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Biomedical subjects

D R Twardzik

Publications and source records attributed to D R Twardzik.

At least 19 recordsLinked to original sources
PubMed1997

Identification of rat prostatic steroid-binding protein as a target antigen of experimental autoimmune prostatitis: implications for prostate cancer therapy.

The long term goal of this study is to develop autoimmune prostatitis as a therapy for prostate cancer. An immune attack capable of destroying normal prostate epithelial cells should also destroy malignant prostate tissue and provide therapeutic benefit in cancer patients. The current study was initiated to identify antigenic targets for experimental autoimmune prostatitis on the assumption that such proteins might also be suitable targets for immunotherapy of prostate cancer. Male Lewis rats were immunized with syngeneic prostate homogenates, and the immune sera were used to screen prostate proteins for immunoreactivity by Western blot analysis. The dominant protein recognized by the immune sera was purified by ion exchange chromatography and reverse phase HPLC. Microsequence analysis of two polypeptide components of this immunodominant protein demonstrated N-terminal sequences identical with two of the three component chains of rat prostatic steroid-binding protein (PSBP). T cell responses to PSBP were also detected in rats immunized with prostate homogenate. Immunizing male rats with purified PSBP induced vigorous Ab and T cell responses. Significant prostate inflammation was observed in some rats immunized with PSBP. Adoptive transfer of T cells immune to PSBP induced rapid and severe destructive autoimmune prostatitis. These results demonstrate that PSBP is a major target Ag of experimental autoimmune prostatitis in a rat model and may serve as a target Ag for vaccine and T cell therapy against prostate cancer.

Animals↗
PubMed1995

The enhancement in wound healing by transforming growth factor-beta 1 (TGF-beta 1) depends on the topical delivery system.

Transforming growth factor-beta 1 (TGF-beta 1) has beneficial effects on wound healing. However, the ideal method for its administration to the wound site remains unknown. Our aim was to analyze the release of TGF-beta 1 from different formulations and to study whether the changes in wound healing by TGF-beta 1 depend on its topical delivery system. For the studies the TGF-beta 1 was incorporated into phosphate-buffered saline, into a polyoxamer gel, into DuoDERM hydroactive paste, and into a poly(ethylene oxide) hydrogel. The release of 125I-labeled TGF-beta 1 from carriers was measured in full-thickness wounds in rats and the healing of the wounds was analyzed by histology and wound area measurements. The TGF-beta 1 was released from all formulations at a different rate and in an active form as determined by growth inhibition assay. Wound size measurements and the analysis on the amount of cellular influx, fibroplasia, and granulation tissue showed that a single dose (1 microgram/wound) of locally administered TGF-beta 1 significantly (P < 0.01) enhanced the wound healing. This effect was most prominent with polyoxamer gel formulation, which provided the most sustained release of TGF-beta 1. Our finding that the enhancement in wound healing by TGF-beta 1 was significantly dependent on the carrier used for its topical delivery to the wound site is novel and shows the importance of using adequate delivery systems when growth factors are used to enhance wound repair.

Administration, Topical↗
PubMed1994

Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta.

