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 patient's, 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.

Genetic Polymorphism and Messenger Ribonucleic Acid Concentrations of TNFα and TGFß Genes in Patients with Chronic Lower Limb Infections.

BACKGROUND: The number of chronic lower limb infections and their complications as venous and diabetic ulcers and chronic calf dermatitis is increasing worldwide. The clinical course and outcome in the immune responses to infection have been shown to be associated with genetic polymorphisms. The aim of study was to investigate frequencies of chosen single nucleotide polymorphisms (SNPs) in TNFα and TGFß genes in patients with chronic lower limb infections and evaluate expression of messenger ribonucleic acid (mRNA) concentrations in chronic leg ulcers. METHODS: Patients were divided into three groups: (group A) chronic venous leg ulcers, (group B) chronic post-traumatic non-healing wounds, and (group C) infected ischemic necrosis of the foot. Blood donors comprised the control group. Detection of polymorphisms was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and gene expression by real-time PCR methods. RESULTS: Patients in all groups showed higher frequency of TNFα gene polymorphism -308GG and lower frequency of -308GA genotypes than controls. The mutated homozygote AA was higher in groups A and B than in controls. The TGFß74GG genotype was represented at highest values in group B. The GC genotype was found in all groups at a similar concentration lower than in controls. Genotypes TGFß29TT and TC were represented at similar concentrations as controls. Analyses showed that the presence of the polymorphic allele -308A of TNFα gene was correlated with an increased concentration of gene expression in patients with chronic leg ulcers (group A). In the case of both TGFß gene polymorphisms the presence of polymorphic allele C resulted in increased TGFß gene expression. CONCLUSIONS: Comparison of genotypes in polymorphic sites in TNFα and TGFß genes with their expression concentrations showed that the presence of polymorphic alleles could predispose to increased production of their proteins. Patients with prolonged non-healing wounds should have their genotypes studied, and in cases of mutation, long-term antibiotic and immune protein supply should be considered.

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.

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.

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-beta1 (TGF-beta1) and type I TGF-beta receptors (TGFbetaR1), 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 TGFbetaRI and decreased expression of interleukin (IL)-10, IL-15, and TGF-beta1 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.

Neurogenic factors in the impaired healing of diabetic foot ulcers.

BACKGROUND: We hypothesize that the reduced innervation of skin can be observed both in clinically neuropathic and non-neuropathic diabetic foot ulcers and can contribute to low inflammatory cell infiltration. MATERIALS AND METHODS: Twenty patients with type 2 diabetes and active foot ulcers, without clinical evidence of peripheral sensory neuropathy (n = 12) and with sensory neuropathy (n = 8) were involved in this study. Biopsies from ulcer margin were examined immunohistochemically. RESULTS: Studies revealed presence of protein gene product 9.5 (PGP9.5)+ nerve endings only in reticular dermis in 3 of 12 non-neuropathic subjects, however, regenerating GAP-43+ endings were seen in dermis of almost all specimens. Lack of substance P+ nerve endings was characteristic for both groups. The reduced distribution of calcitonin gene-related peptide+ nerves in epidermis and dermis was seen mainly in neuropathic group. In neo-epidermis lack of nerve growth factor expression was observed in both groups, whereas neurotrophin 3 immunostaining was characteristic for neuropathic specimens (P < 0.03). Expression of trkA and trkC receptors did not differ significantly between groups. Low inflammatory cell infiltration and moderate presence of fibroblasts was characteristic for all studied specimens. CONCLUSIONS: The observed reduction of foot skin innervation and neurogenic factors expression can be correlated with low inflammatory cell accumulation and subsequently leads to the observed chronicity of diabetic foot ulcer healing process in both neuropathic and non-neuropathic patients.

[The molecular characteristic of Staphylococcus aureus and Staphylococcus epidermidis strains isolated from clinical sources]. / Charakterystyka molekularna szczepów Staphylococcus aureus i Staphylococcus epidermidis wyizolowanych z materialu klinicznego.

The aim of study was the molecular characteristic of S. aureus and S. epidermidis isolates obtained from skin surface, wounds, deep tissues of hospitalized patients and from skin surface of non-hospitalized patients. Genes encoding virulence factors were examined using PCR reaction and specific primers. Genes encoding adhesinsfnbA and cna and gene eta for epidermolytic toxin were mostly present in S. aureus isolates coming from wounds and deep tissues compared to these from skin surface. Gene atlE encoding autolysin of S. epidermidis was detected in all studied isolates, whereas gene icaAB was present in almost all isolates. Comparison of results obtained by PCR and conventional method of the resistance to methicillin estimation showed discrepances suggesting the need for using of both methods in some clinically difficult cases of S. aureus infection.

Expression of natural antimicrobial peptide beta-defensin-2 and Langerhans cell accumulation in epidermis from human non-healing leg ulcers.

