Deferiprone and Gallium-Protoporphyrin Chitogel as an antimicrobial treatment: Preclinical studies demonstrating antimicrobial activity for S. aureus infected cutaneous wounds.

Staphylococcus aureus (S. aureus) is one of the most common wound pathogens with increased resistance towards currently available antimicrobials. S. aureus biofilms lead to increase wound chronicity and delayed healing. Chitosan-dextran hydrogel (Chitogel) loaded with the hydroxypyridinone-derived iron chelator Deferiprone (Def) and the heme analogue Gallium-Protoporphyrin (GaPP) have previously been shown to have antimicrobial effects in clinical sinusitis. In this study, the efficacy of Chitogel loaded with Def, GaPP and a combination of Def and GaPP, were investigated in an S. aureus biofilm infected wound murine model over 10 days of treatment. Bacterial wound burden was monitored daily showing a significant decrease in bacterial bioburden on days 6 and 8 when treated with Def-GaPP Chitogel (log10 1.0 and 1.2 reduction vs control, respectively). The current study demonstrates that the combination of Def-GaPP delivered in a Chitogel in vivo is not only effective in reducing S. aureus biofilm infection, but also improves cutaneous healing via effects on reduced inflammation, promotion of anti-inflammatory macrophage phenotype and marked early collagen deposition in the wound bed. This delivery platform presents a promising alternative non-toxic, antibacterial, wound-promoting treatment as a novel approach for the management of S. aureus wound infections that warrants further clinical investigation.

Delivery of multipotent adult progenitor cells via a functionalized plasma polymerized surface accelerates healing of murine diabetic wounds.

Introduction: Stem cell therapies have been investigated as potential treatment modalities for chronic wounds however there has been limited success to date. Multipotent Adult Progenitor Cells (MAPCs©) have been identified as having potential as an allogenic stem cell product due to their high population doubling number and their characteristic dampening of T-cell proliferation. This helps to prevent autoimmunity and graft/cell rejection. Methods: We have developed a dressing, consisting of medical grade silicone coated with a heptylamine plasma polymer, which supports the growth and transfer of MAPCs to skin. To determine if the dressing can deliver functional stem cells into diabetic wounds, they were loaded with MAPCs and then placed over excisional wounds in both normal and diabetic mice. Results and discussion: Accelerated healing was observed in both the normal and diabetic wounds with wound gape being significantly smaller at day 3 when compared to controls. Wound analysis showed that treatment with the MAPC dressings dampened the inflammatory response with reduced numbers of neutrophils and macrophages observed. Additionally, an increase in pro-angiogenic VEGF and CD31 positive endothelial cells was observed indicating improved new blood vessel formation. The MAPC dressings had no effect on fibrosis with collagen I and III being equally affected in both control and treated wounds. Overall, the functionalized MAPC dressings improve healing responses particularly in diabetic mice with impaired healing responses and therefore, show potential for development as an advanced therapeutic approach for the treatment of chronic diabetic wounds.

Flightless I exacerbation of inflammatory responses contributes to increased colonic damage in a mouse model of dextran sulphate sodium-induced ulcerative colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by cytokine driven inflammation that disrupts the mucosa and impedes intestinal structure and functions. Flightless I (Flii) is an immuno-modulatory protein is a member of the gelsolin family of actin-remodelling proteins that regulates cellular and inflammatory processes critical in tissue repair. Here we investigated its involvement in UC and show that Flii is significantly elevated in colonic tissues of patients with inflammatory bowel disease. Using an acute murine model of colitis, we characterised the contribution of Flii to UC using mice with low (Flii+/-), normal (Flii+/+) and high Flii (FliiTg/Tg). High levels of Flii resulted in significantly elevated disease severity index scores, increased rectal bleeding and degree of colon shortening whereas, low Flii expression decreased disease severity, reduced tissue inflammation and improved clinical indicators of UC. Mice with high levels of Flii had significantly increased histological disease severity and elevated mucosal damage with significantly increased inflammatory cell infiltrate and significantly higher levels of TNF-α, IFN-γ, IL-5 and IL-13 pro-inflammatory cytokines. Additionally, Flii overexpression resulted in decreased ß-catenin levels, inhibited Wnt/ß-catenin signalling and impaired regeneration of colonic crypts. These studies suggest that high levels of Flii, as is observed in patients with UC, may adversely affect mucosal healing via mechanisms involving Th1 and Th2 mediated tissue inflammation and Wnt/ß-catenin signalling pathway.

