Modified High-Molecular-Weight Hyaluronan Promotes Allergen-Specific Immune Tolerance.

The extracellular matrix in asthmatic lungs contains abundant low-molecular-weight hyaluronan, and this is known to promote antigen presentation and allergic responses. Conversely, high-molecular-weight hyaluronan (HMW-HA), typical of uninflamed tissues, is known to suppress inflammation. We investigated whether HMW-HA can be adapted to promote tolerance to airway allergens. HMW-HA was thiolated to prevent its catabolism and was tethered to allergens via thiol linkages. This platform, which we call "XHA," delivers antigenic payloads in the context of antiinflammatory costimulation. Allergen/XHA was administered intranasally to mice that had been sensitized previously to these allergens. XHA prevents allergic airway inflammation in mice sensitized previously to either ovalbumin or cockroach proteins. Allergen/XHA treatment reduced inflammatory cell counts, airway hyperresponsiveness, allergen-specific IgE, and T helper type 2 cell cytokine production in comparison with allergen alone. These effects were allergen specific and IL-10 dependent. They were durable for weeks after the last challenge, providing a substantial advantage over the current desensitization protocols. Mechanistically, XHA promoted CD44-dependent inhibition of nuclear factor-κB signaling, diminished dendritic cell maturation, and reduced the induction of allergen-specific CD4 T-helper responses. XHA and other potential strategies that target CD44 are promising alternatives for the treatment of asthma and allergic sinusitis.

Crosstalk Between T Lymphocytes and Lung Fibroblasts: Generation of a Hyaluronan-Enriched Extracellular Matrix Adhesive for Monocytes.

In immunity and inflammation, T cells are often associated with stromal mesenchymal cells such as fibroblasts. Hyaluronan and proteins that associate with hyaluronan such as versican and tumor necrosis factor-inducible gene-6 (TSG-6) are extracellular matrix (ECM) components that promote leukocyte adhesion, accumulation, and activation. However, the factors responsible for producing this specialized ECM and its impact on inflammatory events are not well understood. In this study, we explored the role of T cells in stimulating lung fibroblasts to produce an ECM that impacts monocyte adhesion. We found that CD3/CD28-activated human CD4+ T cells when co-cultured with human lung fibroblasts stimulated the expression of mRNA for hyaluronan synthase 2 (HAS2) and decreased the expression of hyaluronidase 2 (HYAL2). This led to an increase in the deposition of hyaluronan that formed cable-like structures within the ECM. Co-culturing activated T cells with fibroblasts also led to increased expression and accumulation of TSG-6. Surprisingly, addition of activated CD4+ T cells to the fibroblasts reduced the expression of mRNA for versican, and increased the expression of enzymes that degrade versican, such as ADAMTS4 and ADAMTS9 (a disintegrin and metalloproteinase with a thrombospondin type-1 motif) leading to a decrease in versican in the ECM of the co-cultures. Furthermore, addition of human monocytes to these co-cultures resulted in elevated monocyte adhesion to the cable-like structures in the ECM when compared to controls. These results illustrate the importance of crosstalk between T cells and fibroblasts in promoting the generation of a matrix that is adhesive for monocytes. J. Cell. Biochem. 118: 2118-2130, 2017. © 2016 Wiley Periodicals, Inc.

Active control of acoustic field-of-view in a biosonar system.

Active-sensing systems abound in nature, but little is known about systematic strategies that are used by these systems to scan the environment. Here, we addressed this question by studying echolocating bats, animals that have the ability to point their biosonar beam to a confined region of space. We trained Egyptian fruit bats to land on a target, under conditions of varying levels of environmental complexity, and measured their echolocation and flight behavior. The bats modulated the intensity of their biosonar emissions, and the spatial region they sampled, in a task-dependant manner. We report here that Egyptian fruit bats selectively change the emission intensity and the angle between the beam axes of sequentially emitted clicks, according to the distance to the target, and depending on the level of environmental complexity. In so doing, they effectively adjusted the spatial sector sampled by a pair of clicks-the "field-of-view." We suggest that the exact point within the beam that is directed towards an object (e.g., the beam's peak, maximal slope, etc.) is influenced by three competing task demands: detection, localization, and angular scanning-where the third factor is modulated by field-of-view. Our results suggest that lingual echolocation (based on tongue clicks) is in fact much more sophisticated than previously believed. They also reveal a new parameter under active control in animal sonar-the angle between consecutive beams. Our findings suggest that acoustic scanning of space by mammals is highly flexible and modulated much more selectively than previously recognized.