We have analysed the interactions of three proteoglycans of the decorin family, decorin, biglycan and fibromodulin, with transforming growth factor beta (TGF-beta). The proteoglycan core proteins, expressed from human cDNAs as fusion proteins with Escherichia coli maltose-binding protein, each bound TGF-beta 1. They showed only negligible binding to several other growth factors. Intact decorin, biglycan and fibromodulin isolated from bovine tissues competed with the fusion proteins for the TGF-beta binding. Affinity measurements suggest a two-site binding model with Kd values ranging from 1 to 20 nM for a high-affinity binding site and 50 to 200 nM for the lower-affinity binding site. The stoichiometry indicated that the high-affinity binding site was present in one of ten proteoglycan core molecules and that each molecule contained a low-affinity binding site. Tissue-derived biglycan and decorin were less effective competitors for TGF-beta binding than fibromodulin or the non-glycosylated fusion proteins; removal of the chondroitin/dermatan sulphate chains of decorin and biglycan (fibromodulin is a keratan sulphate proteoglycan) increased the activities of decorin and biglycan, suggesting that the glycosaminoglycan chains may hinder the interaction of the core proteins with TGF-beta. The fusion proteins competed for the binding of radiolabelled TGF-beta to Mv 1 Lu cells and endothelial cells. Affinity labelling showed that the binding of TGF-beta to betaglycan and the type-I receptors in Mv 1 Lu cells and to endoglin in endothelial cells was reduced, but the binding to the type-II receptors was unaffected. TGF-beta 2 and 3 also bound to all three fusion proteins. Latent recombinant TGF-beta 1 precursor bound slightly to fibromodulin and not at all to decorin and biglycan. The results show that the three decorin-type proteoglycans each bind TGF-beta isoforms and that slight differences exist in their binding properties. They may regulate TGF-beta activities by sequestering TGF-beta into extracellular matrix.

Biglycan↗
PubMed1994

Novel delivery system for inducing quiescence in intestinal stem cells in rats by transforming growth factor beta 1.

BACKGROUND/AIMS: Intestinal mucosa, a tissue in a dynamic state of rapid cellular proliferation, is often adversely affected by cytotoxic drugs. The purpose of this study was to develop an oral delivery system targeting transforming growth factor (TGF) beta 1 locally and analyze its effects on the epithelial stem cells of gastrointestinal mucosa. METHODS: Rats were treated with recombinant TGF-beta 1 in alginate beads perorally or with recombinant TGF-beta 1 in phosphate-buffered saline perorally or intraperitoneally. Control animals received phosphate-buffered saline only. The size of the villi was measured. Proliferating and mitotic indices were determined by quantifying immunohistochemical staining for proliferating cell nuclear antigen. RESULTS: Alginate beads released no TGF-beta 1 in acid. However, in pH 7.4, TGF-beta 1 was released in an active form. Histomorphometrical analysis showed a marked reduction in villus height (50%-70%) in the intestinal mucosa of animals treated perorally with recombinant TGF-beta 1 in alginate beads. Also, the proliferating and mitotic indices were significantly reduced (P < 0.01) in these animals as compared with controls and other routes of administration. CONCLUSIONS: This study shows that recombinant TGF-beta 1 administered using a novel oral delivery system induces stem cell quiescence in the intestinal mucosa of the rat.

Administration, Oral↗
PubMed1993

Transforming growth factor beta as a virulence mechanism for Leishmania braziliensis.

Transforming growth factor beta (TGF-beta) has potent down-regulating effects on macrophages and is thus capable of influencing the fate of intramacrophage parasites, including leishmanias. We report the development of a mouse model for the study of the human pathogen Leishmania braziliensis and demonstrate, both in vitro and in vivo, a key regulatory role for TGF-beta in the pathogenesis of infection with this parasite. Recombinant TGF-beta added to cultures of murine peritoneal macrophages led to increased intracellular L. braziliensis replication, whereas addition of neutralizing anti-TGF-beta monoclonal antibody decreased levels of infection. Macrophages infected with L. braziliensis produced biologically active TGF-beta, with a direct correlation between amounts of TGF-beta induced by two parasite isolates and their relative virulence. In vivo, treatment with recombinant TGF-beta rendered avirulent parasites virulent and activated latent L. braziliensis infection. Activation of parasite replication was observed in mice which had been infected with L. braziliensis 15 weeks previously but had not developed lesions or had healed lesions, depending on the parasite isolate used to infect the mice. The exacerbation of L. braziliensis infection in vivo was associated with an increase of interleukin 10 mRNA in the draining lymph node. These results demonstrate that TGF-beta is able to alter the course of in vitro and in vivo infections with L. braziliensis, the latter being characterized by an increase in interleukin 10, an important Th2 helper-T-cell cytokine.

Actins↗
PubMed1993

Inhibition of interleukin-2 synthesis and interleukin-2 receptor alpha expression on T cells by a cell-free factor derived from a CD4+ regulatory T cell clone.

