Transforming growth factor-beta (TGF-beta)-mediated immunosuppression in the tumor-bearing state: enhanced production of TGF-beta and a progressive increase in TGF-beta susceptibility of anti-tumor CD4+ T cell function

The present study deals with the effect of transforming growth factor-beta (TGF-beta) on anti-tumor immune responsiveness at various stages of the tumor-bearing state. Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 1-3 wk after inoculation with CSA1M cells produced interleukin-2 (IL-2) and macrophage-activating factor (MAF)/interferon-gamma (IFN-gamma) upon in vitro culture without addition of exogenous tumor antigens. This lymphokine production was achieved through collaboration between anti-CSA1M CD4+ T cells and antigen-presenting cells that had been pulsed with CSA1M tumor antigens in vivo in the tumor-bearing state. The IL-2-producing capacity of CD4+ T cells reached the maximal level as early as one week after tumor implantation but decreased with the progress of tumor-bearing stages. In contrast, the capacity of CD4+ T cells to produce MAF/IFN-gamma was not affected but was maintained at high levels even late in the tumor-bearing state. The addition of recombinant TGF-beta (rTGF-beta) to cultures of spleen cells from various tumor-bearing stages resulted in the suppression of lymphokine production. However, the magnitude of the TGF-beta-induced suppression varied depending on which tumor-bearing stages of splenic cells were tested as a responding cell population; it was slight in cells from early (1-3 wk) tumor-bearing stages but increased in cells from donor mice at later tumor-bearing stages. Thus, spleen cells from late tumor-bearing stages with weak but significant IL-2-producing and considerable MAF/IFN-gamma producing capacities failed to produce these lymphokines when rTGF-beta was present in cultures. A progressive increase in the TGF-beta susceptibility was also observed for IL-4-producing Th2 as well as IL-2/MAF-producing Th1 cells. In addition, increased levels of TGF-beta were detected in plasma from tumor-bearing mice at late stages. Taken together, these results indicate that tumor-bearing mice exhibit enhanced production of TGF-beta as well as a progressive increase in the susceptibility of anti-tumor CD4+ T cells to TGF-beta-induced suppressive mechanisms.     

Acquired and naturally occurring resistance of thyroid follicular cells to the growth inhibitory action of transforming growth factor-beta 1 (TGF-beta 1)

While the multifunctional proteins of the transforming growth factor-beta (TGF-beta) family have a potent antiproliferative effect on thyroid follicular cell growth, increased expression of TGF-beta in proliferating thyroid cells and in thyroid tumours has recently been described, suggesting a secondary counter-regulatory role of these proteins. We have studied further this apparent paradox in vitro using FRTL-5 cells, 5 continuous cell strains from feline multinodular goitres (MNG) and 29 primary cultures prepared from human MNG. While dose dependent inhibition of FRTL-5 cell growth was confirmed, a variable fraction of these cells was naturally resistant towards TGF-beta 1, thus explaining the large interassay variability of growth inhibition (36 to 98% within 2 days, n = 19). After 40 days of continuous exposure, FRTL-5 cells became fully refractory towards TGF-beta 1 inhibition, due to the selective growth of naturally resistant subclones, as demonstrated for example by microscopic observation of three-dimensionally growing collagen-embedded cell clusters. The refractoriness could still be demonstrated even after several cell passages. In addition, 2 out of 5 feline thyroid cell strains obtained from feline MNG and 18 out of 29 primary cultures from human MNG showed a high degree of refractoriness towards TGF-beta. We conclude that constitutively TGF-beta resistant cells may occur in thyroid glands and that persistent TGF-beta refractoriness may secondarily be acquired. Resistant cells may escape regular growth control mechanisms and hence may contribute to the notorious heterogeneity of thyroid growth and to nodular transformation.     

