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Biomedical subjects

A B Roberts

Publications and source records attributed to A B Roberts.

At least 217 records · Page 12Linked to original sources
PubMed1989

Further studies of the role of transforming growth factor-beta in human B cell function.

This study was designed to address three specific questions in human B cells. First, to determine whether transforming growth factor-beta (TGF-beta)2 has similar biologic effects on B cell function as does TGF-beta 1. Second, to test the hypothesis that TGF-beta 1 is an autocrine growth and differentiation inhibitor. Finally, because multiple receptor species for TGF-beta have been identified on other cell types, to determine by chemical cross-linking and competitive binding studies the nature of the TGF-beta 1 R present on normal and transformed B cells. Exogenous TGF-beta 2 was found to be functionally similar to TGF-beta 1 in its inhibition of factor dependent normal B cell proliferation and Ig secretion. When an antibody, specific for the active form of TGF-beta 1, was added in conjunction with IL-2 to previously stimulated B cell cultures, there was a 14.4 +/- 4.2% increase in B cell proliferation, a 22 +/- 6% increase in IgG production, and a 33 +/- 8.6% increase in IgM production when compared to control cultures. Chemical cross-linking of 125I-TGF-beta 1 to normal B cell membranes identified two major cross-linked species of 65 and 90 kDa. A fivefold excess of unlabeled TGF-beta 1 competitively inhibited the detection of both of these bands while a 50-fold excess of unlabeled TGF-beta 2 did not inhibit the 90-kDa band and only partially inhibited (60%) of the 65-kDa band. Chemical cross-linking of 125I-TGF-beta 1 to transformed B cell membranes identified only a single band of 60 kDa. Scatchard plot analysis of 125I-TGF-beta 1 binding to normal B cells that was competitively inhibited with increasing concentrations of unlabeled TGF-beta 1 revealed both high and low affinity binding sites whereas analysis of 125I-TGF-beta 1 binding in the presence of increasing concentrations of unlabeled TGF-beta 2 revealed only low affinity sites. These findings demonstrate that TGF-beta 2 is as effective as TGF-beta 1 in inhibiting human B cell function, that small amounts of active TGF-beta 1 are present endogenously in in vitro cultures which partially inhibit B cell function, that two major TGF-beta 1 R cross-linked complexes of 65 and 90 kDa are present on normal B cells, and that transformation of B cells may be accompanied by changes in the TGF-beta 1 R.

Adolescent↗
PubMed1989

Transforming growth factor-beta production by synovial tissues from rheumatoid patients and streptococcal cell wall arthritic rats. Studies on secretion by synovial fibroblast-like cells and immunohistologic localization.

The growth of synovial fibroblast-like cells from patients with rheumatoid arthritis and rats with streptococcal cell wall (SCW)-induced arthritis in vitro under anchorage-independent conditions is inhibited by transforming growth factor-beta (TGF-beta). Because this growth factor is present in rheumatoid synovial fluids, we studied whether this cytokine might be secreted by cells in rheumatoid synovial tissue. We show that synovial tissues from patients with rheumatoid arthritis and osteoarthritis, and rats with SCW-induced arthritis, contain TGF-beta-1 mRNA. TGF-beta, predominantly type 1, was spontaneously secreted in vitro by synovial tissue explants and synovial fibroblast-like cells. In addition, TGF-beta could be detected immunohistochemically in cells throughout rheumatoid and SCW-induced arthritic rat synovial tissues. Finally, exogenous TGF-beta induced collagen and inhibited collagenase mRNA levels by cultured synoviocytes. These data support an autocrine role for TGF-beta in the regulation of synoviocytes in rheumatoid arthritis and, in light of its demonstrated effects on the immune system, suggest that TGF-beta might also have important paracrine effects on infiltrating inflammatory cells.

Animals↗
PubMed1989

Promoter sequences of the human transforming growth factor-beta 1 gene responsive to transforming growth factor-beta 1 autoinduction.