Reducing Flightless I expression decreases severity of psoriasis in an imiquimod-induced murine model of psoriasiform dermatitis.

BACKGROUND: Psoriasis is a common chronic skin condition characterized by excessive inflammation and aberrant epidermal proliferation. Flightless I (Flii) is an actin-remodelling protein that regulates these processes, suggesting a possible role in psoriasis. OBJECTIVES: We sought to determine whether a benefit in psoriasiform dermatitis might occur after modulating Flii gene expression or reducing its levels using neutralizing antibodies. METHODS: Biopsies of psoriatic skin lesions from patients were assessed for Flii levels. Psoriasis-like lesions were induced in Flii heterozygous (Flii+/- ), wild-type (Flii+/+ ) and Flii transgenic (FliiTg/Tg ) mice using topical application of imiquimod. Additionally, psoriasis-induced wild-type mice were treated with topical application of Flii neutralizing antibodies and erythema, inflammation and histology were assessed. RESULTS: Flii was elevated in psoriatic lesions from patients with psoriasis compared with normal human skin. Reducing Flii decreased erythema, inflammatory cell infiltrate, proinflammatory cytokines and the thickness of the epidermis. Topical application of Flii neutralizing antibodies to wild-type mice treated with imiquimod resulted in significantly reduced psoriasiform dermatitis. CONCLUSIONS: Flii is a novel target involved in psoriasiform dermatitis and reducing cutaneous Flii could potentially be a new approach for treating patients with psoriasis.

Flightless I is a key regulator of the fibroproliferative process in hypertrophic scarring and a target for a novel antiscarring therapy.

BACKGROUND: Hypertrophic scarring carries a large burden of disease, including disfigurement, pain and disability. There is currently no effective medical treatment to reduce or prevent hypertrophic scarring. Flightless I (Flii), a member of the gelsolin family of actin remodelling proteins, is an important negative regulator of wound repair. OBJECTIVES: The objective of this study was to investigate the role of Flii as a potential regulator of hypertrophic scarring. METHODS: Using human skin samples and an animal model of bleomycin-induced hypertrophic scarring in mice that overexpress or have reduced expression of Flii, we investigated its effect on dermal fibrosis and hypertrophic scarring. RESULTS: Flii expression was increased in human burns and hypertrophic scars. A similar increase in Flii was observed in hypertrophic scars formed in mice post-treatment with bleomycin. However, Flii-deficient (Flii(+/-) ) mice had reduced scarring in response to bleomycin evidenced by decreased dermal thickness, smaller cross-sectional scar areas, fewer myofibroblasts and a decreased collagen I/III ratio. In contrast, bleomycin-treated Flii-overexpressing mice (Flii(Tg/Tg) ) showed increased scar dermal thickness, larger cross-sectional scar areas, more myofibroblasts and an increased collagen I/III ratio. Injecting developing scars with a Flii neutralizing antibody led to a significant reduction in the size of the scars and a reduction in the collagen I/III ratio. CONCLUSIONS: This study identifies Flii as a profibrotic agent that contributes to excessive scar formation. Reducing its activity using neutralizing antibodies is a promising approach for reducing hypertrophic scarring.

Venous ulceration contaminated by multi-resistant organisms: larval therapy and debridement.

A 72-year-old female with venous insufficiency presented to a hospital-based multidisciplinary wound clinic after 20 years of recurrent episodes of venous leg ulcers. Examination showed bilateral leg ulcers with no evidence of arterial insufficiency, but complicated by considerable devitalised tissue, abnormally high bacterial load and the presence of multi-resistant organisms. The ulcers were initially treated with larvae to aid debridement and reduce the bacterial load, prior to skin grafting. Although ulcer free for a period of 4 months, further debridement was required when the skin condition deteriorated. Surgical intervention was chosen as the preferred method by the surgeons for a second acute care admission using hydrosugery, along with supplementary skin grafts and compression. Ongoing management, consisting of regular debridement, skin care and compression therapy, continues.

Attenuation of Flightless I, an actin-remodelling protein, improves burn injury repair via modulation of transforming growth factor (TGF)-beta1 and TGF-beta3.

