedly elevated the level of DNA synthesis in the more depressed control L-FABP-nonexpressing cells and moderately raised it in the less depressed L-FABP-expressing cells. In accord, the control L-FABP-nonexpressing cells needed 10(-6)-10(-5) M linoleic acid to achieve the extent of DNA synthesis attained by the expressing cells in the absence of added fatty acid. At 10 microM linoleic acid, their levels of DNA synthesis were equal. In contrast, five saturated fatty acids had no detectable effect on DNA synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Specific growth stimulation by linoleic acid in hepatoma cell lines transfected with the target protein of a liver carcinogen.

The hepatic carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) was shown previously to interact specifically with its target protein, liver fatty acid binding protein (L-FABP), early during hepatocarcinogenesis in rats. In search of the significance of the interaction, rat L-FABP cDNA in the sense and antisense orientations was transfected into a subline of the rat hepatoma HTC cell line that did not express L-FABP. After the transfections, the basal doubling times of the cells were not significantly different. However, at 10(-5)-10(-7) M, linoleic acid, which is an essential fatty acid, a ligand of L-FABP, and the precursor of many eicosanoids and related lipids, stimulated the incorporation of [3H]thymidine in three randomly isolated and stably transfected cell clones that expressed L-FABP, but virtually did not stimulate the incorporation of [3H]thymidine in three L-FABP-nonexpressing clones transfected with the antisense DNA. Linoleic acid at 10(-6) M increased cell number almost 3-fold (38% vs. 14%; P less than 0.0001) and thymidine incorporation nearly 5-fold (23.2% vs. 4.9%; P less than 0.001) in the L-FABP-expressing cells compared to that in the transfected nonexpressing cells. L-FABP acted specifically and cooperatively with linoleic acid, inasmuch as all the proteins other than L-FABP in the transfected L-FABP nonexpressing cells and four other fatty acids (gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, and palmitoleic acid) were unable to effect a significant elevation or difference in the level of DNA synthesis that was attributable to the transfection. Metabolism of the linoleic acid to oxygenated derivatives was apparently necessary, since the cyclooxygenase inhibitor indomethacin partly inhibited and the antioxidant lipoxygenase inhibitors nordihydroguariaretic acid and alpha-tocopherol completely abolished the growth stimulation. The evidence supports the idea that L-FABP, the target protein of the liver carcinogen, acts specifically in concert with oxygenated metabolites of linoleic acid to modulate the growth of hepatocytes.

Animals↗
PubMed1992

Activation of the prolactin receptor gene by promoter insertion in a Moloney murine leukemia virus-induced rat thymoma.

The prolactin receptor (Prlr) and growth hormone receptor (Ghr) genes and the Moloney murine leukemia virus integration-2 (Mlvi-2) locus were mapped to mouse chromosome 15 and human chromosome 5 bands p12-p14. To examine the potential relationship between Mlvi-2 and the genes encoding the growth hormone receptor and the prolactin receptor, we determined the chromosomal location of all three loci in the rat, using a panel of rat-mouse somatic cell hybrids, and in the mouse, using a panel of (C57BL/6J x Mus spretus)F1 x C57BL/6J interspecific backcross mice. These analyses revealed that Ghr, Prlr, and Mlvi-2 map to chromosome 2 in the rat and to chromosome 15 in the mouse, in close proximity with each other. Pulsed-field gel electrophoresis of rat genomic DNA showed no overlaps between the gene encoding the prolactin receptor and the remaining loci. Moreover, expression of the prolactin receptor was not affected by provirus insertion in Mlvi-2. During these studies, however, we detected one T-cell lymphoma line (2779) in which the prolactin receptor gene was activated by provirus integration. Sequence analysis of polymerase chain reaction-derived cDNA clones showed that the prolactin receptor RNA message initiates at the 5' long terminal repeat and utilizes the splice donor site 5' of the gag gene to splice the viral sequences onto exon 1 of the prolactin receptor. This message is predicted to encode the intact prolactin receptor protein product. Exposure of the T-cell lymphoma line 2779 to prolactin promoted cellular proliferation.

Animals↗
PubMed1990

Column liquid chromatography of endotoxins.

A new, fast and highly reproducible column liquid chromatographic method was elaborated for the analysis and small-scale preparative isolation of endotoxin from Serratia marcescens Bizio (ATCC No. 264). This procedure detects contaminants of such preparations with high sensitivity and it is capable of separating them from endotoxic components. Extensive heterogeneity of both 5% trichloroacetic acid and phenol-water-extracted endotoxin preparations was recorded. Heterogeneity among the endotoxic components of purified preparations could also be detected by this method. Measurements of biological activities, such as Limulus amoebocyte lysate activation, lymphoblastogenesis (mitogenicity) and tumor necrosis factor (TNF) liberation were carried out on the chromatographically separated fractions. During these studies, non-toxic but in vitro TNF-generating components of crude endotoxin extracts were also detected.

Animals↗
PubMed1989

Effects on growth and energy metabolism in untransformed and transformed BALB/c mouse fibroblasts by a novel cytotoxic compound.

