bluuebunny/crossref_metadata_2025 huggingface dataset:
134 Evaluation of Alterations in Gene Expression in Normal and Diabetic Mice Resulting from Dermal Injury
Authors: Rodgers Kathleen E. , Ellefson Dolph D. , Espinoza Theresa , Hsu Ya‐hsuan , DiZerega Gere S. , Mehrian‐Shai Ruty
Published: March 2007

C57Bl/6‐db/db mice are a model of delayed healing after full thickness dermal excision injury. We evaluated the differences in gene expression between C57Bl/6 mice and C57Bl/6‐db/db mice in uninjured skin and within a wound 7 days after injury and observed a number of differences resulting from the diabetic mutation. Cluster analysis indicated differences in genes involved in all structural organization and metabolism. There were 718 genes that had greater than 2 fold difference between these 2 strains of mice in granulation tissue at day 7. In normal and diabetic mice, 884 or 765 genes were altered 2 fold by injury (granulation tissue), respectively. When compared at the 4 fold level, the overlap of genes altered between normal and diabetic mice was approximately 68%. In granulation tissue, changes were observed in genes associated with inflammation, immune response, cell motility, and response to external stimuli. Normal mice had greater immune, stress, inflammatory, and biotic responses. Wounds of diabetic mice had increased responses in energy metabolism and fatty acid metabolism. These data provide a basis for further understanding of the response of skin to injury and the delay that occurs in insulin resistant Type 2 diabetes.
139 Gene Array Profiling of Keloid Fibroblasts to Identify the Target Genes for Therapeutic Evaluations
Authors: Satish Latha , Luo Jian‐Hua , Priya Kulasekaran S , Wells Alan
Published: March 2007

Keloids are scars that overgrow the original boundaries of the injury, involving aberrant functioning of the fibroblasts. We have found that during normal wound healing the ELR‐negative CXC chemokines IP‐9 (CXCL11) and IP‐10 (CXCL10) act to limit fibroblast immigration. Our initial goal was to determine if keloid fibroblasts failed to respond to this ‘stop’ signal. Immunohistochemical and immunocytochemical studies on keloid fibroblasts show that these lesions express excessive amounts of IP‐9 and CXCR3 receptor. The protein levels of CXCR3 receptor was also not dissimilar between normal and keloid fibroblasts. Though, the expression of the protein and the receptor seems to be normal, the fibroblasts derived from these abnormal lesions did not respond to IP‐9 during EGF induced cell migration. We have extended these findings to determine whether other genes are differentially expressed by keloid fibroblasts. The immediate first gene that interest us was the mRNA levels of CXCR3 receptor (GPR9) wherein, we did not find any significant difference between normal and keloid fibroblasts. Further, we also found interestingly, that genes related to tumor progression are up regulated in keloid fibroblasts than the normal fibroblasts. The marker genes like calgranulin, BCL‐2 associated anthogen 4 (BAG4), and dual specificity phosphatase 1 (DUSP1). Certain transcription factors like GATA‐6 and SpiB are also increased. Apart, the classical growth factor and growth factor receptor related genes which includes, bone morphogenetic protein BMP‐1, heparin binding EGF like growth factor, EGF like repeats discoidin1, IGFBP‐5 and 3, EphB1&B2, tumor necrosis factor alpha induced protein, protein tyrosine phosphatase receptor, serine threonine kinase 6 along with chondroitin sulfate proteoglycan brevican and versican and vitronectin are also up regulated. The newl identified genes will open new avenues in targeting keloid lesions by understanding the basic signaling mechanism of these specific proteins.

This work was supported by grants from NIH/NIGMS and PPRTP.
143 HB‐107, A Non‐Antimicrobial Fragment of Cecropin B, Accelerates Murine Wound Repair
Authors: Lee P. H. A. , Harris S. M. , Zhang L. , Falla T. J. , Gallo R. L.
Published: March 2007

Intact skin represents the first line of defense against microbial pathogens. Effectors of innate immunity such as antimicrobial peptides are important when this barrier is breached due to infection or injury. Antimicrobial peptides can kill microbes directly, however several investigations have suggested they can also modify host cell behaviors to promote wound repair. It is unclear whether the ability of these molecules to positively influence wound repair is dependent on their antimicrobial function or on their ability to influence the host. To investigate this, the microbial killing capacity of specific antimicrobial peptide fragments was determined and compared with their ability to affect wound repair in a murine model of aseptic full‐thickness excisional injury. HB‐107, a peptide fragment derived from Cecropin B, lacks antimicrobial activity yet showed up to 64% improvement in wound closure at day13 when compared to either scrambled peptide or vehicle controls. This effect was comparable to that seen after treatment with currently accepted therapy. Histological evaluation of wounds treated with HB‐107 displayed keratinocyte hyperplasia and increased leukocyte infiltration compared to controls. To explore the mechanism for these findings, we tested the ability of HB‐107 to stimulate IL‐8 secretion. HB‐107 peptide was able to stimulate IL‐8 release from cultured dermal microvascular endothelial cells when compared to scrambled peptide control or other peptides derived from Cecropin B. Taken together, this data confirms that antimicrobial peptides can function as important effectors of wound repair independent of antimicrobial function.
146 Enhancing the Repair Quality of Injured Skin on Porcine after Autografting with Bone Marrow Mesenchymal Stem Cells
Authors: Fu Xiaobing , Fang Lijun , Wang Yuxin , Sun Tongzhu , Cheng Biao
Published: March 2007