Bat wing sensors support flight control.

Bats are the only mammals capable of powered flight, and they perform impressive aerial maneuvers like tight turns, hovering, and perching upside down. The bat wing contains five digits, and its specialized membrane is covered with stiff, microscopically small, domed hairs. We provide here unique empirical evidence that the tactile receptors associated with these hairs are involved in sensorimotor flight control by providing aerodynamic feedback. We found that neurons in bat primary somatosensory cortex respond with directional sensitivity to stimulation of the wing hairs with low-speed airflow. Wing hairs mostly preferred reversed airflow, which occurs under flight conditions when the airflow separates and vortices form. This finding suggests that the hairs act as an array of sensors to monitor flight speed and/or airflow conditions that indicate stall. Depilation of different functional regions of the bats' wing membrane altered the flight behavior in obstacle avoidance tasks by reducing aerial maneuverability, as indicated by decreased turning angles and increased flight speed.

ECM components guide IL-10 producing regulatory T-cell (TR1) induction from effector memory T-cell precursors.

We describe a role for ECM as a biosensor for inflammatory microenvironments that plays a critical role in peripheral immune tolerance. We show that hyaluronan (HA) promotes induction of Foxp3- IL-10-producing regulatory T cells (TR1) from conventional T-cell precursors in both murine and human systems. This is, to our knowledge, the first description of an ECM component inducing regulatory T cells. Intact HA, characteristic of healing tissues, promotes induction of TR1 capable of abrogating disease in an IL-10-dependent mouse colitis model whereas fragmentary HA, typical of inflamed tissues, does not, indicating a decisive role for tissue integrity in this system. The TR1 precursor cells in this system are CD4(+)CD62L(-)FoxP3(-), suggesting that effector memory cells assume a regulatory phenotype when they encounter their cognate antigen in the context of intact HA. Matrix integrity cues might thereby play a central role in maintaining peripheral tolerance. This TR1 induction is mediated by CD44 cross-linking and signaling through p38 and ERK1/2. This induction is suppressed, also in a CD44-dependent manner, by osteopontin, a component of chronically inflamed ECM, indicating that CD44 signaling serves as a nexus for fate decisions regarding TR1 induction. Finally, we demonstrate that TR1 induction signals can be recapitulated using synthetic matrices. These results reveal important roles for the matrix microenvironment in immune regulation and suggest unique strategies for immunomodulation.

Regulatory T cells use heparanase to access IL-2 bound to extracellular matrix in inflamed tissue.

Although FOXP3+ regulatory T cells (Treg) depend on IL-2 produced by other cells for their survival and function, the levels of IL-2 in inflamed tissue are low, making it unclear how Treg access this critical resource. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing monoclonal antibody-directed chimeric antigen receptor (mAbCAR) Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their ability to suppress neuroinflammation in vivo. Together, these data identify a role for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.

FOXP3+ regulatory T cells use heparanase to access IL-2 bound to ECM in inflamed tissues.

FOXP3+ regulatory T cells (Treg) depend on exogenous IL-2 for their survival and function, but circulating levels of IL-2 are low, making it unclear how Treg access this critical resource in vivo. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their tolerogenic function in vivo. Together, these data identify novel roles for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.

Extracellular vesicles in fatty liver promote a metastatic tumor microenvironment.

Liver metastasis is a major cause of death in patients with colorectal cancer (CRC). Fatty liver promotes liver metastasis, but the underlying mechanism remains unclear. We demonstrated that hepatocyte-derived extracellular vesicles (EVs) in fatty liver enhanced the progression of CRC liver metastasis by promoting oncogenic Yes-associated protein (YAP) signaling and an immunosuppressive microenvironment. Fatty liver upregulated Rab27a expression, which facilitated EV production from hepatocytes. In the liver, these EVs transferred YAP signaling-regulating microRNAs to cancer cells to augment YAP activity by suppressing LATS2. Increased YAP activity in CRC liver metastasis with fatty liver promoted cancer cell growth and an immunosuppressive microenvironment by M2 macrophage infiltration through CYR61 production. Patients with CRC liver metastasis and fatty liver had elevated nuclear YAP expression, CYR61 expression, and M2 macrophage infiltration. Our data indicate that fatty liver-induced EV-microRNAs, YAP signaling, and an immunosuppressive microenvironment promote the growth of CRC liver metastasis.