Determination of transforming growth factor-beta 1 (TGF-beta 1) and insulin-like growth factor (IGF-1) in bovine colostrum samples

The major growth factors in bovine colostrum are transforming growth factor-beta s (TGF-beta 1 and TGF-beta 2) and insulin-like growth factors (IGF-1 and IGF-2). Recently, TGF-beta 2 content of bovine colostrum was measured using a TGF-beta 2 specific ELISA (1) and now we have validated ELISAs for for bovine TGF-beta 1 and IGF-1. The concentrations of IGF-1 and TGF-beta 1 in the first milking after calving were 248-1850 ng/ml and 12.4-42.6 ng/ml, respectively, and they declined in correlation with total protein concentration to 27.0-101 ng/ml (IGF-1) and 0.80-3.49 ng/ml(TGF-beta 1) by the fifth milkings. The amount of TGF-beta 1 was on average 5.3 +/- 1.4% of that of TGF-beta 2 and there is a high correlation (r = 0.966) between the concentrations of these growth factors in the same samples. No free TGF-beta 1 form of could be detected.     

Transforming growth factor-beta, endothelin-1, and c-fos expression in necrotizing/crescentic IgA glomerulonephritis

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes. Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA. The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a property similar to the eukaryotic type I topoisomerases. The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.     

Transforming growth factor-beta1 in hemangioma of the ovary

OBJECTIVE: Anastomotic pseudoaneurysms continue to be a late complication of vascular surgery, particulary following prosthetic graft procedures. The purpose of this study was to investigate if a previously reported increase in interval between the original operation and the development of pseudoaneurysm was related to a change in incidence. DESIGN: Retrospective study. METHODS: We reviewed the records of 76 patients who presented with 90 femoral pseudo-aneurysms and underwent reconstructive procedures from January 1989 to June 1994. The median age was 69 years (range: 39-83). In the same time period all femoral artery anastomosis operations were recorded. RESULTS: The incidence of femoral pseudo-aneurysms in Copenhagen was approximately 4.3%. CONCLUSIONS: A previously reported increase in interval between primary operation and aneurysms formation was not related to a change in incidence during the same time period.     

Transforming growth factor-beta1 increases mRNA levels of osteoclastogenesis inhibitory factor in osteoblastic/stromal cells and inhibits the survival of murine osteoclast-like cells

Osteoclastogenesis inhibitory factor (OCIF), also termed osteoprotegerin (OPG), is a secreted member of the tumor necrosis factor (TNF) receptor family. It inhibits bone resorption in vivo and osteoclast-like cell (OCL) formation in vitro. To better understand the biological role of OCIF, we first examined the effects of various osteotropic agents on OCIF mRNA levels in mouse calvarial osteoblasts. Northern blot analysis showed that stimulators of OCL formation such as 1,25-(OH)2D3, prostaglandin E2 (PGE2), parathyroid hormone (PTH), and interleukin 1 (IL-1) decreased OCIF mRNA levels. In contrast, transforming growth factor (TGF)-beta1 increased OCIF mRNA levels in primary osteoblasts as well as in osteoblastic/stromal cell lines. Since it was reported that both TGF-beta1 and OCIF not only inhibited OCL formation but also impaired the survival of OCL by inducing apoptosis in vitro, we next examined the possible involvement of OCIF in TGF-beta1-induced impairment of OCL survival. In a mouse bone marrow culture, we confirmed that addition of OCIF or TGF-beta1 decreased the number of surviving OCL. Anti-OCIF IgG, which completely neutralized the effect of OCIF, partially prevented the TGF-beta1-induced decrease in the number of OCL. Our results suggest that (i) downregulation of OCIF expression is one of the mechanisms for the stimulatory effects of 1,25(OH)2D3, PGE2, PTH, and IL-1 on osteoclastogenesis; and (ii) the TGF-beta1-induced apoptosis of OCL is mediated, at least in part, by upregulation of OCIF expression.     