To explore the effects of bone marrow mesenchymal stem cells (MSCs) on the quality healing of porcine skin wounds after burn injury so as to provide a new method for clinical skin repair in the future. Seventy‐two deep‐partial thickness burn wounds were produced on the back of 6 minipigs and randomly divided into 6 groups: saline control, MSCs treatment, MSCs plus bFGF treatment, MSCs plus EGF treatment, bFGF treatment or EGF treatment only. MSCs were isolated from porcine marrow and cultured in vitro. After labeling with BrdU, MSCs were autografted onto the skin wounds. At 7, 14, 21 and 42 days after injury, the area of the wounds were measured and the histological examination was performed to evaluate the velocity and quality of wound healing. At 1, 2 and 4 weeks after transplantation, immunohistochemical examinations were carried out to detect the positive staining of BrdU, cytokeratin and S‐100 to evaluate the wound healing quality. The area of wounds was decreased at day 7 and most of these wounds were healed on day 21 after injury. There was no significant difference on the contraction rate among six groups. Histological examination demonstrated that the number of vessel and the expression density of S‐100 in MSCs plus bFGF treatment wounds were significantly enhanced than that in other groups. MSCs autografting may benefit to enhance the wound healing quality in porcine skin, which may open a new way to reach a “perfect repair” after skin injury.
149 Angiogenesis Induced by Enriched Bone Marrow Cell Transplantation in Ischemic Hindlimb Canine Model
Authors: De La Garza Anabel S. , Padilla Luis , Krötzsch Edgar , Figueroa Siegfred A. , Avila Gerardo , Morales José A. , Glennie German , Di Silvio Mauricio
Published: March 2007

Bone marrow is an important supply for many progenitor cells, among them endothelial precursor cells. They have been already considered as potential therapeutic angiogenesis for ischemic hindlimb. In this work we performed an unilateral chronic ischemic hindlimb model in 20 dogs, we were dissected and ligated the middle sacra artery and external right iliac artery preserving the internal iliac artery, after one week we removed de femoral artery performing a proximal and distal ligation and inserted into the gracilis muscle 3 Silastic tubes (0.8 mm diameter) in a middle circle form in order to created fibrocollagenous tunnels. Fifteen days later, we obtained bone marrow (30 mL) and mononuclear cells were separated; at the same time, all tubes were removed and bone marrow cells were transplanted into the fibrocollagenous tunnels, only in the third and fourth groups. During the 8 at 12 days we administered subcutaneously saline solution to the first and third groups and G‐CSF to the second and fourth groups. Finally, after 30 days we performed contrasted angiographies from both limbs and was calculated mean angiographic score (MAS) from the number of vascular intersections, also was taken a biopsy from the central portion of right gracilis muscle for vascular assessment, as well as for vascular proliferating cells by immunohistochemistry.

Results demonstrated that white bone marrow cell transplantation enrichment by G‐CSF administration stimulates angiogenesis in ischemic hindlimb, twice than controls, and it is better than white bone marrow cell transplantation alone. White bone marrow cell is a potential therapy for ischemic hindlimb, because stem cells are growth factor provider, as well as an important source for endothelial precursors capable to form vessels de novo, mainly when this stem cells are deposited in an appropriate extracellular matrix, such as the fibrocollagenous tunnels in this model.
151 Engineering a Human ‘Skin” with Adult Primary Cells.
Authors: Martins‐Green M. , Li QiJing , Yao Min
Published: March 2007

The inability to experiment in humans creates a great need to develop culture systems that mimic human tissues/organs. Skin is arguably the simplest human tissue and therefore provides an excellent prototype for tissue engineering. Moreover, skin is the body’s first line of defense.

Currently available skin replacements have been classified into four categories: (i) those that are composed completely of epidermal cells; (ii) those consisting of dermal components derived from processing of cadaver skin or from collagen and other matrix molecules; (iii) those containing both dermal and epidermal components and (iv) those that contain dermal, epidermal and vascular components. All of these substitutes have drawbacks. Therefore, there is a need for skin replacements that: (a) are prepared with pertinent primary human cells but yet can be ready “off” the shelf; (b) can be prepared rapidly; (c) contain stable structures in particular microvessels that can rapidly connect with the patient’s vasculature in this manner establish circulation in the “graft” increasing the chances of survival; (d) can be tailored for specific wound impairments (e) are long lasting. We have developed a new generation human “skin” that can fulfil these requirements and can potentially be used as a “living bandage”. We start with three primary human cell types and a collagen matrix that self‐assemble into a connective tissue containing a network of mature microvessels, is covered with a stratified epidermis, expresses biochemical markers, matrix molecules, and cytokines characteristic of normal human skin and matures in 10–15 days. Moreover, two additional cell types, pericytes and monocytes, differentiate in situ adjacent to and within microvessels, respectively providing stability to the micriovessels and the epidermis expresses keratins that are typical of mature skin and not those characteristically produced in response to injury such as keratins 6, 16, 17. This tissue can potentially be developed into a skin replacement for patients with impaired healing. In addition, this tissue responds normally to bological stimuli, providing a powerful vehicle to investigate mechanisms of skin development and regeneration, understand pathological processes, and test drugs and treatments for skin diseases
152 Blocking IL‐6 Receptors Increases Matrix Metalloproteinase‐3 Production in Hypertrophic Scar Fibroblasts
Authors: Mohan R.K.Dasu , Barrow RE , Hawkins HK , McCauley RL , Herndon DN
Published: March 2007

Introduction: We hypothesize that interleukin‐6 receptor (IL‐6r) blockade with IL‐6r antibody will increase matrix metalloproteinase‐3 (MMP‐3) production in hypertrophic scar fibroblasts.

