Is population genetic structure of vascular plants shaped more by ecological or geographic factors? A study case on the Mediterranean endemic Centaurea filiformis (Asteraceae).

All known populations of the Sardinian endemic Centaurea filiformis Viv. (Asteraceae) were studied in order to understand the impact of both geographic and ecological factors on the genetic structuring of this species. Fourteen populations and 234 individuals were sampled. The demographic structure of the populations and the reproductive ecology were estimated in 28 plots. Population genetic analyses were based on SSR markers. Genetic structure was investigated by spatial Bayesian methods. Average densities of 0.51 individuals m-2 were detected, with a prevalence of adults. Ten species of pollinators were identified; C. filiformis ability to self-pollinate and myrmecochory were demonstrated experimentally. The populations displayed an average heterozygosity value of He  = 0.576 and high genetic differentiation (overall FST  = 0.218). Bayesian analysis suggests that five is the most probable number of gene pools of origin. A strong correlation between geographic distances and genetic distances among populations was highlighted. The demographic population structure of C. filiformis is dominated by adults, suggesting that it is a stable-regressive or senile species, investing more in local persistence than colonisation ability. Despite the scattered distribution, the populations studied do not present evidence of genetic erosion. The analysis of genetic differentiation reveals very high differentiation levels among populations, thus indicating that effective barriers exist against gene flow. A general conclusion is that population distribution results in a clear genetic structure for the populations studied, and that geography and not ecology is shaping the present distribution of this species.

Hydrosurgery as a safe and efficient debridement method in a clinical wound unit.

OBJECTIVE: Hydrosurgical debridement allows removal of non-viable tissue, preserving healthy tissues. This study was designed to analyse whether hydrosurgery, used in a clinical wounds unit, is an effective and safe method that may reduce debridement time. METHODS: Patients' wounds had the following characteristics: wounds with devitalised tissue needing rapid debridement, wounds with cavities, or non-healing wounds. Hydrosurgical debridement uses a pressurised stream of saline (0.9% sodium chloride) and a vacuum around this stream to remove the devitalised tissue of the wound, preserving healthy surrounding tissues. RESULTS: This prospective study comprised of 53 wounds from 39 patients. The wound aetiology included 39.7% arterial insufficiency, 22.6% pressure ulcers (PUs), 15.1% diabetic foot ulcers (DFUs), 9.4% venous leg ulcers (VLUs), and 13.2% from other aetiologies. The percentage of wounds according the size was the following: 32.1% (<10cm2), 43.4% (10-49cm2), 15.1% (50-99cm2), and 9.4% (≥100cm2). Superficial wounds were 43.4% of the total and 56.6% of wounds had cavities. Pain associated with the hydrosurgery was mild to moderate. There were no hydrosurgery-related adverse events. For effective debridement, the required sessions were as follows: one procedure (73.6%), two procedures (18.9%) and three procedures (7.5%). There was a statistical significant direct correlation (r=0.307) between the number of required sessions and wound size. All patients improved in a week (>80% of granulation tissue). CONCLUSION: We demonstrate that hydrosurgery is an effective and rapid debridement method that can be used safely in the outpatient setting.

Low expression of Abelson interactor-1 is linked to acquired drug resistance in Bcr-Abl-induced leukemia.

The basis for persistence of leukemic stem cells in the bone marrow microenvironment remains poorly understood. We present evidence that signaling cross-talk between α4 integrin and Abelson interactor-1 (Abi-1) is involved in the acquisition of an anchorage-dependent phenotype and drug resistance in Bcr-Abl-positive leukemia cells. Comparison of Abi-1 (ABI-1) and α4 integrin (ITGA4) gene expression in relapsing Bcr-Abl-positive CD34+progenitor cells demonstrated a reduction in Abi-1 and an increase in α4 integrin mRNA in the absence of Bcr-Abl mutations. This inverse correlation between Abi-1 and α4 integrin expression, as well as linkage to elevated phospho-Akt and phospho-Erk signaling, was confirmed in imatinib mesylate -resistant leukemic cells. These results indicate that the α4-Abi-1 signaling pathway may mediate acquisition of the drug-resistant phenotype of leukemic cells.

