Library

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: 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:)

Notes: This study was conducted to further explore the mechanism of transforming growth factor β1 (TGF-β1) activation, which plays a critical role in many physiological and pathological conditions. We have previously shown that the large (270 kDa), but not small (40 kDa), mannose 6-phosphate receptors facilitate the cellular response to latent TGF-β1 released from genetically modified cells. In this study, we explored the role of cell membrane associated transglutaminase and plasmin in mannose 6-phosphate receptor induced latent TGF-β activation using MS and MS-9 cells bearing either no receptors or the 270 kDa mannose 6-phosphate/insulin-like growth factor II receptors, respectively. As a source of latent TGF-β1, PA317 cells were transfected with either pLin-TGF-β1 vector or pLin retroviral vector with no TGF-β1 insert using calcium phosphate precipitation. The latency and bioactivity of TGF-β1 in conditioned medium derived from transfected PA317 cells were evaluated by enzyme-linked immunosorbent assay and mink lung epithelial cell growth inhibition assay, respectively. The level of latent TGF-β1 was 13-fold higher (20.1 ± 0.4 vs. 1.5 ± 0.03 ng/ml) in conditioned medium from pLin-TGF-β1 transfected cells than that of control. The latency and bioactivity of TGF-β1 released from pLin-TGF-β1 transfected cells were confirmed by evaluation of 3H-thymidine incorporation in Mv1Lu epithelial cells treated with non- and heat-activated 10% conditioned medium. The results showed a significantly lower 3H-thymidine incorporation in Mv1Lu epithelial cells treated with heat-activated PA317 conditioned medium (4% of control) relative to those treated with either control or nonheated conditioned medium. This inhibition was abrogated by addition of 40 μg/ml of TGF-β1 neutralizing antibody. The level of 3H-thymidine incorporation was then evaluated in MS-9 cells receiving Dulbecco's modified Eagle medium containing either 0% 10%, 30% or 50% volumes of nonactivated PA317 conditioned medium for 24 hours. The results showed a markedly lower proliferation in response to 30% and 50% conditioned medium used in MS-9 cells. Under similar experimental conditions, addition of only mannose 6-phosphate, but not fructose 6-phosphate or mannose 1-phosphate, at 1 mM concentration restored the MS-9 cell proliferative response to latent TGF-β1. The inhibitory effects of latent TGF-β1 on MS-9 cell proliferation were restored by addition of either TGF-β1 neutralizing antibody or cystamine, a transglutaminase inhibitor. In contrast, addition of aprotinin, a plasmin inhibitor, had a marginal influence on inhibitory effects of latent TGF-β1 on MS-9 cell proliferation. Interestingly, a mixture of latent TGF-β1 + MS-9 cell membranes, but not MS cell membranes, also inhibited the mink lung epithelial cell proliferation (34% of control). These findings indicate that mannose 6-phosphate/insulin-like growth factor II receptors are involved in latent TGF-β activation and that is at least partly dependent on cell membrane associated transglutaminase, but not on plasmin.

Notes: Transforming growth factor-β (TGF-β) is known to affect nearly every aspect of wound repair. Many of the effects have been extensively investigated; however, the primary effect of endogenously derived TGF-β on wound reepithelialization is still not completely understood. To examine this, two types of wounds were made on a transgenic mouse over-expressing TGF-β1. Full-thickness back wounds were made to compare the wound healing process in the presence of compensatory healing mechanisms. Superficial partial-thickness ear wounds involving only the epidermis were made to determine the effect of TGF-β on reepithelialization. In the partial-thickness ear wounds, at later time points, the transgenic group had smaller epithelial gaps than the wild-type mice. A greater number of actively proliferating cells, as determined by bromodeoxyuridine incorporation, was also found in the transgenic mice at post-injury day 8. These results show that TGF-β1 stimulates the rate of reepithelialization at later time points in partial-thickness wounds. However, in the full-thickness back wounds, the transgenic animals exhibited a slower reepithelialization rate at all time points and the number of bromodeoxyuridine-positive cells was fewer. Our findings would suggest that the overexpression of TGF-β1 speeds the rate of wound closure in partial-thickness wounds by promoting keratinocyte migration. In full-thickness wounds, however, the overexpression of TGF-β1 slows the rate of wound reepithelialization.

Notes: Abstract  Growth of Listeria monocytogenes in a Listeria enrichment broth (LEB) was automatically monitored by electrochemical cyclic voltammetric scan using a gold working electrode. Changes in cyclic voltammograms were observed during growth of L. monocytogenes in LEB. The reduction peak at −0.4 V (vs Ag/AgCl) corresponding to the reduction of oxygen dissolved in LEB on cyclic voltammograms was decreasing with proliferation of L. monocytogenes, and disappeared eventually. A pair of reversible redox peaks appeared during growth of L. monocytogenes in LEB. These cyclic voltammetric characteristics can be used to detect L. monocytogenes in various samples. Threshold values (detection time) obtained from the oxygen consumption curves were inversely related to the concentrations of L. monocytogenes in the broth. A calibration curve was obtained by plotting initial cell concentrations (CFU/mL) determined by conventional plate counting, as a function of the detection time. A linear response was found on the calibration curve for L. monocytogenes between 1 ∼ 9 times 100 and 1 ∼ 9 times 105 cells/mL. The detection time was approximately 17 and 6 h for 1 ∼ 9 times 100 and 1 ∼ 9 × 105 cells/mL of viable L. monocytogenes in the broth, respectively. The method was evaluated in detection of L. monocytogenes in milk samples.

Notes: A bienzyme (tyrosinase and horseradish peroxidase) electrochemical biosensor was developed for detection of Salmonella typhimurium, and evaluated for application in a flow injection system coupled with immunomagnetic separation for food samples. Parameters for immunomagnetic separation, enzymatic reaction, flow injection and electrochemical detection were determined using pure culture samples. The selectivity was tested in the presence of Listeria monocytogenes, Campylobacter jejuni and E. coli 0157:H7. The results showed a linear relationship for logarithmic values between peak current ratio and the cell number of S. typhimurium in the range of 103 105 cfu/mL, with R2= 0.99. The detection limit of this method was 1.09 × 103 cfu/mL for S. typhimurium and the detection time was 2.5 h. Samples of chicken carcass wash water and ground beef were used to evaluate the biosensor. The results demonstrated that this biosensor has a potential for rapid detection of different pathogens in various food samples.