Vitamin C regulates keratinocyte viability, epidermal barrier, and basement membrane in vitro, and reduces wound contraction after grafting of cultured skin substitutes

Cultured skin substitutes have become useful as adjunctive treatments for excised, full-thickness burns, but no skin substitutes have the anatomy and physiology of native skin. Hypothetically, deficiencies of structure and function may result, in part, from nutritional deficiencies in culture media. To address this hypothesis, vitamin C was titrated at 0.0, 0.01, 0.1, and 1.0 mM in a cultured skin substitute model on filter inserts. Cultured skin substitute inserts were evaluated at 2 and 5 wk for viability by incorporation of 5-bromo-2'-deoxyuridine (BrdU) and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion. Subsequently, cultured skin substitute grafts consisting of cultured human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates were incubated for 5 wk in media containing 0.0 mM or 0.1 mM vitamin C, and then grafted to athymic mice. Cultured skin substitutes (n = 3 per group) were evaluated in vitro at 2 wk of incubation for collagen IV, collagen VII, and laminin 5, and through 5 wk for epidermal barrier by surface electrical capacitance. Cultured skin substitutes were grafted to full-thickness wounds in athymic mice (n = 8 per group), evaluated for surface electrical capacitance through 6 wk, and scored for percentage original wound area through 8 wk and for HLA-ABC-positive wounds at 8 wk after grafting. The data show that incubation of cultured skin substitutes in medium containing vitamin C results in greater viability (higher BrdU and MTT), more complete basement membrane development at 2 wk, and better epidermal barrier (lower surface electrical capacitance) at 5 wk in vitro. After grafting, cultured skin substitutes with vitamin C developed functional epidermal barrier earlier, had less wound contraction, and had more HLA-positive wounds at 8 wk than without vitamin C. These results suggest that incubation of cultured skin substitutes in medium containing vitamin C extends cellular viability, promotes formation of epidermal barrier in vitro, and promotes engraftment. Improved anatomy and physiology of cultured skin substitutes that result from nutritional factors in culture media may be expected to improve efficacy in treatment of full-thickness skin wounds.     

Intraarterial digital subtraction arteriographic evaluation of extremity tumors: comparison with conventional arteriography

Conventional arteriography and intraarterial digital subtraction arteriography (IADSA) were compared in 36 patients with primary bone or soft-tissue tumors of the extremities. The sensitivity of IADSA was at least equal to conventional arteriography for demonstrating normal or abnormal major arteries and feeding arteries, equal to or superior for depicting tumor stains or draining veins, but slightly inferior for revealing minute tumor vessels. An increase of the matrix size from 256 X 256 to 512 X 512 improved these sensitivities. IADSA with 15% diatrizoate contrast material eliminated the contrast material-induced pain in all patients. With a computer-controlled iris setting, an average of 5 minutes of procedure time and 1.7 R of radiation (0.44 mC kg) per examination could be saved. IADSA reduced the cost of an examination by an average of $67. The results indicate that IADSA was diagnostic in all instances and can replace conventional arteriography for the evaluation of extremity tumors.     

Improved outcome for high-risk acute myeloid leukemia patients using autologous bone marrow transplantation and monoclonal antibody-purged bone marrow

We have conducted a 9-year multicenter trial of autologous bone marrow transplantation (ABMT) for acute myeloid leukemia (AML). Remission BM was purged in vitro using monoclonal antibodies (MoAbs; PM-81, AML-2- 23) and complement targeting myeloid differentiation antigens (CD15, CD14). In 1988, the preparative regimen changed from 60 mg/kg/d cyclophosphamide x 2 and fractionated total body irradiation (TBI) total dose, 1,200 cGy (Cy/fTBI), to 4 mg/kg/d busulfan x 4 and 60 mg/kg/d Cy x 2 (Bu/Cy2). Recent analysis (October 1, 1993) shows that the Bu/Cy2 regimen along with the same MoAb purging method yields an improved outcome. Seven first complete-remission (CR) (CR1), 45 second- or third-CR (CR2/3), and 11 first-relapse (R1) patients were treated with chemotherapy and TBI or chemotherapy alone followed by ABMT with MoAb-purged BM. Median age at ABMT for those patients in CR 2/3 and R1 patients was 36 years. Twenty-nine CR 2/3 and R1 patients were conditioned with Cy/fTBI, and 27 CR2/3 and R1 patients were conditioned with Bu/CY. Using the Kaplan-Meier method, the CY/fTBI, CR2/3, and R1 patients have a 3-year disease-free survival (DFS) of 21%. On the other hand, the Bu/Cy2, CR2/3, and R1 patients have a 3-year DFS of 48%. Nineteen CR2/3 and R1 patients relapsed post-ABMT. On analysis by conditioning regimen, those treated with Cy/fTBI have a 3-year relapse rate (RR) of 58%, whereas the patients conditioned with Bu/Cy2 have a 39% 3-year RR. Long-term DFS can be achieved in about 50% of patients with advanced remissions and relapsed AML using Bu/Cy2 with MoAb-purged BM.     