Two distinct regions of the transforming growth factor (TGF)-beta 1 promoter are responsive to autoregulation. Sequences located between nucleotides -454 to -323 and between the two major transcriptional start sites have positive regulatory activities and are induced by TGF-beta 1 in A-549 cells. The chloramphenicol acetyltransferase activity of the upstream human TGF-beta 1 promoter-chloramphenicol acetyltransferase gene is increased 8- to 10-fold by treatment of cells with TGF-beta 1, whereas that of the second promoter is increased approximately 3- to 4-fold. Using an S1 nuclease protection assay of chloramphenicol acetyl-transferase mRNA, we found that the steady-state expression of chloramphenicol acetyltransferase mRNA also is markedly increased. Seven distinct factors present in nuclear extracts from A-549 cells interact with the sequences between -454 and -323, strongly supporting the involvement of sequence-specific transcription factors in the transcriptional autoactivation of the human TGF-beta 1 gene.

DNA-Binding Proteins↗
PubMed1989

Characterization of the promoter region of the human transforming growth factor-beta 1 gene.

The 5'-end of the human transforming growth factor-beta 1 gene (TGF-beta 1) was isolated from a human leukocyte genomic DNA library. Analysis of the transcriptional start sites of human TGF-beta 1 mRNAs by S1 mapping and primer extension revealed two major start sites 271 nucleotides from one another; several minor sites were also identified. DNA sequence analysis showed that the promoter region contains neither a "TATA" box nor a "CAAT" box, is very G+C rich, and contains 11 CCGCCC repeats. Seven putative binding sites for the transcription factor Sp1 were also identified. To determine the location of sites that may be important for the function of the TGF-beta 1 promoter, we joined the 5'-end of the TGF-beta 1 gene to the coding region for chloramphenicol acetyltransferase. The chimeric gene produced high levels of chloramphenicol acetyltransferase activity in transfected HT-1080, AKR-2B, and A-549 cells. Sequences responsible for both promotion and inhibition of transcription were located in the region extending from 1400 to 300 base pairs upstream of the first major TGF-beta 1 transcriptional start site. The 130-base pair fragment located between 453 and 323 base pairs upstream of this start site contains positive regulatory activity in all cells tested. A second promoter activity was identified in the region between the two major transcriptional start sites. These findings revealed a complex pattern of regulation of human TGF-beta 1 gene expression.

Animals↗
PubMed1989

Transforming growth factor beta. A biologic chorioretinal glue.

Transforming growth factor beta (TGF-beta) stimulates fibrosis. We studied its possible role as a bioactive substance for inducing localized chorioretinal wound healing along the edge of a retinal tear. The TGF-beta was applied to induced retinal tears that were examined histopathologically. One day after surgery, neither control nor TGF-beta-treated eyes developed chorioretinal wound healing. Four days, two weeks, and two months after surgery, the control eyes still had not developed chorioretinal wound healing. In contrast, the edges of the retinal tear treated with TGF-beta were adherent to the underlying Bruch's membrane via localized fibrous tissue without apparent effects elsewhere. These results demonstrate intraocular in vivo bioactivity of TGF-beta and suggest that TGF-beta may have a potential role as an alternative means for inducing a chorioretinal adhesion in the treatment of retinal tears.

Animals↗
PubMed1989

Immunodetection and quantitation of the two forms of transforming growth factor-beta (TGF-beta 1 and TGF-beta 2) secreted by cells in culture.

Transforming growth factor beta (TGF-beta), a potent modulator of cell growth, differentiation, and the expression of extracellular matrix components in a variety of cell types, exists as two distinct homodimers (TGF-beta 1 and TGF-beta 2), sharing 71% sequence homology. Radioreceptor and previously described radioimmunological assays using rabbit antibodies have not been able to distinguish between these two forms. We have developed antisera in turkeys against native TGF-beta 1 and TGF-beta 2, each of which specifically blocks both the receptor binding and biological activity of each of these peptides. With these immunological reagents we describe sensitive and specific immunological assays for TGF-beta 1 and TGF-beta 2 in complex biological fluids. Using these assays we show that both TGF-beta 1 and TGF-beta 2 are secreted by a variety of cultured cells, but that some cells secrete predominantly either TGF-beta 1 or TGF-beta 2 while others secrete both peptides in nearly equal amounts. Our results demonstrate that the expression of each of the two forms of TGF-beta is independently regulated.

Animals↗
PubMed1989

Fetal liver length in normal and isoimmunized pregnancies.

