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.

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.

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.

Postoperative pressure-induced alopecia: report of a case and discussion of the role of apoptosis in non-scarring alopecia.

We report a case of postoperative pressure induced alopecia in a 21-year-old black female after multiple intraoperative procedures. The histopathology is distinctive and demonstrated features in common with trichotillomania and alopecia areata, including the presence of pigment casts, catagen follicles, melanophages and apoptotic bodies. External hair manipulation is considered the primary event in the etiology of pigment casts, however, our present case demonstrated numerous pigment casts despite a complete lack of evidence of external hair manipulation. We performed pattern analysis and in situ end-labeling in 19 cases of non-scarring alopecia. Pigment casts were seen in postoperative alopecia (1 case), alopecia areata (1 case) and trichotillomania (5 cases). These forms of alopecia have in common the sudden termination of the anagen phase of the hair cycle. When the anagen portion of the hair cycle is prematurely disrupted hairs enter into catagen. Pigment casts may represent a non-specific reaction pattern of follicles that are suddenly transformed from anagen to catagen. We therefore propose that hair manipulation is not uniquely responsible for the formation of pigment casts. The primary pathophysiology resulting in the formation of pigment casts more correctly reflects the sudden termination of the anagen phase of the hair cycle.

Histological comparison of postoperative wound care regimens for laser resurfacing in a porcine model.

BACKGROUND: The use of short-pulsed CO2 lasers for skin resurfacing is routinely performed, but few studies have examined postsurgical care. OBJECTIVE: To determine which postoperative treatments are most beneficial in promoting optimal healing after laser resurfacing. METHODS: Four pigs received laser resurfacing. The laser sites were randomly left untreated or treated with petroleum-based ointment or dressed with 1 of the following occlusive dressings: hydrocolloid, hydrogel or foam. Biopsies were taken from each treatment group on Days 2, 3, 4, 5, 8, 12, and 19. All samples were stained with hematoxylin and eosin. Each histological slide was evaluated by a blinded investigator. RESULTS: Differences were observed between treatment groups in the amount of cellular infiltrate, presence of necrotic tissue, progression of the epidermal sheet, maturation of the epidermis, presence of rete ridges, and appearance of new collagen. CONCLUSION: Postoperative treatments after laser resurfacing vary in their ability to influence the quality of healing.

TGF-beta3 stimulates and regulates collagen synthesis through TGF-beta1-dependent and independent mechanisms.

In contrast to the TGF-beta1 and beta2 isoforms, TGF-beta3 has shown the ability to downregulate scarring and fibrosis in vivo under certain experimental conditions. In this study, we determined the direct effects of TGF-beta3 on cultures of human dermal fibroblasts. TGF-beta3 (0.1 to 100 pg per ml) increased DNA synthesis up to 50% (p < 0.01, r = 0.970), collagen protein synthesis up to 200% (dose range of 0.1 to 5 ng per ml, p < 0.001, r = 0.990), and increased alpha1(I) procollagen mRNA levels (r = 0.999), with maximal effects (200% of control) observed by 24 h. Collagen lattice contraction was increased by more than 50% in response to TGF-beta3 (p < 0.001), and to a similar extent as the TGF-beta1 isoform. Stimulation of collagen synthesis and of alpha1(I) procollagen mRNA levels in response to TGF-beta3 was partially blocked by a TGF-beta1-specific anti-sense oligonucleotide but was still detectable (35% greater than baseline) when TGF-beta3 was added to dermal fibroblasts from TGF-beta1 knock-out mice. In contrast with these stimulatory effects, however, downregulation of alpha1(I) procollagen, alpha1(III) procollagen, and TGF-beta1 mRNA levels toward baseline occurred when TGF-beta3 (0.1 to 5 ng per ml) was added simultaneously and in combination with TGF-beta1. We conclude that stimulation of collagen synthesis by TGF-beta3 occurs through TGF-beta1-dependent and independent pathways. By downregulating the response to TGF-beta1 and by shifting from one pathway to the other, TGF-beta3 can dampen and provide fine-tuning to the overall TGF-beta's induced program of collagen deposition.

Dermal fibroblasts from venous ulcers are unresponsive to the action of transforming growth factor-beta 1.