We have shown that certain CD4+ T cell lines can function as suppressor cells in a cell culture system. In this context, the CD4+ T cells (AS-9) cloned from the peripheral blood lymphocytes (PBL) of a melanoma patient are capable of suppressing the induction of cytolytic response in autologous PBL in coculture. Here we show that a trypsin-sensitive cell-free culture supernatant factor from the AS-9 cells, AS-9 SF, interferes with IL-2 synthesis by T cells when they are stimulated. AS-9 SF also selectively blocks the expression of interleukin-2 receptor alpha (IL-2R alpha) on T cells during activation. Expression of transferrin receptors and the CD3 molecules is not down-regulated by this factor. The AS-9 SF consequently blocks proliferation of T cells when they are stimulated by lectin or activated through the T cell receptors. AS-9 SF suppresses the IL-2R alpha induction and the T cell proliferation at the induction phase only because it has no suppressive effect on preactivated T cells. Interleukin-2, IL-2R alpha, and beta messages are not down-regulated by the AS-9 SF and the suppressive effect of the AS-9 SF on IL-2R alpha expression and on T cell proliferation is not neutralized by the addition of exogenous recombinant IL-2. The factor does not appear to be IL-4, IL-10, or TGF-beta, three known cytokines possessing regulatory properties on T cell activation.

CD4-Positive T-Lymphocytes↗
PubMed1993

The effect of sterilization on transforming growth factor beta isolated from demineralized human bone.

Growth factors have been identified as the primary cause of osteoinduction in bone healing. Transforming growth factor beta (TGF-beta) has been shown to promote bone formation and is present in bone in high quantities. The aims of the present study were to isolate TGF-beta from human bone, demonstrate its biologic activity, and analyze the effects of conventional sterilization techniques on activity. Bone, obtained from femoral heads of five patients (mean age, 70 years) was ground, demineralized, and freeze-dried, and samples from each patient were divided into three groups: no treatment, sterilization with 1.60 to 1.94 Mrad of 60Co irradiation, and sterilization with ethylene oxide (ETO). Carrier-free recombinant TGF-beta control was also treated and was totally inactivated by ETO but not by irradiation (p < 0.01). TGF-beta activity in demineralized bone was not significantly diminished (p > 0.1) by either sterilization procedure, and substantial amounts of active TGF-beta were recovered in all bone samples: 1.04 +/- 0.77 ng per mg of protein in irradiated samples, 0.67 +/- 0.26 ng per mg in ETO-treated samples, and 1.04 +/- 0.33 in untreated samples, respectively (mean +/- SD). Although a recent report demonstrated that the osteoinductive activity of bone morphogenetic protein in bone powder is diminished considerably by ETO and by 2.5 Mrad of irradiation sterilization of bone powder, these data demonstrate that TGF-beta activity, with its osteoinductive properties, was not destroyed in more coarsely ground, demineralized bone by ETO or by lower doses of irradiation. These findings support the use of human bone allografts in clinical instances involving impaired bone formation.

Aged↗
PubMed1993

A 60-kD protein mediates the binding of transforming growth factor-beta to cell surface and extracellular matrix proteoglycans.

The biological activity of many cytokines is regulated by binding proteins present at the cell surface, in extracellular matrices or in soluble phase. We describe here a TGF-beta binding protein that is both an extracellular matrix and a cell surface protein. When intact extracellular matrices of HEP-G2 cells were affinity cross-linked with 125I-TGF-beta 1, two major binding components were seen: a 250-kD, proteoglycan-like molecule, presumed to be betaglycan, and a 60-kD protein. The 60-kD TGF-beta-binding protein was also present at the cell surface. It could be released from the cell surface by treating cells with high salt, heparin, chondroitin sulfate, heparitinase, or chondroitinase, indicating that it is bound to heparan sulfate and chondroitin sulfate proteoglycans. The 60-kD protein bound TGF-beta 1 with an apparent dissociation constant of 1.6 nM, and there were 30,000 binding sites per cell at the cell surface. In addition to the HEP-G2 cells and another hepatoma cell line, the 60-kD protein was also found in a human colon carcinoma (HT-29) cell line but not in rat kidney (NRK-49F) or human fibroblast (HUT-12) cell lines. The 60-kD protein could be extracted from cells containing it and transferred to the surface of previously negative cells. The 60-kD protein may serve to regulate the binding of TGF-beta to its signal transducing receptors by targeting TGF-beta to appropriate locations in the microenvironment of cells.