The length of the right lobe of the fetal liver was measured by means of ultrasound examined on 53 occasions in 21 isoimmunized pregnancies. Fetal blood samples were taken in all patients within 24 hours. Comparisons were made with 350 measurements of liver length in normal pregnancies. A good correlation was found between liver length and fetal hemoglobin level (r = 0.794, p less than 0.001) and between liver length and reticulocyte count (r = 0.721, p less than 0.001). At the time of first sample, all fetuses with a hemoglobin of less than 100 gm/L had a liver length that was greater than the ninetieth percentile. Liver length measurement seems to be a useful indicator of the degree of fetal anemia in isoimmunized pregnancies.

Cell Count↗
PubMed1989

Transforming growth factor-beta 1: histochemical localization with antibodies to different epitopes.

We have localized transforming growth factor-beta (TGF-beta) in many cells and tissues with immunohistochemical methods, using two polyclonal antisera raised to different synthetic preparations of a peptide corresponding to the amino-terminal 30 amino acids of TGF-beta 1. These two antibodies give distinct staining patterns; the staining by anti-CC(1-30) is intracellular. This differential staining pattern is consistently observed in several systems, including cultured tumor cells; mouse embryonic, neonatal, and adult tissues; bovine fibropapillomas; and human colon carcinomas. The extracellular staining by anti-CC(1-30) partially resembles that seen with an antibody to fibronectin, suggesting that extracellular TGF-beta may be bound to matrix proteins. The intracellular staining by anti-LC(1-30) is similar to that seen with two other antibodies raised to peptides corresponding to either amino acids 266-278 of the TGF-beta 1 precursor sequence or to amino acids 50-75 of mature TGF-beta 1, suggesting that anti-LC(1-30) stains sites of TGF-beta synthesis. Results from RIA and ELISAs indicate that anti-LC(1-30) and anti-CC(1-30) recognize different epitopes of this peptide and of TGF-beta 1 itself.

Animals↗
PubMed1989

Expression of transforming growth factor-beta 1 in specific cells and tissues of adult and neonatal mice.

We have used immunohistochemical techniques to detect transforming growth factor-beta 1 (TGF-beta 1) in many tissues of adult and neonatal mice. Each of two antibodies raised to the amino-terminal 30 amino acids of TGF-beta 1 selectively stained this molecule in either intracellular or extracellular locations. Strong intracellular staining was found in adrenal cortex, megakaryocytes and other cells of the bone marrow, cardiac myocytes, chondrocytes, renal distal tubules, ovarian glandular cells, and chorionic cells of the placenta. Marked staining of extracellular matrix was found in cartilage, heart, pancreas, placenta, skin, and uterus. Staining was often particularly intense in specialized cells of a given tissue, suggesting unique roles for TGF-beta within that tissue. Levels of expression of mRNA for TGF-beta 1 and its histochemical staining did not necessarily correlate in a given tissue, as in the spleen. The present data lend further support to the concept that TGF-beta has an important role in controlling interactions between epithelia and surrounding mesenchyme.

Adrenal Glands↗
PubMed1989

Regulation of endothelial cell growth, architecture, and matrix synthesis by TGF-beta.

Transforming growth factor-beta (TGF-beta) has profound effects on all cell types making up the vasculature, including endothelial cells, smooth muscle cells, and adventitial connective tissue. As such, it plays a prominent role not only in the physiologic vasculogenesis and angiogenesis characteristic of embryogenesis and inflammation and repair but also in vascular disorders such as the arterial thickening associated with pulmonary hypertension. The actions of TGF-beta on these vascular cells in vitro and in vivo are extremely complex. In vitro, TGF-beta inhibits both the proliferation and migration of endothelial cells in monolayer culture, but it promotes organization of the cells into tubelike structures in three-dimensional culture in collagen gels. TGF-beta also increases synthesis of fibronectin and decreases secretion of proteases by both endothelial cells and fibroblasts; the resultant changes in matrix composition could mediate the effects of TGF-beta on both the growth and phenotype of these cells, and overexpression could contribute to fibrosis. TGF-beta also regulates the synthesis by endothelial cells of platelet-derived growth factor, which can stimulate growth of vascular smooth muscle cells. In vivo, TGF-beta stimulates neovascularization at local sites of injection and also is angiogenic when assayed in the rabbit cornea or on the chick chorioallantoic membrane. However, because angiogenesis involves the participation of many different cell types, effects of TGF-beta on inflammatory cells must also be considered. Thus, the ability of TGF-beta to chemoattractant macrophages and to increase expression by the cells of mRNAs for several growth factors known to act on endothelial cells, smooth muscle cells, and fibroblasts must also be considered.

