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Notes: Fibrocytes are a unique leukocyte subpopulation implicated in wound healing. They are derived from peripheral blood mononuclear cells, display fibroblast-like properties, and synthesize extracellular matrix macromolecules. This study investigated whether fibrocytes are present in healing burn wounds and whether the number of fibrocytes in tissue correlates with the degree of burn injury and the development of hypertrophic scar. Proteins extracted from cultured fibrocytes and nonadherent lymphocytes were found to be similar using two-dimensional gel electrophoresis and quite distinct from those obtained from fibroblasts. However, one protein, identified as leukocyte-specific protein 1 using mass spectrometric peptide mapping, was found in significantly larger amounts in fibrocytes than in lymphocytes but was undetectable in fibroblasts. Double immunostaining with antibodies to leukocyte-specific protein-1 and to the N-terminal propeptide of type I collagen was performed on cryosections of hypertrophic scar, mature scar, and normal skin. Fibrocytes were seen in scar tissue as dual-labeled spindle-shaped cells but were absent from normal skin. Moreover, the number of fibrocytes was higher in hypertrophic than in mature scar tissue. We conclude that fibrocytes, which have been reported to be antigen-presenting cells, are recruited to wounds following extensive burn injury and could potentially upregulate the inflammatory response and synthesize collagen and other matrix macromolecules, thus contributing to the development of hypertrophic scarring.

Notes: Introduction: Pseudomonas aeruginosa, remains a serious cause of nosocomial infection and septic mortality in burn patients particularly when nosocomially acquired. Our purpose is to investigate the morbidity and mortality associated with nosocomial infection with an aminoglycoside resistant Pseudomonas and associated costs compared to a group of patients with similar severity of burn injury that did not acquire resistant Pseudomonas during hospitalization. Methods: Using a TRACS burn database, patients treated at our institution with Pseudomonas resistant to gentamicin were identified and case-matched to controls for age (±5 years), TBSA(±5%), admission year (±5 years) and presence of inhalation injury. Patients who died 〈48 hours after injury were excluded. Data examined included demographics, number of days to onset of positive Pseudomonas culture and gentamicin resistance, as well as antibiotic use and cost. Results: 42 patients admitted to our unit between 1980 and 2001 were identified with gentamicin resistant Pseudomonas. Patients in the resistant Pseudomonas(Ps) group were similar in age (39.2 ± 3.2 vs 40.4 ± 3.3 years), TBSA (47.5 ± 3.7 vs 48.9 ± 3.7%), extent of full thickness injury (37.0 ± 3.9 vs 30.3 ± 3.2%) and presence of inhalation injury (62.8% vs 55.0%) compared to controls. There was a significant increase in the mortality rate in the Ps group (39.5 vs 5.0%, p 〈 0.001)(paired t test) compared to controls and the morbidity in terms of length of stay, increased in the Ps group (73.1 ± 13.2 vs 55.8 ± 8.1 days). Ventilatory days (22.6 ± 5.1 vs 8.2 ± 2.4, p 〈 0.05), number of surgical procedures (4.5 ± 0.6 vs 2.9 ± 2.5, p 〈 0.05), and amount of blood products used (packed cells 47.9 ± 7.8 vs 18.6 ± 3.3, p 〈 0.01)(platelets 10.5 ± 2.9 vs 0.5 ± 0.3, p 〈 0.01) were all significantly higher in the Ps group compared to control. Costs associated with antibiotic requirements were also significantly higher in the Ps group ($3,191.90 ± 848.00 vs $613.60 ± 145.50, p 〈 0.01). Conclusions: Our data demonstrate that nosocomial infection in burn patients with aminoglycoside-resistant Pseudomonas is associated with significantly higher morbidity and mortality and cost of care. Increased resource consumption in terms of length of stay, number of surgical procedures, amount of blood products, and antibiotic costs did not prevent significantly higher mortality rates when compared to control patients who avoided infection with the resistant organism. Thus, prevention, identification and eradication of nosocomial Pseudomonas infection are critical for cost-effective, successful burn care.

