Investigating the effects of Pirfenidone on TGF-ß1 stimulated non-SMAD signaling pathways in Dupuytren's disease -derived fibroblasts.

BACKGROUND: Dupuytren's disease (DD) is a progressive, debilitating condition of the hand that can eventually cause contractures of the affected fingers. Transforming growth factor- ß1 (TGF-ß1) has been reported to play a key role in DD pathology. Increased expression of TGF-ß1 has shown to be the main stimulator of myofibroblast activity and in DD contractures. Pirfenidone (PFD), a small active molecule possess the ability to inhibit TGF-ß1-mediated action in various fibrotic disorders. Our recent published findings show that PFD reduced TGF-ß1-mediated cellular functions implicated in DD through SMAD signaling pathways. In the present study, the effect of PFD on TGF-ß1-mediated non-SMAD signaling pathways were investigated in both carpal tunnel (CT) - and DD-derived fibroblasts. METHODS: Fibroblasts harvested from Dupuytren's disease (DD) and carpal tunnel (CT) tissues were cultured in the presence or absence of TGF-ß1 (10 ng/ml) and/or PFD (800 µg/ml). Cell lysates were analyzed using Western blots. Equal amounts of proteins were loaded to determine the phosphorylation levels of phosphatidylinositol-3 kinase (PI3K/AKT), extracellular regulated kinases (ERK1/2), p38 mitogen-activated protein kinase and Rho family related myosin light chain (MLC). RESULTS: We show that the TGF-ß1-induced phosphorylation of AKT was significantly decreased by the addition of PFD (800 µg/mL) in both CT- and DD-derived fibroblasts. Interestingly, there was no significant difference in the phosphorylation levels of both ERK and p38 on TGF-ß1- induced cells in both CT-and DD-derived fibroblasts. But, PFD significantly decreased the TGF- ß1-induced phosphorylation levels of ERK1/2 in both CT- and DD- cells. In contrast, PFD significantly decreased the basal and TGF- ß1-induced phosphorylation levels of p38 in DD-derived fibroblasts. TGF- ß1-induced phosphorylation levels of MLC was decreased by PFD in DD-derived fibroblasts. CONCLUSIONS: These in-vitro results indicate for the first time that PFD has the potential to inhibit TGF-ß1-induced non-SMAD signaling pathways in both CT- and DD-derived fibroblasts but pronounced statistically significant inhibition on all molecules was observed only in DD-derived fibroblasts. Our previous studies show that PFD can inhibit TGF-ß1- induced SMAD signaling pathway proteins, namely p- SMAD2/SMAD3. These broad and complementary actions suggest PFD as a promising candidate to inhibit the TGF-ß1- mediated molecular mechanisms leading to DD fibrosis.

Pericytes reduce inflammation and collagen deposition in acute wounds.

BACKGROUND: Pericytes have been shown to have mesenchymal stromal cell-like properties and play a role in tissue regeneration. The goal of this study was to determine whether the addition of a pericyte sheet to a full-thickness dermal wound would enhance the healing of an acute wound. METHODS: Human muscle-derived pericytes and human dermal fibroblasts were formed into cell sheets, then applied to full-thickness excisional wounds on the dorsum of nu/nu mice. Histology was performed to evaluate epidermal and dermal reformation, inflammation and fibrosis. In addition, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine cytokine response. RESULTS: Pericytes were detected in the wounds until day 16 but not fibroblasts. Decrease in wound size was noted in pericyte sheet-treated wounds. Enhanced neo-vascularization and healthy granulation tissue formation were noted in the pericyte-treated wounds. Expression of type I collagen messenger RNA (mRNA) was significantly higher in the fibroblast-treated group, whereas Type III collagen mRNA showed significant increase in the pericyte group at days 3, 6 and 9 compared with the fibroblast and no-cell groups. Trichrome staining revealed thick unorganized collagen fibrils in the fibroblast-treated wounds, whereas pericyte-treated wounds contained thinner and more alligned collagen fibrils. Tumor necrosis factor (TNF)-α mRNA levels were increased in the fibroblast-treated wounds compared with pericyte-treated wounds. DISCUSSION: The addition of pericytes may confer beneficial effects to wound healing resulting in reduced recruitment of inflammatory cells and collagen I deposition, potential to enhance wound closure and better collagen alignment promoting stronger tissue.

