A Novel Approach to Negative Pressure Wound Therapy: Use of High Suction Capillary Device to Improve Wound Healing.

INTRODUCTION: Negative Pressure Wound Therapy (NPWT) is a procedure used for nonhealing wounds. In NPWT, a special sealed dressing of large cell foam (>400 µm) or gauze is connected to a pump. Most commonly, negative pressures between -10 and -125 millimeters of mercury (mm Hg) are used. The mechanism of healing is unknown but maybe attributable to removal of the exudate and bacteria, and the stimulation of tissue repair through microdeformation. Reticulated foams with micron-size open cells, Capillary Suction Devices (CSD; 100 to 5 µm) exert capillary suction between 10 and 70 mm of Hg with a multilayered foam dressing. MATERIALS AND METHODS: Yorkshire pigs received 5 surgical excision wounds, 3 cm2, on each side of the back. The wounds were covered with a NPWT dressing (110 mm Hg negative pressure by a pump), CSD with capillary suctions of 30 mm Hg (CSD-30) and 70 mm Hg (CSD-70), and a conventional gauze dressing. The wounds were measured on day 2, and then every 4-5 days thereafter; the total fluid collected by the various dressing over time. RESULTS: By post-wound day 20, the wounds treated with CSD-70 and NPWT were 100% closed while the wounds treated with CSD-30 and gauze were 65% and 45%, respectively. This indicated comparable wound closure efficacies for CSD-70 and NPWT. The average total fluid uptake measured in grams dry weight were similar for CSD-70 and NPWT, 36 and 38 g, respectively, while the values were 24 g for CSD-30 and 12 g for gauze. However, the maximum fluid uptake observed at day 2 indicated that CSD-70 and CSD 30, 24 and 14 g, respectively, were superior to NPWT and gauze 12 and 7 g, respectively. CONCLUSION: This data indicate comparable wound closure efficacies for CSD-70 and NPWT. It is felt that CSD is an effective, safe, and lower cost alternative to vacuum-assisted NPWT.

Significance of the melanocortin 1 receptor in the DNA damage response of human melanocytes to ultraviolet radiation.

Activation of the melanocortin 1 receptor (MC1R) by α-melanocortin (α-MSH) stimulates eumelanin synthesis and enhances repair of ultraviolet radiation (UV)-induced DNA damage. We report on the DNA damage response (DDR) of human melanocytes to UV and its enhancement by α-MSH. α-MSH up-regulated the levels of XPC, the enzyme that recognizes DNA damage sites, enhanced the UV-induced phosphorylation of the DNA damage sensors ataxia telangiectasia and Rad3-related (ATR) and ataxia telangiectasia mutated (ATM) and their respect-ive substrates checkpoint kinases 1 and 2, and increased phosphorylated H2AX (γH2AX) formation. These effects required functional MC1R and were absent in melanocytes expressing loss of function (LOF) MC1R. The levels of wild-type p53-induced phosphatase 1 (Wip1), which dephosphorylates γH2AX, correlated inversely with γH2AX. We propose that α-MSH increases UV-induced γH2AX to facilitate formation of DNA repair complexes and repair of DNA photoproducts, and LOF of MC1R compromises the DDR and genomic stability of melanocytes.

Oral tungstate (Na2WO4) exposure reduces adaptive immune responses in mice after challenge.