Antibiotic-refractory Lyme arthritis is associated with HLA-DR molecules that bind a Borrelia burgdorferi peptide.

An association has previously been shown between antibiotic-refractory Lyme arthritis, the human histocompatibility leukocyte antigen (HLA)-DR4 molecule, and T cell recognition of an epitope of Borrelia burgdorferi outer-surface protein A (OspA163-175). We studied the frequencies of HLA-DRB1-DQA1-DQB1 haplotypes in 121 patients with antibiotic-refractory or antibiotic-responsive Lyme arthritis and correlated these frequencies with in vitro binding of the OspA163-175 peptide to 14 DRB molecules. Among the 121 patients, the frequencies of HLA-DRB1-DQA1-DQB1 haplotypes were similar to those in control subjects. However, when stratified by antibiotic response, the frequencies of DRB1 alleles in the 71 patients with antibiotic-refractory arthritis differed significantly from those in the 50 antibiotic-responsive patients (log likelihood test, P = 0.006; exact test, P = 0.008; effect size, Wn = 0.38). 7 of the 14 DRB molecules (DRB1*0401, 0101, 0404, 0405, DRB5*0101, DRB1*0402, and 0102) showed strong to weak binding of OspA163-175, whereas the other seven showed negligible or no binding of the peptide. Altogether, 79% of the antibiotic-refractory patients had at least one of the seven known OspA peptide-binding DR molecules compared with 46% of the antibiotic-responsive patients (odds ratio = 4.4; P < 0.001). We conclude that binding of a single spirochetal peptide to certain DRB molecules is a marker for antibiotic-refractory Lyme arthritis and might play a role in the pathogenesis of the disease.

Evidence for molecular mimicry between human T cell epitopes in rotavirus and pancreatic islet autoantigens.

In type 1 diabetes, insulin-producing beta cells in the islets of the pancreas are destroyed by autoreactive T cells. Rotavirus (RV) has been implicated in the pathogenesis of type 1 diabetes. Peptides in VP7, a major immunogenic protein of RV, have high sequence similarity to T cell epitope peptides in the islet autoantigens tyrosine phosphatase-like insulinoma Ag 2 (IA2) and glutamic acid decarboxylase 65 (GAD65). We aimed to educe evidence for the hypothesis that molecular mimicry with RV promotes autoimmunity to islet autoantigens. Peptides in RV and their sequence-similar counterparts in IA2 and GAD65 were assayed for binding to HLA molecules associated with type 1 diabetes and for the ability to elicit T cell proliferative responses in HLA-typed individuals. T cells expanded or cloned to epitopes in IA2 or RV were then tested for cross-reactivity with these epitopes. Peptides in RV-VP7, similar to T cell epitopes in IA2 and GAD65, bound strongly to HLA-DRB1*04 molecules that confer susceptibility to type 1 diabetes and were also T cell epitopes in humans at risk for type 1 diabetes. The proliferative responses of T cells to the similar peptides in RV and islet autoantigens were significantly correlated. T cells expanded to the IA2 epitope could be restimulated to express IFN-gamma by the similar peptide in RV-VP7, and T cell clones generated to this RV-VP7 peptide cross-reacted with the IA2 epitope. Our findings are consistent with the hypothesis that molecular mimicry with RV could promote autoimmunity to islet Ags.

Trypanosoma brucei Acyl-Protein Thioesterase-like (TbAPT-L) Is a Lipase with Esterase Activity for Short and Medium-Chain Fatty Acids but Has No Depalmitoylation Activity.