Carrier Proteins↗
PubMed1992

Transforming growth factor-beta in leishmanial infection: a parasite escape mechanism.

The course of infection with the protozoan parasite Leishmania is determined in part by their early replication in macrophages, the exclusive host cells for these organisms. Although factors contributing to the survival of Leishmania are not well understood, cytokines influence the course of infection. Transforming growth factor-beta (TGF-beta) is a multipotential cytokine with diverse effects on cells of the immune system, including down-regulation of certain macrophage functions. Leishmanial infection induced the production of active TGF-beta, both in vitro and in vivo. TGF-beta was important for determining in vivo susceptibility to experimental leishmanial infection.

Actins↗
PubMed1992

Transforming growth factor beta has neurotrophic actions on sensory neurons in vitro and is synergistic with nerve growth factor.

Transforming growth factor beta (TGF beta) influences the growth and differentiation of a wide variety of nonneuronal cells (nnc) during embryogenesis and in response to wounding. In the present study TGF beta 1 and TGF beta 2 were examined for their neurotrophic actions on neonatal rat dorsal root ganglion (DRG) neurons with ganglionic nnc in dissociated cultures. TGF beta 1 and TGF beta 2 each increased both neuronal survival and levels of the peptide neurotransmitter substance P (SP) expressed per neuron as well as per culture. TGF beta 1 was maximally effective at a concentration of 40 pM, whereas TGF beta 2 was about 10-fold less potent. Survival effects promoted by simultaneous treatment with both factors were not additive. TGF beta 1 also changed the morphology and distribution of DRG nnc which resulted in clustering of DRG neurons on top of the nnc. Cotreatment of the cultures with two different anti-nerve growth factor (NGF) antibodies eliminated the neurotrophic effects of TGF beta 1. However, treatment with TGF beta 1 did not alter NGF mRNA expression in the cultures nor did it change the amount of NGF in the medium. Further, TGF beta 1 greatly enhanced survival effects and SP stimulation promoted by exogenous NGF at concentrations up to 100 ng/ml. The neurotrophic effects of TGF beta 1 were significantly attenuated by decreasing the proportion of the ganglionic nnc, suggesting a role for these cells in mediating TGF beta 1 action on the neurons. It is hypothesized that the neurotrophic activity of TGF beta depended upon the presence of molecules immunologically related to NGF and that the effects of TGF beta were synergistic with NGF. These observations suggest that TGF beta may play a role in the differentiation and regeneration of DRG neurons in vivo.

Animals↗
PubMed1992

Transforming growth factor alpha, but not epidermal growth factor, promotes the survival of sensory neurons in vitro.

Transforming growth factor alpha (TGF alpha) is a mitogenic polypeptide that is structurally homologous to epidermal growth factor (EGF) and appears to bind to the same receptor in all systems tested previously. In the present study, TGF alpha was found to enhance survival and neurite outgrowth of cultured neonatal rat dorsal root ganglion (DRG) neurons in a dose-dependent manner. This effect was observed with TGF alpha concentrations as low as 17.8 pM. By contrast, EGF at concentrations up to 83 nM was ineffective. Moreover, EGF did not antagonize the TGF alpha survival-promoting effect unless present in large excess (500-fold the concentration for which TGF alpha is effective); even in this case, only partial antagonism was achieved. Survival of neurons from nodose, trigeminal, and sympathetic ganglia was not increased by TGF alpha. Both a subpopulation of DRG neurons and of macrophages in the cultures bound iodinated TGF alpha. This binding was inhibited by excess unlabeled TGF alpha but not EGF. Our data are consistent with the possibilities that the actions of TGF alpha on DRG neurons occur indirectly via unidentified neurotrophic molecules other than NGF as well as directly on the neurons themselves. Thus, TGF alpha, in contrast to EGF, may act as a survival or maintenance factor for a subset of rat sensory neurons. Mediation of this neurotrophic effect appears to occur via a new form of TGF alpha receptor.

