John M. McPherson

TGF-β1 stimulates cultured human fibroblasts to proliferate and produce tissue-like fibroplasia: A fibronectin matrix-dependent event

During wound repair, fibroblasts accumulate in the injured area until any defect is filled with stratified layers of cells and matrix. Such fibroplasia also occurs in many fibrotic disorders. Transforming growth factor‐β (TGF‐β), a promotor of granulation tissue in vivo and extracellular matrix production in vitro, is expressed during the active fibroplasia of wound healing and fibroproliferative diseases. Under usual tissue culture conditions, normal fibroblasts grow to confluence and then cease proliferation. In this study, culture conditions with TGF‐β1 have been delineated that promote human fibroblasts to grow in stratified layers mimicking in vivo fibroplasia. When medium supplemented with serum, ascorbate, proline, and TGF‐β was added thrice weekly to normal human dermal fibroblasts, the cells proliferated and stratified up to 16 cell layers thick within the culture dish, producing a tissue‐like fibroplasia. TGF‐β stimulated both DNA synthesis as measured by 1H‐thymidine uptake and cell proliferation as measured by a Hoechst dye DNA assay in these postconfluent cultures. The stratification was dependent on fibronectin assembly, as demonstrated by anti‐fibronectin antibodies which inhibited both basal and TGF‐β‐stimulated cell proliferation and stratification. Suppression of collagen matrix assembly in cell layers with β‐amino‐proprionitrile (BAPN) did not inhibit basal or TGF‐β stimulated in vitro fibroplasia. BAPN did not interfere with fibronectin matrix assembly as judged by immunofluorescence microscopy. Thus, in concert with serum factors, TGF‐β stimulates postconfluent, fibronectin matrix‐dependent, fibroblast growth creating a fibroplasia‐like tissue in vitro. J Cell Physiol 170:69–80, 1997

TGF-β in the central nervous system: Potential roles in ischemic injury and neurodegenerative diseases

The Transforming Growth Factor-betas (TGF-beta) are a group of multifunctional proteins whose cellular sites of production and action are widely distributed throughout the body, including the central nervous system (CNS). Within the CNS, various isoforms of TGF-beta are produced by both glial and neural cells. When evaluated in either cell culture or in vivo models, the various isoforms of TGF-beta have been shown to have potent effects on the proliferation, function, or survival of both neurons and all three glial cell types, astrocytes, microglia and oligodendrocytes. TGF-beta has also been shown to play a role in several forms of acute CNS pathology including ischemia, excitotoxicity and several forms of neurodegenerative diseases including multiple sclerosis, Parkinsons disease, AIDS dementia and Alzheimers disease.

Targeting the Transforming Growth Factor-β pathway inhibits human basal-like breast cancer metastasis

BackgroundTransforming Growth Factor β (TGF-β) plays an important role in tumor invasion and metastasis. We set out to investigate the possible clinical utility of TGF-β antagonists in a human metastatic basal-like breast cancer model. We examined the effects of two types of the TGF-β pathway antagonists (1D11, a mouse monoclonal pan-TGF-β neutralizing antibody and LY2109761, a chemical inhibitor of TGF-β type I and II receptor kinases) on sublines of basal cell-like MDA-MB-231 human breast carcinoma cells that preferentially metastasize to lungs (4175TR, 4173) or bones (SCP2TR, SCP25TR, 2860TR, 3847TR).ResultsBoth 1D11 and LY2109761 effectively blocked TGF-β-induced phosphorylation of receptor-associated Smads in all MDA-MB-231 subclones in vitro. Moreover, both antagonists inhibited TGF-β stimulated in vitro migration and invasiveness of MDA-MB-231 subclones, indicating that these processes are partly driven by TGF-β. In addition, both antagonists significantly reduced the metastatic burden to either lungs or bones in vivo, seemingly independently of intrinsic differences between the individual tumor cell clones. Besides inhibiting metastasis in a tumor cell autonomous manner, the TGF-β antagonists inhibited angiogenesis associated with lung metastases and osteoclast number and activity associated with lytic bone metastases. In aggregate, these studies support the notion that TGF-β plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-β signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-β pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer.ConclusionsIn aggregate, these studies support the notion that TGF-β plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-β signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-β pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer.

Recombinant Human Thyroid Stimulating Hormone: Development of a Biotechnology Product for Detection of Metastatic Lesions of Thyroid Carcinoma