Application of Laser Capture Microdissection to Craniofacial Biology: Characterization of Anatomically Relevant Gene Expression in Normal and Craniosynostotic Rabbit Sutures.

OBJECTIVE: Fusion of the cranial sutures is thought to depend on signaling among perisutural tissues. Mapping regional variations in gene expression would improve current models of craniosynostosis. Laser capture microdissection (LCM) isolates discrete cell populations for gene expression analysis. LCM has rarely been used in the study of mineralized tissue. This study sought to evaluate the potential use of LCM for mapping of regional gene expression within the cranial suture. DESIGN: Coronal sutures were isolated from 10-day-old wild-type and craniosynostotic (CS) New Zealand White rabbits, and LCM was used to isolate RNA from the sutural ligament (SL), osteogenic fronts (OF), dura mater, and periosteum. Relative expression levels for Fibroblast Growth Factor 2 (FGF2), Fibroblast Growth Factor Receptor 2 (FGFR2), Transforming Growth Factor Beta 2 (TGFß-2), Transforming Growth Factor Beta 3 (TGFß-3), Bone Morphogenetic Protein 2 (BMP-2), Bone Morphogenetic Protein 4 (BMP-4), and Noggin were determined using quantitative real-time PCR. RESULTS: A fivefold increase in TGFß2 expression was detected in the CS SL relative to wild type, whereas 152-fold less TGFß-3 was detected within the OF of CS animals. Noggin expression was increased by 10-fold within the CS SL, but reduced by 13-fold within the CS dura. Reduced expression of FGF2 was observed within the CS SL and dura, whereas increased expression of FGFR2 was observed within the CS SL. Reduced expression of BMP-2 was observed in the CS periosteum, and elevated expression of BMP-4 was observed in the CS SL and dura. CONCLUSIONS: LCM provides an effective tool for measuring regional variations in cranial suture gene expression. More precise measurements of regional gene expression with LCM may facilitate efforts to correlate gene expression with suture morphogenesis and pathophysiology.

Local Probiotic Therapy with Lactobacillus plantarum Mitigates Scar Formation in Rabbits after Burn Injury and Infection.

BACKGROUND: Infection is the most common complication in burn-injured patients and is believed to contribute to the hypertrophic scarring frequently observed in such injury. Pseudomonas aeruginosa is a common pathogen in burn wound infection. We examined the effect of local probiotic therapy with Lactobacillus plantarum on the severity of the scarring following burn wounding and infection with P. aeruginosa in a rabbit model. METHODS: Full-thickness burn wounds were inoculated with control vehicle or L. plantarum; wounds were then challenged with bioluminescent P. aeruginosa. The time course of the ensuing infection was monitored by quantification of the emitted light. After allowing wounds to contract to near completion, they were harvested and analyzed for markers of scar formation. RESULTS: Application of L. plantarum curtailed both the severity and the length of the pseudomonal infection. Probiotic therapy significantly reduced both Type I collagen mRNA concentrations and total collagen protein accumulation in infected wounds, consistent with reduced scarring. Surprisingly, the probiotic showed a nearly equivalent effect in uninfected wounds. Masson's trichrome staining confirmed these findings histologically. CONCLUSIONS: Lactobacillus plantarum shows exciting potential as a therapeutic agent to both counteract burn wound infection and to alleviate scarring even in the absence of infection.

Anti-fibrotic action of pirfenidone in Dupuytren's disease-derived fibroblasts.