Notes: JCR:LA-cp/cp obese rats and their lean controls were evaluated as a type 2 diabetic wound healing model and the healing quality was characterized. This model of insulin resistance has been used extensively to study atherosclerosis but has not previously been used to study wound healing. Six circular excisional wounds were made on the dorsum of each rat and followed to day 21.Tracings of the wounds were made and used to assess the rate of wound closure. Planimetry showed a significantly diminished contraction of wounds in obese rats, but no significant difference in reepithelialization was observed. Collagen content was determined from the hydroxyproline content in wounded and unwounded skin. There were significantly lower levels of hydroxyproline in the wounds of obese compared to lean animals at day 21. Histology showed adipose tissue in place of dermal tissue in the JCR:LA-cp/cp rat in both unwounded tissue and in the wound at day 21. Active transforming growth factor-β1 (TGF-β1) was measured in the serum using the plasminogen activator inhibitor-1/luciferase assay and serum total TGF-β was measured using an enzyme-linked immunosorbent assay. Active TGF-β was significantly higher in the serum of obese animals compared with lean animals, while total TGF-β1 was not significantly different between the groups. Both active and total TGF-β was measured in tissue sections using the plasminogen activator inhibitor-1/luciferase assay. There was no significant difference in active TGF-β between genotypes, while obese rats had significantly higher levels of total TGF-β at day 21. These results indicate a deficiency in wound healing in obese animals characterized by decreased wound contraction, decreased collagen production, and changes in histology. The JCR:LA-cp rat develops insulin resistance, atherosclerosis and early type 2 diabetes and may be a good model for impairment of wound healing in humans with metabolic syndrome.

Notes: T-helper (Th) cells can be classified into at least three subsets based on their cytokine profiles: Th0, Th1, and Th2. The functional significance of each subset of Th cells can be determined in isolated human peripheral blood mononuclear cells (PBMC). Using two- or three-color cytometric detection of intracellular cytokines. These analyses have been limited by the requirement for fresh cells making sequential samples and longitudinal studies difficult. Cryopreservation of PBMC in liquid nitrogen for up to 1 year was evaluated to determine whether the Th1/Th2 ratio remained unchanged in cryopreserved lymphocytes. Aliquots of human PBMC from normal volunteers analyzed for activation using phorbol myristate acetate and evaluated using morphology showed that the surface marker expression was unchanged in fresh and frozen cells. Cytokine expression was measured using intracellular cytokine staining and three-color flow cytometric analysis. The percentages of cells producing interferon (IFN)-γ or interleukin (IL)-4 were determined after 16 hours of phorbol myristate acetate and ionomycin stimulation in the presence of brefeldin A. No significant difference was found in cytokine production between fresh and frozen cells. The percentage of IFN-γ and IL-4 producing CD3-positive fresh T cells was 19.2 ± 5.8 percent and 0.9 ± 0.4 percent vs. 17.6 ± 0.75 percent and 0.9 ± 0.3 percent, respectively, for frozen PBMC. The effects of thermal injury on the Th1/Th2 cytokine ratio and the development of hypertrophic scarring were then determined. Twelve burn patients examined 4 weeks postburn showed a significant shift in the Th1/Th2 ratio, compared with 13 normal human volunteers used as controls. IL-4 levels in the patient group were significantly higher than controls at 1 month postburn (12.7 ± 2.6 percent vs. 3.9 ± 0.5 percent, p 〈 0.01) and IFN-γ levels were significantly lower (9.3 ± 1.7 percent vs. 15.3 ± 2.3 percent, p 〈 0.05). Thus, PBMC can be cryopreserved for up to 1 year, enabling investigation of chronologic changes in Th1/Th2 profiles. It is suggested that a “locked on” Th2 profile may contribute to the development of hypertrophic scarring after burn injury.

