INDEXES OF CYTOKINE PROFILE OF BLOOD IN PATIENTS WITH COMPLICATED ERYSIPELAS.

The cytokine blood profile in patients with complicated erysipelas was investigated. It was found that in patients with complications of erysipelas (gangrene, phlegmon, abscess, thrombophlebitis of the subcutaneous veins of the shin) levels of pro-inflammatory cytokines IL-1ß, TNF-α, IL-2, IL-6 in serum significantly increase and level of anti-inflammatory cytokine IL-4 increases slightly, as well as was found a significant increase in coefficients reflecting the ratio of pro-inflammatory and anti-inflammatory cytokines, which indicates the prevalence in the blood of examined patients with complications of erysipelas an anti-inflammatory properties. A more significant increase in pro-inflammatory cytokines serum levels is typical for patients with destructive forms of erysipelas - phlegmonous and gangrenous, a slight increase - for patients without purulent-necrotic component of complication (thrombophlebitis of the subcutaneous veins of the shin). In the future we plan to study pharmacological correction of shifts in cytokine blood profile with drugs with immunomodulating properties in patients with complicated erysipelas.

Analysis of Serum Cytokines and Single-Nucleotide Polymorphisms of SOD1, SOD2, and CAT in Erysipelas Patients.

Increased free radical production had been documented in group A (ß-hemolytic) streptococcus infection cases. Comparing 71 erysipelas patients to 55 age-matched healthy individuals, we sought for CAT, SOD1, and SOD2 single polymorphism mutation (SNPs) interactions with erysipelas' predisposition and serum cytokine levels in the acute and recovery phases of erysipelas infection. Whereas female patients had a higher predisposition to erysipelas, male patients were prone to having a facial localization of the infection. The presence of SOD1 G7958, SOD2 T2734, and CAT C262 alleles was linked to erysipelas' predisposition. T and C alleles of SOD2 T2734C individually were linked to patients with bullous and erythematous erysipelas, respectively. G and A alleles of SOD1 G7958A individually were associated with lower limbs and higher body part localizations of the infection, respectively. Serum levels of IL-1ß, CCL11, IL-2Rα, CXCL9, TRAIL, PDGF-BB, and CCL4 were associated with symptoms accompanying the infection, while IL-6, IL-9, IL-10, IL-13, IL-15, IL-17, G-CSF, and VEGF were associated with predisposition and recurrence of erysipelas. While variations of IL-1ß, IL-7, IL-8, IL-17, CCL5, and HGF were associated with the SOD2 T2734C SNP, variations of PDFG-BB and CCL2 were associated with the CAT C262T SNP.

[INDICATORS OF PHAGOCYTIC ACTIVITY OF MONOCYTES AND CYTOKINE PROFILE IN PATIENTS WITH ERYSIPELAS OF VARYING SEVERITY].

Erysipelas (PB) ­ a serious disease that is accompanied by severe complications and high lethality. The immunological investigations were carry out in 51 patients who were treated at the clinic for erysipelas with an immunomodulator application. Marked changes in the indices of phagocytic activity of monocytes (PHAM) and the cytokine profile in patients with erysipelas were note. Application in complex treatment of immunomodulator give the positive effect on the indices of PHAM.

HMGB1 in severe soft tissue infections caused by Streptococcus pyogenes.

Extracellular High Mobility Group Box 1 (HMGB1) has been associated with acute and chronic inflammatory conditions. However, little is known about HMGB1 in necrotizing bacterial infections. We hypothesized that the local HMGB1 response is excessive in severe soft tissue infections (STIs), which are characterized by necrosis and hyperinflammation. To explore this, tissue biopsies were collected from patients with varying severity of Streptococcus pyogenes skin and STIs, including erysipelas, cellulitis, and necrotizing fasciitis. Tissue sections were immunostained for HMGB1, S. pyogenes, and inflammatory cell infiltrates and results quantified by acquired computerized image analysis (ACIA). HMGB1 expression increased in parallel to disease severity and was significantly higher in necrotizing fasciitis than in erysipelas (p = 0.0023). Confocal microscopy of sections co-stained for HMGB1 and cell markers revealed both extracellular and cytoplasmic HMGB1, the latter of which was found predominantly in macrophages. To further verify macrophages as main source of activation triggered HMGB1 release, human macrophages were infected with clinical S. pyogenes isolates. The results demonstrated infection triggered release of HMGB1. Dual staining's visualized HMGB1 in areas close to, but not overlapping, with neutrophils, indicating a potential chemotactic role. In vitro transmigration experiments showed a chemotactic effect of HMGB1 on neutrophils. The data furthermore provided in vivo support that HGMB1 may form immunostimulatory complexes with IL-1ß. Taken together, the findings provide the first in vivo evidence that HMGB1 is abundant at the local site of severe bacterial STIs and its levels correlated to severity of infections; hence, indicating its potential value as a biomarker for tissue pathology.