Tungstate (WO²â»4) has been identified as a ground water contaminant at military firing ranges and can be absorbed by ingestion. In this study, C57BL6 mice were exposed to sodium tungstate (Na2WO4·2H2O) (0, 2, 62.5, 125, and 200 mg/kg/day) in their drinking water for an initial 28-day screen and in a one-generation (one-gen) model. Twenty-four hours prior to euthanasia, mice were intraperitoneally injected with Staphylococcal enterotoxin B (SEB) (20 µg/mouse) or saline as controls. After euthanasia, splenocytes and blood were collected and stained with lymphocyte and/or myeloid immunophenotyping panels and analyzed by flow cytometry. In the 28-day and one-gen exposure, statistically significant reductions were observed in the quantities of activated cytotoxic T-cells (TCTL; CD3(+)CD8(+)CD71(+)) and helper T-cells (TH; CD3(+)CD4(+)CD71(+)) from spleens of SEB-treated mice. In the 28-day exposures, CD71(+) TCTL cells were 12.87 ± 2.05% (SE) in the 0 tungstate (control) group compared to 4.44 ± 1.42% in the 200 mg/kg/day (p < 0.001) group. TH cells were 4.85 ± 1.23% in controls and 2.76 ± 0.51% in the 200 mg/kg/day (p < 0.003) group. In the one-gen exposures, TCTL cells were 7.98 ± 0.49% and 6.33 ± 0.49% for P and F1 mice after 0 mg/kg/day tungstate vs 1.58 ± 0.23% and 2.52 ± 0.25% after 200 mg/kg/day of tungstate (p < 0.001). Similarly, TH cells were reduced to 6.21 ± 0.39% and 7.20 ± 0.76%, respectively, for the 0 mg/kg/day P and F1 mice, and 2.28 ± 0.41% and 2.85 ± 0.53%, respectively, for the 200 mg/kg/day tungstate P and F1 groups (p < 0.001). In delayed-type hypersensitivity Type IV experiments, tungstate exposure prior to primary and secondary antigen challenge significantly reduced footpad swelling at 20 and 200 mg/kg/day. These data indicate that exposure to tungstate can result in immune suppression that may, in turn, reduce host defense against pathogens.

The abundance of Rad51 protein in mouse embryonic stem cells is regulated at multiple levels.

DNA double-strand breaks (DSBs) in embryonic stem (ES) cells are repaired primarily by homologous recombination (HR). The mechanism by which HR is regulated in these cells, however, remains enigmatic. To gain insight into such regulatory mechanisms, we have asked how protein levels of Rad51, a key component of HR, are controlled in mouse ES cells and mouse embryo fibroblasts (MEFs). The Rad51 protein level is about 15-fold higher in ES cells than in MEFs. The level of Rad51 mRNA, however, is only ~2-fold higher, indicating that the differences in mRNA levels due to rates of transcription or mRNA stability are not sufficient to account for the large difference in the abundance of Rad51 protein. Comparison of Rad51 half-lives between ES cells and MEFs also did not explain the elevated level of Rad51 protein in the ES cells. A comparative assessment of the Rad51 translation level demonstrated that it is translated with much greater efficacy in ES cells than in MEFs. To determine whether this high level of translation in ES cells is a general phenomenon in these cells or whether it is a characteristic of specific proteins, such as those involved with recombination and cell cycle progression, we compared mechanisms that regulate the level of Pcna in ES cells with those that regulate Rad51. The half-life of Pcna and its rate of synthesis were considerably different from those of Rad51 in ES cells, demonstrating that regulation of Rad51 abundance cannot be generalized to other ES cell proteins and not to proteins involved in DNA replication and cell cycle control. Finally, we show that only a small proportion of the abundant Rad51 protein population is activated under basal conditions in ES cells and recruited to DNA DSBs and/or stalled replication forks.

Defining MC1R regulation in human melanocytes by its agonist α-melanocortin and antagonists agouti signaling protein and ß-defensin 3.

The melanocortin 1 receptor (MC1R), a G(s) protein-coupled receptor, has an important role in human pigmentation. We investigated the regulation of expression and activity of the MC1R in primary human melanocyte cultures. Human ß-defensin 3 (HBD3) acted as an antagonist for MC1R, inhibiting the α-melanocortin (α-melanocyte-stimulating hormone (α-MSH))-induced increase in the activities of adenylate cyclase and tyrosinase, the rate-limiting enzyme for melanogenesis. α-Melanocortin and forskolin, which activate adenylate cyclase, and 12-O-tetradecanoylphorbol-13-acetate, which activates protein kinase C, increased, whereas exposure to UV radiation reduced, MC1R gene and membrane protein expression. Brief treatment with α-MSH resulted in MC1R desensitization, whereas continuous treatment up to 3 hours caused a steady rise in cAMP, suggesting receptor recycling. Pretreatment with agouti signaling protein or HBD3 prohibited responsiveness to α-MSH, but not forskolin, suggesting receptor desensitization by these antagonists. Melanocytes from different donors expressed different levels of the G protein-coupled receptor kinases (GRKs) 2, 3, 5, and 6, as well as ß-arrestin 1. Therefore, in addition to the MC1R genotype, regulation of MC1R expression and activity is expected to affect human pigmentation and the responses to UV.