Identification of novel type VII collagen gene mutations resulting in severe recessive dystrophic epidermolysis bullosa.

In this work, we studied the proband in a small nuclear family of Chinese and Dutch/German descent and identified two novel mutations in the type VII collagen gene leading to recessive dystrophic epidermolysis bullosa, Hallopeau-Siemens variant (HS-RDEB). The maternal mutation is a single base pair deletion of a cytosine nucleotide in exon 26, designated 3472delC, resulting in a frameshift and a premature termination codon (PTC) within the same exon, 7 bp downstream of the site of the mutation. The paternal mutation is a G-->A transition located at the 5' donor splice site within intron 51, designated IVS51 + 1G-->A. This mutation leads to the activation of a cryptic splice site, 32 bp downstream of the mutation site and to subsequent aberrant out-of-frame splicing, resulting in two alternative mRNA transcripts and a downstream PTC. To our knowledge, these two mutations have not been previously reported. These findings extend the body of evidence for compound heterozygous mutations leading to HS-RDEB and provide the basis for prenatal diagnosis in this family.

The marrow stem cell: the continuum.

The marrow hematopoietic stem cell is currently being redefined as to all aspects of its phenotype and its total differentiation capacity. This redefinition now includes its plasticity as to production of nonhematopoietic and hematopoietic cell types, the determinants of its in vivo engraftment potential and its expression of stem cell functional characteristics.

Cholesterol crystal embolization: an atherosclerotic disease with frequent and varied cutaneous manifestations.

In this paper the basic pathogenesis of cholesterol crystal embolization (CCE) is described, its clinical characteristics are presented and diagnosis and therapy are discussed. The main focus will be on the cutaneous manifestations; however, considering that CCE is a systemic illness, findings in other organs will also be highlighted, particularly the commonly involved renal and gastrointestinal systems.

Marrow stem cell potential within a continuum.

On the basis of our studies of the fluctuation of the hematopoietic stem cell phenotype with cell cycle trnsit, we hypothesize that the ability of marrow stem cells to convert to nonhematopoietic cells will also vary at different points in the cell cycle. The new biology of stem cells has an impact on many fields including developmental biology and stem cell biology and the clinical potential is enormous.

Systemic 5-fluorouracil producing an inflammatory response in porokeratosis.

Topical 5-fluorouracil has been used as an effective treatment for porokeratosis. Upon its treatment, an inflammatory effect occurs with the topical 5-fluorouracil. We report a case of a patient with disseminated superficial actinic porokeratosis displaying a comparable inflammatory process following therapy with systemic 5-fluorouracil used to manage a metastatic breast cancer.

Slow release iodine preparation and wound healing: in vitro effects consistent with lack of in vivo toxicity in human chronic wounds.

BACKGROUND: Antiseptic agents, particularly slow-release preparations, are increasingly being used in the management of chronic wounds. One such agent, cadexomer iodine, carries iodine (0.9% weight/weight) immobilized in beads of dextrin and epichlorhydrin and has been demonstrated to be highly effective in promoting healing of exudative wounds. However, there have been no studies directly assessing the potential lack of toxicity of cadexomer iodine on human cutaneous tissues. OBJECTIVES: To determine if, within a certain concentration range, cadexomer iodine is non-toxic to human cells and cutaneous tissue and to assess histologically human chronic exudative wounds that are being treated with cadexomer iodine. METHODS: We examined the effects of varying concentrations of cadexomer iodine on the viability of human fibroblasts in culture (by trypan blue exclusion). The morphology, cellular proliferation capacity (measured by [3H]thymidine uptake), ability to produce alpha 1(I) procollagen chain mRNA, and cell outgrowth from neonatal foreskin explants were also evaluated in human fibroblasts after incubation with various concentrations of cadexomer iodine. Moreover, biopsies of chronic exudative wounds concurrently treated with cadexomer iodine were stained with haematoxylin and eosin or a Gram stain and evaluated microscopically. RESULTS: At concentrations of up to 0.45%, cadexomer iodine was found to be non-toxic to fibroblasts in vitro; there were no changes in viability, morphology, cellular proliferation, ability to produce collagen, and cell outgrowth from explants. In vivo, skin biopsies of chronic exudative wounds being treated with cadexomer iodine demonstrated no evidence of cell necrosis, displayed re-epithelialization, and revealed bacteria within the cadexomer beads. CONCLUSIONS: These studies demonstrate that cadexomer iodine has definite non-toxic concentration ranges for fibroblasts in vitro, which are consistent with a lack of cellular toxicity in human chronic exudative wounds treated with cadexomer iodine. Cadexomer iodine may also have the additional property of trapping microorganisms.