To test this hypothesis, MMP‐3 protein levels were measured in fibroblasts cultured from normal skin and hypertrophic scar with and without IL‐6 receptor blockade.

Methods: Primary cell cultures of normal skin (NSF) and hypertrophic scar fibroblasts (HTS) were obtained from the same patient (n = 5) and seeded into 6 well plates (40,000 cells/cm2) in complete media. Cells were treated with and without IL‐6 receptor antibody and IgG (10 ng/ml) for 6 hours. Protein concentrations of MMP‐3 are determined in equal volume of supernatants by ELISA.

Statistical analysis was by unpaired t‐test(*P < 0.05).

Results: Blocking IL‐6 receptors with its antibody showed significant increase in MMP‐3 protein concentration in HTS compared to NSF.

MMP‐3(ng/ml)

MMP‐3(ng/ml)

NSF
1.6 ± 0.13
HTS
0.7 ± 0.37

NSF + IL‐6r
0.3 ± 0.15
HTS + IL‐6r
2.3 ± 0.5*

NSF + IgG
0.26 ± 0.16
HTS + IgG
0.75 ± 0.14

Conclusion: IL‐6 receptor mediated signaling is involved in the pathogenesis of hypertrophic scar formation and its blockade may reduce hypertrophic scar formation.
153 Kinetic Evaluation of Scar after Full Thickness Incision Injury in the Rat
Authors: Rodgers Kathleen E. , Ellefson Dolph D. , Espinoza Theresa , Roda Norma , Maldonado Sonia , DiZerega Gere S.
Published: March 2007

Previous studies have shown that angiotensin peptides, NorLeu3‐A(1–7) in particular, accelerate dermal healing and reduce scar formation. In this report, the effect of this peptide on scar formation is more fully delineated. The effect of surgical day, time after injury and observer on the clinical appearance of the incision was determined. Clinical observations of incision site included inflammation, dehiscence of the injury and appearance of scar were conducted by two blinded observers (two observations per time point) twice weekly. The scores correlated between observers and for the same observer within an observation day. Dehiscence of the incision occurred in 35% to 40% of incisions early (days 4 and 7) after injury. Administration of NorLeu3‐A(1–7) at the time of injury reduced the incidence of dehiscence at day 7 to approximately 20%. Further, the length of the wound opening was significantly reduced in the peptide‐treated incisions at day 7. Starting on day 14 after injury, scar formation was evaluated. Up to 80–90% of control animals had observable scars starting on day 14. Thereafter, the scar remodeled with fewer incisions having visible scar on day 28. With administration of NorLeu3‐A(1–7), significantly fewer incisions had observable scars starting on day 14 and throughout the study. As few as 20% of the incisions had observable scars on day 28. The histological appearance of the healing wound was also evaluated at weekly intervals starting on day 7 and continuing until day 42. At day 7, the maximal number of fibroblasts at the wound site was observed. Thereafter, the number gradually reduced, plateauing at day 28. The administration of peptide had no effect on fibroblast number at the incision site. A similar pattern was observed in the thickness of the epidermis with the resolution of te hyperplastic phase at day 21. Administration of the peptide significantly increased epidermal height at day 7. Blood vessel formation peaked on day 21 and 28 in control wounds and was further enhanced by peptide administration during the neovascularization phase. After day 28, blood vessel number was comparable between control and treated incisions. Collagen deposition and remodeling were are increased by the administration of NorLeu3‐A(1–7) at the time of injury. This paper describes the kinetics of scar formation in the rat by clinical and histological observation and further described the beneficial effect of NorLeu3‐A(1–7) on scar formation.
005 Cyclodextrin Polymer Based Biocompatible Matrix for Local Gene Delivery
Authors: Kang D.W. , Bellocq N.C. , Schluep T. , Looper L.D. , Gu D‐L. , Quijano E.M. , Wen S.F. , Wang X. , Jensen G.S. , Pun S.H. , Zepeda M.L. , Davis M.E.
Published: March 2007

Biocompatible matrices, such as bovine collagen, have demonstrated usefulness in delivering gene therapy vectors that express growth factors to local environments for tissue repair. Unlike animal derived matrices, we have developed a new synthetic matrix consisting of a linear cyclodextrin‐polyethyleneglycol co‐polymer that is non‐covalently cross‐linked with di‐adamantane‐polyethyleneglycol via inclusion complex formation between adamantane and cyclodextrin (CD‐Ada). We performed both in vitro and in vivo experiments for biocompatibility and localized transgene expression using a recombinant adenovirus (rAd) vector containing either the reporter gene, GFP, or the therapeutic gene, PDGF‐B. In vitro results demonstrated cell migration, adenoviral transduction, and gene expression with no visible signs of toxicity in human skin fibroblasts. Qualitative gene expression from the CD‐Ada containing rAd was delayed by approximately two days when compared to collagen, but the level of expression was greater over a longer period of time. In vivo experiments demonstrated gene expression after local delivery to mouse skin using rAd‐GFP in CD‐Ada. Again, the expression was slightly delayed but duration of expression was comparable to collagen. Expression studies using rAd‐PDGF‐B, were performed in the rat polyvinyl alcohol sponge model and showed comparable quantitative DNA and RNA levels between CD‐Ada and collagen (DNA: 4.1 × 1010 and 4.5 × 1010 MEQ of PDGF‐B/assayed sponge, respectively; RNA: 7.0 × 108 and 3.2 × 108 MEQ of PDGF‐B/assayed sponge, respectively). Additionally, we explored the use of plasmid DNA with the CD‐Ada matrix and observed PDGF‐B expression in vivo. Our results show that this new delivery system provides a safe, efficient, and adaptable medium for both viral and non‐viral gene delivery.
008 Cross – Talk between Fibroblasts and Keratinocytes in 3‐d Fibrin Clots. In Vitro Studies
Authors: Sese N. , Cole M. , Tawil B.
Published: March 2007