Transforming growth factor-alpha promotes mammary tumorigenesis through selective survival and growth of secretory epithelial cells

Transforming growth factor (TGF)-alpha stimulates the growth and development of mammary epithelial cells and is implicated in the pathogenesis of human breast cancer. In this report we evaluate the consequences of overexpressing TGF-alpha in the mammary gland of transgenic mice and examine associated cellular mechanisms. When operating on a FVB/N genetic background (line MT100), TGF-alpha induced the stochastic development of mammary adenomas and adenocarcinomas f secretory epithelial origin in 64% of multiparous females. In contrast, tumors were exceedingly rare in virgin MT100 females, MT100 males, and multiparous FVB/N females. In MT100 females multiple foci of hyperplastic secretory lesions preceded the development of frank tumors; these initial lesions appeared during the involution period after the first lactation. Serial transplantation of these hyperplasias indicated an absence of proliferative immortality. Nevertheless, they gave rise to tumors at a low frequency and after a prolonged latency in virgin hosts; in multiparous hosts, tumors developed earlier and at a high incidence. The TGF-alpha transgene was highly expressed in hyperplasias and tumors but not in virgin and nonlesion-bearing tissue, suggesting that TGF-alpha overexpression provides a selective growth advantage. TGF-alpha also induced at lactation a 6.4-fold increase in DNA synthesis in MT100 epithelial cells, many of which were binucleated. MT100 mammary tissue experienced an obvious delay in involution, resulting in the postlactational survival of a significant population of unregressed secretory epithelial cells. In contrast, another line of transgenic mice on a CD-1 genetic background (MT42), in which TGF-alpha overexpression induced liver but not mammary tumors, failed to demonstrate postlactational epithelial cell survival. These data show that TGF-alpha promotes mammary tumorigenesis in multiparous MT100 mice by stimulating secretory epithelial cell proliferation during lactation and prolonging survival during involution. These points support the notion that TGF-alpha can act as a mitogen and also as a differentiation factor in mammary epithelium.     

Retinoids potentiate transforming growth factor-beta activity in bovine endothelial cells through up-regulating the expression of transforming growth factor-beta receptors

The present paper reports data on secular trends in the stature of Brazilian Navy recruits born from 1940 to 1965. The final sample included 3269 individuals aged 18.00-18.99. Statistics performed were: ANOVA (one-way and two-way), Sheffe test, simple linear regression between stature and year of birth, and multiple linear regression adjusting for level of schooling (beta coefficient) and chi-square. Results indicated a progressive growth trend in stature of 0.1 cm/yr. for the country as a whole. The trend was also observed for nearly all regions and two out of three levels of schooling and can be explained by improvement in some of the country's health indicators. One important characteristic was a higher level of schooling observed among Navy recruits, suggesting that these individuals represent a highly select group, and that therefore data on the Navy cannot be applied directly to the Brazilian population as a whole.     

IL-4-dependent regulation of TGF-alpha and TGF-beta1 expression in human eosinophils

TGFs play important roles in wound healing and carcinogenesis. We have previously demonstrated that eosinophils infiltrating into different pathologic processes elaborate TGF-alpha and TGF-beta1. Eosinophils infiltrating hamster cutaneous wounds were found to express TGFs sequentially. In this study, we examined the biologic mediators that may regulate the expression of TGF-alpha and -beta1 by eosinophils. Eosinophils were isolated from the peripheral blood of healthy donors and cultured in the absence or presence of IL-3, IL-4, and IL-5. Cells were analyzed by in situ hybridization and immunohistochemistry. Supernatants from these cultures were assayed for secreted TGF-alpha and TGF-beta1 using TGF-specific ELISAs. IL-3, IL-4, and IL-5 independently up-regulated TGF-beta1 mRNA and product expression by eosinophils in all donors. Interestingly, TGF-alpha production by eosinophils was up-regulated by IL-3 and IL-5 but was down-regulated by IL-4. Consistent with the ability of IL-4 to regulate eosinophil responses, IL-4 signaling molecules are present in human eosinophils. The observation that IL-4 can differentially regulate the expression of TGF-alpha and TGF-beta1 suggests that IL-4 may serve as a physiologic molecular switch of TGF expression by the infiltrating eosinophils in wound healing and carcinogenesis.     