Dynamic post-translational modifications allow the rapid, specific, and tunable regulation of protein functions in eukaryotic cells. S-acylation is the only reversible lipid modification of proteins, in which a fatty acid, usually palmitate, is covalently attached to a cysteine residue of a protein by a zDHHC palmitoyl acyltransferase enzyme. Depalmitoylation is required for acylation homeostasis and is catalyzed by an enzyme from the alpha/beta hydrolase family of proteins usually acyl-protein thioesterase (APT1). The enzyme responsible for depalmitoylation in Trypanosoma brucei parasites is currently unknown. We demonstrate depalmitoylation activity in live bloodstream and procyclic form trypanosomes sensitive to dose-dependent inhibition with the depalmitoylation inhibitor, palmostatin B. We identified a homologue of human APT1 in Trypanosoma brucei which we named TbAPT-like (TbAPT-L). Epitope-tagging of TbAPT-L at N- and C- termini indicated a cytoplasmic localization. Knockdown or over-expression of TbAPT-L in bloodstream forms led to robust changes in TbAPT-L mRNA and protein expression but had no effect on parasite growth in vitro, or cellular depalmitoylation activity. Esterase activity in cell lysates was also unchanged when TbAPT-L was modulated. Unexpectedly, recombinant TbAPT-L possesses esterase activity with specificity for short- and medium-chain fatty acid substrates, leading to the conclusion, TbAPT-L is a lipase, not a depalmitoylase.

The insulin A-chain epitope recognized by human T cells is posttranslationally modified.

The autoimmune process that destroys the insulin-producing pancreatic beta cells in type 1 diabetes (T1D) is targeted at insulin and its precursor, proinsulin. T cells that recognize the proximal A-chain of human insulin were identified recently in the pancreatic lymph nodes of subjects who had T1D. To investigate the specificity of proinsulin-specific T cells in T1D, we isolated human CD4(+) T cell clones to proinsulin from the blood of a donor who had T1D. The clones recognized a naturally processed, HLA DR4-restricted epitope within the first 13 amino acids of the A-chain (A1-13) of human insulin. T cell recognition was dependent on the formation of a vicinal disulfide bond between adjacent cysteine residues at A6 and A7, which did not alter binding of the peptide to HLA DR4. CD4(+) T cell clones that recognized this epitope were isolated from an HLA DR4(+) child with autoantibodies to insulin, and therefore, at risk for T1D, but not from two healthy HLA DR4(+) donors. We define for the first time a novel posttranslational modification that is required for T cell recognition of the insulin A-chain in T1D.

Adaptive behavior for texture discrimination by the free-flying big brown bat, Eptesicus fuscus.

This study examined behavioral strategies for texture discrimination by echolocation in free-flying bats. Big brown bats, Eptesicus fuscus, were trained to discriminate a smooth 16 mm diameter object (S+) from a size-matched textured object (S-), both of which were tethered in random locations in a flight room. The bat's three-dimensional flight path was reconstructed using stereo images from high-speed video recordings, and the bat's sonar vocalizations were recorded for each trial and analyzed off-line. A microphone array permitted reconstruction of the sonar beam pattern, allowing us to study the bat's directional gaze and inspection of the objects. Bats learned the discrimination, but performance varied with S-. In acoustic studies of the objects, the S+ and S- stimuli were ensonified with frequency-modulated sonar pulses. Mean intensity differences between S+ and S- were within 4 dB. Performance data, combined with analyses of echo recordings, suggest that the big brown bat listens to changes in sound spectra from echo to echo to discriminate between objects. Bats adapted their sonar calls as they inspected the stimuli, and their sonar behavior resembled that of animals foraging for insects. Analysis of sonar beam-directing behavior in certain trials clearly showed that the bat sequentially inspected S+ and S-.

Restricted autoantigen recognition associated with deletional and adaptive regulatory mechanisms.

Autoimmune diabetes (T1D) is characterized by CD4(+) T cell reactivity to a variety of islet-associated Ags. At-risk individuals, genetically predisposed to T1D, often have similar T cell reactivity, but nevertheless fail to progress to clinically overt disease. To study the immune tolerance and regulatory environment permissive for such autoreactive T cells, we expressed TCR transgenes derived from two autoreactive human T cells, 4.13 and 164, in HLA-DR4 transgenic mice on a C57BL/6-derived "diabetes-resistant" background. Both TCR are responsive to an immunodominant epitope of glutamic acid decarboxylase 65(555-567), which is identical in sequence between humans and mice, is restricted by HLA-DR4, and is a naturally processed self Ag associated with T1D. Although both TCR use the identical Valpha and Vbeta genes, differing only in CDR3, we found stark differences in the mechanisms utilized in vivo in the maintenance of immune tolerance. A combination of thymic deletion (negative selection), TCR down-regulation, and peripheral activation-induced cell death dominated the phenotype of 164 T cells, which nevertheless still maintain their Ag responsiveness in the periphery. In contrast, 4.13 T cells are much less influenced by central and deletional tolerance mechanisms, and instead display a peripheral immune deviation including differentiation into IL-10-secreting Tr1 cells. These findings indicate a distinct set of regulatory alternatives for autoreactive T cells, even within a single highly restricted HLA-peptide-TCR recognition profile.