BACKGROUND: Dupuytren's disease (DD) is a complex fibro-proliferative disorder of the hand that is often progressive and eventually can cause contractures of the affected fingers. Transforming growth factor beta (TGF-ß1) has been implicated as a key stimulator of myofibroblast activity and fascial contraction in DD. Pirfenidone (PFD) is an active small molecule shown to inhibit TGF-ß1-mediated action in other fibrotic disorders. This study investigates the efficacy of PFD in vitro in inhibiting TGF-ß1-mediated cellular functions leading to Dupuytren's fibrosis. METHODS: Fibroblasts harvested from (DD) and carpal tunnel (CT)- tissues were treated with or without TGF-ß1 and/or PFD and were subjected to cell migration, cell proliferation and cell contraction assays. ELISA; western blots and real time RT-PCR assays were performed to determine the levels of fibronectin; p-Smad2/Smad3; alpha-smooth muscle actin (α-SMA), α2 chain of type I collagen and α1 chain of type III collagen respectively. RESULTS: Our results show that PFD effectively inhibits TGF-ß1-induced cell migration, proliferation and cell contractile properties of both CT- and DD-derived fibroblasts. TGF-ß1-induced α-SMA mRNA and protein levels were inhibited at the higher concentration of PFD (800 µg/ml). Interestingly, TGF-ß1 induction of type I and type III collagens and fibronectin was inhibited by PFD in both CT- and DD- derived fibroblasts, but the effect was more prominent in DD cells. PFD down-regulated TGF-ß1-induced phosphorylation of Smad2/Smad3, a key factor in the TGF-ß1 signaling pathway. CONCLUSION: Taken together these results suggest the PFD can potentially prevent TGF-ß1-induced fibroblast to myofibroblast transformation and inhibit ECM production mainly Type I- and Type III- collagen and fibronectin in DD-derived fibroblasts. Further in-vivo studies with PFD may lead to a novel therapeutic application in preventing the progression or recurrence of Dupuytren's disease.

Local Application of Probiotic Bacteria Prophylaxes against Sepsis and Death Resulting from Burn Wound Infection.

OBJECTIVE: To determine if local prophylactic application of probiotic bacteria to burn wounds will prevent death in a mouse model of burn wound sepsis. BACKGROUND: Infection remains the most common complication after burn injury and can result in sepsis and death, despite the use of topical and systemic antibiotics. Pseudomonas aeruginosa is a frequently implicated pathogen. Local application of probiotics directly to burn wounds is an attractive novel intervention that avoids the pitfalls of standard antibiotic therapies. METHODS: A burn-sepsis model was established using a sub-eschar injection of bioluminescent P. aeruginosa; infection was tracked using a charge-coupled camera. Full-thickness burn injuries were placed on the dorsums of adult mice; the injured sites were then treated with vehicle (burn wound control), probiotics (Lactobacillus plantarum only), pathogenic bacteria (Pseudomonas aeruginosa only), or probiotics plus pathogen (Lactobacillus plus Pseudomonas). Animals were monitored until death/moribundity or for one week, then sacrificed. Harvested tissues were subjected to imaging and molecular assays. RESULTS: Control and probiotic-only animals showed no mortality (100% survival) at one week. Pseudomonas-only animals showed > 90% mortality within 40 hours of infection. In contrast, animals treated with probiotics plus Pseudomonas showed less than 10% mortality. Use of bioluminescent Pseudomonas bacteria demonstrated that probiotic therapy inhibited septicemic accumulation of the pathogen in remote organs. In addition, probiotic therapy successfully suppressed the infection-dependent induction of TNF-α and interleukins 6 and 10 in the liver. CONCLUSIONS: Local probiotic therapy shows great potential as a valuable adjunct in the management of complicated burn injury.

Expression analysis of human adipose-derived stem cells during in vitro differentiation to an adipocyte lineage.