We have genetically engineered a cell line, and developed a reproducible process, for the expression and purification of biologically active recombinant human thyroid stimulating hormone (rhTSH). rhTSH was expressed by co-transfecting a human α-subunit cDNA with a human β-subunit partial genomic clone into Chinese Hamster Ovary (CHO) cells. Stable transfectants which expressed high levels of rhTSH were selected, and subsequently cultured on microcarrier beads. The rhTSH-containing media, produced under serum-free conditions, was clarified and purified by a combination of ion exchange, dye and gel filtration chromatographies. Individual step recoveries were greater than 90% with the exception of a very conservative pooling of the final gel filtration step (78% recovery) that resulted in a cumulative yield of 54% for the purification process. Purity of the final bulk material was judged to be >99% by SDS polyacryl-amide gel electrophoresis (SDS-PAGE), reverse phase HPLC, and size exclusion chromatography. Initial characterization of the oligosaccharide composition indicated the presence of partially sialylated bi- and triantenary complex oligosaccharides. Purified rhTSH was active in a thyroid membrane bioactivity assay with a specific activity of 8.2 IU/mg. The in vivo activity of rhTSH in cynomolgus monkeys appeared to be equal to or greater than that reported for bovine TSH (bTSH) in human subjects. The rapid clearance phase half-life of rhTSH was approximately 35 minutes while the post-distribution phase half life was approximately 9.8 hours. Furthermore, the monkeys showed cumulative increases in minimum plasma rhTSH levels when given three daily intramuscular (IM) rhTSH injections; a phenomenon not observed when bTSH had been administered to humans. The rhTSH showed no evidence of toxic or adverse effects when administered at doses up to 7.2 IU/kg and 0.52 IU/kg in rat and monkey, respectively. These are 50X and 4X multiples of the bTSH doses of 0.143 IU/kg (10 IU/70kg) previously administered to humans.

Intermittent recombinant TSH injections prevent ovariectomy-induced bone loss

We recently described the direct effects of thyroid-stimulating hormone (TSH) on bone and suggested that the bone loss in hyperthyroidism, hitherto attributed solely to elevated thyroid hormone levels, could at least in part arise from accompanying decrements in serum TSH. Recent studies on both mice and human subjects provide compelling evidence that thyroid hormones and TSH have the opposite effects on the skeleton. Here, we show that TSH, when injected intermittently into rodents, even at intervals of 2 weeks, displays a powerful antiresorptive action in vivo. By virtue of this action, together with the possible anabolic effects shown earlier, TSH both prevents bone loss and restores the lost bone after ovariectomy. Importantly, the osteoclast inhibitory action of TSH persists ex vivo even after therapy is stopped for 4 weeks. This profound and lasting antiresorptive action of TSH is mimicked in cells that genetically overexpress the constitutively active ligand-independent TSH receptor (TSHR). In contrast, loss of function of a mutant TSHR (Pro → Leu at 556) in congenital hypothyroid mice activates osteoclast differentiation, confirming once again our premise that TSHRs have a critical role in regulating bone remodeling.

Synergistic Enhancement of CD8+ T Cell–Mediated Tumor Vaccine Efficacy by an Anti–Transforming Growth Factor-β Monoclonal Antibody

Purpose: Transforming growth factor-β (TGF-β) is an immunosuppressive cytokine, having direct suppressive activity against conventional CD4+ and CD8+T cells and natural killer cells, thereby inhibiting tumor immunosurveillance. Here, we investigated possible synergy between anti–TGF-β (1D11) and a peptide vaccine on induction of antitumor immunity, and the mechanisms accounting for synergistic efficacy. Experimental Design: The effect of combination treatment with a peptide vaccine and anti–TGF-β was examined in a subcutaneous TC1 tumor model, as well as the mechanisms of protection induced by this treatment. Results: Anti–TGF-β significantly and synergistically improved vaccine efficacy as measured by reduction in primary tumor growth, although anti–TGF-β alone had no impact. The number of tumor antigen–specific CTL with high functional avidity as measured by IFN-γ production and lytic activity was significantly increased in vaccinated mice by TGF-β neutralization. Although TGF-β is known to play a critical role in CD4+Foxp3+ Treg cells, Treg depletion/suppression by an anti-CD25 monoclonal antibody (PC61) before tumor challenge did not enhance vaccine efficacy, and adding anti–TGF-β did not affect Treg numbers in lymph nodes or tumors or their function. Also, TGF-β neutralization had no effect on interleukin-17–producing T cells, which are induced by TGF-β and interleukin-6. Absence of type II NKT cells, which induce myeloid cells to produce TGF-β, was not sufficient to eliminate all sources of suppressive TGF-β. Finally, the synergistic protection induced by anti–TGF-β vaccine augmentation was mediated by CD8+ T cells since anti-CD8 treatment completely abrogated the effect. Conclusions: These results suggest that TGF-β blockade may be useful for enhancing cancer vaccine efficacy. (Clin Cancer Res 2009;15(21):6560–9)

Thyroid-stimulating hormone restores bone volume, microarchitecture, and strength in aged ovariectomized rats.

We show the systemic administration of low levels of TSH increases bone volume and improves bone microarchitecture and strength in aged OVX rats. TSHs actions are mediated by its inhibitory effects on RANKL‐induced osteoclast formation and bone resorption coupled with stimulatory effects on osteoblast differentiation and bone formation, suggesting TSH directly affects bone remodeling in vivo.