A lipid, recently discovered as a contaminant of endotoxin and White-Type polysaccharide preparations from an unidentified isolate of Serratia marcescens, was named DCX for its direct cytotoxic activity. Previously we have shown that DCX is cytotoxic to several transplantable tumors and transformed cell lines in culture at nanomolar concentrations, while normal fibroblasts were significantly more resistant. In this study the selectivity and mechanism of DCX-mediated cytotoxicity was examined more thoroughly with the use of clonally related cell lines. DCX is growth inhibitory but is not cytotoxic to a BALB/c embryonal cell line. Transformation by Simian Virus 40 renders a cloned derivative cell line sensitive to the cytotoxicity of DCX. Treatment of both cell lines with DCX increased 2-deoxyglucose uptake and lactate production, and decreased ATP levels. Electron micrographs show extensive damage to mitochondria in cells treated with DCX. The results indicate that the growth inhibitory activity of DCX in these cells is due to effects on mitochondria resulting in depletion of cellular ATP. The data suggest that the basis for selective cytotoxicity is in how the different cells are able to cope with lower energy levels.

Adenosine Triphosphate↗
PubMed1989

Incidence and pattern of bladder tumor recurrence following combined suprapubic prostatectomy and excision of a bladder tumor.

We studied retrospectively 36 patients who had undergone combined suprapubic transvesical prostatectomy for benign prostatic hypertrophy (BPH) and excision of a simultaneous bladder tumor. The follow-up period ranged from 1 to 7 years (average 3 years) and included periodic cystoscopy and voided urine cytology. Thirteen recurrences out of 60 (21.7%) occurred at the bladder neck, in 8 out of 23 patients (34.8%) with recurrent tumors. We relate the high recurrence rate located at the bladder neck to the concurrent prostatectomy. We conclude that combined surgery for BPH and bladder tumor is not recommended since it predisposes the bladder neck to tumor implantation at surgery.

Aged↗
PubMed1988

Thin-layer chromatography of endotoxins, their derivatives and contaminants.

Thin-layer chromatographic (TLC) separation techniques were used to analyze the heterogeneity of various preparations which included smooth and rough endotoxins (ET), Lipid A precipitates and synthetic Lipid A samples and a novel cytotoxic bacterial lipid. Furthermore, carbohydrate-rich split products (PS) of ET were also separated on commercial silica-coated plates. Satisfactory results were obtained by two-dimensional TLC or by the combination of chromatography followed by high-voltage electrophoresis in the separation of PS of ET cleaved by mild acetic hydrolysis. Several spray reagents were found which were eminently suitable to detect carbohydrate containing compounds. Less specific but generally useful spray reagents were also developed which gave strong color reactions with lipids, proteinaceous and carbohydrate containing split products of the ET preparations. Improved chromatographic resolution has also revealed substantial heterogeneity in both rough and smooth ET samples. Three biological activities of the separated components could be determined. These were antigenicity detected by reactivity with monoclonal antibodies on the TLC plates, endotoxicity, determined by the Limulus amoebocyte lysate (LAL) test and direct cytotoxicity of P815 cells in vitro. Considerable amounts of non-endotoxic and non-antigenic contaminants could be detected in all preparations tested. Significant amounts of free Lipid A were also found in smooth ETs. Thus a new level of complexity is recognized by TLC within these preparations.

Animals↗
PubMed1988

Isolation of a nonendotoxic antitumor preparation from Serratia marcescens.

White-type polysaccharide preparation (WPS) obtained from Serratia marcescens bacteria by hot 0.2 N acetic acid extraction was shown to have antitumor effects. These were manifested by enhanced resistance to the take of TA3 transplantable murine adenocarcinoma and by the induction of regression of Meth A sarcoma in mice. Optimal conditions for the liberation and isolation of these substances were sought to achieve the highest antitumor activity and the lowest endotoxin (ET) content. Simultaneously, the activities of the WPS preparations were tested in various tests which are frequently used as in vitro correlates of in vivo antitumor effects, such as the activation of macrophage cytotoxicity, activation of natural killer (NK) cells, and tumor necrosis factor (TNF) generation. We found that the enhanced resistance to the take of TA3 tumor correlated with ET content of the WPS preparations. Preparations with reduced or no ET content showed diminishing activity in this assay or were without any measurable effect. The induction of TNF production and NK activation did not show such close relationship with the ET content. This was particularly evident if testing WPS samples obtained after 60 or 120 min hydrolysis at 90 degrees C. The greatest discrepancy was found between ET content and the Meth A regression induction. Samples with no detectable ET content and no activity in the macrophage, NK, or TNF tests were potent inducers of Meth A regression. Partial purification of such WPS samples could be achieved and a preparation was obtained with high Meth A regression capacity. Preliminary chemical analysis of this preparation showed 25.5% amino acid, 53.7% neutral carbohydrate, less than 0.4% KDO, 0.8% hexosamine, less than 0.1% phosphorous, and less 1.0% long-chain carboxylic acid content. The above chemical analytical data are not consistent with designating such preparations as ET or ET derivatives, such as Lipid A or its split products. This conclusion was confirmed by the lack of endotoxic properties as determined by biological assays on this preparation.