Failure to reepithelialize is the major clinical problem in venous ulcers. It is not clear whether the problem resides with keratinocytes or with inadequate and improper formation of extracellular matrix. In this study, we characterized the biosynthetic activity and response to transforming growth factor-beta 1 (TGF-beta) of dermal fibroblast cultures isolated from biopsies of venous ulcers and from normal thigh skin (controls) of seven patients. We found that baseline 3H-proline incorporation was similar in fibroblasts from venous ulcers and control skin (p= 0.1716). No difference was detected by ELISA between ulcer and control fibroblasts in the synthesis of total TGF-beta (p = 0.2309), and the mRNA levels for alpha 1(I) procollagen and TGF-beta were comparable in both groups. However, TGF-beta (0.1-5 ng/ml) enhanced collagen protein synthesis by more than 60% and in a dose-dependent manner (r = 0.997) in control fibroblast cultures, while failing to stimulate collagen production by venous ulcer fibroblasts (p = 0.0001). This unresponsiveness to TGF-beta was associated with up to a fourfold decrease in TGF-beta Type II receptors. We conclude that fibroblasts from the edge of non-healing venous ulcers are unresponsive to the action of TGF-beta, and that this blunted response may cause faulty deposition of the extracellular matrix needed for reepithelialization and wound healing.

Retrovirally mediated gene transfer in a skin equivalent model of chronic wounds.

Given that treatment for chronic wounds is unsatisfactory, it is likely that gene therapy may be tested as a therapeutic modality in this difficult clinical problem. Actively proliferating cells in wounds are also a good target for retroviral transduction, an increasingly useful method for gene therapy. However, it is unclear how gene therapy may best be used in chronic wounds, and experimental models are urgently needed to study and manipulate gene transfer in the context of chronic wounds. In this report, partial- and full-thickness wounds were made in vitro in a human living skin equivalent (LSE) consisting of fully differentiated keratinocytes layered over a collagen matrix seeded with fibroblasts. To mimic a chronic wound situation, we used tissue culture conditions which, as in a chronic wound, allowed fibroblast but not keratinocyte proliferation or migration. The wounded LSE was then placed over a transduced cell line (PA317) which produced a replication defective retrovirus containing as a histological marker the bacterial beta galactosidase gene. Using this close and direct exposure to the virus-producing cell line, distinct staining for beta-galactosidase was observed in partial-thickness wounds, and was limited to fibroblasts away from the upper site of injury and immediately overlying the retrovirus-producing cell monolayer. Expression of beta-galactosidase was uniformly present at the wound edges and along the base of the entire partial thickness wound. These studies demonstrate that, in in vivo conditions mimicking a chronic wound, an intimate apposition of the injured LSE with the virus-producing cell line is needed for gene transfer. Using this in vitro model system, gene transfer protocols may be optimized prior to beginning in vivo studies in chronic wounds.

An in vivo analysis of c-myc and c-fos expression during terminal erythroid differentiation in mouse spleen progenitors.

Using thiamphenicol and scheduled bleeding, we were able to induce an adequate number of erythroid stem cells (CFU-e) in mice in order to conduct an in vivo study of the changing expression of c-myc and c-fos oncogenes during erythropoiesis. Results indicated that c-myc and c-fos are active in erythropoiesis and have a similar pattern of expression. A large decrease in expression of both c-myc and c-fos occurs when erythroid cells begin the biochemical transition into mature phenotypes, i.e., when RNA synthesis is down-regulated.

Stimulation of erythropoietin gene transcription during hypoxia and cobalt exposure.

Erythropoietin, a plasma glycoprotein produced primarily by the kidney, is a growth and differentiation factor for erythroid progenitor cells. Production of renal erythropoietin is regulated by modulation of mRNA levels in response to changes in tissue oxygenation. Exposure to cobalt, a nonphysiologic stimulus for erythropoietin production, also acts by inducing mRNA accumulation. To determine whether variations in erythropoietin mRNA levels result from enhanced transcription of the erythropoietin gene, in vitro transcription reactions were performed using isolated rat kidney cell nuclei. Quantitation of specific nuclear RNAs labeled during in vitro transcription revealed active erythropoietin gene transcription in kidney nuclei from anemic-hypoxic and cobalt-treated animals while erythropoietin transcriptional activity was undetectable in normal kidney nuclei. Time course studies showed that stimulation of transcription begins between two and four hours following cobalt treatment and parallels the kinetics of mRNA and plasma erythropoietin accumulation. These results indicate that tissue hypoxia and cobalt exposure specifically enhance erythropoietin gene expression. This increase in erythropoietin production is regulated at least in part at the level of gene transcription.