Keratin expression in cultured skin substitutes suggests that the hyperproliferative phenotype observed in vitro is normalized after grafting

Cultured skin substitutes, consisting of fibroblasts and keratinocytes in a biopolymer matrix, are an adjunctive treatment for full thickness burn wounds. Previous studies revealed that cultured skin substitutes in vitro exhibit a gene expression profile similar to hyperproliferative skin or wounded normal skin. In the present study, we sought to determine whether this hyperproliferative phenotype is maintained after healing of grafted cultured skin in vivo. Immunohistochemistry was used to localize multiple keratin proteins in native human skin, and in cultured skin substitutes in vitro and after grafting to athymic mice. Keratin 6, keratin 16, and keratin 17, which are known to be upregulated during keratinocyte activation and in hyperproliferative epidermis, were highly expressed in cultured skin substitutes in vitro. These proteins were low or undetectable in native human skin, and were reduced in cultured skin after grafting. Conversely, keratin 15, which is downregulated in activated keratinocytes, was not detected in cultured skin substitutes in vitro but was upregulated after grafting to mice. The results confirm previous observations suggesting a hyperproliferative or activated phenotype in cultured skin substitutes in vitro, similar to wounded native skin. After grafting to athymic mice, the expression patterns suggest a normalization of cultured skin substitutes to a phenotype more closely resembling uninjured human skin.     

Complementary use of ultrasound and computed tomography in studies of the pancreas and kidney

113 cases of pancreatic and renal disease studied by both ultrasound and computed tomography (CT) were analyzed retrospectively. CT provided a diagnosis when pancreatic ultrasound was unsuccessful due to overlying bowel gas or obesity and when renal ultrasound was unsuccessful due to obesity, reverberations from ribs, small lesions, or multiple lesions. Conversely, ultrasound provided a diagnosis when CT was unsuccessful due to lack of fat planes or respiratory motion. CT usualy distinguished carcinoma from pancreatitis when ultrasound showed a focal echogenic mass. CT resolved renal cyst from neoplasm when ultrasound showed a mixed echo pattern mass.     

Characterization of nonlymphoid cells derived from rat peripheral lymph

Mesenteric lymphadenectomy in rats is followed by union of peripheral and central lymphatics, allowing the collection of intestine-derived peripheral lymph cells via the thoracic duct for several days. These cells include a proportion of nonlymphoid cells (NLC) that show irregular and heterogeneous surface morphology including long pseudopodia and veils. They stain variably for nonspecific esterase and acid phosphatase and are ATPase-positive. Their nuclei are irregular and some contain cytoplasmic inclusions, some of which show peroxidase activity and/or contain DNA. NLC have a range of densitites generally lower than that of lymphocytes. Freshly collected NLC express the leukocyte-common antigen (defined by monoclonal antibody MRC Ox 1) and Ia antigens (I-A and I-E subregion products defined by monoclonal antibodies) but they show a relative lack of other surface markers normally found on rat B or T lymphocytes (W3/13, W3/25, MRC Ox 12 (sIg), MRC Ox 19) or rat macrophages (FcR, C’R, mannose R, W3/25). In general NLC are only weakly adherent to glass or plastic. Although a subpopulation of NLC appear to have had a phagocytic past, freshly collected NLC fail to phagocytose a variety of test particles in vitro. NLC also appear incapable of pinocytosis in vitro. This heterogeneity may represent distinct subpopulations of NLC or different stages in the development of a single cell lineage. Direct cannulation of mesenteric lacteals shows that the majority of NLC are derived from the small intestine and their precursors appear to be present both in lamina propria and Peyer's patches. Kinetic studies, following irradiation or intravenous tritiated thymidine, show that the majority of NLC turn over rapidly in the intestine with a modal time of 3-5 d. Studies with bone marrow chimeras show that they are derived from a rapidly dividing precursor present in normal bone marrow. NLC occur at very low frequencies in normal thoracic duct lymph at all times following cannulation. The evidence presented suggests that NLC closely resemble mouse lymphoid dendritic cells. This conclusion is supported by evidence already obtained showing that NLC are potent stimulators of the semi-allogeneic rat primary mixed leukocyte reaction. In addition to the ceils resembling dendritic cells rare monocytoid cells are found in thoracic duct lymph of lymphadenectomized specific pathogen-free rats. The proportion of these cells increases greatly when the animals are conventionally housed. It seems probable that the physiological function of NLC is to act as accessory cells in the lymph nodes to which they normally drain. Methods for enriching NLC and thus facilitating analysis of their functions are discussed.     

