Jan R. Mekkes

Causes, investigation and treatment of leg ulceration

Summary Chronic ulceration of the lower leg is a frequent condition, with a prevalence of 3–5% in the population over 65 years of age. The incidence of ulceration is rising as a result of the ageing population and increased risk factors for atherosclerotic occlusion such as smoking, obesity and diabetes. Ulcers can be defined as wounds with a ‘full thickness depth’ and a ‘slow healing tendency’. In general, the slow healing tendency is not simply explained by depth and size, but caused by an underlying pathogenetic factor that needs to be removed to induce healing. The main causes are venous valve insufficiency, lower extremity arterial disease and diabetes. Less frequent conditions are infection, vasculitis, skin malignancies and ulcerating skin diseases such as pyoderma gangrenosum. But even rarer conditions exist, such as the recently discovered combination of vasculitis and hypercoagulability. For a proper treatment of patients with leg ulcers, it is important to be aware of the large differential diagnosis of leg ulceration.

Cultured fibroblasts from chronic diabetic wounds on the lower extremity (non-insulin-dependent diabetes mellitus) show disturbed proliferation.

Abstract Patients with diabetes mellitus experience impaired wound healing often resulting in chronic foot ulcers. Hospital discharge data indicate that 6–20% of all diabetic individuals hospitalized (mostly with type 2 diabetes) have a lower extremity ulcer. Maintaining glucose levels at acceptable levels (below 10 mmol/l) is considered to be an important part of the clinical treatment, but the exact mechanism by which diabetes delays wound repair is not yet known. We studied this phenomenon by determining the potential of fibroblasts isolated from the ulcer sites of four patients with non-insulin-dependent diabetes mellitus to proliferate in vitro. Controls were fibroblasts isolated from normal skin of the upper leg of five healthy age-matched volunteers and of six non-insulin-dependent diabetes patients. Proliferative capacity was analysed by evaluation of plates after trypsinization and [ 3 H]thymidine incorporation. Fibroblast morphology was studied by light and transmission electron microscopy. Diabetic ulcer fibroblasts, measured by [ 3 H]thymidine incorporation, proliferated significantly more slowly than the nonlesional control fibroblasts ( P < 0.00047) and age-matched control fibroblasts ( P < 0.00003). After culturing the fibroblasts for a prolonged period in high-glucose (27.5 m M ) and low-glucose (5.5 m M , i.e. physiological) medium, this difference in proliferation rate between diabetic ulcer fibroblasts and nonlesional diabetic fibroblasts remained ( P < 0.0001 for high-glucose and P < 0.0009 for low-glucose on day 7). Fibroblast proliferation in all three groups was slightly lower in high-glucose than in low-glucose medium, although not significantly at any time-point. Light microscopy showed diabetic ulcer fibroblasts to be large and widely spread. Transmission electron microscopy of cultured diabetic ulcer fibroblasts and nonlesional diabetic skin fibroblasts revealed a large dilated endoplasmic reticulum, a lack of microtubular structures and multiple lamellar and vesicular bodies. These results show a diminished proliferative capacity and abnormal morphology of fibroblasts derived from diabetic ulcers of non-insulin-dependent diabetes patients.

Fibroblasts derived from chronic diabetic ulcers differ in their response to stimulation with EGF, IGF-I, bFGF and PDGF-AB compared to controls.