Fibrin Sealant products such as TisseelR have been used over the last 25 years to reach hemostasis during surgery or to seal tissues. Moreover, there were many studies on the use of fibrin sealant in cell and bioactive substance delivery. Our In Vitro and In Vivo studies, over the last 2 years, have focused on the use of fibrin sealant to deliver human fibroblasts or keratinocytes to overcome the healing deficiency in chronic wounds. We have shown that some fibrin formulations supported a high fibroblast proliferation and other fibrin formulations supported a high proliferation of human keratinocytes. In this study, we examined the use of fibrin sealant in the co‐delivery of both human – derived keratinocytes and fibroblasts. The study report examined the cell proliferation of these two cell types in various formulations of fibrin sealant. Fibroblasts and keratinocytes were mixed with various dilutions of the Sealer Protein solution and added to culture plates before adding the Thrombin solution to form fibrin clots containing both cell types. We found that a low to medium sealer protein concentration (1–34 mG/mL) and a very low thrombin concentration (1 U/mL) in the final fibrin clots provided for an optimal cell proliferation for both cell types within these fibrin clots. This profile of proliferation was different from that seen when keratinocytes alone or fibroblasts alone were incorporated in the fibrin clots. Morphologically, it was difficult to determine the cell type from examining cell morphology, thus, it was difficult to determine the cell distribution. In conclusion, we found that various modified formulations of fibrin sealant may be chosen when co‐ delivering fibroblasts and keratinocytes. Moreover, there seems to be a positive feedback between keratinocytes and fibroblasts when they were co – introduced in the fibrin clots. This feedback could be carried by growth factors. Future studies will determine his signaling process.
023 The Use of Cilostazol in the Treatment of Raynaulds Disease with Digital Ischemia and Tissue Loss
Authors: Serena T. , Probst J.
Published: March 2007

Background: More than three quarters of patients with a connective tissue disorder will develop secondary Raynauld’s disease. This more severe form of the Raynauld’s can lead to digital ischemia, tissue loss and may even result in the need for amputation of one or more digits. Cilostazol is a phosphodiesterase inhibitor currently approved for use in vascular disease of the lower extremities.

Case History: In August of 2002 a 62 year old female pianist with scleroderma presented to the wound clinic with a two year history of intractable digital pain secondary to ischemic ulcerations of the index, long and ring fingers of the right hand. She suffered from secondary Raynauld’s disease. Prior unsuccessful treatments included warming the hands, calcium channel blockade, anticoagulation, topical xylocaine and narcotic analgesics. In August of 2002 cilostazol was started at 100mg BID. Wound care consisted of serial debridement, moist wound healing and topical rhPDGF.

Results: Four weeks into treatment the patient’s narcotic requirement decreased substantially and the fingers had a pink appearance. The ring finger healed after eight weeks. At three months the patient discontinued narcotic pain medication. Nine months into treatment the index finger had healed and she resumed playing the piano. The long finger finally healed one year after she initially presented to the wound clinic.

Conclusion: Cilostazol may be an important adjunct in the treatment of secondary Raynauld’s disease particularly when digital ischemia has developed.

Acknowledgements: Funding from Otsuka Pharmaceuticals.
012 Molecular Mechanisms of Thrombin‐Induced Interleukin‐8 Expression in Human Macrophages
Authors: Zheng L. , Martins‐Green M.
Published: March 2007

Wound healing involves a series of overlapping, highly coordinated events. Macrophages play multiple crucial roles in these processes including the secretion of chemokines. Interleukin‐8 (IL‐8) is an inducible chemokine that is expressed in a variety of cell types present at the wound site, including macrophages, and is known to contribute to chemotaxis, angiogenesis, and tissue remodeling during wound healing. One of the potent natural inducers of IL‐8 is thrombin, a multifunctional enzyme that is released upon wounding. Little is known about how this enzyme stimulates IL‐8 in macrophages. Therefore, we used a variety of cellular and molecular approaches to investigate the signal transduction mechanism of thrombin‐induced IL‐8 expression in THP‐1‐differentiated macrophages. We show that stimulation of IL‐8 by thrombin occurs in a rapid manner that is time‐ and dose‐dependent and can be abolished by addition of hirudin, a specific inhibitor of thrombin. At the mRNA level, IL‐8 peaks ∼4 hrs after treatment and declines by 9 hrs. A variety of inhibitors were used to delineate the signal transduction pathways leading to IL‐8 expression. Application of AG1478, a potent EGFR inhibitor, abolishes thrombin‐induced IL‐8 production suggesting EGFR transactivation. We further show that MEK1/2 and p38 are important signal mediators; their inhibition abolishes IL‐8 production. Contrarily, PKC inhibitors do not abolish, but rather enhance production of IL‐8, whereas activators inhibit the thrombin stimulation, suggesting that PKC is a negative regulator of IL‐8 expression. In summary, these results suggest that thrombin stimulates IL‐8 expression by transactivating the EGFR leading to activation of MEK1/2 and p38, and that PKC is a negative regulator of this stimulation process.
013 Variable p53 Response in a 3D Matrix
Authors: Carlson M.A. , Longaker M.T.
Published: March 2007