Healing of venous ulcers of long duration with a bilayered living skin substitute: results from a general surgery and dermatology department.

BACKGROUND: A laboratory-grown bilayered living skin substitute (LSS) has been shown to accelerate the healing of venous ulcers. However, issues related to optimal wound bed preparation prior to the application of LSS have not been addressed. OBJECTIVE: When combined with standard compression therapy and near elimination of wound exudate, bioengineered skin can achieve complete closure of venous ulcers which have been present for more than a year and which are difficult to heal. METHODS: In the general surgery (center A) and dermatology (center B) departments at two separate medical centers, LSS was used to treat venous ulcers of more than 1 year's duration and which had been unresponsive to conventional therapy. Wound bed preparation at both centers had as common goals the removal of necrotic tissue, optimal formation of granulation tissue, and elimination of wound exudate. RESULTS: There was great comparability between the two centers in the patients being treated, wound size and duration, and number of LSS applications. Both centers achieved a frequency of complete wound closure of greater than 70% within 6 months. CONCLUSION: At two separate clinical and specialty sites having a common goal of optimal wound preparation, treatment with LSS was associated with a high rate of complete closure of hard to heal venous ulcers.

Short-contact topical tretinoin therapy to stimulate granulation tissue in chronic wounds.

BACKGROUND: The use of retinoids in wound healing is increasing. It has been shown that retinoic acid reverses the inhibitory effects of glucocorticoids on wound healing and accelerates the formation of healthy granulation tissue. Pretreatment with tretinoin before epidermal injury such as chemical peeling and dermabrasion has shown accelerated wound healing. Enhanced healing of full-thickness skin wounds has also been demonstrated in early wound healing studies. However, tretinoin therapy can be quite irritating. OBJECTIVE: Our purpose was to observe the clinical and histologic effects of topical tretinoin solution 0.05% applied directly to the wound beds of chronic leg ulcerations. METHODS: We report on the cases of 5 patients with long-standing leg ulcerations. All were treated with topical tretinoin solution 0.05% applied directly to the wound bed. The tretinoin solution was left in contact with the ulcer bed for a maximum of 10 minutes daily and then rinsed with normal saline. Punch biopsy specimens were obtained from the wound beds at baseline and mid therapy. Standard wound care was continued throughout the study. RESULTS: In this study we found that as early as 1 week after treatment with topical tretinoin solution 0.05%, there was increased granulation tissue first noted at the wound's edge. After 4 weeks of therapy with tretinoin, there was further stimulation of granulation tissue, new vascular tissue, and new collagen formation. CONCLUSION: Short-contact tretinoin therapy is a novel modality in which to treat chronic ulcers and stimulate the formation of granulation tissue.

Gene therapy.

With recent advances in molecular biology, the ability to transfer genes to patients is becoming a reality. Ongoing clinical trials using gene transfer techniques have illustrated the potential and pitfalls of this new therapeutic modality for the treatment of a wide variety of disorders. While these techniques are not currently a part of routine clinical practice, it is only a matter of time until some form of gene therapy is approved for general use in the clinic. This review highlights some of the basic methods used in current gene therapy protocols. The objective of this review is to familiarize practitioners with these concepts so they can more effectively follow the progress of this emerging technology and better inform their patients.