BACKGROUND: Adipose tissue-derived stromal stem cells (ASCs) represent a promising regenerative resource for soft tissue reconstruction. Although autologous grafting of whole fat has long been practiced, a major clinical limitation of this technique is inconsistent long-term graft retention. To understand the changes in cell function during the transition of ASCs into fully mature fat cells, we compared the transcriptome profiles of cultured undifferentiated human primary ASCs under conditions leading to acquisition of a mature adipocyte phenotype. METHODS: Microarray analysis was performed on total RNA extracted from separate ACS isolates of six human adult females before and after 7 days (7 days: early stage) and 21 days (21 days: late stage) of adipocyte differentiation in vitro. Differential gene expression profiles were determined using Partek Genomics Suite Version 6.4 for analysis of variance (ANOVA) based on time in culture. We also performed unsupervised hierarchical clustering to test for gene expression patterns among the three cell populations. Ingenuity Pathway Analysis was used to determine biologically significant networks and canonical pathways relevant to adipogenesis. RESULTS: Cells at each stage showed remarkable intra-group consistency of expression profiles while abundant differences were detected across stages and groups. More than 14,000 transcripts were significantly altered during differentiation while ~6000 transcripts were affected between 7 days and 21 days cultures. Setting a cutoff of +/-two-fold change, 1350 transcripts were elevated while 2929 genes were significantly decreased by 7 days. Comparison of early and late stage cultures revealed increased expression of 1107 transcripts while 606 genes showed significantly reduced expression. In addition to confirming differential expression of known markers of adipogenesis (e.g., FABP4, ADIPOQ, PLIN4), multiple genes and signaling pathways not previously known to be involved in regulating adipogenesis were identified (e.g. POSTN, PPP1R1A, FGF11) as potential novel mediators of adipogenesis. Quantitative RT-PCR validated the microarray results. CONCLUSIONS: ASC maturation into an adipocyte phenotype proceeds from a gene expression program that involves thousands of genes. This is the first study to compare mRNA expression profiles during early and late stage adipogenesis using cultured human primary ASCs from multiple patients.

Developing an animal model of Dupuytren's disease by orthotopic transplantation of human fibroblasts into athymic rat.

BACKGROUND: Dupuytren's disease (DD) is a slow, progressive fibroproliferative disorder affecting the palms of the hands. The disease is characterized by the formation of collagen rich- cords which gradually shorten by the action of myofibroblasts resulting in finger contractures. It is a disease that is confined to humans, and a major limiting factor in investigating this disorder has been the lack of a faithful animal model that can recapitulate its distinct biology. The aim of this study was to develop such a model by determining if Dupuytren's disease (DD)- and control carpal tunnel (CT)-derived fibroblasts could survive in the forepaw of the nude rats and continue to exhibit the distinct characteristics they display in in vitro cultures. METHODS: 1x10(7) fluorescently labeled DD- and CT-derived fibroblasts were transplanted into the left and right forepaws of nude rats respectively. Cells were tracked at regular intervals for a period of two months by quantifying emitted fluorescent signal using an IVIS imaging system. After a period of 62 days rat forepaw connective tissues were harvested for histology and total RNA was isolated. Human-specific probes were used to perform real time RT-PCR assays to examine the expression patterns of gene products associated with fibrosis in DD. Rat forepaw skin was also harvested to serve as an internal control. RESULTS: Both CT- and DD-derived fibroblasts survived for a period of 62 days, but DD-derived cells showed a significantly greater level of persistent fluorescent signal at the end of this time than did CT-derived cells. mRNA expression levels of α-smooth muscle actin (α-SMA), type I- and type III- collagens were all significantly elevated in the forepaw receiving DD cord-derived fibroblasts in comparison to CT-derived fibroblasts. Masson's trichrome stain confirmed increased collagen deposition in the forepaw that was injected with DD cord-derived fibroblasts. CONCLUSIONS: For the first time we describe an animal model for Dupuytren's disease at the orthotopic anatomical location. We further show that gene expression differences between control (CT) and diseased (DD) derived fibroblasts persist when these cells are transplanted to the forepaw of the nude rat. These preliminary findings indicate that, with further refinements, this animal model holds promise as a baseline for investigating novel therapeutic regimens to determine an effective strategy in treating DD.

Direct demonstration of bacterial biofilms on prosthetic mesh after ventral herniorrhaphy.

BACKGROUND: Prosthetic mesh is employed routinely in the treatment of ventral and parastomal hernias, but its use can lead to major complications, including infection, extrusion, and fistula. Bacterial biofilms have been posited to play a role in mesh-related infection, but although bacteria have been noted to form biofilms on mesh surfaces in vitro, they have never been visualized directly in biofilms on mesh recovered from patients experiencing infectious complications. METHODS: Five patients who developed complications after ventral hernia repair with prosthetic mesh were operated on again. Explanted mesh was examined for biofilm with confocal laser scanning microscopy (CLSM) and fluorescence in situ hybridization (FISH). In two cases, a novel molecular assay (the Ibis T5000) was used to characterize the biofilm-forming bacteria. RESULTS: The CLSM examination demonstrated adherent biofilms on mesh surfaces in all five patients. Biofilms also were noted on investing fibrous tissue. The FISH study was able to discriminate between bacterial species in polymicrobial biofilms. In two patients the Ibis T5000 detected more species of constituent biofilm bacteria than did standard culture. Removal of the mesh and reconstruction with autologous tissues or biologic materials resolved the presenting complaints in all cases. CONCLUSION: Bacterial biofilms should be considered an important contributor to the pathology and complications associated with prosthetic mesh implanted in the abdominal wall. If biofilms are present, complete removal of the mesh and repair of the resulting defect without alloplastic materials is an effective intervention.