The burn wound inflammatory response is influenced by midazolam.

Burn patients requiring hospitalization are often treated for anxiety with benzodiazepines (BDZs). Benzodiazepines are reported to influence immune system function. Immune system alterations are a major cause of burn-induced mortality. We wanted to determine whether the BDZ, midazolam given daily at an anxiolytic dose, had any influence on the burn injury-induced inflammatory response in the blood and wound. Mice received a 15% total body surface area flame burn and received either midazolam 1 mg/kg i.p. or saline 0.1 ml daily. Blood and skin wounds were harvested 24 h after injection on post-burn day 2, 3, 7, or 8. Mice treated with midazolam had significantly lower serum IL-1ß (p=0.002), TNF-α (p=0.002), IL-6 (p=0.016), IL-10 (p=0.009), and TGF-ß (p=0.004) than saline-treated mice, with little impact on serum chemokine levels. In the wound, TNF-α and IL-10 were the only cytokines significantly influenced by the drug, being lower (p=0.018) and higher (p=0.006), respectively. The chemokines in the wound influenced significantly by midazolam were MIP-1α, MIP-1ß, and MIP-2 while MCP-1 and KC were not. There were more inflammatory cells at the burn wound margin in midazolam-treated mice on post-burn day 3. Although serum nitrate/nitrite was significantly increased by midazolam (p=0.03), both eNOS and iNOS mRNA expression in the wound were similar to the saline group. We found that midazolam given daily after burn injury significantly influenced the inflammatory response. The clinical implications of these findings on wound healing and shock following burn injury, especially larger burns, deserve further investigation.

The human DEK oncogene regulates DNA damage response signaling and repair.

The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms. Here we report that DEK is important for DNA double-strand break repair. DEK depletion in human cancer cell lines and xenografts was sufficient to induce a DNA damage response as assessed by detection of γH2AX and FANCD2. Phosphorylation of H2AX was accompanied by contrasting activation and suppression, respectively, of the ATM and DNA-PK pathways. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end joining (NHEJ) when compared to their wild-type counterparts. Taken together, the data demonstrate new molecular links between DEK and DNA damage response signaling pathways, and suggest that DEK contributes to DNA repair.

Epidermal keratinocytes from light vs. dark skin exhibit differential degradation of melanosomes.

Modification of skin complexion coloration has traditionally been accomplished by interruption or attenuation of melanogenesis and/or melanosome transfer. Post-transfer modification of pigmented melanosomes provides an attractive and distinct avenue of modulating skin pigmentation. The processing of melanosomes during keratinocyte (KC) terminal differentiation and the degradative variability observed between light and dark skin (LS and DS) remains enigmatic. To evaluate this, we developed a model system to investigate the loss of fluorescently labeled and isolated melanosomes by cultured human KCs. The extent of melanosome loss has been qualitatively assessed using transmission electron microscopy and indirect immunofluorescence with confocal microscopy, and quantitatively assessed using flow cytometry analysis. Results show that melanosomes are incorporated into the cytoplasm of both light and dark keratinocytes (LKCs and DKCs) and trafficked to a perinuclear region. Within 48 hours, confocal microscopy images suggest that LKCs display accelerated melanosome loss. This time-dependent decrease in carboxyfluorescein diacetate (CFDA) fluorescence was then quantitatively analyzed using flow cytometry. Consistent with the results of the confocal analysis, over a 48-hour time frame, LKCs appear to lose melanosomes more efficiently than DKCs. These experiments show that melanosomes are more rapidly lost in KCs derived from LS as opposed to DS.

Mismatch and base excision repair proficiency in murine embryonic stem cells.