Growth inhibition of primary keratinocytes following transduction with a novel TGFbeta-1 containing retrovirus.

Growth and migration of keratinocytes are known to be affected by the addition of exogenous cytokines, such as TGFbeta-1, to culture media. We have developed a retroviral vector, LNTbeta-1, that confers constitutive expression of human TGFbeta-1 to transduced cells. Keratinocytes were exposed to retroviral particles generated in serum-free media, and infected cells were selected for with Geneticin. Transduced keratinocytes remained in culture as single cells instead of a normally grouped growth pattern. While these transduced keratinocytes survived in culture for several weeks, they did not proliferate and seemed arrested in their growth. Keratinocytes transduced with retrovirus not containing the TGFbeta-1 gene appeared normal in their growth pattern. These findings indicate that high-level endogenous expression of TGFbeta-1 in keratinocytes can at least inhibit, and possibly arrest, growth.

Graftskin, a human skin equivalent, is effective in the management of noninfected neuropathic diabetic foot ulcers: a prospective randomized multicenter clinical trial.

OBJECTIVE: We assessed in a randomized prospective trial the effectiveness of Graftskin, a living skin equivalent, in treating noninfected nonischemic chronic plantar diabetic foot ulcers. RESEARCH DESIGN AND METHODS: In 24 centers in the U.S., 208 patients were randomly assigned to ulcer treatment either with Graftskin (112 patients) or saline-moistened gauze (96 patients, control group). Standard state-of-the-art adjunctive therapy, which included extensive surgical debridement and adequate foot off-loading, was provided in both groups. Graftskin was applied at the beginning of the study and weekly thereafter for a maximum of 4 weeks (maximum of five applications) or earlier if complete healing occurred. The major outcome of complete wound healing was assessed by intention to treat at the 12-week follow-up visit. RESULTS: At the 12-week follow-up visit, 63 (56%) Graftskin-treated patients achieved complete wound healing compared with 36 (38%) in the control group (P = 0.0042). The Kaplan-Meier median time to complete closure was 65 days for Graftskin, significantly lower than the 90 days observed in the control group (P = 0.0026). The odds ratio for complete healing for a Graftskin-treated ulcer compared with a control-treated ulcer was 2.14 (95% CI 1.23-3.74). The rate of adverse reactions was similar between the two groups with the exception of osteomyelitis and lower-limb amputations, both of which were less frequent in the Graftskin group. CONCLUSIONS: Application of Graftskin for a maximum of 4 weeks results in a higher healing rate when compared with state-of-the-art currently available treatment and is not associated with any significant side effects. Graftskin may be a very useful adjunct for the management of diabetic foot ulcers that are resistant to the currently available standard of care.

Accelerated healing of pyoderma gangrenosum treated with bioengineered skin and concomitant immunosuppression.

Pyoderma gangrenosum is a rare, destructive, neutrophilic dermatosis, the origin of which remains largely obscure. The ulcerative variant of this inflammatory disorder causes painful, necrotic, rapidly enlarging ulcers. Because of pathergy, many clinicians avoid managing these nonhealing ulcers with aggressive surgical debridement and autologous grafts. This article proposes that the application of an allogeneic cultured human skin equivalent (Graftskin) not only circumvents this problem, but also hastens re-epithelialization of the ulcer bed. An added benefit of the possible improvement of the cosmetic appearance of the final scar by preventing severe wound contracture is also postulated. We report a newly diagnosed case of ulcerative pyoderma gangrenosum; the use of bioengineered skin as an adjunct to concurrent immunosuppressive therapy with cyclosporine hastened the healing and diminished pain in a rapidly enlarging leg ulcer. Within 2 weeks, the ulcer was 30% to 40% healed, achieving 100% re-epithelialization within 6 weeks.

Randomized trial of topically applied repifermin (recombinant human keratinocyte growth factor-2) to accelerate wound healing in venous ulcers.