BACKGROUND: The pathophysiological mechanisms involved in burn injury repair are still not fully understood but include processes involving cellular proliferation, migration and adhesion. The actin cytoskeleton is intricately involved in these key wound repair processes. Flightless I (Flii), an actin-remodelling protein and transcriptional regulator, is an important regulator of wound healing. OBJECTIVES: To investigate the function of Flii gene expression in burn injury repair. METHODS: Partial-thickness scald wounds were created on Flii heterozygous (Flii(+/-)), wild-type (WT) and Flii transgenic (Flii(Tg/+)) mice. Burns were assessed using histology and immunohistochemistry, real-time quantitative polymerase chain reaction and biochemical analysis. RESULTS: Flii expression, while upregulated in burn injuries, was significantly lower in the wounds of Flii(+/-) vs. WT vs. Flii(Tg/+) mice and healing was improved in Flii(+/-) mice with their burns healing faster than WT and Flii(Tg/+). Pro-scarring transforming growth factor (TGF)-beta1 protein and gene expression were reduced in Flii(+/-) burns while antiscarring TGF-beta3 was significantly elevated. Anti-alpha-smooth muscle actin (alpha-SMA) was decreased in Flii(+/-) burns suggesting a decrease in contractile myofibroblasts in the developing scars. Although Flii is primarily a nuclear and cytoplasmic protein it is also released by wounded cells. Intradermal injection of Flii-neutralizing antibodies (FliAbs) to WT burn wounds significantly improved their healing, indicating a potential novel approach for treating burns. Decreased TGF-beta1 and elevated TGF-beta3 expression were observed in FliAb-treated burns, which may contribute to their observed improvement in healing. CONCLUSIONS: Strategies aimed at reducing Flii expression, for example using neutralizing antibodies, may lead to improved burn outcomes.

Flightless I: an actin-remodelling protein and an important negative regulator of wound repair.

Flightless I (FliI) is a member of the gelsolin family of actin-remodelling proteins, and has been identified as having two functional protein family domains: a leucine rich repeat (LRR) domain and a gelsolin-like domain. This unique structure allows FliI to act as an actin-remodelling protein as well as a nuclear receptor co-activator with ability to interact with various other proteins important in cellular signaling. The actin cytoskeleton is an integral component of all cells and the effect of FliI protein on actin remodelling is a vital part of cellular motility, contraction and adhesion. The product of the FliI gene is expected to provide a vital link between the molecules of yet unidentified signal transduction pathways and the actin cytoskeleton. Exact signaling pathways and mechanisms underpinning FliI effects in wound healing are yet to be fully identified however strong research evidence clearly identifies this molecule as a possible new therapeutic target whose manipulation may greatly improve wound healing and could lead to potential innovative medical applications.

Flightless I deficiency enhances wound repair by increasing cell migration and proliferation.

Wound healing disorders are a therapeutic problem of increasing clinical importance involving substantial morbidity, mortality, and rising health costs. Our studies investigating flightless I (FliI), a highly conserved actin-remodelling protein, now reveal that FliI is an important regulator of wound repair whose manipulation may lead to enhanced wound outcomes. We demonstrate that FliI-deficient + /- mice are characterized by improved wound healing with increased epithelial migration and enhanced wound contraction. In contrast, FliI-overexpressing mice have significantly impaired wound healing with larger less contracted wounds and reduced cellular proliferation. We show that FliI is secreted in response to wounding and that topical application of antibodies raised against the leucine-rich repeat domain of the FliI protein (FliL) significantly improves wound repair. These studies reveal that FliI affects wound repair via mechanisms involving cell migration and proliferation and that FliI might represent an effective novel therapeutic factor to improve conditions in which wound healing is impaired.

Collagen loss and impaired wound healing is associated with c-Myb deficiency.