Animals↗
PubMed1989

Anchorage-independent growth of synoviocytes from arthritic and normal joints. Stimulation by exogenous platelet-derived growth factor and inhibition by transforming growth factor-beta and retinoids.

Exuberant tumor-like synovial cell proliferation with invasion of periarticular bone is a feature of rheumatoid arthritis in humans and of streptococcal cell wall (SCW)-induced arthritis in rats. These histologic observations prompted us to examine synoviocytes from arthritic joints for phenotypic characteristics of transformed cells. The capacity to grow in vitro under anchorage-independent conditions is a characteristic that correlates closely with potential in vivo tumorigenicity. In medium supplemented with 20% serum or in basal media supplemented with platelet-derived growth factor (PDGF), early passage synoviocytes from both SCW-induced and rheumatoid arthritic joints formed colonies in soft agarose. Epidermal growth factor (EGF), interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and transforming growth factor-beta (TGF-beta) did not support growth, although EGF enhanced PDGF-dependent growth. On the other hand, TGF-beta, as well as all-trans-retinoic acid, inhibited colony growth. Early passage normal rat and human synoviocytes also grew under the same conditions, but lung, skin, and late-gestation embryonic fibroblast-like cells did not. Considered in the context of other published data our findings provide cogent evidence that synoviocytes, but not other types of fibroblast-like cells, readily acquire phenotypic characteristics commonly associated with transformed cells. Expression of the transformed phenotype in the inflammatory site is likely regulated by paracrine growth factors, such as PDGF and TGF-beta.

Animals↗
PubMed1989

Correlation of fibrosis and transforming growth factor-beta type 2 levels in the eye.

Approximately 1 out of every 10 eyes undergoing surgery for retinal detachment develops excessive intraocular fibrosis that can lead to traction retinal detachment and ultimate blindness. This disease process has been termed proliferative vitreoretinopathy (PVR). The ability to monitor and grade this fibrotic response accurately within the eye as well as the ability to aspirate vitreous cavity fluid bathing the fibrotic tissue makes this an ideal setting in which to investigate the development of fibrosis. Although laboratory studies have recently shown that transforming growth factor-beta (TGF-beta) can enhance fibrosis, little clinical evidence is yet available correlating the level of this or other growth factors with the degree of fibrosis in a clinical setting. We have found that vitreous aspirates from eyes with intraocular fibrosis associated with PVR have more than three times the amount of TGF-beta (1,200 +/- 300 pM [SEM]) found in eyes with uncomplicated retinal detachments without intraocular fibrosis (360 +/- 91 pM [SEM]). Using an in vitro assay, 84-100% of the TGF-beta activity could be blocked with specific antibodies against TGF-beta 2, whereas only 10-21% could be blocked by specific antibodies against TGF-beta 1. TGF-beta 1 was used in an animal model of traction retinal detachment. Since beta 1 and beta 2 have essentially identical biologic effects and only human beta 1 was available in quantities required, beta 1 was chosen for these in vivo studies. The injection of TGF-beta1 plus fibronectin (FN) but not TGF-beta1 alone into the vitreous cavity of rabbits resulted in the increased formation of intraocular fibrosis and traction retinal detachments as compared to control eyes. In previous studies, intravitreal FN levels were also found to be elevated in eyes with intraocular fibrosis.

Adolescent↗
PubMed1989

The effects of TGF beta on haemopoietic cells.

Transforming growth factor beta (TGF beta) suppresses the growth of differentiation inducible, murine IL-3-dependent multipotential cell lines but has no growth inhibitory effect upon an IL-3-independent (leukaemic) cell line arising from one of them, nor on IL-3-dependent cell lines that are unable to undergo differentiation. TGF beta inhibits in vitro colony formation by normal multipotential haemopoietic progenitor cells. Bipotential progenitors recruited by GM-CSF are, however, more resistant to the inhibitory effects of TGF beta, whereas progenitors recruited by the lineage restricted factor, M-CSF, are sensitive to the inhibitory effects. These data indicate that responsiveness to TGF beta is differentiation linked and studies with the cell lines suggest that response (or lack of response is not determined solely by levels of expression of TGF beta receptors. Furthermore, the effects of TGF beta 2 on haemopoietic progenitors are very similar to those induced by TGF beta.