About 600,000 people in the United States are estimated to be affected by venous ulcers. The cornerstone of care of chronic venous ulcers involves the application of compression bandages. Other therapies include treatment of associated infection, treatment for edema and inflammation, and debridement when necessary. Repifermin, a recombinant human KGF-2 (fibroblast growth factor-10), exerts a proliferative effect on epithelial cells, in vitro and in vivo, and has been shown to accelerate wound healing in several experimental animal models. A randomized, double-blind, parallel-group, placebo-controlled, multicenter study was conducted to evaluate the safety and efficacy of topical repifermin treatment, for 12 weeks, in the healing of chronic venous ulcers in 94 patients. Repifermin was shown to accelerate wound healing, with significantly more patients achieving 75% wound closure with repifermin than with placebo. The treatment effect appeared more marked for a subgroup of patients with initial wound areas < or = 15 cm2 and wound ages of < or = 18 months. A longer duration of treatment (e.g., 26 weeks) may allow better differentiation of the benefit of repifermin compared with placebo, particularly with respect to complete wound closure. The safety assessment showed that repifermin was well tolerated.

Lack of co-ordinate expression of the alpha1(I) and alpha1(III) procollagen genes in fibroblast clonal cultures.

BACKGROUND: Several extracellular matrix genes, most notably alpha1(I) and alpha1(III) procollagen, are reported to be co-ordinately expressed in cultures of dermal fibroblasts. However, it remains unclear whether the expression of these genes is truly co-ordinate or whether it may be the result of averaging the phenotypic expression of different fibroblast subpopulations present within each culture. Objectives To determine by Northern analysis the correlation between alpha1(I) and alpha1(III) procollagen mRNA levels in clonal populations of human dermal fibroblasts. METHODS: As previously described, clonal cultures were derived from parent strains of human dermal fibroblasts by a microscopically controlled dilution technique and by stimulation of single cells with low oxygen tension in the early phases of clonal growth. RESULTS: In agreement with previous reports, we found that baseline steady-state levels of alpha1(I) procollagen mRNA were co-ordinately regulated with the alpha1(III) procollagen mRNA in 26 parent strains (r = 0. 9003; P < 0.0001). However, this close correlation between the expression of these two procollagen chains was absent in a total of 40 unselected clonal strains derived from four of the parent cultures (r = 0.5745; P < 0.0001). Moreover, this intrachain heterogeneity in alpha1(I) and alpha1(III) procollagen mRNA levels in clonal cultures was statistically significant from that measured in parent strains (P = 0.0016). CONCLUSIONS: alpha1(I) and alpha1(III) procollagen mRNA levels in clonal cultures do not show the tight co-ordinate regulation observed in non-clonal cultures, suggesting that these two genes operate under different sets of regulatory controls. This clonal heterogeneity may provide additional flexibility to the process of tissue repair and fibroblast clonal expansion.

Tissue engineering in wound repair.

Tissue-engineering products can cover wounds and provide a microenvironment that stimulates their repair. To date, Graftskin (APLIGRAF, Organogenesis Inc, Canton, MA, and Novartis Pharmaceuticals Corporation, East Hanover, NJ) is the most advanced bioengineered skin product. Graftskin is a bilayered living skin construct consisting of a dermis and a well-differentiated epidermis. The epidermal cells (keratinocytes) and dermal cells (fibroblasts) are obtained from neonatal foreskin. The efficacy of Graftskin in healing venous ulcers was evaluated in a prospective, randomized study of 240 patients at 14 centers over a 6-month period. Patients received either compression therapy plus Graftskin or compression therapy alone (active control). Clinical efficacy was defined as complete wound closure. Treatment with Graftskin was more effective than compression alone as measured by time to complete wound closure and frequency of complete wound closure by 6 months. When applied to hard-to-heal wounds (> 1 year's duration), Graftskin was 3 times more effective than compression therapy alone in achieving complete wound closure at 8 weeks (32% vs 10%, P = .008) and 2 times more effective by 24 weeks (47% vs 19%, P = .002). Graftskin is highly effective in healing venous ulcers, particularly those of long duration, that have proved hard to heal with conventional modalities.