Collagen type I serves as an abundant structural and signalling component of skin. It is also an established target gene of the transcription factor, c-Myb. When c-myb-/- embryos were examined it was observed that their skin was markedly thinner than normal. Importantly, immunohistochemical investigation showed complete absence of collagen type I. Although these homozygous knock-out embryos fail to develop beyond day 15, fibroblasts established from these embryos (mouse embryonic fibroblasts [MEFs]) show defective proliferative responses. Furthermore, in vitro scratch wound assays demonstrated that these c-myb-/- MEFs also exhibit slower closure than their wild-type counterparts. Embryonic lethality has meant that examination of the role of c-Myb in adult mouse skin has not been reported to date. However, in view of the abundance of collagen type I in normal skin, its role in skin integrity and the in vitro data showing proliferative and migration defects in c-myb-/- MEFs, we investigated the consequences of heterozygous c-myb loss in adult mice on the complex process of skin repair in response to injury. Our studies clearly demonstrate that heterozygous c-myb deficiency has a functional effect on wound repair, collagen type I levels and, in response to wounding, transforming growth factor-beta1 (an important collagen stimulating factor) induction expression is aberrantly high. Manipulation of c-Myb may therefore provide new therapeutic opportunities for improving wound repair while uncontrolled expression may underpin some fibrotic disorders.

Effect of healing on the expression of transforming growth factor beta(s) and their receptors in chronic venous leg ulcers.

The transforming growth factor betas are of major importance in the wound repair process; however, no studies to date have investigated the role of the transforming growth factor beta receptors in chronic venous leg ulcers or what effect healing has on these proteins. To determine whether the transforming growth factor beta peptides and their receptors are expressed in chronic venous wounds, we used immunofluorescent analysis and quantitative competitive reverse transcription polymerase chain reaction to identify the protein and mRNA expression, respectively. Biopsy samples from wounds and normal skin were collected from 12 patients with chronic venous leg ulcers and three patients undergoing reconstructive surgery, respectively. Additionally four of the chronic venous leg ulcer patients were re-biopsied between 2 and 8 wk after the first biopsy when the wounds had entered the healing phase. The tissue excised from the ulcers included the surrounding intact skin, the ulcer edge, and the ulcer base. Immunofluorescent staining for transforming growth factors beta1, beta2, and beta3 was observed within the epidermis of the skin surrounding the chronic venous ulcers and in fibroblasts and inflammatory cells of the dermis, although this staining was not as strong as that seen in normal unwounded skin. Very little staining could be seen within the ulcers for any of the ligands, however. In contrast the transforming growth factor beta type I receptor was observed throughout the ulcers and the normal unwounded skin biopsies, particularly in the basal epidermal cells. No immunofluorescence for the type II transforming growth factor beta receptor was observed in any of the ulcer biopsies investigated, although it was observed throughout the epidermis and in fibroblasts and inflammatory cells in the surrounding skin. Quantitative, competitive reverse transcription polymerase chain reaction was used to analyze mRNA expression for transforming growth factor beta1 and the type II receptor in the nonhealing ulcers and normal unwounded skin biopsies. These studies revealed that transforming growth factor beta1 and transforming growth factor beta receptor II mRNA was expressed in all the chronic nonhealing ulcers albeit at very low levels for the type II receptor. In marked contrast to the staining observed in nonhealing chronic ulcers, positive immunostaining was observed for the transforming growth factor betas and both the type I and type II receptors in healing ulcers. These results suggest that the absence of a viable receptor complex for the transforming growth factor betas in nonhealing chronic venous ulcers may contribute to wound chronicity.

Hepatocyte growth factor and macrophage-stimulating protein are upregulated during excisional wound repair in rats.

Hepatocyte growth factor (HGF) and macrophage-stimulating protein (MSP) are structurally related molecules that stimulate epithelial cell proliferation and migration. MSP also acts directly as a chemoattractant for resident macrophages. These activities are integral to the wound repair processes of inflammation, epithelialization and tissue remodelling. To begin to examine the involvement of HGF and MSP in healing of cutaneous wounds we have mapped the temporal expression of these two molecules and their receptors, MET and RON respectively, in adult rat excisional wounds. Four 2x2-cm full-thickness excisional wounds were created on the dorsum of 18 rats, and biopsies were taken through the wounds at 3, 5, 7, 14, 21, and 28 days postwounding. These biopsies were analyzed using immunofluorescent staining and in situ hybridization (ISH). The number of cells staining positively for HGF and MET significantly increased in response to wounding. HGF staining and mRNA peaked at 7 days postwounding whereas MET was upregulated earlier, peaking after 3 days. Both HGF and MET protein were observed in fibroblasts of the dermis and in the newly forming granulation tissue. ISH studies also revealed that fibroblasts at the wound edges and within the newly forming granulation tissue also expressed HGF and c-met mRNA. Immunofluorescent staining revealed both MSP and RON within the wound, with maximum staining occurring between 7 and 21 days for both the ligand and receptor. In addition, MSP co-localized with a small subset of ED1-positive cells (monocytes). In contrast, ED2-positive cells (macrophages) did not co-localize with MSP. Thus, increased expression of HGF, MSP and their receptors MET and RON respectively was observed in response to wounding. Furthermore, MSP co-localization with a subset of monocytes may confirm a role for MSP in the activation of mature macrophages, which may be important in tissue remodelling.