Detachment of an attached (stressed) fibroblast‐populated collagen matrix (FPCM) induces fibroblast apoptosis; this may be secondary to increased p53 after detachment. We have noted, however, that the p53 response after detachment of an attached FPCM (i.e., stress‐release) varies among cells strains. We hypothesized that modulation of p53 after FPCM stress‐release may be related to the fibroblast population density at the time of release. Human foreskin fibroblasts (initially seeded at 0.5 × 106/ml in 0.2 ml) were cultured for 48 hr in attached collagen matrices prior to detachment for 0–6 hr; p53 levels and lysate DNA concentration (used as a measure of fibroblast population density) were determined with immunoblot densitometry and a spectrophotometer, respectively. The experiment was performed with 15 different strains of fibroblasts. The p53 level increased (defined as ≥100% increase in baseline p53 level) during the 6 hr detachment period in 6/15 (40%) of fibroblast strains (responders), and downregulated or unchanged in the remaining strains (nonresponders). The mean DNA concentration in the responders vs. nonresponders was 1.56 μg/ml (median 1.63, range 0.89–2.25)vs. 2.57 (median 1.94, range 1.38–5.09), respectively (p < 0.05, Wilcoxon rank sum test). Stress‐release of the FPCM resulted in a variable p53 response which appeared to have a relationship with the fibroblast population density; a lower density was associated with p53 upregulation after detachment. This variable p53 response may in turn be related to varying proliferative capacity in the collagen matrix, as there was a broad range (nearly an order of magnitude) of DNA concentration among the 15 cell strains after 48 hr of attached FPCM cuture.
014 Macrophage Suspensions Treatment of Infected Sternal Wounds Following Cabg Surgery
Authors: Danon David , Zuloff‐Shani Adi , Kachel Erez , Mohr Raphael , Shinar Eilat , Orenstein Arie
Published: March 2007

Macrophages serve as the coordinators of the wound healing process. Since 1998 following the Israeli Ministry of health authorization, macrophage suspensions have been used for the treatment of ulcers, in more than 800 elderly and paraplegic patients suffering from decubital chronic ulcers.

As previously published, a significant number of genes showed increased levels of expression in hypo‐osmotic shock activated cells, using DNA microarrays technique. The majority of these genes are considered to be directly involved in the macrophage function and in the wound healing process.

Macrophge suspensions are prepared from a whole blood unit of healthy, young volunteer blood donors in a closed, sterile system, as previously described. The activated cells are applied to the wounds either by local injection or by direct deposition to the wound.

Between January 2000 and October 2003, 112 patients with postoperative sternal wound infection were treated with macrophage suspension. Full closure of the wounds was achieved in 104 (93%) of the patients.

Original wound surface area
8–175 cm2 (mean 93)

Days until treatment
6–180 (mean 47)

Days until 50% closure
6–60 (mean 21)

Days until full closure
10–138 (mean 49)

No side effects were noted.

The use of macrophage suspension is a safe and effective therapeutic strategy that reduces risk of complications and morbidity and improves the quality of life for long ‐suffering patients. Length and cost of hospital stay may be reduced, as the treatment requires no hospitalization.
015 Impact of Hydration on MMP‐Activity
Authors: Tandara Andrea A. , Mustoe Thomas A. , Mogford Jon E.
Published: March 2007

Hydration of keratinocytes modifies the levels of cytokines they secrete, which in turn impacts the secretory behaviour of dermal fibroblasts. In an in vitro coculture model, conditioned media (CM) collagen content was decreased 44% when keratinocytes were hydrated. We hypothesized that this is partly due to increased MMP‐activity. We used the same coculture model to study changes in MMP‐activity and TIMP secreted by keratinocytes as well as by fibroblasts in monoculture and in coculture in relation to air‐treatment or hydration of keratinocytes. Stratified human epidermal keratinocytes (HEK) and confluent human dermal fibroblasts (HDF) were cocultured for 72 h under serum‐free conditions. HEK were either kept at the air‐interface or hydrated. CM was assayed for MMP‐1, −2, −9, TIMP‐1 and −2 were assayed using zymograms, western blotting, and ELISA. MMP‐1, secreted by both cell types, increased significantly in cocultures compared to monocultures (4‐fold in the air‐treated group, 26‐fold in the hydrated group). MMP‐2, secreted mainly by HDFs, was significantly increased by coculture (hydration: 2.4‐fold, air: 2.8‐fold). MMP‐9, predominantly secreted by air‐treated HEKs and was significantly decreased in hydrated monoculture (76%) and coculture. HEK‐monoculture hydration also significant decreased MMP‐1 (86%) and MMP‐2 (81%) activity. HDF‐secreted TIMP‐1 expression was significantly increased by coculture and was unaffected by hydration. Our findings demonstrate that paracrine interactions between HEK and HDF modify MMP activity and that HEK hydration significantly effects on MMP activity. The findings provide insight into the role of hydration on HEK and HDF ctivity during the wound healing process.
016 TNF‐alpha Mediated Induction of MMP‐9 is Modulated by P21‐activated Kinase (PAK)
Authors: Han Yuan‐Ping , Zhou Ling , Garner Warren L.
Published: March 2007