Animals↗
PubMed1989

Sandwich enzyme-linked immunosorbent assays (SELISAs) quantitate and distinguish two forms of transforming growth factor-beta (TGF-beta 1 and TGF-beta 2) in complex biological fluids.

We have developed sandwich enzyme-linked immunosorbent assays (SELISAs) for TGF-beta 1 and TGF-beta 2 using both turkey and rabbit neutralizing polyclonal antibodies against native TGF-beta s. Each assay is based on the binding of two different antibodies to distinct epitopes of a single TGF-beta molecule. With these assays, TGF-beta types 1 and 2 can each be specifically quantitated in complex biological fluids, with detection limits of 2-5 pg. TGF-beta 3 and TGF-beta 5 either do not cross-react or cross-react very poorly in these assays. TGF-beta 1.2 heterodimer, although 50-80% neutralized by either TGF-beta 1 or TGF-beta 2 antibodies, shows only a 1.5 and 3.7% cross-reactivity in the TGF-beta 1 and TGF-beta 2 SELISAs, respectively. The SELISAs reported here represent the most specific, rapid, and precise assays for TGF-beta 1 and TGF-beta 2 reported thus far.

Animals↗
PubMed1989

Transforming growth factor-beta and suppression of carcinogenesis.

Transforming growth factor-beta (TGF-beta) plays an important role in controlling proliferation or differentiation in almost all epithelial tissues. The pathophysiology of TGF-beta during carcinogenesis is now an important area of investigation, since it appears that as the process of carcinogenesis progresses, epithelial cells often become refractory to the growth-regulatory actions of TGF-beta. In this article we consider the possible cellular and molecular bases for this phenomenon, and then discuss some pharmacological approaches to enhancing the synthesis or activity of TGF-beta. These approaches may provide new modalities for prevention of carcinogenesis, if they can be applied during the early stages of the disease process, before cells become refractory. We give particular attention to tamoxifen and retinoic acid, since it has been shown that these agents, which are of known efficacy for prevention of cancer, can markedly enhance the secretion of specific isotypes of TGF-beta by several types of cells.

Amino Acid Sequence↗
PubMed1988

cDNA cloning of porcine transforming growth factor-beta 1 mRNAs. Evidence for alternate splicing and polyadenylation.

Most eukaryotic cells encode principally a 2.5-kilobase (kb) transforming growth factor (TGF)-beta 1 mRNA. However, we have found two major TGF-beta 1 RNA species, 3.5 and 2.5 kb long, in porcine tissues. The 3.5-kb species has a longer 3'-untranslated sequence generated by the selection of an alternate polyadenylation site. There is a 117-nucleotide sequence within this unique 3' region, which is similar to the PRE-1 repetitive sequence of unknown function, reported earlier in the porcine genome. We have also cloned and characterized an alternately spliced mRNA species specific for the TGF-beta 1 gene, in which exons IV and V of the corresponding human TGF-beta 1 gene are deleted. The nucleotide sequence of this cDNA clone predicts a putative precursor protein of 256 amino acids; the N-terminal 211 amino acids of this putative protein are identical to the TGF-beta 1 precursor protein (exons I, II, and III of the human TGF-beta 1 gene), but the C-terminal 45 amino acids are distinct, due to a frameshift in the translation of exons VI and VII. In addition we provide data for the existence of other mRNA species generated in a tissue-specific manner either by alternate splicing or by heterogeneous 5' leader sequences.

Amino Acid Sequence↗
PubMed1988

Transforming growth factor beta regulates the metabolism of proteoglycans in bovine cartilage organ cultures.

The effect of transforming growth factor beta (TGF-beta) has been studied in a bovine articular cartilage organ culture. The peptide stimulates synthesis of proteoglycans in a dose-dependent manner, reaching saturation at 10 ng/ml. This dose gave an approximate 7-fold increase in synthesis over basal controls. In addition, the peptide decreased the rates of catabolism of proteoglycans with an approximately 2-fold maximal effect seen at 5 ng/ml. At the latter concentration, TGF-beta prevented the 4-fold loss of proteoglycans which occurred in cultures maintained under basal conditions over the course of 3 weeks. There was no increase in cell (DNA) content of the cartilage explants under these conditions of TGF-beta treatment, and the net collagen content of the explants remained constant.

Animals↗