Waldemar L. Olszewski

Low recruitment of immune cells with increased expression of endothelial adhesion molecules in margins of the chronic diabetic foot ulcers.

Diabetic foot skin close to an ulcer shows only a few infiltrating cells compared to nondiabetic inflamed tissues. Diabetes is characterized by thickened basement membrane of the blood arterioles and capillaries. This may affect the transcapillary transport of immune humoral factors and cells to the extravascular space. We analyzed by immunohistochemistry the phenotype and expression pattern of adhesion molecules on leukocyte, dermal fibroblast, and endothelial cells in diabetic foot ulcers. Although there was accumulation of granulocytes on the surface and superficial layers of the granulation tissue, rare perivascular granulocyte infiltrates in the dermis were seen. Moreover, lack of macrophage and CD3+ T cell infiltrates was observed. In contrast, there was increased intensity of CD1a staining of Langerhans cells in the epidermis and papillary dermis (pu2003<u20030.05). Fibroblasts revealed increased presence in the ulcer margins compared with normal skin (pu2003<u20030.05). Skin endothelial cells expressed stronger von Willebrand factor and E‐selectin compared with normal skin (pu2003<u20030.05). Our study provides evidence that increased expression of endothelial cell adhesion molecules responsible for immunocyte extravasation is not associated with increased inflammatory cell infiltration of the ulcerated diabetic foot tissue. We suggest that the healing process of diabetic foot ulcers may be hampered by mechanisms decreasing accumulation of leukocytes. This implies that pharmacological or biological stimulation of leukocyte extravasation into the ulcer tissue should be tried.

Keratinocyte and dermal vascular endothelial cell capacities remain unimpaired in the margin of chronic venous ulcer

The role of endogenously produced cytokines and growth factors in the impaired healing of chronic leg ulcers remains uncertain. The aim of this study was to determine the functional capacity of skin cells in ulcer bed tissue compared to those in the edge of ulcers and skin distal to ulcers. Biopsies from leg ulcers of ten randomly selected patients were examined immunohistochemically for cytokines and growth factors produced by keratinocytes (KC) and vascular endothelial cells (EC). The phenotype of leukocytes infiltrating venous ulcers and the expression of vascular adhesion molecules responsible for extravasation were also studied. The expression of cytokines and growth factors by KC was similar in areas adjacent and remote from an ulcer. In the dermis adjacent to an ulcer, the expression of IL-1α, IL-1β, IL-1Ra, EGF and PDGFa by EC was higher than the levels of expression in EC from the distant dermis. The expression of IL-6, TNFα and GM-CSF was comparable to that in cells from intact dermis. For all these factors staining was cytoplasmic, suggesting production in these areas. Ulcer bed tissue contained few fibroblasts and blood capillaries showing a high staining intensity for CD62E and CD106 EC adhesion molecules but no FGF2 expression (P<0.05). The intensity of staining for scavenging CD15+elastase+ granulocytes and CD35+ (C3bR) activated macrophages in the ulcer bed was comparable to that in the margin but higher than that in the distant dermis (P<0.05), whereas staining for CD68+, HLA DR+, TGFβ+ and CD54+ dermal macrophages was similar in all areas. There was reduced staining for CD4+ and CD8+ cells in the ulcer bed (P<0.05). There were no CD1a+ Langerhans cells in the epidermis encroaching upon the granulation tissue and there was reduced CD1a staining in the adjacent epidermis (P<0.05). In conclusion, there is chronic accumulation of scavenging cells with lack of remodeling of the granulation tissue and, at the same time, preserved cytokine and growth factor secretory potential of KC and dermal EC in non-healing venous leg ulcers.

The Influence of Tissue Fluid/Lymph Cytokines and Growth Factors on Human Keratinocyte Proliferation and Differentiation

Cultured keratinocytes (KC) are needed for transplantation to the surface of large burn wounds and ulcers. They can be cultured in artificial media. However, the yield is always limited, viability is low, and proliferation and migration after grafting are slow. The question arose whether tissue fluid/lymph, which is a natural humoral environment for epidermal and dermal cells, contains cytokine(s) specifically regulating KC proliferation and could be used to culture large numbers of cells for transplantation. Culturing of skin keratinocytes in dermal tissue fluid/lymph containing keratinocyte growth factor, interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and transforming growth factor-beta revealed its strong stimulatory effect on the expression of p63 stem cell marker and proliferation but not differentiation of KC. Neutralizing these cytokines with antibodies resulted in decreased percentages of mitotic figures. None of the individual cytokines showed a dominant effect on proliferation. This observation suggests that either there may be other (so far undetected) specific cytokines or that the proliferation and differentiation of keratinocytes is an effect of the combined action of all investigated cytokines.

Hyperkeratosis in human lower limb lymphedema: the effect of stagnant tissue fluid/lymph

Hyperkeratosis of skin in lower limb lymphedema is one of the sequelae of tissue fluid/lymph (TF/L) stasis, but its mechanisms remain unknown. It is noteworthy, nonetheless, that human TF/L contains high levels of growth factors and cytokines, and may serve as the physiological environment for keratinocyte (KC) proliferation.