Matrix metalloproteinase‐9 (MMP‐9) transiently expresses in acute wound. In non‐healed wounds, MMP‐9 together with other proteinases persistently elevate, which may lead excessive ECM degradation and failure of wound closure. To understand the molecular regulation of MMP‐9 we investigated the signal transduction for TNF‐alpha mediated induction of MMP‐9 by dermal fibroblasts. TNF‐alpha initiates three major signal pathways including NF‐11B, JUN N‐terminal kinase (JNK), and p38 MAPK. On the other hand, Rho‐GTPase plays an important role in a variety of cellular functions including cell morphogenesis, motility, survival, angiogenesis, and mitosis. It remains unknown if the “cross talk” of these signals having a role in regulation of matrix metalloproteinases (MMPs). In this study we found that over expression of the p21‐activated kinase (PAK) specifically attenuates TNF‐alpha mediated induction of MMP‐9. However, TNF‐alpha mediated induction of MMP‐3 and proMMP‐2 activation was intact. NF‐κB signal is regarded as a common pathway for many MMPs. Indeed, PAK did not affect TNF‐alpha mediated degradation of Ikappa B, suggesting additional signal is targeted by PAK. In contrast, MMP‐3 but not MMP‐9 expression is specifically blocked by p38 MAK. Thus TNF‐alpha induced expression of multiple MMPs in wound healing may utilize different intracellular signal pathways.
018 Healing Rate for Diabetic Neuropathic Foot Ulcer
Authors: Margolis DJ , Allen‐Taylor L , Hoffstad O , Berlin JA
Published: March 2007

The goal of this study was to benchmark by year the likelihood that an individual with a diabetic neuropathic foot ulcer (DNFU) will heal. We observed these rates over a more than 10 year period (1988–2000). For this study, we designed a cohort study within the multi‐center Curative Health System wound care network of diabetics who have DNFU and whether they healed by the 20th week of care stratified by calendar year.

In total, we evaluated 27, 193 individuals with a DNFU. For, example, between 1988 and 1990 approximately 66% of patients did not heal. By 1999 this percentage had decreased to 49%. Most of the change in improvement occurred before 1998. We noted that the change in the rate of failure to heal was very closely associated with an increase over time in the proportion of patients seen with wounds identified as prognostically favorable as determined by a previously published prognostic model (i.e., individuals with wounds ≤ 2 cm2, wound less ≤2 months old, and wound of grade less ≤ 2). Among those most likely to heal, the likelihood of failing to heal went from 62% in 1990 to 32% in 2000. Therefore, the overall decrease in likelihood that a patient with a DNFU would not heal is not just due to the enrollment of less severe cases. There was, however, no real improvement in the likelihood that those with wounds that were unlikely to heal over time and fewer patients with these wounds sought care over time.

In conclusion, those with a DFFU seeking care are more likely to heal today than 10 years ago. This improvement is related to the fact that individuals are seeking care when their wounds are most easily treated and these wounds are now more likely to heal. Unfortunately, those with the most severe wounds are not anymore likely to heal today than they were 10 years ago.
019 Effectiveness of Oasis® Wound Matrix Versus Regranex® in Treating Diabetic Wounds
Authors: Niezgoda J. A.
Published: March 2007

Background: Current care for chronic diabetic wounds involves maintaining wound hydration, providing protection from infection through regular dressing changes, and providing appropriate pressure relief. Even with the best care available, such therapies lead to effective wound healing in only 24% of ulcers within 12 weeks. Treatment of chronic ulcers with recombinant growth factors only moderately improves 12‐week healing rates, to 34%. A new, bioactive material, the Oasis® Wound Matrix, comprises a full complement of structural extracellular matrix components and associated growth factors and may be more effective in managing chronic wounds than other current alternatives. Method: Interim data from 88 evaluable patients in this multicenter, prospective, randomized clinical trial are presented. Patients diagnosed with chronic, full‐thickness, diabetic ulcers were enrolled and randomly assigned to receive either weekly treatments of Oasis Wound Matrix or daily treatments of Regranex® becaplermin gel. All wounds were carefully cleaned and irrigated weekly for the duration of the study, with debridement performed as needed; wound areas were recorded to track the rate of wound closure. Incidence of complete wound healing by 12 weeks was evaluated.  Results: Study groups were balanced with respect to patient age and gender. Currently, 53%(24/45) of patients receiving Oasis wound matrix are considered healed versus 35%(15/43) of patients receiving treatment with Regranex. Conclusion: These data clearly demonstrate Oasis wound matrix is at least as effective as becaplermin gel in managing chronic, full‐thickness, diabetic ulcers.
The effect of maternal depression and marital adjustment on young children's internalizing and externalizing behaviour problems
Authors: Trapolini T. , McMahon C. A. , Ungerer J. A.
Published: March 2007

Background  The present study aimed to clarify the circumstances under which maternal depression is associated with adverse outcomes in pre‐school‐aged children, and to explore the additional impact of the marital relationship in a socio‐economically low‐risk sample.