Accumulation of mutations in embryonic stem (ES) cells would be detrimental to an embryo derived from these cells, and would adversely affect multiple organ systems and tissue types. ES cells have evolved multiple mechanisms to preserve genomic integrity that extend beyond those found in differentiated cell types. The present study queried whether mismatch repair (MMR) and base-excision repair (BER) may play a role in the maintenance of murine ES cell genomes. The MMR proteins Msh2 and Msh6 are highly elevated in mouse ES cells compared with mouse embryo fibroblasts (MEFs), as are Pms2 and Mlh1, albeit to a lesser extent. Cells transfected with an MMR reporter plasmid showed that MMR repair capacity is low in MEFs, but highly active in wildtype ES cells. As expected, an ES cell line defective in MMR was several-fold less effective in repair level than wildtype ES cells. Like proteins that participate in MMR, the level of proteins involved in BER was elevated in ES cells compared with MEFs. When BER activity was examined biochemically using a uracil-containing oligonucleotide template, repair activity was higher in ES cells compared with MEFs. The data are consistent with the suggestion that ES cells have multiple mechanisms, including highly active MMR and BER that preserve genetic integrity and minimize the accumulation of mutations.

Lumican is required for neutrophil extravasation following corneal injury and wound healing.

An important aspect of wound healing is the recruitment of neutrophils to the site of infection or tissue injury. Lumican, an extracellular matrix component belonging to the small leucine rich proteoglycan (SLRP) family, is one of the major keratan sulfate proteoglycans (KSPGs) within the corneal stroma. Increasing evidence indicates that lumican can serve as a regulatory molecule for several cellular processes, including cell proliferation and migration. In the present study, we addressed the role of lumican in the process of extravasation of polymorphonuclear leukocytes (PMNs) during the early inflammatory phase present in the healing of the corneal epithelium following debridement. We used Lum(-/-) mice and a novel transgenic mouse, Lum(-/-),Kera-Lum, which expresses lumican only in the corneal stroma, to assess the role of lumican in PMN extravasation into injured corneas. Our results showed that PMNs did not readily invade injured corneas of Lum(-/-) mice and this defect was rescued by the expression of lumican in the corneas of Lum(-/-),Kera-Lum mice. The presence of lumican in situ facilitates PMN infiltration into the peritoneal cavity in casein-induced inflammation. Our findings are consistent with the notion that in addition to regulating the collagen fibril architecture, lumican acts to aid neutrophil recruitment and invasion following corneal damage and inflammation.

Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation.

The melanocortin 1 receptor gene is a main determinant of human pigmentation, and a melanoma susceptibility gene, because its variants that are strongly associated with red hair color increase melanoma risk. To test experimentally the association between melanocortin 1 receptor genotype and melanoma susceptibility, we compared the responses of primary human melanocyte cultures naturally expressing different melanocortin 1 receptor variants to α-melanocortin and ultraviolet radiation. We found that expression of 2 red hair variants abolished the response to α-melanocortin and its photoprotective effects, evidenced by lack of functional coupling of the receptor, and absence of reduction in ultraviolet radiation-induced hydrogen peroxide generation or enhancement of repair of DNA photoproducts, respectively. These variants had different heterozygous effects on receptor function. Microarray data confirmed the observed differences in responses of melanocytes with functional vs. nonfunctional receptor to α-melanocortin and ultraviolet radiation, and identified DNA repair and antioxidant genes that are modulated by α-melanocortin. Our findings highlight the molecular mechanisms by which the melanocortin 1 receptor genotype controls genomic stability of and the mutagenic effect of ultraviolet radiation on human melanocytes.

Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks.

Embryonic stem (ES) cells give rise to all cell types of an organism. Since mutations at this embryonic stage would affect all cells and be detrimental to the overall health of an organism, robust mechanisms must exist to ensure that genomic integrity is maintained. To test this proposition, we compared the capacity of murine ES cells to repair DNA double-strand breaks with that of differentiated cells. Of the 2 major pathways that repair double-strand breaks, error-prone nonhomologous end joining (NHEJ) predominated in mouse embryonic fibroblasts, whereas the high fidelity homologous recombinational repair (HRR) predominated in ES cells. Microhomology-mediated end joining, an emerging repair pathway, persisted at low levels in all cell types examined. The levels of proteins involved in HRR and microhomology-mediated end joining were highly elevated in ES cells compared with mouse embryonic fibroblasts, whereas those for NHEJ were quite variable, with DNA Ligase IV expression low in ES cells. The half-life of DNA Ligase IV protein was also low in ES cells. Attempts to increase the abundance of DNA Ligase IV protein by overexpression or inhibition of its degradation, and thereby elevate NHEJ in ES cells, were unsuccessful. When ES cells were induced to differentiate, however, the level of DNA Ligase IV protein increased, as did the capacity to repair by NHEJ. The data suggest that preferential use of HRR rather than NHEJ may lend ES cells an additional layer of genomic protection and that the limited levels of DNA Ligase IV may account for the low level of NHEJ activity.