CD44 costimulation promotes FoxP3+ regulatory T cell persistence and function via production of IL-2, IL-10, and TGF-beta.

Work by our group and others has demonstrated a role for the extracellular matrix receptor CD44 and its ligand hyaluronan in CD4(+)CD25(+) regulatory T cell (Treg) function. Herein, we explore the mechanistic basis for this observation. Using mouse FoxP3/GFP(+) Treg, we find that CD44 costimulation promotes expression of FoxP3, in part through production of IL-2. This promotion of IL-2 production was resistant to cyclosporin A treatment, suggesting that CD44 costimulation may promote IL-2 production through bypassing FoxP3-mediated suppression of NFAT. CD44 costimulation increased production of IL-10 in a partially IL-2-dependent manner and also promoted cell surface TGF-beta expression. Consistent with these findings, Treg from CD44 knockout mice demonstrated impaired regulatory function ex vivo and depressed production of IL-10 and cell surface TGF-beta. These data reveal a novel role for CD44 cross-linking in the production of regulatory cytokines. Similar salutary effects on FoxP3 expression were observed upon costimulation with hyaluronan, the primary natural ligand for CD44. This effect is dependent upon CD44 cross-linking; while both high-molecular-weight hyaluronan (HA) and plate-bound anti-CD44 Ab promoted FoxP3 expression, neither low-molecular weight HA nor soluble anti-CD44 Ab did so. The implication is that intact high-molecular weight HA can cross-link CD44 only in those settings where it predominates over fragmentary LMW-HA, namely, in uninflamed tissue. We propose that intact but not fragmented extracellular is capable of cross-linking CD44 and thereby maintains immunologic tolerance in uninjured or healing tissue.

Nanocarrier-enhanced intracellular delivery of benznidazole for treatment of Trypanosoma cruzi infection.

Chagas disease is caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi), an intracellular pathogen that causes significant morbidity and death among millions in the Americas from Canada to Argentina. Current therapy involves oral administration of the nitroimidazole benznidazole (BNZ), which has serious side effects that often necessitate cessation of treatment. To both avoid off-target side effects and reduce the necessary dosage of BNZ, we packaged the drug within poly(ethylene glycol)-block-poly(propylene sulfide) polymersomes (BNZ-PSs). We show that these vesicular nanocarriers enhanced intracellular delivery to phagocytic cells and tested this formulation in a mouse model of T. cruzi infection. BNZ-PS is not only nontoxic but also significantly more potent than free BNZ, effectively reducing parasitemia, intracellular infection, and tissue parasitosis at a 466-fold lower dose of BNZ. We conclude that BNZ-PS was superior to BNZ for treatment of T. cruzi infection in mice and that further modifications of this nanocarrier formulation could lead to a wide range of custom controlled delivery applications for improved treatment of Chagas disease in humans.

Autoantigen-specific regulatory T cells induced in patients with type 1 diabetes mellitus by insulin B-chain immunotherapy.

There is a growing body of evidence to suggest that the autoimmunity observed in type 1 diabetes mellitus (T1DM) is the result of an imbalance between autoaggressive and regulatory cell subsets. Therapeutics that supplement or enhance the existing regulatory subset are therefore a much sought after goal in this indication. Here, we report the results of a double blind, placebo controlled, phase I clinical trial of a novel antigen-specific therapeutic in 12 subjects with recently diagnosed T1DM. Our primary objective was to test its safety. The study drug, human insulin B-chain in incomplete Freund's adjuvant (IFA) was administered as a single intramuscular injection, with subjects followed for 2 years. All subjects completed therapy and all follow-up visits. The therapy was generally safe and well-tolerated. Mixed meal stimulated C-peptide responses, measured every 6 months, showed no statistical differences between arms. All patients vaccinated with the autoantigen, but none who received placebo, developed robust insulin-specific humoral and T cell responses. Up to two years following the single injection, in peripheral blood from subjects in the experimental arm, but not the control arm, insulin B-chain-specific CD4+ T cells could be isolated and cloned that showed phenotypic and functional characteristics of regulatory T cells. The induction of a lasting, robust immune response generating autoantigen-specific regulatory T cells provides strong justification for further testing of this therapy in type 1 diabetes. (clinicaltrials.gov identifier NCT00057499).