Patients with diabetes mellitus experience impaired wound healing, often resulting in chronic foot ulcers. Healing can be accelerated by application of growth factors like platelet-derived growth factor (PDGF). We investigated the mitogenic responses, measured by 3[H]thymidine incorporation, of fibroblasts cultured from diabetic ulcers, non-diabetic ulcers, and non-lesional diabetic and age-matched controls, to recombinant human PDGF-AB, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and insulin-like growth factor (IGF-I). We determined the optimal concentration of these factors and investigated which single factor, or combination of factors, added simultaneously or sequentially, induced the highest mitogenic response. For single growth factor additions, in all fibroblast populations significant differences in mitogenic response to different growth factors were observed, with PDGF-AB consistently inducing the highest response and IGF-I the lowest (p < 0.043). IGF-I produced only a 1.7-fold stimulation over control in diabetic ulcer fibroblasts, versus 2.95-fold for chronic ulcer, 3.2-fold for non-lesional (p = 0.007) and 5-fold for age-matched fibroblasts (p = 0.007). The highest mitogenic response induced by EGF was significantly less for chronic ulcer fibroblasts compared with age-matched and nonlesional controls (p < 0.03), chronic ulcer fibroblasts also needed significantly more EGF to reach this optimal stimulus (p < 0.02 versus age-matched and non-lesional controls). The simultaneous addition of FGF-IGF-I, PDGF-IGF-I and FGF-PDGF to diabetic ulcer fibroblasts always produced a higher stimulatory response than sequential additions (p < or = 0.05). Also the addition of bFGF, PDGF-AB and EGF prior to IGF-I induced a higher 3[H]thymidine uptake in all fibroblasts compared to the combination of each in reverse order. Significant differences were observed when comparing the combinations of growth factors with the highest stimulatory responses (PDGF-IGF-I, FGF-PDGF and EGF-PDGF added simultaneously) to a double dose of PDGF, with the highest mean rank for the combination PDGF-IGF-I (p = 0.018). In conclusion, combinations such as PDGF-AB and IGF-I may be more useful than PDGF-AB alone for application in chronic diabetic wounds.

Review and expert opinion on prevention and treatment of infliximab-related infusion reactions

Infliximab (Remicade®; Schering‐Plough, Kenilworth, NJ, U.S.A.) is a chimeric monoclonal antibody that acts as a tumour necrosis factor‐α inhibitor. Infliximab is registered for the treatment of rheumatoid arthritis, psoriatic arthritis, Crohn disease, ulcerative colitis, ankylosing spondylitis and plaque‐type psoriasis. Like other foreign protein‐derived agents, infliximab may lead to infusion reactions during and after infusion. Infusion reactions occur in 3–22% of patients with psoriasis treated with infliximab. Most of these reactions are mild or moderate and only few are severe. Nevertheless, they may lead to discontinuation of treatment. As infliximab for psoriasis is prescribed as a last resort and is in most cases very effective, discontinuation of treatment is undesirable. With proper care and prevention of the infusion reactions the need to discontinue treatment with infliximab can be diminished. The objective of this article is to present a guideline for the management of infliximab‐related infusion reactions, based on the best available evidence. This guideline can be used in patients with psoriasis as well as in dermatology patients receiving infliximab for off‐label indications such as hidradenitis suppurativa or pyoderma gangrenosum.

Long-term efficacy of a single course of infliximab in hidradenitis suppurativa

Background  Hidradenitis suppurativa is a chronic inflammatory skin disease characterized by abscess formation, predominantly in the axillae and groins. The disease is difficult to treat and has a severe impact on quality of life. Recently, several case reports have been published describing successful treatment of hidradenitis suppurativa with infliximab and other tumour necrosis factor α inhibitors.

Quantification of cutaneous sclerosis with a skin elasticity meter in patients with generalized scleroderma

BACKGROUND The skin score, a subjective assessment of skin elasticity, is widely used in patients with systemic sclerosis. Although this scoring method is regarded as a validated and accepted tool, the interobserver and intraobserver reproducibility is relatively poor. OBJECTIVE Our purpose was to investigate whether the recently developed SEM 474 cutometer, which exerts a controlled vacuum force to the skin, can measure skin elasticity more objectively than the skin score. METHODS Skin elasticity was measured in 74 different body areas in patients with systemic sclerosis and compared with the skin score obtained from the same areas. RESULTS The cutometer produced quantitative and reproducible data. A large-diameter (8 mm) measuring probe was superior to a small probe. The interobserver intraclass correlation coefficient (ICC) was 0.92; the intraobserver ICC was 0.94. A linear correlation was found with the clinical skin score; the Spearman rank correlation test was 0.69. CONCLUSION The correlation with the skin score was reasonable, despite the observation that regional differences in skin elasticity were detected by the cutometer but not by the human observer, who automatically compensates for these factors and integrates them into the skin score. The high interobserver and intraobserver ICC makes the cutometer more suitable for quantifying changes in skin thickness than the subjective skin score.

Image processing in the study of wound healing

All wound care products that are said to be suitable for wound cleaning should preferably be evaluated with an observer-blind, quantitative system, as described above.