Biofilms in periprosthetic orthopedic infections.

As the number of total joint arthroplasty and internal fixation procedures continues to rise, the threat of infection following surgery has significant clinical implications. These infections may have highly morbid consequences to patients, who often endure additional surgeries and lengthy exposures to systemic antibiotics, neither of which are guaranteed to resolve the infection. Of particular concern is the threat of bacterial biofilm development, since biofilm-mediated infections are difficult to diagnose and effective treatments are lacking. Developing therapeutic strategies have targeted mechanisms of biofilm formation and the means by which these bacteria communicate with each other to take on specialized roles such as persister cells within the biofilm. In addition, prevention of infection through novel coatings for prostheses and the local delivery of high concentrations of antibiotics by absorbable carriers has shown promise in laboratory and animal studies. Biofilm development, especially in an arthoplasty environment, and future diagnostic and treatment options are discussed.

Bacterial biofilms on implanted suture material are a cause of surgical site infection.

BACKGROUND: Surgical site infection (SSI) has been estimated to occur in up to 5% of all procedures, accounting for up to 0.5% of all hospital costs. Bacterial biofilms residing on implanted foreign bodies have been implicated as contributing or causative factors in a wide variety of infectious scenarios, but little consideration has been given to the potential for implanted, submerged suture material to act as a host for biofilm and thus serve as a nidus of infection. METHODS: We report a series of 15 patients who underwent open Roux-en-Y gastric bypass (with musculofascial closure with permanent, multifilament sutures) who developed longstanding and refractory SSIs in the abdominal wall. Explanted suture material at subsequent exploration was examined for biofilm with confocal laser-scanning microscopy (CLSM) and fluorescence in situ hybridization (FISH). RESULTS: All 15 patients at re-exploration were found to have gross evidence of a "slimy" matrix or dense reactive granulation tissue localized to the implanted sutures. Confocal laser-scanning microscopy revealed abundant biofilm present on all sutures examined; FISH was able to identify the presence of specific pathogens in the biofilm. Complete removal of the foreign bodies (and attendant biofilms) resulted in all cases in cure of the SSI. CONCLUSION: Bacterial biofilms on implanted suture material can manifest as persistent surgical site infections that require complete removal of the underlying foreign body substrata for resolution.

Cloning of TgfßR1 and TgfßR2 and Likely Exclusion as Loci of Origin in a Rabbit Craniosynostotic Model.

OBJECTIVE: To determine whether TgfßR1 or TgfßR2 cause the craniosynostotic phenotype in a rabbit model of nonsyndromic craniosynostosis. DESIGN: Full-length TgfßR1 and TgfßR2 cDNAs were sequenced and real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed to measure TgfßR1 and TgfßR2 transcripts in sutural tissue from wild type (WT) and craniosynostotic (CS) rabbits. Single nucleotide polymorphisms (SNP) were identified within TgfßR1 and TgfßR2 and were assayed for segregation with disease phenotype in 22 craniosynostotic animals. RESULTS: No structural mutations in TgfßR1 and TgfßR2 were identified in the craniosynostotic rabbits. Real-time RT-PCR quantification of TgfßR1 and TgfßR2 mRNA showed no significant difference in TgfßR1 expression between CS and WT animals, while TgfßR2 showed 50% elevation in the CS animals compared to WT (P < .05). SNP analysis within the TgfßR1 and TgfßR2 genes suggested that neither locus is linked to the craniosynostotic phenotype because no allelic combination showed any specific correlation with disease phenotype for either TgfßR1 or TgfßR2. CONCLUSIONS: Our data indicate that the craniosynostotic phenotype in this rabbit model does not arise from any structural mutation in TgfßR1 or TgfßR2, and SNP analysis also likely excludes these genes more broadly as the site of causative mutation.

