Hanna Galkowska

Chemokines, cytokines, and growth factors in keratinocytes and dermal endothelial cells in the margin of chronic diabetic foot ulcers

Keratinocytes and dermal endothelial cells, excluding leukocytes that infiltrate wounds, are the main source of soluble factors regulating healing of skin ulcers. We used immunohistochemistry to analyze the expression of various chemotactic and growth factors and their receptors in the margin of diabetic foot ulcers and in normal nondiabetic foot skin. Our study found significantly elevated expression of transforming growth factor‐β1 (TGF‐β1) and type I TGF‐β receptors (TGFβR1), granulocyte macrophage colony‐stimulating factor (GM‐CSF), and epidermal growth factor (EGF) in keratinocytes in the ulcer margin (p<0.05). Significantly increased expression of monocyte chemotactic protein‐1, GM‐CSF, CXCR1, and TGFβRI and decreased expression of interleukin (IL)‐10, IL‐15, and TGF‐β1 were observed in ulcer dermal endothelial cells (p<0.05). There was a lack of up‐regulation of IL‐8, CCR2A, IL‐10 receptor, GM‐CSF receptor, platelet‐derived growth factors and their receptors, vascular endothelial growth factor and its type II receptor, EGF receptor, insulin‐like growth factor‐1, and nitric oxide synthase‐2 in both KCs and endothelial cells in the ulcer. Finally, there was a lack of up‐regulation of IL‐10 and IL‐15 in keratinocytes and of EGF, basic fibroblast growth factor, and nitric oxide synthase‐3 in endothelial cells in the ulcer margins. The enhanced expression of some factors responsible for KC behavior could suggest an unimpaired capacity of keratinocytes to reepithelialize the margin of diabetic foot ulcers. However, lack of up‐regulation of some angiogenic and leukocyte chemotactic factors, associated with the reduced influx of immune cells, may account for a poor formation of granulation tissue and chronicity of ulcer epithelialization.

Reactivity of antibodies directed against human antigens with surface markers on canine leukocytes

A panel of anti-human antibodies was used in order to investigate their cross-reactivity with canine leukocytes. The labeling was carried out at the microscopic level by immunocytochemical staining. Of 50 antibodies 22 cross-reacted with canine leukocytes from afferent lymph and peripheral blood. All leukocytes reacted with MHM23 (CD18). Two anti-HLA DR antibodies, DK22 and L243, reacted mostly with veiled cells and with PHA-stimulated lymphocytes, whereas TAL1B5 was cross-reactive with all canine lymphocytes. The activation markers CD25, Ki-67, PCNA were identified on PHA-stimulated lymphocytes with ACT1, Ki-67 and PC10 clone produced antibodies. Canine eosinophils reacted with MHM6 (CD23) antibody. A large number of antibodies reacted with canine lymph veiled cells. Canine granulocytes and a subset of lymphocytes were stained with the anti-CD15 antibody only after treatment of cytospins with neuraminidase.

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.

Epidemiology and prevalence of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis in patients with diabetic foot ulcers: focus on the differences between species isolated from individuals with ischemic vs. neuropathic foot ulcers.

We examined whether foot ischemia or neuropathy with diabetic foot ulcer (DFU) promote selection of staphylococci species, evaluated frequency of MRSA and MRSE among strains yielded from patients with DFU and assessed multidrug resistance of isolates. Patients with DFU and foot osteomyelitis were divided into ischemic foot ulcer (IFU, n=21) and neuropathic foot ulcer (NFU, n=29) groups. Frequency of Staphylococcus epidermidis yielded from curettage of IFU was higher compared with NFU (P<0.05). S. epidermidis was also more frequently isolated from the toe web surface of patients with IFU compared with NFU (55% vs. 17.9%, respectively) and healthy volunteers (HV, n=20) (17.6%, P<0.05). These mostly MRSE strains (83.3-100%) originating from DFU patients were multidrug resistant (88.8%). Also, most of MRSA isolates were multidrug resistant (70.3%). Higher rates of MSSA from DFU patients than HV showed resistance to antimicrobials. This is the first report indicating that diabetic patients with IFU differ with NFU patients in higher frequency of S. epidermidis skin colonization and ulcer infection. We suggest that IFU should be defined as separate disease state of DFU and S. epidermidis should be appreciated as a nosocomial pathogen.

Slime production by Staphylococcus aureus and Staphylococcus epidermidis strains isolated from patients with diabetic foot ulcers

Slime production is a very important factor related to biofilm formation. The objective of the present study was to determine the frequency of slime production by Staphylococcus aureus and Staphylococcus epidermidis strains recovered from 50 patients with diabetic foot ulcers. Slime production was determined using the Congo red agar (CRA) method and compared with immunocytochemistry for the production of polysaccharide intercellular adhesin (PIA). Out of 55 S. aureus strains, 69% produced slime as shown by the CRA method. Of them, 84.2% also produced PIA. Of 17 CRA-negative strains, 70.6% produced PIA. Out of 20 S. epidermidis strains, 75% were CRA positive and 93.3% produced PIA. All CRA-negative S. epidermidis produced PIA. In conclusion, PIA production is a very common trait of S. aureus and S. epidermidis isolates obtained from diabetic foot ulcer patients.