Monoallelic expression of Krt12 gene during corneal-type epithelium differentiation of limbal stem cells.

PURPOSE: The purpose of this study was to characterize a Krt12-Cre knock-in mouse line for corneal epithelium-specific gene ablation and to analyze the allelic selection of the keratin 12 (Krt12) gene during corneal type-epithelium differentiation. METHODS: Knock-in mice were generated by gene targeting. The authors examined the expression patterns of several reporter genes in the corneas of bitransgenic Krt12cre/+/ROSA(EGFP), Krt12Cre/+/ZEG, and Krt12Cre/+/ZAP mouse lines. Krt12 and cre recombinase (Cre) immunostaining was performed. Corneal epithelial cells from bitransgenic Krt12Cre/+/ROSA(EGFP) mice were examined by fluorescence-activated cell sorter. RESULTS: Mosaic and spiral expression patterns of EGFP were observed in young and adult bitransgenic Krt12Cre/+/ZEG mice, respectively. Immunostaining revealed that Cre- cells were also Krt12 negative in the corneal epithelia of Krt12Cre/-/ZAP mice. Using FACS analysis, 60% to 70% of the corneal epithelial cells from Krt12Cre/+/ROSAEGFP mice were EGFP positive, whereas 20% to 30% were negative. RT-PCR revealed that EGFP+ cells express both Krt12Cre and Krt12+ alleles, whereas EGFP- cells express only Krt12+. In the Krt12Cre/- cornea, the number of epithelial cells expressing Cre is the same as that found in Krt12Cre/Cre, which can be explained by the fragility of corneal epithelial cells that did not produce Krt12 because the Krt12Cre allele was not transcribed. CONCLUSIONS: These observations are consistent with the notion that clonal limbal stem cells randomly activate Krt12 alleles in the process of terminal differentiation. The authors suggest that this selection is advantageous for retaining epithelial cells expressing the Krt12+ allele and that it allows tolerance to structural mutations of Krt12.

Sodium tungstate (Na2WO4) exposure increases apoptosis in human peripheral blood lymphocytes.

The potential for adverse health effects of using tungsten and its alloys in military munitions are an important concern to both civilians and the US military. The toxicological implications of exposure to tungsten, its alloys, and the soluble tungstate (Na(2)WO(4)) are currently under investigation. To examine tungstate toxicity, a series of experiments to determine its in vitro effects on cells of the immune system were performed. We identified alterations in isolated human peripheral blood lymphocytes (PBL) treated in vitro with sodium tungstate (0.01, 0.1, 1.0, and 10 mM). Analyses of apoptosis with annexin V and propidium iodide revealed a dose- and time-dependent increase in the quantity of cells in early apoptosis after tungstate exposure. Reductions in the number of cells entering into the cell cycle were also noted. Exposure of PBL to tungstate (1 mM) and Concanavalin A (ConA) for 72 h reduced the number of cells in S and G(2)/M phases of the cell cycle. There were alterations in the numbers of cells in G(0)/G(1), S, and G(2)/M phases of the cell cycle in long-term THP-1 (acute leukemic monocytes) cultures treated with tungstate (0.01, 0.1, 1.0, and 10 mM). Gel electrophoresis, silver staining, and LC-MS/MS showed the cytoplasmic presence of histone H1b and H1d after 72 h of tungstate exposure. The addition of tungstate to cultures resulted in significant reductions in the quantity of interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha), and IL-6 produced by stimulated [CD3/CD28, ConA, or lipopolysaccharide (LPS)] and tungstate-treated lymphocytes. Taken together, these data indicate that tungstate increases apoptosis of PBL, alters cell cycle progression, reduces cytokine production, and therefore warrants further investigation.