Alternative delivery of keratinocytes using a polyurethane membrane and the implications for its use in the treatment of full-thickness burn injury

The Epicel ASAProgram service generates autologous keratinocyte grafts used for the closure of full-thickness wounds in moderately and severely burned patients. The manufacturing process used to generate Epicel service autografts (ESA) is based upon the keratinocyte co-culture technique described by Rheinwald and Green which employs murine Swiss 3T3/J2 fibroblasts as feeder cells. Recently, a technique has been described that employs a polyurethane wound dressing, HydroDerm (HD, Innovative Technologies, Ltd), as a delivery vehicle for cultured keratinocytes intended for autologous grafting. We have examined the practical feasibility of this technique and report on testing the ability of HD to support keratinocyte growth and epithelium formation in vitro, at the air-liquid interface (ALI), and in vivo, after grafting to full-thickness wounds created on the backs of athymic (Swiss Nu/Nu) mice. The results demonstrate that keratinocytes grow on the HD dressing in Gibco SFM at a rate that is approximately 15 per cent of that observed when cells are cultivated on tissue culture (TC) plastic using standard techniques, yet the cells retain their proliferative capacity and form an epithelium in vitro when cultivated at the ALI on a dermal substrate. Keratinocyte-seeded HD membranes were also transferred to full-thickness wounds in athymic mice. Animals grafted with cells seeded to HD developed human epithelium, as revealed by species-specific detection of involucrin and evolved a normal attachment to the wound substratum, as demonstrated through the expression of dermally opposed laminin and alpha 6 beta 4 integrin. The ability of keratinocytes to maintain proliferative potential after seeding onto HD and their ability to form a properly oriented epithelium in vitro and in vivo suggests that this wound dressing may be useful as a vehicle for autologous keratinocyte grafting and help to provide earlier epithelial coverage to the burned patient. However, because of the slow proliferation rate of keratinocytes on HydroDerm, timely graft delivery would be best achieved by combining cell expansion via the Rheinwald and Green culture system, followed by the seeding of cells onto HydroDerm in a reduced calcium medium for subsequent autologous grafting.

Collagen Fibrillogenesis In Vitro: A Characterization of Fibril Quality as a Function of Assembly Conditions

Pepsin-solubilized bovine corium collagen was reconstituted by rapid neutralization in dilute phosphate buffer at temperatures ranging from 10 degrees C-25 degrees C. The resultant fibrils were harvested by centrifugation and resuspended in physiological buffer to a constant protein concentration. The optical density of such suspensions, measured at 410 nm in a 1 mm path length cuvette, exhibited a strong inverse correlation with temperature of fibrillogenesis. The absorbance values of fibrillar suspensions prepared from intact collagen were greater than those observed with suspensions prepared from pepsin-solubilized collagen under similar conditions and demonstrated a reduced dependence on temperature of fibril assembly. The nature of the variation in opacity of fibrillar suspensions prepared from pepsin-solubilized material was further investigated using transmission electron microscopy, trypsin sensitivity, SDS gel electrophoresis and polarimetry. Reconstitution conditions that favored more rapid precipitation (e.g., higher incubation temperatures) tended to produce fibril suspensions of lower opacity (translucent). These translucent suspensions exhibited fibrils that were small in diameter when compared to fibril suspensions of higher opacity. Translucent preparations also contained higher levels of a trypsin sensitive, early melting component and displayed a higher proportion of peptides migrating faster than alpha 2(I) on SDS polyacrylamide gels. Collagen preparations depleted of the early melting component continued to demonstrate the correlation between increased temperature and decreased fibrillar opacity, suggesting that the two phenomena were independent. It is proposed that the unstable components are nicked or shortened collagen helices, presumably generated by pepsinization or the action of endogenous proteases of the bovine corium, which are differentially incorporated into fibrils depending on the conditions of fibril assembly.

BIOCHEMICAL AND PHARMACOLOGICAL CHARACTERIZATION OF DIFFERENT RECOMBINANT ACID α-GLUCOSIDASE PREPARATIONS EVALUATED FOR THE TREATMENT OF POMPE DISEASE

Pompe disease results in the accumulation of lysosomal glycogen in multiple tissues due to a deficiency of acid alpha-glucosidase (GAA). Enzyme replacement therapy for Pompe disease was recently approved in Europe, the U.S., Canada, and Japan using a recombinant human GAA (Myozyme, alglucosidase alfa) produced in CHO cells (CHO-GAA). During the development of alglucosidase alfa, we examined the in vitro and in vivo properties of CHO cell-derived rhGAA, an rhGAA purified from the milk of transgenic rabbits, as well as an experimental version of rhGAA containing additional mannose-6-phosphate intended to facilitate muscle targeting. Biochemical analyses identified differences in rhGAA N-termini, glycosylation types and binding properties to several carbohydrate receptors. In a mouse model of Pompe disease, glycogen was more efficiently removed from the heart than from skeletal muscle for all enzymes, and overall, the CHO cell-derived rhGAA reduced glycogen to a greater extent than that observed with the other enzymes. The results of these preclinical studies, combined with biochemical characterization data for the three molecules described within, led to the selection of the CHO-GAA for clinical development and registration as the first approved therapy for Pompe disease.