Expression of TGF-beta and its receptors in murine fetal and adult dermal wounds.

The transforming growth factor-betas (TGF-betas) are of major importance in wound healing and have been implicated in the scar-less wound repair observed in fetuses. Few studies have characterised the role of TGF-beta in fetal wound repair and to date no studies have characterised the expression of its receptors within non-scarring fetal wounds. We have localised the TGF-beta isoforms beta1, beta2 and beta3 and its two receptors, TGF-betaRI and TGF-betaRII in both adult and fetal dermal murine wounds. We observed low level immunofluorescence of TGF-beta1 and TGF-beta2 in fetal wounds and although TGF-beta3 staining was observed in the epidermis of fetal skin, there was no upregulation in response to injury. By contrast, all three isoforms were strongly expressed in adult wounds. Similar to its ligands, TGF-beta receptor expression was increased post-wounding in the adult wounds. However, in contrast, no mRNA or protein for either of the TGF-beta receptors was observed in response to wounding in the fetal dermis although there was both mRNA and protein expression of both the receptors localised within the fetal alimentary tract, one of the few fetal organs which does scar post-injury. The differences that we observed in the expression of TGF-beta and its receptors in adult and fetal wounds could be important in the absence of scar formation that is observed in the fetus.

Mitogenic whey extract stimulates wound repair activity in vitro and promotes healing of rat incisional wounds.

The ability of single growth factors to promote healing of normal and compromised wounds has been well described, but wound healing is a process requiring the coordinated action of multiple growth factors. Only the synergistic effect on wound healing of combinations containing at most two individual growth factors has been reported. We sought to assess the ability of a novel milk-derived growth factor-enriched preparation ¿mitogenic bovine whey extract (MBWE), which contains six known growth factors, to promote repair processes in organotypic in vitro models and incisional wounds in vivo. MBWE stimulated the contraction of fibroblast-populated collagen lattices in a dose-dependent fashion and promoted the closure of excisional wounds in embryonic day 17 fetal rat skin. Application of MBWE increased incisional wound strength in normal animals on days 3, 5, 7, and 10 and reversed the decrease in wound strength observed following steroid treatment. Wound histology showed increased fibroblast numbers in wounds from normal and steroid-compromised animals. These data suggest the mixture of factors present in bovine milk exerts a direct action on the cells of cutaneous wound repair to enhance both normal and compromised healing.

Endogenous inflammatory response to dermal wound healing in the fetal and adult mouse.

The recruitment of inflammatory cells to a wound may play an important role in the resulting cellular processes and ultimately the quality of the healing response in the fetus (scar-free healing) or the adult (scar-forming healing). Using a range of antibodies to monocytes and macrophages and also to different activation markers of activated macrophages, we have compared the inflammatory profile of scar-free healing E16 mouse fetal wounds to those of scarring adult wounds. In the fetal wound, small numbers of monocyte derived cells (MOMA-2 and F4/80 positive) are recruited to the wound by 3 hr post-wounding. No Mac-1 positive cells indicative of activated macrophages were observed until 18 hr post-wounding. Eventually all types of macrophages studied were recruited to both adult and fetal wound sites but the numbers and persistence of these cells are lower in the fetus than in the adult. B cells were detected in healing adult wounds but not in the fetal wounds. This absence of H-21-A positive (B) cells in murine fetal wounds could be associated with the low levels of activated Mac-1 positive macrophages at the murine fetal wound site. Activated macrophages in addition to releasing growth factors may also release signals to recruit B cells. Thus, the E16 mouse fetus can mount an inflammatory response to wounding. This response differs from that of the adult in the numbers of inflammatory cells recruited to the wound and the subpopulations of activated cells found at the wound site. This study indicates that there are complex differences between the inflammatory responses elicited in adult and fetal murine dermal wounds. These differences may determine the profile of growth factors and cytokines released at fetal and adult wound sites. Manipulation of either the numbers or the activation states of inflammatory cells at the adult wound site may be an approach to the control of scarring during adult wound healing.