Adenocarcinoma↗
PubMed1987

Cytotoxicity of a novel lipid-like bacterial product.

A highly cytotoxic, lipid-like compound was isolated from a Serratia marcescens strain currently under identification. We have named the compound DCX for its direct cytotoxic activity on various cell types in culture. DCX was purified by preparative thin layer chromatography from chloroform: methanol = 4:1 extracts of whole bacteria, and is chromatographically homogeneous. The effect of DCX on cells is dose, time, and temperature dependent. DCX is particularly toxic to the mastocytoma cell line P815 (TD50 = 75 pg/ml). Three other malignant or transformed murine cell lines were sensitive to the cytotoxic action of DCX. The effect of DCX was also tested on normal cells (human gingival fibroblasts), which showed greater resistance to DCX than the other cells tested.

Animals↗
PubMed1987

Distinctive immunomodulatory effects of endotoxin and nontoxic lipopolysaccharide derivatives in lymphoid cell cultures.

Endotoxin derived from Serratia marcescens, as well as a hydrolyzed polysaccharide-rich derivative from the bacteria similar to the White-type polysaccharide (WPS), and hydrolyzed split products of endotoxin, including Lipid A and the nontoxic polysaccharide (PS), were studied in terms of their ability to induce mitogenic proliferation of murine spleen cells in vitro and enhancement of antibody formation to sheep erythrocytes. The intact endotoxin and the Lipid A component induced marked proliferative activity of normal lymphoid cells, but the PS components obtained after a 30 min or longer mild hydrolysis of the endotoxin had much less mitogenic activity. The WPS preparation retained mitogenic activity after 30 min but lost it during continued hydrolysis. Nevertheless, all of the components were marked adjuvants for murine spleen cells in vitro in terms of enhancing the antibody response to sheep red cells. Both the intact endotoxin as well as the Lipid A component induced interleukin-1 and interferons, including alpha/beta and gamma, following stimulation of spleen cell cultures for 24 h in vitro. Although only the endotoxin and Lipid A, but not the PS components, were mitogenic for murine splenocytes, all of these preparations had immune adjuvant effects and induced spleen cells to produce or release interferons. However, the PS-rich derivative did not induce tumor necrosis factor. These distinct properties of components of endotoxin indicate that the nontoxic PS-rich derivative, as compared to the toxic endotoxin or Lipid A, has some immunostimulatory activities but lacks others.

Adjuvants, Immunologic↗
PubMed1986

Metachromatic assay for the quantitative determination of bacterial endotoxins.

A metachromatic dye, 1,9-dimethylmethylene blue (DMB) reacts with bacterial endotoxins. This interaction results in a shift of the absorption maximum of DMB to shorter wavelengths. The findings indicate that the negatively charged lipid moiety of the endotoxic lipopolysaccharide reacts with DMB. The lowest amount of endotoxin detectable by the procedure described here is approximately one microgram. The dye could not be used in the presence of serum components. DMB mixed to column chromatographic effluent showed good resolution in continuous monitoring of endotoxin components leaving the column.

Chromatography, Ion Exchange↗
PubMed1984

Plasmin degradation of cartilage proteoglycan.

Employing agarose gel electrophoresis, physiological concentrations of plasmin have been shown to degrade purified proteoglycan monomers and aggregates isolated from bovine articular cartilage. Proteoglycan degradation was (1) proportional to plasmin concentration, (2) dependent on the conversion of plasminogen to plasmin by plasminogen activator, (3) not displayed by plasminogen activator alone, and (4) inhibited by a serine proteinase inhibitor. These results, coupled with other findings, provide further support for a possible role of plasmin/plasminogen activator in cartilage destruction associated with rheumatoid arthritis.

Animals↗
PubMedDate not supplied

Fc gamma RI blockade and modulation for immunotherapy.

Splenectomy and corticosteroids are the treatment of choice for patients with immune thrombocytopenic purpura (ITP). However, for the 10%-15% of patients who do not respond to conventional therapy, high-dose i.v. IgG can induce life-saving transient responses. The benefits of i.v. IgG have been attributed to Fc receptor blockade; however, the involvement of the individual Fc receptors for IgG (Fc gamma R) in ITP remain to be more completely defined. Recently a mAb, designated mAb H22, which recognizes an epitope on Fc gamma RI (CD64) outside the ligand-binding domain, was humanized. Because mAb H22 is a human IgG1 and Fc gamma RI has a high affinity for human IgG1 antibodies, we predicted that mAb H22 would bind to the Fc gamma RI ligand-binding site through its Fc domain and to its external Fc gamma RI epitope through both Fab domains. These studies demonstrate that mAb H22 blocked Fc gamma RI-mediated phagocytosis of opsonized red blood cells more effectively than an irrelevant IgG. Moreover, cross-linking Fc gamma RI with mAb H22 down-modulated Fc gamma RI expression on monocytes, an effect seen within 2 h.

Antibodies, Monoclonal↗