Notes: We have previously shown that the expression of collagenase mRNA and activity are suppressed in fibroblasts derived from postburn hypertrophic scar. Although it is known that differential synthesis of collagen and collagenase in postburn hypertrophic scar is one of the main reasons for the excessive accumulation of collagen, it is not clear why the expression of collagenase in hypertrophic scar fibroblasts is suppressed relative to that in normal fibroblasts. In this study, we hypothesized that dermal fibroblasts from deeper layers of skin, which normally migrate toward the wound site, have a different capacity to express key extracellular matrix proteins such as collagenase and types I and III procollagen. To test this hypothesis, we established four different pairs of hypertrophic and site-matched normal cell strains from four different patients with postburn hypertrophic scarring. In another set of experiments, ten different cell strains from two normal human skin samples that were horizontally sectioned into five different layers (layers 1–5 correspond to upper to deeper layers, respectively) were established in culture. Cells at the same passages were harvested, total RNA was extracted, and Northern analysis was conducted to determine the level of collagenase and types I and III procollagen mRNA expression in each cell strain. The results of Northern blot analysis showed two transcripts each for the pro α1(I) collagen chain (5.8 and 4.8 kb) and for the pro α1(III) collagen chain (5.4 and 4.8 kb) in all cell strains examined. The intensity of the pro α1(I) chain of type I procollagen mRNA varied, ranging from 50.8 to 137.1 and 44.4 to 131.5 densitometry units among either normal or hypertrophic scar cells examined, respectively. Similarly, the relative quantity of type III procollagen transcript also varied, ranging from 10.4 to 91.1 and 28.7 to 116.1 among normal and hypertrophic scar cells, respectively. When five different skin layers from each tissue sample were evaluated for functional cell heterogeneity, the results showed a marked variation in expression of mRNA for type I and III procollagen. Although the level of collagenase mRNA also varied among different cell strains examined, the expression of collagenase mRNA was lower in fibroblasts derived from deeper layers of each skin sample. In conclusion, cells from different layers of normal skin samples are heterogeneous in their constitutive expression of some key extracellular matrix components such as collagenase and types I and III procollagen.

Notes: Introduction:  Fibrocytes are a unique bone marrow origin leukocyte subpopulation which display fibroblast-like properties and are antigen-presenting cells that are up-regulated systemically after burn injury. This study investigated their role in FPD of the skin using leukocyte-specific protein-1 (LSP1) specific for fibrocytes in HTS, keloids, and mature scar.Materials and Methods:  Fibrocytes were cultured from PBMC isolated from burn patients and normal individuals. Skin biopsies were taken from burn patients with hypertrophic (3) and mature scar (3) and normal skin (20) as well as patients with keloids (3). Double immunostaining (IHC) with antibodies to LSP1 and to the C-propeptide of type I collagen were performed on cryosections to identify fibrocytes in tissue. Extraction of fibrocytes from keloids, HTS, mature scar, and normal skin allowed quantification of fibrocytes by flow cytometry.Results:  LSP1 was found in both fibrocytes and lymphocytes, but not in fibroblasts. Western blot analysis showed that LSP1 expression was significantly higher in fibrocytes than in lymphocytes obtained from all five patients examined. Fibrocytes in hypertrophic scar were seen as duallabeled spindle-shaped cells. In normal skin, fibrocytes were present in the upper layers of the dermis and 〈1% of cells extracted, whereas, approximately threefold more fibrocytes were found in HTS and primarily in the papillary dermis. Preliminary data suggests that normal dermis contains 0.51% ± 0.35 fibrocytes, mature scar 0.30% ± .031, keloids 1.18 ± 0.22, and HTS 1.12%.Conclusions:  Fibrocytes are up-regulated systemically after injury and in FPD in skin where they appear to be up-regulated contributing to the development of keloids and hypertrophic scarring. More investigation is required to determine the functional roles of these bone marrow derived cells in fibroproliferative disorders.