Recurrence of autoreactive antigen-specific CD4+ T cells in autoimmune diabetes after pancreas transplantation.

CD4+ T cells specific for the diabetes-associated autoantigen GAD65 were analyzed using peripheral blood samples after pancreas transplantation in subjects with T1D with clinical evidence of recurrent autoimmune diabetes. MHC class II tetramers facilitated the identification and cloning of antigen-specific autoreactive cells, which were found at several time points over a multiyear span, in spite of chronic immunosuppression of the subjects. Comparisons of TCR clonotypes by cDNA sequencing revealed that identical T cells were present in the circulation, separated by long time intervals, consistent with a persistent memory response associated with recurrent autoimmunity.

Autoreactive T cells in a partially humanized murine model of T1D.

Glutamic acid decarboxylase (GAD65) and insulin are implicated as target antigens in the pathogenesis of human diabetes through correlative measurements of humoral and cellular reactivity to them in diabetics and at-risk diabetic individuals. Recently, an age-dependent loss of tolerance to one of several naturally processed epitopes of GAD65 (555-567) has been observed to precede diabetes in diabetes-prone mice transgenic for diabetes-correlated human class II genes. Extended studies in these mice (RIP-B7/DR0404) now show that tolerance is maintained to another DR4-restricted naturally processed region within GAD65. While tolerance is lost to GAD65 (555-567) in B7/DR0404 mice prior to diabetes, these mice remain T cell-tolerant to GAD65 (273-286). Prediabetes loss of tolerance to GAD65 (555-567) has now been shown to correlate with an impaired response to exogenous glucose in an intraperitoneal (i.p.) glucose tolerance test. In addition, these mice exhibit a T cell response to insulin A(6-21) at the hyperglycemic state. Investigating a possible cause-and-effect relationship between T cell reactivity to GAD65 and diabetes pathogenesis, GAD65 (555-567) T cell receptor (TcR) transgenic mice have been generated and future work is aimed at understanding the importance of T cell GAD65 reactivity and its role in diabetes progression.

Use of versican variant V3 and versican antisense expression to engineer cultured human skin containing increased content of insoluble elastin.

Skin substitutes for repair of dermal wounds are deficient in functional elastic fibres. We report that the content of insoluble elastin in the dermis of cultured human skin can be increased though the use of two approaches that enhance elastogenesis by dermal fibroblasts, forced expression of versican variant V3, which lacks glycosaminoglycan (GAG) chains, and forced expression of versican antisense to decrease levels of versican variant V1 with GAG chains. Human dermal fibroblasts transduced with V3 or anti-versican were cultured under standard conditions over a period of 4 weeks to produce dermal sheets, with growth enhanced though multiple seedings for the first 3 weeks. Human keratinocytes, cultured in supplemented media, were added to the 4-week dermal sheets and the skin layer cultured for a further week. At 5 weeks, keratinocytes were multilayered and differentiated, with desmosome junctions thoughout and keratin deposits in the upper squamous layers. The dermal layer was composed of layered fibroblasts surrounded by extracellular matrix of collagen bundles and, in control cultures, small scattered elastin deposits. Forced expression of V3 and versican antisense slowed growth, decreased versican V1 expression, increased tropoelastin expression and/or the deposition of large aggregates of insoluble elastin in the dermal layer, and increased tissue stiffness, as measured by nano-indentation. Skin sheets were also cultured on Endoform Dermal Template™, the biodegradable wound dressing made from the lamina propria of sheep foregut. Skin structure and the enhanced deposition of elastin by forced expression of V3 and anti-versican were preserved on this supportive substrate. Copyright © 2014 John Wiley & Sons, Ltd.