Dermal regeneration in native non-cross-linked collagen sponges with different extracellular matrix molecules.

Collagenous dermal templates can prevent scarring and wound contraction in the healing of full‐thickness defects. In a porcine wound model, full‐thickness wounds were substituted by reconstituted and native collagen sponges in combination with autologous split‐skin mesh grafts and covered with a semipermeable wound membrane. Native collagen sponges were also linked with either hyaluronic acid, elastin, or fibronectin. Reconstituted collagen matrixes, composed of cross‐linked small collagen fibrils, disintegrated within a week and did not contribute to dermal regeneration, whereas native collagen matrixes, composed of intact collagen fibers, disintegrated within 2 weeks and did contribute to dermal regeneration. Addition of extracellular matrix proteins retarded the disintegration to 4 weeks. However, fibronectin‐treated matrixes caused aberrant epithelization. When hyaluronic acid was added, matrixes were invaded by more fibroblasts and myofibroblasts. This process correlated with fibrosis and wound contraction. In contrast, the native collagen/elastin matrix reduced the amount of fibroblasts and myofibroblasts. This latter matrix resulted in optimal dermal regeneration and little wound contraction.

Extracorporeal photochemotherapy (photopheresis) induces apoptosis in lymphocytes : a possible mechanism of action of PUVA therapy

Abstract— The mechanism of action of psoralen plus UVA (PUVA) and photopheresis is not entirely understood. These therapies are assumed to be immunomodulating partly by gradually decreasing leukocyte viability. We investigated whether this delayed form of cell death was due to apoptosis. Untreated and treated (PUVA exposed) leukocytes obtained from six patients with systemic sclerosis and (untreated) leukocytes from healthy control individuals were studied. Qualitative gel electrophoresis and quantitative in situ nick translation analysis of DNA fragmentation was performed. Apoptosis of the treated cells did occur (gel electrophoresis) after 24 h. At t = 0 h, immediately after exposure to PUVA, there was no evidence of DNA fragmentation in the treated cells. The percentage of treated cells undergoing apoptosis was 20–55% at t = 24 h (in situ nick translation). The untreated leukocytes of the patients and the healthy individuals showed no distinctive rise in apoptotic cells. Apoptosis of the leukocytes after PUVA or photopheresis treatment might be a mechanism of action and might explain the therapeutic response.

Allogeneic fibroblasts in dermal substitutes induce inflammation and scar formation.

In the present study, we compared the use of autologous versus allogeneic fibroblasts in dermal skin substitutes in a porcine wound model. The allogeneic fibroblast populations were isolated from female and a male pig (allo‐1, ‐ 2 and ‐ 3) and the controls, autologous fibroblasts, from female graft‐recipient pigs (control). The histocompatibility of the three donor pigs with the recipient pigs was determined with a mixed lymphocyte reaction. In two pigs, full‐thickness wounds were treated with the fibroblast‐seeded dermal substitutes (n=5 per animal) and immediately overgrafted with meshed split‐skin autografts. After 6 weeks, wound contraction was measured by planimetry and scar formation was scored. At 2, 4, and 6 weeks biopsies were taken and evaluated for the presence of inflammatory reactions, myofibroblasts, and scar formation. The mixed lymphocyte reaction of both recipient pigs showed the highest responses on peripheral blood mononuclear cells of the allo‐3 donor pig, and was low or negative for allo‐1 and allo‐2. In all “allogeneic” wounds, more inflammatory cells were observed over time along with inflammatory foci consisting of a mix of lymphocytes and granulomatous cells. After 4 weeks, myofibroblasts were absent in the control wounds, whereas in “allogeneic” wounds, myofibroblasts colocalized with inflammation foci. The final scar tissue of the “allogeneic” wounds showed granulating areas with thin, immature collagen bundles. In contrast, the control wounds showed a dermal tissue with mature collagen bundles organized randomly like in normal skin. The wounds treated with allo‐3 fibroblasts showed in both pigs a significant increase in scar formation and wound contraction when compared with control wounds. In conclusion, for optimal restoration of dermal skin function with minimal scar formation, skin substitutes containing autologous fibroblasts are preferred over skin substitutes with allogeneic fibroblasts. (WOUND REP REG 2002;10:)