Chronic wounds like venous calf and diabetic foot ulcers are frequently contaminated and colonized by bacteria and it remains unclear whether there is sufficient expression of defensins and recruitment of epidermal Langerhans cells in the margin of ulcer compared to normal skin. The aim of this study was to examine immunohistochemically the expression of beta-defensin-2 (hBD2), GM-CSF, VEGF growth factors and accumulation of CD1a+ Langerhans cells (LC) in epidermis from chronic skin ulcers and to compare it to normal skin from the corresponding areas. Studies were carried out in 10 patients with diabetic foot, 10 patients with varicous ulcers of the calf and 10 patients undergoing orthopedic surgery (normal skin for control). Biopsy specimens were immunostained using specific primary antibodies, LSAB+ kit based on biotin-avidin-peroxidase complex technique and DAB chromogen. Results were expressed as a mean staining intensity. Statistical analysis of staining showed significantly higher staining of hBD2 in both normal and ulcerated epidermis from foot sole skin compared to calf skin (normal and ulcerated, p < 0.05). Chronic ulcers showed the same expression of hBD2 as normal skin. There was significantly lower accumulation of CD1a+ LC in normal epidermis from foot sole skin compared to normal calf skin (p<0.05). Accumulation of CD1a+ LC and GM-CSF upregulation at the border area of diabetic foot ulcer and reduction of LC concentration at the margin of venous calf ulcer compared to normal skin were observed. It seems that normal calf and sole epidermis is, unlike in the mechanisms of innate immunity, influenced by the different keratinocyte turnover and bacterial flora colonizing these regions. Insufficient upregulation of hBD2 in both diabetic foot and venous calf ulcers may suggest the pathological role of this protein in the chronicity of ulcers.

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 (p < 0.05). Fibroblasts revealed increased presence in the ulcer margins compared with normal skin (p < 0.05). Skin endothelial cells expressed stronger von Willebrand factor and E-selectin compared with normal skin (p < 0.05). 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-1alpha, IL-1beta, 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, TNFalpha 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+, TGFbeta+ 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.

Dendritic cells as regulators of immune reactivity: implications for skin transplantation.

Skin allografts, in contrast to other organ transplants, are acutely rejected despite intensive and toxic for the graft recipient immunosuppressive therapy. Long-term immunosuppression increases the risk for life-threatening infections and cancers. This is why clinical skin allografting practically does not exist. Skin Langerhans' (dendritic) cells play a crucial role in the process of alloantigen recognition, its processing and initiation of the rejection reaction. These cells mature and migrate from the epidermis toward the dermal initial lymphatic vessels and further with afferent lymph, as veiled cells, they flow to the regional lymph nodes. Since a major goal in transplantation research is to understand and exploit the immunogenic properties of "passenger cells" as well as the tolerogenic properties of immature dendritic cells, studies concerning migrating less matured veiled cells obtained from afferent lymph draining skin seem to be relevant. Knowledge of mechanisms responsible for immunological synapse formation by veiled cells upon stimulation with allogeneic and bacterial antigens and of immunosuppressive drugs effect on this process, as well as of localization of Langerhans' cells in skin epidermis and dermis in the inflammatory foci, would facilitate a rational approach for the therapeutic protocols enabling the prolongation of skin allograft survival time.

Expression of apoptosis- and cell cycle-related proteins in epidermis of venous leg and diabetic foot ulcers.

BACKGROUND: Epithelialization of cutaneous ulcers is a long-lasting process. To study the pathomechanism of impaired epithelialization, we evaluated the role of cell cycle- and apoptosis-related proteins in the regenerating epidermis. We characterized immunohistochemically the expression of cell cycle regulators p63, CD29, PCNA, p53, pro- and antiapoptotic proteins bcl2, bax, caspase 3 and DNA breaks, as well as keratin 10, 16 and 17. METHODS: Studies were carried out in 12 patients with diabetic foot, and 10 patients with varicose ulcers of the calf. Skin biopsy specimens were obtained from the border area of ulcers and the topographically corresponding sites of normal skin of patients undergoing orthopedic surgery. Biopsy specimens were stained by use of specific primary antibodies, a kit based on biotin-avidin-peroxidase complex technique, and DAB substrate. Results were expressed as a mean staining intensity. RESULTS: At the edge of both types of ulcers, keratinocytes were p63+, CD29+, PCNA+ and p53-. The mean intensity of p63 and CD29 staining was slightly higher than in controls. The intensity of bcl2 staining was higher at the edge of diabetic ulcers compared with venous ulcers, whereas the intensity of bax staining was similar. The expression of caspase 3 was lower at the edge of venous ulcers and higher in diabetic ulcers and the intensity of TUNEL staining was lower at the edge of both types of ulcers compared with controls. Keratinocytes at the edge and distally to both types of ulcers expressed cytokeratin 16 and 17. There was no expression of cytokeratin 10 at the edge of ulcers. Together, there was a slight tendency for higher expression of cell cycle-related proteins in venous and of apoptosis-related proteins in diabetic ulcers epidermis; however, the differences were minor. CONCLUSIONS: The impaired epithelialization of chronic leg ulcers is not caused by an inadequate epidermal stem cell proliferation, differentiation, or apoptosis. It may rather reflect the distorted organization of wound bed, caused by infection and impaired nutrition supply, altering keratinocyte migration. To accelerate healing of an ulcer, modeling of the granulation tissue by regulatory cytokines but not stimulation of keratinocyte growth seems to be indicated.