Methods  Ninety‐two mothers recruited into a longitudinal study were assessed for symptoms of depression when their children were 4, 12 and 15 months, and later at 4 years of age. At 4 years of age, mothers were also asked to report on marital adjustment. Mothers, fathers and pre‐school teachers were asked to report on children's internalizing and externalizing behaviour problems.

Results  Children exposed to chronic maternal depression were rated by their parents as significantly more problematic on internalizing and externalizing behaviours. This association was not mediated or moderated by low marital satisfaction. No parent‐reported effects were evident for children exposed to transient depression. Ten per cent of the children had internalizing scores in the clinical range, and 7% had externalizing scores in the clinical range. Almost all of the children with clinically elevated scores had been exposed to chronic maternal depression. Teacher reports of internalizing behaviours corroborated parent reports; however, teachers tended to report significantly lower levels of behaviour problems. Although there was a tendency for teachers to rate children exposed to any depression as more problematic on internalizing behaviours, the subgroup differences were not significant.

Conclusions  Findings confirm that depression chronicity is important in determining child behavioural outcomes, and that depression effects cannot be accounted for by low marital adjustment. Results are discussed with reference to transmission of risk models, and treatment implications are considered.
The costs of key worker support for disabled children and their families
Authors: Beecham J. , Sloper P. , Greco V , Webb R.
Published: March 2007

Background  Key workers provide a single point of contact for disabled children and their families, supporting them and facilitating access to other services. Key worker services appear to generate good outcomes for families and provision is encouraged through central government policy. Recent increases in key worker provision have been accompanied by a proliferation of delivery models yet there is very little information on their costs.

Methods  Within a study identifying which service characteristics generate good outcomes for families, unit costs for seven key worker services were estimated. Informed by economic theory, the estimates drew on data from managers, finance departments and other publicly available sources. Data on the frequency and duration of contact with key workers were obtained from families and combined with the service‐specific unit costs to arrive at the contact cost per family. The variations in the costs of supporting families were explored.

Results  On average, key worker contact with disabled children and their families cost £151 over a 3‐month period. Contact costs were found to vary with the level of the child's disability and the number of role aspects that key workers performed. This latter variable was associated with improved outcomes.

Conclusion  The low response rate to the questionnaire and absence of data on the costs of other elements of the families' support packages may limit generalization from this study. Even so, such findings can help inform managers and planners about allocation of their scarce resources. Good‐quality services appear to cost more but the relationship between cost, quality and outcomes is not a simple one.
The impact of percutaneous endoscopic gastrostomy feeding in children; the parental perspective
Authors: Brotherton A. M. , Abbott J. , Aggett P. J.
Published: March 2007

Background  The prevalence of percutaneous endoscopic gastrostomy (PEG) feeding of children in the UK has increased. This study explored how gastrostomy feeding affects the daily lives of children and their families, from the parents' perspectives and developed a conceptual framework for assessing the impact of feeding on family life.

Methods  A semi‐structured interview approach was used to obtain the parents' views regarding the impact of feeding on daily lives; both of the children and their family. The study was a cross‐sectional qualitative design employing purposive sampling. Twenty‐four interviews were conducted and data were analysed descriptively and thematically.

Results  Difficulties arising from PEG feeding included vomiting (71%), diarrhoea (33%), infection of the PEG site (46%) and leakage (54%). The key issues that emerged included delayed and disturbed sleep, restricted ability to go out, difficulties finding a place to feed, childcare problems, negative attitudes of others towards feeding and family divisions. Parents primarily raised key issues regarding the impact of PEG feeding on themselves or the family rather than on the child.

Conclusions  An assessment framework has been developed, based on the parents perceptions, for use in assessing the impact of feeding upon family life and identifying the support required for parents of children receiving PEG feeding. Provision of adequate support is likely to require a network of healthcare professionals, social care and support groups, all working together to meet identified needs.
Diagnostic significance of ‘benign pairs’ and signet ring cells in fine needle aspirates (FNAs) of the breast
Authors: Sturgis C. D. , Sethi S. , Cajulis R. S. , Hidvegi D. F. , Yu G. H.
Published: March 2007

sturgis c. d., sethi s., cajulis r. s., hidvegi d. f. and yu g. h. (1998) Cytopathology9, 308–319

Diagnostic significance of ‘benign pairs’ and signet ring cells in fine needle aspirates (FNAs) of the breast

Most breast mass lesions are readily characterized by FNA with cytodiagnosis. Occasionally, benign but markedly proliferative lesions are diagnostically difficult to separate from well differentiated malignancies. We present information pertaining to the diagnostic significance of two cytologic findings observed in breast aspiration specimens, namely pairs of stripped bipolar nuclei and signet ring cells (SRC). We have evaluated aspirate smears from 219 cases of histologically proven benign (n= 114) and malignant (n= 105) breast lesions. Both singlets and pairs of bipolar nuclei and SRC were enumerated, and their numbers were correlated to histological diagnosis. Closely associated pairs of stripped bipolar nuclei were found in 68% of benign lesions compared with only 3.8% of carcinomas, establishing their presence as a highly specific indicator of a benign process. Large numbers of such ‘benign pairs’ also favoured the diagnosis of fibroadenoma. SRC were identified in 66% of histologically proven carcinomas (67% of ductal and 70% of lobular). SRC were also present in 10% of histologically benign cases. In the malignant cases, SRC were most frequently noted in a single cell distribution or within small, loosely cohesive tissue fragments. In the benign instances, SRC were most commonly noted within large fragments, and many of these cells were proved by immunohistochemical reactions to be vacuolated myoepithelial cells. We conclude that the presence of bipolar nuclei in closely associated pairs suggests benignity and aids in the subclassification of benign breast masses. In addition, the presence of SRC does not aid in the classification of tumour subtype (ductal vs lobular), and the occurrence of such cells in the proper context should prompt surgical biopsy.
Density‐related effects of deer browsing on the regeneration dynamics of boreal forests
Authors: TREMBLAY JEAN‐PIERRE , HUOT JEAN , POTVIN FRANÇOIS
Published: March 2007