Mice treated with a benzodiazepine had an improved survival rate following Pseudomonas aeruginosa infection.

Psychological stress has a high incidence after burn injury, therefore, anxiolytic drugs are often prescribed. Unfortunately, to date, no burn study has investigated the effects of anxiolytic drugs on the ability to fight infection. This study was undertaken to determine if psychological stress, anxiety-modulating drugs, or both, alter survival following an infection. On day 0, 7-week-old male C57Bl/6 mice either received a 15% full-thickness flame burn or were sham treated (anesthesia and shaved), whereas controls received no treatment. Mice received midazolam (1 mg/kg intraperitoneally) or saline daily and were stressed by exposure to rat in a guinea pig cage or placed in an empty cage for 1 hour a day, beginning on postburn day 1. For the survival experiments, mice either received bacteria after 2 or 8 consecutive days of predator exposure and drug treatment, which continued daily for 7 days after inoculation. In a separate set of experiments, after eight daily injections of midazolam, mice were given lipopolysaccharide, bacteria, or saline and were killed 12 hours later. Mice that received midazolam had improved survival rates when compared with their saline-treated counterparts, and the protective effect was more significant the more days they received the drug. For most of the cytokines, the bacteria-induced increase was significantly attenuated by midazolam as was the amount of bacteria in the liver. The protective effect seems to be independent of the drug's anxiolytic activity as there were no significant differences in survival between the predator-stressed and the nonstressed mice. The mechanisms responsible for the protective effect remain to be elucidated.

Employing a recombinant HLA-DR3 expression system to dissect major histocompatibility complex II-thyroglobulin peptide dynamism: a genetic, biochemical, and reverse immunological perspective.

Previously, we have shown that statistical synergism between amino acid variants in thyroglobulin (Tg) and specific HLA-DR3 pocket sequence signatures conferred a high risk for autoimmune thyroid disease (AITD). Therefore, we hypothesized that this statistical synergism mirrors a biochemical interaction between Tg peptides and HLA-DR3, which is key to the pathoetiology of AITD. To test this hypothesis, we designed a recombinant HLA-DR3 expression system that was used to express HLA-DR molecules harboring either AITD susceptibility or resistance DR pocket sequences. Next, we biochemically generated the potential Tg peptidic repertoire available to HLA-DR3 by separately treating 20 purified human thyroglobulin samples with cathepsins B, D, or L, lysosomal proteases that are involved in antigen processing and thyroid biology. Sequences of the cathepsin-generated peptides were then determined by matrix-assisted laser desorption ionization time-of-flight-mass spectroscopy, and algorithmic means were employed to identify putative AITD-susceptible HLA-DR3 binders. From four predicted peptides, we identified two novel peptides that bound strongly and specifically to both recombinant AITD-susceptible HLA-DR3 protein and HLA-DR3 molecules expressed on stably transfected cells. Intriguingly, the HLA-DR3-binding peptides we identified had a marked preference for the AITD-susceptibility DR signatures and not to those signatures that were AITD-protective. Structural analyses demonstrated the profound influence that the pocket signatures have on the interaction of HLA-DR molecules with Tg peptides. Our study suggests that interactions between Tg and discrete HLA-DR pocket signatures contribute to the initiation of AITD.

Generation of mice with a conditional allele for transforming growth factor beta 1 gene.

Transforming growth factor beta1 (TGFbeta1) is a multifunctional growth factor involved in wound healing, tissue fibrosis, and in the pathogenesis of many syndromic diseases (e.g., Marfan syndrome, Camurati-Engelmann disease) and muscular, neurological, ophthalmic, cardiovascular and immunological disorders, and cancer. Since the generation of Tgfb1 knockout mice, there has been extraordinary progress in understanding its physiological and pathophysiological function. Here, we report the generation of a conditional knockout allele for Tgfb1 in which its exon 6 is flanked with LoxP sites. As proof of principle, we crossed these mice to LckCre transgenic mice and specifically disrupted Tgfb1 in T cells. The results indicate that T-cell-produced TGFbeta1 is required for normal in vivo regulation of peripheral T-cell activation, maintenance of T-cell homeostasis, and suppression of autoimmunity.