Molecular Analysis of Twist1 and FGF Receptors in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

Craniosynostosis is the premature fusion of the cranial vault sutures. We have previously described a colony of rabbits with a heritable pattern of nonsyndromic, coronal suture synostosis; however, the underlying genetic defect remains unknown. We now report a molecular analysis to determine if four genes implicated in human craniosynostosis, TWIST1 and fibroblast growth factor receptors 1-3 (FGFR1-3), could be the loci of the causative mutation in this unique rabbit model. Single nucleotide polymorphisms (SNPs) were identified within the Twist1, FGFR1, and FGFR2 genes, and the allelic patterns of these silent mutations were examined in 22 craniosynostotic rabbits. SNP analysis of the Twist1, FGFR1, and FGFR2 genes indicated that none were the locus of origin of the craniosynostotic phenotype. In addition, no structural mutations were identified by direct sequence analysis of Twist1 and FGFR3 cDNAs. These data indicate that the causative locus for heritable craniosynostosis in this rabbit model is not within the Twist1, FGFR1, and FGFR2 genes. Although a locus in intronic or flanking sequences of FGFR3 remains possible, no direct structural mutation was identified for FGFR3.

Increased CCT-eta expression is a marker of latent and active disease and a modulator of fibroblast contractility in Dupuytren's contracture.

Dupuytren's contracture (DC) is a fibroproliferative disorder of unknown etiology characterized by a scar-like contracture that develops in the palm and/or digits. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta) is increased in fibrotic wound healing, and is essential for the accumulation of α-smooth muscle actin (α-SMA) in fibroblasts. The purpose of this study was to determine if CCT-eta is similarly implicated in the aberrant fibrosis seen in DC and to investigate the role of CCT-eta in the behavior of myo/fibroblasts in DC. Fibroblasts were obtained from DC-affected palmar fascia, from adjacent phenotypically normal palmar fascia in the same DC patients (PF), and from non-DC palmar fascial tissues in patients undergoing carpal tunnel (CT) release. Inherent contractility in these three populations was examined using fibroblast-populated collagen lattices (FPCLs) and by cell traction force microscopy. Expression of CCT-eta and α-SMA protein was determined by Western blot. The effect of CCT-eta inhibition on the contractility of DC cells was determined by deploying an siRNA versus CCT-eta. DC cells were significantly more contractile than both matching palmar fascial (PF) cells and CT cells in both assays, with PF cells demonstrating an intermediate contractility in the FPCL assay. Whereas α-SMA protein was significantly increased only in DC cells compared to PF and CT cells, CCT-eta protein was significantly increased in both PF and DC cells compared to CT cells. siRNA-mediated depletion of CCT-eta inhibited the accumulation of both CCT-eta and α-SMA protein in DC cells, and also significantly decreased the contractility of treated DC cells. These observations suggest that increased expression of CCT-eta appears to be a marker for latent and active disease in these patients and to be essential for the increased contractility exhibited by these fibroblasts.

Scarless integumentary wound healing in the mammalian fetus: molecular basis and therapeutic implications.

Adult mammals respond to injury of their skin/integument by forming scar tissue. Scar is useful in rapidly sealing an injured area, but can also lead to significant morbidity. Mammals in fetal life retain the ability to heal integumentary wounds regeneratively, without scar. The critical molecular mechanisms governing this remarkable phenomenon have been a subject of great interest, in the hopes that these could be dissected and recapitulated in the healing adult wound, with the goal of inducing scarless healing in injured patients. Multiple lines of investigation spanning decades have implicated a number of factors in distinguishing scarless from fibrotic wound healing, including most prominently transforming growth factor-ß and interleukin-10, among others. Therapeutic interventions to try to mitigate scarring in adult wounds have been developed out of these studies, and have reached the level of clinical trials in humans, although as yet no FDA-approved treatment exists. More recent expressomic studies have revealed many more genes that are differentially expressed in scarlessly healing fetal wounds compared with adult, and microRNAs have also been identified as participating in the fetal wound healing response. These represent an even greater range of potential therapeutics (or targets for therapy) to translate the promise of scarless fetal wound healing to the injured adult patient.

Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture.

BACKGROUND: Dupuytren's contracture (DC) is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC. METHODS: To achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group). These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments). Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02), hierarchical clustering, concordance mapping and Venn diagram. RESULTS: We found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that a collagen-rich environment differentially alters gene expression in these cells. In addition, Ingenuity pathway analysis of the specific biological pathways that differentiate DC-derived cells from carpal tunnel-derived cells has identified the potential involvement of microRNAs in this fibroproliferative disorder. CONCLUSIONS: These data show that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected palmar fascia in DC patients are highly similar, and differ significantly from the transcriptomic profiles of fibroblasts from the palmar fascia of patients undergoing carpal tunnel release.

Towards diagnostic guidelines for biofilm-associated infections.

Biofilms associated with the human body, particularly in typically sterile locations, are difficult to diagnose and treat effectively because of their recalcitrance to conventional antibiotic therapy and host immune responses. The study of biofilms in medicine today requires a translational approach, with examination of clinically relevant biofilms in the context of specific anatomic sites, host tissues, and diseases, focusing on what can be done to mitigate their pathologic consequences. This review, which grew out of a discussion session on clinical biofilms at the 5th ASM Biofilm Conference in Cancun, Mexico, is designed to give an overview of biofilm-associated infections (BAI) and to propose a platform for further discussion that includes clinicians, medical microbiologists, and biofilm researchers who are stakeholders in advancing the scientific pursuit of better diagnosis and treatment of BAI to mitigate their human and healthcare costs. It also highlights the need for better diagnostic markers, which exploit the difference between planktonic and biofilm cells.

Kinetics and morphology of polymicrobial biofilm formation on polypropylene mesh.

We examined the ability of three clinical bacterial isolates to form mixed biofilms on surgical polypropylene mesh (PPM) in vitro. The three strains--Staphylococcus aureus, Enterococcus faecalis, and Enterobacter cloacae--were isolated from a patient with an infected PPM. Staphylococcus aureus and E. faecalis (alone and in combination) were inoculated into culture containing squares of PPM and allowed to attach and propagate into mature biofilms. Enterococcus faecalis initially attached to the mesh in greater numbers; however, 7 days postinoculation, there were more S. aureus cells attached, indicating that in vitro S. aureus is the out-competing species. All three isolates were then co-cultured to form mature biofilms on mesh, and the biofilms were examined by confocal microscopy using both Live/Dead staining and fluorescent in situ hybridization (FISH). Imaging revealed a dense biofilm structure with interstitial voids and channels; rods and cocci were interspersed throughout the biofilm, indicating bacterial coexistence in close proximity. FISH revealed staphylococci and enterococci adjacent to each other and also to the Enterobacter, distinguishable by its rod morphology. These studies show that different species can co-operatively form mature biofilms on mesh but that the relative abundance of a species within the biofilm may vary over time.

Considering hidradenitis suppurativa as a bacterial biofilm disease.

Hidradenitis suppurativa (HS) is a chronic inflammatory disease of the skin that results in a relapsing course of painful draining sinuses and abscesses. The disease manifests largely in the apocrine gland-bearing regions of the body (axillary, inguinal and anogenital areas) and is usually treated by antibiotics and/or surgery. The exact pathogenesis of HS is still in dispute, but likely multifactorial; in some instances, a genetic component has been demonstrated. While much attention has been given to the cellular and molecular biology of the host tissues affected by HS, rather less has been given to the bacteria involved (most commonly Staphylococci or Streptococci). We note that the characteristics of HS comport exactly with the features of bacterial biofilm-based infections, and examined a case where HS of the buttocks had progressed to an advanced stage. Physical examination of the sinus tracks at surgery revealed a mucinous accumulation consistent with biofilm formation. Confocal microscopic examination using Live/Dead staining revealed clusters of bacteria attached to the sinus luminal surfaces. The paradigmatic clinical features of HS, coupled with the adherent bacterial communities we observe here, suggest that HS should be considered in the expanding spectrum of bacterial biofilm-based disorders.