Notes: Scar contraction following the healing of deep partial-thickness or full-thickness dermal injury is a leading cause of functional and cosmetic morbidity. The therapeutic use of interferon for the treatment of fibroproliferative disorders associated with scar contraction, including hypertrophic scar, has been suggested because of its antifibrotic properties. Treatment of fibroblasts with interferon has been shown to reduce the rate and extent of contraction using the in vitro fibroblast-populated collagen lattice model. In order to establish the effect of interferon-α2b on full-thickness wound contraction in vivo, osmotic pumps loaded with interferon or sterile saline were implanted intraperitoneally in guinea pigs. Seven days following implantation, six full-thickness punch biopsy wounds were created and were monitored by daily assessment of the wound. There was a significant reduction in the rate of wound contraction in the interferon-treated animals after day 3 (p 〈 0.01). Western blot analysis was used to quantitate selected cytoskeletal proteins in the normal skin and tissue biopsied from the wound at days 7, 14, and 21 postinjury. The amount of vimentin in the contracted wound increased following injury as compared with the amount present in normal skin (p 〈 0.0001); however, the relative amounts of the myofibroblast-associated cytoskeletal proteins α-smooth muscle actin and smooth muscle myosin were less than those found in normal, uninjured skin. By using vimentin to adjust the levels of cytoskeletal proteins for the increase in cellularity in the wounds, both α-smooth muscle actin and smooth muscle myosin significantly increased after closure of the wounds on day 14, as compared with the open-wound stage (day 7), before further reductions occurred with remodeling on day 21. Measurement of apoptotic cells using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay revealed an increase in apoptosis in the interferon-α2b-treated animals at 21 days following wounding (p 〈 0.001), which did not colocalize with α-smooth muscle actin staining. Taken together, these findings suggest that interferon-α2b inhibits wound contraction in vivo, not through an appreciable alteration in myofibroblast number or cytoskeletal protein expression, but possibly through a reduction in fibroblast cellularity by the induction of apoptosis.

Notes: Hypertrophic scars, which commonly occur after thermal and traumatic injury of the skin, are a fibroproliferative disorder of the dermal matrix wherein components of the inflammatory process, including the fibrotic growth factor, transforming growth factor-β, appear to activate dormant fibroblasts leading to cellular proliferation and excessive matrix synthesis. To investigate the potential beneficial role and mechanism of interferon alfa-2b in controlling excessive collagen production in hypertrophic scar, we measured dose response, time of onset, and duration of action in hypertrophic scar fibroblasts in vitro and compared them with those of site-matched normal fibroblasts obtained from four patients after thermal injury. Interferon alfa-2b reduced collagen protein synthesis and type I messenger RNA levels in both hypertrophic scar and normal fibroblasts after treatment, but these changes were apparent only after approximately 72 hours. Significant reductions in collagen synthesis occurred in four pairs of normal and hypertrophic scar fibroblasts (p 〈 0.05), accompanied by significant reductions in type I (p 〈 0.05) but not type III procollagen messenger RNA. Hypertrophic scar fibroblasts recovered completely from the effects of interferon alfa-2b on procollagen type I messenger RNA within 48 hours of cessation of treatment in contrast to normal skin fibroblasts, in which the reduction in type I procollagen messenger RNA by interferon alfa-2b persisted beyond 72 hours after treatment. These data suggest that interferon alfa-2b reduces collagen synthesis in both normal and hypertrophic fibroblasts but the hypertrophic fibroblast may remain less sensitive to its effects.