Animals↗
PubMed1991

Regulation of Trypanosoma cruzi infections in vitro and in vivo by transforming growth factor beta (TGF-beta).

The effects of transforming growth factor beta (TGF-beta) on interferon gamma-mediated killing of the intracellular protozoan parasite Trypanosoma cruzi and on the course of T. cruzi infection in mice were investigated. Spleen cells from mice with acute T. cruzi infections were found to produce elevated levels of biologically active TGF-beta in vitro, and the possibility that TGF-beta may mediate certain aspects of T. cruzi infection was then addressed. When mouse peritoneal macrophages were treated with TGF-beta in vitro, the ability of IFN-gamma to activate intracellular inhibition of the parasite was blocked. This occurred whether cells were treated with TGF-beta either before or after IFN-gamma treatment. TGF-beta treatment also blocked the T. cruzi-inhibiting effects of IGN-gamma on human macrophages. Additionally, treatment of human macrophages with TGF-beta alone led to increased parasite replication in these cells. The effects of TGF-beta on T. cruzi infection in vivo were then investigated. Susceptible C57BL/6 mice developed higher parasitemias and died earlier when treated with TGF-beta during the course of infection. Resistant C57BL/6 x DBA/2 F1 mice treated with TGF-beta also had increased parasitemias, and 50% mortality, compared with no mortality in infected, saline-treated controls. A single dose of TGF-beta, given at the time of infection, was sufficient to significantly decrease resistance to infection in F1 mice and to exacerbate infection in susceptible C57BL/6 mice. Furthermore, a single injection of TGF-beta was sufficient to counter the in vivo protective effects of IFN-gamma. We conclude that TGF-beta, produced during acute T. cruzi infection in mice, is a potent inhibitor of the effects of macrophage activating cytokines in vivo and in vitro and may play a role in regulating infection.

Animals↗
PubMed1991

Production of transforming growth factor beta 1 during repair of arterial injury.

Repair of arterial injury produced by balloon angioplasty leads to the formation of a neointima and a narrowing of the vascular lumen. In this study, we examined the possibility that smooth muscle cells (SMC) in injured rat carotid arteries are stimulated to produce type-1 transforming growth factor-beta (TGF-beta 1) during neointima formation in vivo. Levels of TGF-beta 1 transcripts (2.4 kb) were significantly increased within 6 h after carotid injury and reached a maximum (five to sevenfold) by 24 h. Regenerating left carotids had sustained increases in TGF-beta 1 mRNA levels (about fivefold) over the next 2 wk, during which time a substantial neointimal thickening was formed. No changes in basal TGF-beta 1 mRNA levels were found in contralateral uninjured carotids at any of the times examined. Immunohistochemical studies showed that a large majority of neointimal SMC were stained for TGF-beta 1 protein in an intracellular pattern, consistent with active TGF-beta 1 synthesis in this tissue. Neointima formation and TGF-beta 1 immunoreactivity were correlated with increases in fibronectin, collagen alpha 2(I), and collagen alpha 1(III) gene expression. Infusion of purified, recombinant TGF-beta 1 into rats with a preexisting neointima produced a significant stimulation of carotid neointimal SMC DNA synthesis. These results suggest that TGF-beta 1 plays an important role as an endogenous growth regulatory factor produced by neointimal SMC themselves during progressive neointimal thickening after balloon angioplasty.

Angioplasty, Balloon↗
PubMed1991

Latent forms of transforming growth factor-beta (TGF beta) derived from bone cultures: identification of a naturally occurring 100-kDa complex with similarity to recombinant latent TGF beta.