The proliferative responses of porcine thyroid follicular cells to epidermal growth factor and thyrotrophin reflect the autocrine production of transforming growth factor-beta 1.

The present study has investigated an involvement of autocrine transforming growth factor-beta 1 (TGF-beta 1) in regulating the proliferative response of porcine thyroid follicular cells (TFCs) to epidermal growth factor (EGF) and TSH. Primary monolayer TFC cultures exposed to EGF over the range 0-0.4 nmol/l showed a dose-dependent increase in [methyl-3H]thymidine incorporation, whereas higher EGF doses were associated with a reduction in the level of [methyl-3H]thymidine incorporation. TGF-beta immunoneutralisation had little effect on the stimulatory action of low EGF doses, but led to an increase in [methyl-3H]thymidine incorporation at higher EGF levels. In TFC cultures exposed to TSH, the level of [methyl-3H]thymidine incorporation attained at a dose of 1 U TSH/1 was enhanced in the presence of TGF-beta 1 antiserum, although the similar stimulatory effect of 8-bromo cAMP was unaffected. Treatment of TFCs with phorbol 12-myristate 13-acetate (8 nmol/l) to activate protein kinase C (PKC) led to an enhanced incorporation of [methyl-3H]thymidine which was increased further after neutralisation of endogenous TGF-beta 1. While confirming, therefore, a role for autocrine TGF-beta 1 in maintaining control of TFC DNA synthesis in vitro, these findings provide evidence that an increase in the availability of autocrine TGF-beta 1 effected by EGF and TSH may play an instrumental role in limiting the cellular hyperplasia induced by these factors within the thyroid follicular microenvironment. Moreover, the present data also suggest that the availability of active autocrine TGF-beta 1 to TFCs under such conditions may be dependent upon a PKC-mediated mechanism.

Iodide-dependent regulation of thyroid follicular cell proliferation: a mediating role of autocrine insulin-like growth factor-I.

An inhibitory action of intracellular iodide on the autocrine production of insulin-like growth factor-I (IGF-I) by thyroid follicular cells (TFCs) in vitro has been investigated as a possible mechanism underlying the iodide-dependent control of TFC proliferation. IGF-I release from primary monolayer cultures of porcine TFCs increased 5-fold between 24 and 168 h of incubation. Confirmation of a mediating role of IGF-I in TFC proliferation was obtained by exposing TFCs to an immunoadsorbing IGF-I antiserum, which led to a significant (P < 0.05) decline in [methyl-3H]thymidine incorporation, relative to TFCs exposed to preimmune serum. Exposure of TFCs to sodium iodide (NaI; 0.1-100 mumol/l) led to an attenuation of the IGF-I content of the cell-conditioned medium. This was accompanied by a reduction in [methyl-3H]thymidine incorporation that was affected by IGF-I immunoneutralization. The inhibitory effect of NaI on IGF-I production and [methyl-3H]thymidine incorporation were reversed by the thionamide compound methimazole (MMI; 1 mmol/l), exposure to which also led to significant (P < 0.001) increases above control values. However, a residual suppressive effect of NaI on [methyl-3H]thymidine incorporation suggested that certain of the TFC growth-attenuating effects of iodide may not be dependent upon organification. While providing evidence, therefore, for a direct relationship between iodide exposure, suppression of autocrine IGF-I production and a regulation of TFC proliferation, the present studies also suggest that suppression of TFC proliferation by iodide may be partially mediated by MMI-insensitive events.

Porcine thyroid follicular cells in monolayer culture activate the iodide-responsive precursor form of transforming growth factor-beta 1.