CYTOKINES AND ADHERENCE MOLECULES INVOLVED IN SPONTANEOUS DENDRITIC CELL-LYMPHOCYTE CLUSTERING IN SKIN AFFERENT LYMPH

The skin afferent lymph dendritic cell (DC) spontaneously forms clusters with autologous T cells. The role of adhesion molecules and cytokines in this process was investigated. Analysis of the expression of adhesion receptors on the canine peripheral lymph DC revealed the presence of CD54, CD58, CD18 as well as CD49d and CD49e molecules and cell surface fibronectin. The CD54 and CD58 molecules were found to play a key role in the ‘spontaneous’ lymph cell clustering. Antibody against fibronectin, a substrate for CD49d and CD49e receptors, reduced DC‐lymphocyte binding. Analysis of the effect of cytokines revealed that the pro‐inflammatory IL1β rather than ILlα, and TNFα may be responsible for the enhanced lymph cell in vitro clustering. The IL6 had no such augmenting effect. The enhancing effect of endogenous ILlβ present in lymph was reduced by the ILlβ neutralizing antibody. The effect of exogenously added ILlβ was also limited by the IL1 receptor antagonist. The ILlRa alone had no effect on cell binding, even when used in the high doses. Neutralizing of ILlRa in lymph with the specific antibody brought about augmented cluster formation. The enhancing properties of TNFα on cell binding were reduced by the TNFα neutralizing antibody. The IL10 significantly limited lymph DC cluster formation with T cells. In conclusion, these data demonstrate that the present in lymph IL1β and TNFα may be responsible for the observed in vitro enhanced cluster formation of lymph DC with autologous T lymphocytes. Cell binding can be reduced by ILlRa and by IL10. It provides insight into the potential clinical use of these inhibitors.

Spontaneously active suppressive cells in canine peripheral blood

The phenomenon of the unusually high spontaneous suppressive activity of cells in peripheral blood of dogs was analysed. The m/c (mitomycin C)-treated population of peripheral blood leucocytes (PBL) contained cells able to reduce the responsiveness of autologous cells by 48 +/- 15% (P less than 0.01) and their activity was not indomethacin dependent. Thoracic duct lymphocytes (TDL) did not reduce the response of PBL to PHA, neither did cell crowding. The supernatants from 24-h cultures of m/c-treated PBL did not affect the response to PHA, and parallelly precultured cells inhibited the proliferation of PBL to a lesser degree (24 +/- 9%) than the fresh cells (50 +/- 16%, P less than 0.05). Addition of m/c-treated polymorphonuclear cells at PMN to PBL ratios of 1:4 and 1:1 progressively inhibited PBL reactivity to PHA, from 29.5 +/- 3.5% to 68.5 +/- 9%, respectively, and the supernatants from 24-h cultures of PMN reduced the proliferation by 48 +/- 2.8%. The neutrophil-derived inhibitory factor(s) was non-cytotoxic and reduced the formation of blasts to 61.5 +/- 3.5% of the control values. These results indicate that dog PBL from Lymphoprep gradient contain a population of non-recirculating, short-lived, spontaneously suppressive cells, mainly PMN, which modulate T cell reactivity in vitro, suggesting that neutrophils may be able to exert a regulatory effect in vivo.

Inhibition of formation of synapses between dendritic cells and lymphocytes in skin lymph in an allogeneic reaction by cyclosporine and tacrolimus.

Skin, an important component of composite tissue allografts is considered to be among the most immunogenic of tissues. The mechanisms of resistance of skin allografts to pharmacological immunosuppression remain unknown. We investigated this problem at the level of antigen presentation by graft dendritic cells (DC) to recipient lymphocytes (L). Cells obtained from lymph draining skin were examined for formation of synapses, necessary for antigen presentation, in the presence of cyclosporine (CsA) or tacrolimus (FK 506). In culture the frequency of DC-L synapses was greater in allogeneic than syngeneic combinations. Cells treated with FK 50% showed a decreased rate of formation of autologous or allogeneic DC-L synapses and lower expression of CD49d. The suppressive effect of FK 506 on DC-L synapse formation may explain the effectiveness of this drug for skin allograft survival.

A single step centrifugation method for the enrichment of veiled cells from canine afferent lymph

Veiled cells (VC) present in the afferent lymph of dogs with chronic lymphoedema could be enriched from 6% to about 50% VC by density gradient centrifugation on 15% metrizamide or discontinuous Percoll gradients. The recovery of VC was about 40% from 0.22 +/- 0.07 X 10(6) VC/ml of lymph. The cells were strongly Ia positive and had cytoplasmic S 100 protein. They were also strongly ATP-ase positive and showed heterogeneity in acid phosphatase, peroxidase and non-specific esterase activity. Low density VC from canine afferent lymph were able to stimulate both blood and lymphatic lymphocytes in autologous mixed leukocyte reaction when present at concentration as low as 5% of cultured cells.

Virulence of Bacteria Colonizing Vascular Bundles in Ischemic Lower Limbs

BACKGROUND We 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. METHODS We identified virulence genes in bacteria cultured from deep tissue of ischemic legs of 50 patients using a polymerase chain reaction technique. RESULTS The 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. CONCLUSIONS These 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.