Transforming growth factor-beta (TGF beta) is produced by most tissues, including bone, as a complex that is biologically inert. Release of TGF beta homodimer from this latent complex is necessary for TGF beta to exert effects on target cells. Thus, the nature of the latent complex and the mechanisms responsible for TGF beta release are the key to understanding TGF beta actions. We have found that murine calvarial bone cultures secrete multiple latent forms of TGF beta. Using analytical chromatography and Western blot analysis, we have compared bone latent TGF beta with the previously characterized latent complex present in platelets and with simian TGF beta precursor, which is stably expressed in a latent form by Chinese hamster ovarian (CHO) cells. A major component of the bone material appears to be a latent complex of 100 kDa, consisting of mature TGF beta (25-kDa homodimer). Like the recombinant TGF beta precursor, it elutes from a Mono-Q fast pressure liquid chromatography anion exchange column at 0.2 M NaCl and shows a very similar banding pattern on Western blots. Thus, this bone complex closely resembles recombinant TGF beta precursor expressed in a latent form by CHO cells and differs from the naturally occurring platelet complex, which has an additional 135-kDa binding protein that is bound through disulfide bonds to the precursor proregion. Western blot analysis also indicates that, like CHO cells, which express recombinant TGF beta precursor, but unlike other cell types, the bone cultures secrete detectable amounts of uncleaved TGF beta precursor. The bone calvarial culture is the first example of a naturally occurring system that expresses the 100-kDa latent TGF beta complex.

Animals↗
PubMed1990

Autologous melanoma-induced activation of regulatory T cells that suppress cytotoxic response.

The host immune response toward autologous human cancer is subject to regulation by the immunoregulatory network. We show that certain CD4+ T cell clones, derived from melanoma involved lymph node lymphocytes and from PBL stimulated by autologous melanoma cells, selectively down-regulated the induction of cytotoxic immune response of PBL against the respective autologous melanoma cells in two autologous systems. In both systems, only the generation of cytotoxic response against the autologous melanoma cells were suppressed. Cytotoxic response against EBV-infected autologous lymphoblastoid cell line in one case and cytotoxic responses against allogeneic targets in the other were not affected. In addition to suppressor activity selectively expressed against the autologous melanoma cells, the T cell clones up-regulated their Tac receptors when cocultured with the autologous melanoma cells and APC. These results support the existence of a putative tumor Ag-driven activation of regulatory T cells that affect cytotoxic immune response, in vitro, against autologous human melanoma.

CD4-Positive T-Lymphocytes↗
PubMed1990

Localization of cells containing transforming growth factor-alpha precursor immunoreactivity in the basal ganglia of the adult rat brain.

Transforming growth factor-alpha-like immunoreactivity (TGF-alpha-ir) was visualized in the adult rat forebrain using three antisera directed against carboxyterminal sequences in the TGF-alpha precursor. Using immunoperoxidase and immunofluorescence techniques with all three antisera, TGF-alpha-ir was found to be present in a subpopulation of astrocytes in the forebrain. Striatal and pallidal regions of the basal ganglia were studied in detail. In the striatum, there was an uneven distribution of astrocytes containing TGF-alpha-ir, with the greatest number in the dorsal medial third of the caudate-putamen and the overlying corpus callosum/external capsule. In addition, the region of the caudate-putamen bordering the globus pallidus contained numerous clusters of TGF-alpha-ir astrocytes. The globus pallidus itself contained numerous and more evenly distributed TGF-alpha-ir astrocytes. Other pallidal structures--including the ventral pallidum, entopeduncular nucleus, and substantia nigra pars reticulata--contained moderate numbers of TGF-alpha-ir astrocytes. These results suggest that TGF-alpha precursor is present and, perhaps, synthesized in astrocytes. A related growth factor, epidermal growth factor (EGF), has also been reported to be present in pallidal regions of rat brain. Therefore, the TGF-alpha/EGF family of trophic factors may play a role in the function of the central nervous system.

Animals↗