The density of large herbivores is a major driver of forest ecosystem structure and function in conjunction with episodic disturbances, especially in forests with a regeneration strategy based on shade‐tolerant seedlings capable of re‐establishing canopy dominance (advance regeneration). Yet, uncertainty about the relationships between forest regeneration, herbivore density and other disturbances makes it difficult to set population goals. Using an innovative controlled browsing experiment, we investigated the relationships between the regeneration dynamics of balsam firAbies balsamea, the density of white‐tailed deerOdocoileus virginianusand timber harvesting.

We hypothesize that advance tree regeneration either: (i) recovers approximately linearly as deer density is reduced; (ii) recovers exponentially; or (iii) does not recover because factors other than browsing control advance regeneration. We tested these alternatives through manipulation of deer densities (0, 7·5, 15 deer km−2andin situlocal densities) and forest cover (clearcut and uncut forest).

Balsam fir seedling mortality decreased exponentially with decreasing deer density in clearcut and approximately linearly in uncut forest. Independently of deer density, the recruitment of seedlings in clearcut dropped from 56 ± 5% to 7 ± 1% within 3 years.

Seedling growth increased exponentially with decreasing deer density in clearcut whereas no height growth was observed in uncut forest.

Overall, the abundance of fir saplings recovered exponentially in clearcut but remained low and independent of deer density in uncut forest. The abundance of sprucePiceaspp. saplings was unrelated to deer density and increased with time.

Synthesis and applications. Forest disturbance from selective browsing at high deer densities over an extended period of time leads to recruitment failure following a canopy disturbance such as a clearcut. Indirect competitive advantage given to species resistant to browsing can shift forest composition. Nonlinear relationships between fir regeneration and deer densities imply that the level of culling required to reach herbivore densities compatible with natural regeneration of native forest is larger than expected if tree regeneration was proportional to deer density. In the boreal forest of Anticosti Island, local densities < 15 deer km−2achieved within 3 years following clearcut are compatible with the maintenance of native forest.
Plant colonization in heterogeneous landscapes: an 80‐year perspective on restoration of broadleaved forest vegetation
Authors: BRUNET JÖRG
Published: March 2007

Afforestation of agricultural land has become an important issue in Europe during the past two decades. In particular, broadleaf plantations have been promoted as multifunctional forests for biodiversity, timber production and recreation. The aim of this study was to analyse how the colonization of ground‐layer plants in recently planted woodlands is affected by stand age, spatial isolation, former land‐use type and canopy species.

Colonization was studied in 50 plantations of pedunculate oak Quercus robur and 16 plantations of European sycamore Acer pseudoplatanus on former arable land and open pastures in the Torup–Skabersjö area of southern Sweden. Twelve oak plantations and 10 sycamore plantations on ancient woodland sites were used as reference areas.

Woodland species richness increased continuously with stand age in plantations contiguous with ancient woodland. The oldest plantations (70–80 years) approached the species richness of ancient oak woodland. Species richness decreased with increasing distance from ancient woodlands, and there was no significant increase in species richness between stands aged 20 and 80 years in isolated plantations. Most species that regularly colonized isolated plantations had adhesive seeds or small, wind‐dispersed diaspores.

Ordination analysis showed clear differentiation between former land‐use categories and spatiotemporal stand categories (recently isolated, recently contiguous and ancient). No consistent differences were found in vegetation patterns in oak and sycamore stands. Woodland species richness was similar on former arable land and open pastures. Pasture plantations were characterized by the presence of more acid‐tolerant species, whereas the frequency of some acid‐sensitive species was higher on former fields.

Synthesis and applications. This study demonstrates that new woodland should build out from cores of ancient woodland for optimal ground vegetation development. A species‐rich ground vegetation can be achieved by spontaneous colonization within 70–80 years when plantations are contiguous with ancient woods. Formerly cultivated soils are generally well suited for understorey plants of forests on base‐rich loamy soils as long as closed canopy conditions are maintained. However, many typical forest species are not able to disperse across open fields and their establishment in isolated stands will require sowing or planting.
The TB pandemic: an old problem seeking new solutions
Authors: Meya D. B. , McAdam K. P. W. J.
Published: March 2007

Tuberculosis (TB) continues to kill more than 2 million people globally each year. Annual TB case notification rates have risen up to fourfold since the mid‐1980s, with the highest rate of 1000/100 000 around Cape Town, South Africa. There is an urgent need for novel diagnostic methods and preventive vaccines to control this epidemic. The rising incidence of TB has been attributed to HIV co‐infection especially in developing countries. The threat of drug resistance arising from ineffective TB treatment programmes is looming and could potentially lead to loss of any gains made in controlling the disease globally.