Modulation of IL-12 by transforming growth factor-beta (TGF-beta) in Mycobacterium tuberculosis-infected mononuclear phagocytes and in patients with active tuberculosis

In humans, tuberculosis is associated with suppression of T-cell responses to antigens of Mycobacterium tuberculosis. Recently, the macrophage product, transforming growth factor-beta (TGF-beta) has been implicated in suppression of T-cell proliferation and cytokine production during tuberculosis. We studied the effect of TGF-beta on production of IL-12, and on the augmentation of M. tuberculosis-induced IFN gamma production by IL-12, in patients with pulmonary tuberculosis and by M. tuberculosis. Induction of IL-12 p35, but not IL-12 p40, by M. tuberculosis in monocytes was dependent on prior priming of the cells with IFN gamma. Expression of both IL-12 p40 and p35, however, was suppressed by TGF-beta. Further, TGF-beta interfered with the bioactivity of IL-12 in the enhancement of M. tuberculosis-induced IFN gamma mRNA expression and cytokine production. However, in mononuclear cells from patients with tuberculosis the main effect of TGF-beta on IL-12 appeared to be counter action to IL-12 induced IFN gamma production in response to M. tuberculosis.     

Transforming growth factor-beta receptors on human endometrial cells: identification of the type I, II, and III receptors and glycosyl-phosphatidylinositol anchored TGF-beta binding proteins

BACKGROUND: Mutation of the p53 tumor suppressor gene is the most commonly found genetic alteration in human cancer. The E6 gene product of human papillomavirus (HPV) 16 and 18 can inactivate the p53 protein by promoting its degradation. Because most HPV-positive cervical carcinoma cell lines contain wild-type p53 whereas HPV-negative cell lines have point mutations in the p53 gene, a major role in the development of HPV-negative cervical cancer has been attributed to p53. Recent studies, however, have observed no consistent presence of p53 mutation in HPV-negative primary cervical carcinomas. The MDM2 oncogene, which forms an autoregulatory loop with the wild-type p53 protein, has been found amplified in a high percentage of human sarcomas, thus abolishing the antiproliferative function of p53. METHODS: Forty-three primary cervical carcinomas and 10 autopsy-derived distant metastases from one patient were examined for p53 mutation and MDM2 amplification. These tumors had been selected from 238 cervical cancers that had been HPV-typed by Southern blot hybridization and polymerase chain reaction as a representative sample for their HPV status and their clinicopathologic characteristics. Seventeen of the cases had a remarkably good or poor clinical outcome. Human papillomavirus DNA sequences had been detected in 30 of these 43 primary tumors and 13 were negative for HPV by both methods. p53 mutation in the highly conserved exons 5-8 was studied by single-strand conformation polymorphism analysis and direct sequencing. MDM2 amplification was analyzed by Southern blot hybridization. RESULTS: Only two missense point mutations and one nucleotide sequence polymorphism were detected: a TAC-->TGC transition in codon 234 in exon 7, resulting in a Tyr-->Lys substitution, a CGT-->TGT transition in codon 273 in exon 8, resulting in an Arg-->Cys substitution and a polymorphism (CGA-->CGG) in codon 213 in exon 6. Both tumors revealing the point mutations were HPV-negative carcinomas. Amplification of the MDM2 gene was observed in 1 of the 53 specimens tested. CONCLUSIONS: In contrast to data derived from cultured cervical carcinoma cell lines and primary sarcomas, these results indicate that p53 mutation and amplification of the MDM2 oncogene are rare even in HPV-negative primary cervical carcinomas. However, to the authors; knowledge, this is the first observation of MDM2 amplification in humans outside sarcomas and neuroepithelial tumors.