Semantic memory retrieval: cortical couplings in object recognition in the N400 window

To characterize the regional changes in neuronal couplings and information transfer related to semantic aspects of object recognition in humans we used partial-directed EEG-coherence analysis (PDC). We examined the differences of processing recognizable and unrecognizable pictures as reflected by changes in cortical networks within the time-window of a determined event-related potential (ERP) component, namely the N400. Fourteen participants performed an image recognition task, while sequentially confronted with pictures of recognizable and unrecognizable objects. The time-window of N400 as indicative of object semantics was defined from the ERP. Differences of PDC in the beta-band between these tasks were represented topographically as patterns of electrical couplings, possibly indicating changing degrees of functional cooperation between brain areas. Successful memory retrieval of picture meaning appears to be supported by networks comprising left temporal and parietal regions and bilateral frontal brain areas.     

Evaluation of cytotoxicity and antimicrobial activity of Acticoat Burn Dressing for management of microbial contamination in cultured skin substitutes grafted to athymic mice

Cultured skin substitutes (CSS) have become a useful adjunctive treatment for closure of burn wounds, but CSS are avascular and remain susceptible to microbial destruction longer than split-thickness skin grafts. Irrigation of CSS grafted to burn wounds with a topical antimicrobial solution (TAS) has been shown to promote engraftment of CSS, but TAS usage has potential limitations. Acticoat Burn Dressing (Acticoat; Westaim Biomedical, Exeter, NH) is a silver-coated barrier dressing reported to exhibit antimicrobial activity and to reduce infection in partial-thickness and full-thickness wounds. This study evaluated the cytotoxicity of Acticoat with CSS and the efficacy of Acticoat for the management of microbial contamination in CSS grafted to full-thickness wounds in athymic mice. The cytotoxicity of Acticoat was assessed in preliminary studies after 1 week of exposure to CSS during in vitro maturation or healing on wounds in athymic mice. Histologies were analyzed and cellular viability in the CSS was determined by MTT conversion on days 0, 1, and 7 of Acticoat exposure. At 1, 2, 3, and 4 weeks after grafting, wounds were traced, and areas of healing CSS were calculated by image analysis. At 4 weeks, wound biopsies were evaluated and scored for engraftment of human cells. In a subsequent study, wounds were inoculated with strain SBI-N of Pseudomonas aeruginosa at 1 x 10(5) cfu/wound before the application of CSS or inoculated onto the surface of Acticoat. At 4 weeks, swab cultures were collected from the surface of CSS and scored for the presence of SBI-N. Statistical significance was accepted at the 95% confidence level (P <.05). The data show that exposure in vitro of CSS to Acticoat was cytotoxic within 1 day, but 1 week of exposure in vivo did not injure CSS or inhibit wound healing. Contaminated wounds treated with Acticoat healed similarly to control treatments, with comparable rates of engraftment, and detection of SBI-N on the surface of only one graft. No SBI-N was detected on CSS after inoculation onto the surface of Acticoat. These results suggest that Acticoat may be suitable as a protective dressing to reduce environmental contamination of CSS, if used in conjunction with additional antimicrobials to control organisms present in the wound.