Notes: After severe thermal injury, hypertrophic scarring which is associated with accumulation of extracellular matrix proteins including fibronectin, frequently develops. We have recently demonstrated that interferon alfa-2b significantly reduces the level of type 1 procollagen messenger RNA expressed by both hypertrophic and normal dermal fibroblasts. In this report, we provide evidence that this cytokine also significantly decreases the expression of fibronectin messenger RNA in human hypertrophic scar and normal dermal fibroblasts. Four dermal fibroblast cell strains were established in cell culture from four human postburn hypertrophic scar tissues with the use of normal dermal fibroblasts from the same patients as controls. These cells were then treated with 2000 U/ml interferon alfa-2b in culture medium at various times. The results of Northern analysis of interferon-treated dermal fibroblasts indicate that this cytokine reduced the expression of fibronectin messenger RNA as early as 12 hours after treatment and reached its lowest level (24% relative to untreated fibroblasts) after 96 hours. When the expression of fibronectin messenger RNA was quantified by densitometry for each individual paired cell strain, a differential response to interferon treatment was found among cell strains. The level of fibronectin messenger RNA expression decreased from 17.2% to 69% in hypertrophic scar fibroblasts and 47% to 83.7% in normal fibroblasts relative to that of untreated control values. Although this decrease was less pronounced in normal fibroblasts than in hypertrophic scar fibroblasts, this reduction was significant in both interferon alfa-2b treated hypertrophic scar fibroblasts (6.39 ± 0.71 versus 2.88 ± 0.9, n = 4, p 〈 0.05) and normal cells compared with untreated controls (5.47 ± 0.89 versus 3.64 ± 0.99, n = 4, p 〈 0.05) as assessed with Student's paired t test. Rehybridization of the RNA blot prepared from interferon alfa-2b treated and untreated hypertrophic scar fibroblasts with a complementary DNA for the tissue inhibitor of metalloproteinase type 2 gelatinase inhibitor showed no significant changes in abundance of this transcript. This result suggests that this cytokine selectively suppresses the expression of fibronectin messenger RNA and that this reduction is not due to RNA loading. A dot blot analysis of total RNA extracted from these tissues was carried out to compare the expression of fibronectin messenger RNA between human hypertrophic scar tissues and normal dermis obtained from the same patients. The blot was initially hybridized with fibronectin complementary DNA and subsequently with a complementary DNA for the tissue inhibitor of metalloproteinase type 2 to correct for RNA loading. When the ratio of fibronectin to tissue inhibitor or metalloproteinase type 2 messenger RNA expression for each hypertrophic scar tissue was compared with its normal control, this ratio was fourfold higher in human hypertrophic scar tissues relative to normal controls. In contrast, the expression of this message in cultured hypertrophic scar fibroblasts was not significantly different from that in normal fibroblasts. The results of this study suggest that hypertrophic scarring developing after thermal injury is associated with an overexpression of fibronectin messenger RNA, and interferon alfa-2b may be of therapeutic value to down-regulate the expression of this transcript.

Notes: Transforming growth factor-β1 is a fibrogenic cytokine that is important in the development of fibroproliferative disorders of the skin after injury. To investigate the role of transforming growth factor-β1 produced by keratinocytes during wound healing, a plasmid with the human transforming growth factor-β1 gene coupled with the keratin 14 promoter (pG3Z: K14-TGF-β1) was constructed. The construct was tested successfully in vitro before being used to generate transgenic animals, which were subsequently bred into homozygous and heterozygous lines. Genotype screening of founders and progeny was performed by Southern blotting and targeting of the transgene to the epidermis by the keratin 14 promoter was shown by reverse transcription polymerase chain reaction. The major phenotypic change observed in the transgenic animals was “scruffiness” of the fur attributed to transgene expression in the skin, seen primarily in the homozygous line. A significant reduction in the rate of reepithelialization of full-thickness excisional wounds of dorsal skin was seen in homozygous animals compared with normal litter-mate controls at day 7 (p 〈 0.05, Fisher's Exact test) and day 9 (p 〈 0.01) postwounding. Wounds in heterozygous animals also healed more slowly at day 9 (p 〈 0.01). Northern analysis of mRNA extracted from the wounds showed increased human transforming growth factor-β1 message levels in homozygous and heterozygous animals, maximal at day 5. Significant increases in transforming growth factor-β1 activity in healing wounds measured using the plasminogen activator inhibitor-1/luciferase assay were found in the transgenic strains at day 9 postinjury as compared with the normal litter-mate control mice (p 〈 0.001, ANOVA). Type I procollagen mRNA expression was higher in the homozygous and heterozygous animals, with the highest levels reached at day 9. By day 5 postwounding, biopsies of both homozygous and heterozygous tissues were significantly higher in collagen as compared with wounds in control animals (p 〈 0.05, ANOVA). Based on these data, the K14-TGF-β1 transgenic mouse shows that excessive latent transforming growth factor-β1 produced in the epidermal layer of the skin delays reepithelialization in excisional wounds but subsequently the cells of the epidermis stimulate dermal fibroblasts leading to fibrosis through a paracrine mechanism. (WOUND REP REG 2002;10:)