Virulence of Bacteria Colonizing Vascular Bundles in Ischemic Lower Limbs

BACKGROUNDnWe documented previously the presence of bacterial flora in vascular bundles, lymphatics, and lymph nodes of ischemic lower limbs amputated because of multifocal atheromatic changes that made them unsuitable for reconstructive surgery and discussed their potential role in tissue destruction. The question arose why bacterial strains inhabiting lower limb skin and considered to be saprophytes become pathogenic once they colonize deep tissues. Bacterial pathogenicity is evoked by activation of multiple virulence factors encoded by groups of genes.nnnMETHODSnWe identified virulence genes in bacteria cultured from deep tissue of ischemic legs of 50 patients using a polymerase chain reaction technique.nnnRESULTSnThe staphylococcal virulence genes fnbA (fibronectin-binding protein A), cna (collagen adhesin precursor), and ica (intercellular adhesion) were present in bacteria isolated from both arteries and, to a lesser extent, skin. The IS256 gene, whose product is responsible for biofilm formation, was more frequent in bacteria retrieved from the arteries than skin bacteria. Among the virulence genes of Staphylococcus epidermidis encoding autolysin atlE, icaAB (intercellular adhesion), and biofilm insert IS256, only the latter was detected in arterial specimens. Bacteria cultured from the lymphatics did not reveal expression of eta and IS256 in arteries. The Enterococcus faecalis asa 373 (aggregation substance) and cylA (cytolysin activator) frequency was greater in arteries than in skin bacteria, as were the E. faecium cyl A genes. All Pseudomonas aeruginosa virulence genes were present in bacteria cultured from both the skin and arteries. Staphylococci colonizing arterial bundles and transported to tissues via ischemic limb lymphatics expressed virulence genes at greater frequency than did those dwelling on the skin surface. Moreover, enterococci and Pseudomonas isolated from arterial bundles expressed many virulence genes.nnnCONCLUSIONSnThese findings may add to the understanding of the mechanism of development of destructive changes in lower limb ischemic tissues by the patients, but not hospital-acquired, bacteria, as well as the generally unsatisfactory results of antibiotic administration in these cases. More aggressive antibiotic therapy targeted at the virulent species should be applied.

A novel method for long-lasting preservation of arterial grafts.

BACKGROUNDnAutologous venous grafts generally give best results for arterial bypass grafting in cases of arterial stenosis. When no suitable venous graft can be found, synthetic prosthetic graft may be an alternative. Prostheses are easily accessible but susceptible to infection. In these cases, the replacement of infected prosthesis by the human arterial allograft is the best treatment option. The question arises whether we could prepare a graft meeting mechanical conditions of an artery immunologically inert and resistant to bacterial infection.nnnMATERIALS AND METHODSnLEW and BN rat aortic segments were placed in dehydrated sodium chloride and stored for 1 to 12xa0mo. Then, they were transplanted orthotopically as aortic grafts for 3 to 15xa0mo in syngenic and allogenic combination. No immunosuppression was used. Patency, pulsation, and frequency of development of aneurysms were studied. The tensile strength and maximum intraluminal pressures were measured. Morphology of grafts was evaluated on histology and electron microscopy. The endothelial and infiltrating cells were identified.nnnRESULTSnTransplanted allogeneic aortic grafts preserved in anhydrous sodium chloride up to 12xa0mo remained patent for 15xa0mo. Hypertrophy of intima with endothelial cells lining the inner surface and single muscle cells between elastic fibers were seen. Normal structure of collagen and elastic fibers was maintained. Only minor-host mononuclear infiltrates were seen around the preserved allografts.nnnCONCLUSIONSnRat aortas preserved in anhydrous sodium chloride retain patency and function even 15xa0mo after transplantation. Such grafts retain their wall structure and evoke only minor recipient reaction. Our results confirm that anhydrous sodium chloride may be used for arterial grafts preservation. Low immunogenicity is additional advantage.

Transplantation of Keratinocyte Spore-Like Stem Cells

BACKGROUNDnWound granulation tissue should be covered by epidermal cells migrating from the basal layer of the epidermis or hair bulge of the wound edge. However, new epidermal islands are frequently formed on the granulation tissue remote from the wound edge. Thus, current theory of bulge-originating stem cells does not necessarily correspond to the histological pictures of the healing wound. We took imprints of a leg ulcer surface and found single dispersed, large nucleated cells, some of them in mitosis. These cells resembled those from epidermal spinosum layer. The question arouse as to whether these cells might be the spore-like stem cells creating epidermal island. We found similarly shaped cells among the keratinocyte preserved in pulverized sodium chloride as the only surviving population in culture and revealing enzymatic activity. The aim of this work was to study whether the population of human keratinocytes surviving sodium chloride preservation and transplanted to SCID mice may form epidermis.nnnMETHODSnThe 12-month sodium chloride-preserved and cultured keratinocytes (KC) were transplanted to the wound on the dorsum of SCID mice for 14 and 21 days.nnnRESULTSnNinety-five percent of cultured KC were enzymatically active large cells; they did not express p63 and CD29 claimed as specific for stem cells, and they did not proliferate. Transplanted to the center of the wound, they formed small KC islands and became confluent after 14 days.nnnCONCLUSIONSnThe large epidermal keratinocytes survived the 12-month preservation in anhydrous sodium chloride. Transplanted to the wound, they formed epidermal islands of human phenotype. These cells may be the so-called spore-like stem cells.