The release of latent transforming growth factor-beta 1 (TGF-beta 1), and conversion to the biologically active peptide, has been investigated in porcine thyroid follicular cells maintained in primary monolayer culture. Analysis by radioreceptor assay of medium conditioned for 72 h by subconfluent thyroid monolayers showed that a high proportion of the expressed TGF-beta 1 peptide was in the active form. Medium conditioned by iodide (10 mumol/l)-treated follicular cells contained higher levels of both active and total TGF-beta 1 than were present in medium conditioned by untreated cells. Exposure of cells to iodide also led to a marked decrease in [methyl-3H]thymidine incorporation that was relieved by immunoadsorption with a neutralizing antiserum against the active form of TGF-beta 1. Inclusion of a low dose (80 units/l) of porcine plasmin led to a small increase in incorporation of [methyl-3H]thymidine, while higher doses of plasmin (1250-5000 units/l) or plasminogen (100 mg/l) significantly reduced [methyl-3H]thymidine incorporation. This inhibition was effectively reversed by immunoadsorption of TGF-beta 1 from the medium during the test incubations. The study therefore provides direct evidence for a stimulatory role of thyroidal iodide in enhancing the release of latent TGF-beta 1 peptide, and suggests that in normal thyroid follicular cells, as in other TGF-beta 1 producing epithelia, post-secretory processing to the biologically active molecule occurs through an endogenous cellular mechanism. It appears likely that plasmin, generated locally within the thyroid follicular microenvironment, may play a fundamental role in effecting this conversion.

Transforming growth factor-beta 1 synthesis in human thyroid follicular cells: differential effects of iodide and plasminogen on the production of latent and active peptide forms.

The present study has investigated the relative levels and interconversion of latent and active forms of transforming growth factor-beta 1 (TGF-beta 1) in human thyroid follicular cell cultures derived from sporadic non-toxic goitres. Northern blotting of RNA extracted from 72-h cultures revealed a 2.5 kb mRNA transcript hybridizing with a cDNA probe for latent TGF-beta 1, the intensity of which was doubled in cells exposed to NaI (10 mumol/l). Radioreceptor assay of follicular cell-conditioned medium for TGF-beta 1 content confirmed a similar enhancing effect of iodide. The endogenous active component of TGF-beta 1 present in conditioned medium represented only a minor fraction of the total TGF-beta 1 content, this fraction was not enhanced by exposure of follicular cells to iodide. The low level of endogenous active TGF-beta 1 in medium conditioned by either control or iodide-treated cells was confirmed by immunoadsorption with a precipitating antiserum against active TGF-beta 1, when such cells failed to show a reversal of the iodide-induced decrease in [methyl-3H]thymidine incorporation into trichloroacetic acid-precipitable material. In contrast to the inhibitory effect of iodide on de novo DNA synthesis, which appeared not to reflect an increase in active TGF-beta 1, the inhibitory effects of plasminogen (100 mg/l) or plasmin (2000 U/l) on [methyl-3H]thymidine incorporation into thyroid cells were reversible by TGF-beta 1 immunoadsorption. This provides evidence that the plasmin-mediated inhibition of DNA synthesis in thyroid follicular cells may be attributed to the growth-regulating action of an increased level of activated TGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta 1 production in porcine thyroid follicular cells: regulation by intrathyroidal organic iodine.

The present studies have demonstrated the production of transforming growth factor-beta 1 (TGF-beta 1) by porcine thyroid follicular cells (TFCs) maintained in vitro as subconfluent monolayers, and have confirmed a stimulatory effect of iodide on thyroidal TGF-beta 1 mRNA and peptide release. RNA extracted from TFCs maintained in the absence of iodide contained a 2.5 kb transcript which hybridized specifically with a cDNA probe for human TGF-beta 1, and which showed an approximate doubling in intensity in cells exposed to 10 mumol NaI/1. In the presence of the anti-thyroid thionamide drug methimazole (MMI; 1 mmol/l), the action of iodide on TGF-beta 1 mRNA was attenuated, although MMI alone had no effect on the control level of TGF-beta 1 mRNA. The TGF-beta 1 peptide content of TFC-conditioned media (TFC-CM) was assessed using the fetal mink lung cell line Mv1Lu, in which activated TGF-beta 1 specifically suppresses trichloroacetic acid-precipitable [methyl-3H]thymidine incorporation. Newly conditioned TFC-CM stimulated [methyl-3H]thymidine incorporation into Mv1Lu cells, but after heat treatment to inactivate growth stimulators and activate the latent TGF-beta 1 component this medium inhibited [methyl-3H]thymidine incorporation. This inhibitory effect was prevented by immunoadsorption of TFC-CM with a TGF-beta 1-neutralizing antiserum, confirming the specificity of the inhibitory response. The inhibitory activity of TFC-CM was increased when the TFCs were preincubated with 10 mumol NaI/1, and lost when TFCs were exposed to MMI. In conclusion, TFCs produce TGF-beta 1 mRNA and TGF-beta 1 peptide, which are both increased by iodide treatment in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)