Acquisition of complement factor H is important for pathogenesis of Streptococcus pyogenes infections: evidence from bacterial in vitro survival and human genetic association.

Streptococcus pyogenes (or group A streptococcus [GAS]) is a major human pathogen causing infections, such as tonsillitis, erysipelas, and sepsis. Several GAS strains bind host complement regulator factor H (CFH) via its domain 7 and, thereby, evade complement attack and C3b-mediated opsonophagocytosis. Importance of CFH binding for survival of GAS has been poorly studied because removal of CFH from plasma or blood causes vigorous complement activation, and specific inhibitors of the interaction have not been available. In this study, we found that activation of human complement by different GAS strains (n = 38) correlated negatively with binding of CFH via its domains 5-7. The importance of acquisition of host CFH for survival of GAS in vitro was studied next by blocking the binding with recombinant CFH5-7 lacking the regulatory domains 1-4. Using this fragment in full human blood resulted in death or radically reduced multiplication of all of the studied CFH-binding GAS strains. To study the importance of CFH binding in vivo (i.e., for pathogenesis of streptococcal infections), we used our recent finding that GAS binding to CFH is diminished in vitro by polymorphism 402H, which is also associated with age-related macular degeneration. We showed that allele 402H is suggested to be associated with protection from erysipelas (n = 278) and streptococcal tonsillitis (n = 209) compared with controls (n = 455) (p < 0.05). Taken together, the bacterial in vitro survival data and human genetic association revealed that binding of CFH is important for pathogenesis of GAS infections and suggested that inhibition of CFH binding can be a novel therapeutic approach in GAS infections.

[Cutaneous infections]. / Les infections cutanées.

The epidermis, the human being's primary protection against infection and aggression, can crack or split, and result in microbial bacteria penetrating it. Infections may, however, stem from other sources, such as viruses which enter the body via the respiratory tract, and then appear on the skin.

[Time course of changes in serum cytokine levels and the erysipelas clinical pattern].

The time course of changes in the values of proinflammatory tumor necrosis factor-alpha (TNF-alpha) and IL-8 and the anti-inflammatory cytokine IL-4 has been studied in 78 patients with erysipelas of different frequencies and pathomorphological forms of the disease. In erysipelas and its erythematous form, there is an increase in the levels of TNF-alpha and IL-8 in early phase of the disease, a decrease in these indices during convalescence, and a rise in the content of the cytokine IL-4, which are typical of an acute cyclic infectious process with a good outcome. In recurrent erysipelas and its most severe bullous-hemorrhagic form, there is imbalance in the production of cytokines that are suggestive of that there might be a disease recurrence. In erysipelas patients with a TNF-alpha value of 2.5 pg/ml or less at the onset of the disease, the relative risk for prolonged signs of systemic and local inflammatory reactions increases as compared with those who have a TNF-alpha level of more than 2.5 pg/ml.

Erysipelas caused by group A streptococcus activates the contact system and induces the release of heparin-binding protein.

Bacterial skin infections, such as erysipelas or cellulitis, are characterized by fever and a painful erythematous rash. Despite the high prevalence of these infections, little is known about the underlying pathogenic mechanisms. This is partly due to the fact that a bacterial diagnosis is often difficult to attain. To gain insight into the pathogenesis of erysipelas, we investigated the samples obtained from infected and noninfected areas of skin from 12 patients with erysipelas. Bacterial cultures, detection of specific streptococcal antibodies in convalescent sera, and immunohistochemical analyses of biopsies indicated group A streptococcal etiology in 11 of the 12 patients. Also, electron micrographs of erythematous skin confirmed the presence of group A streptococcal cells and showed a limited solubilization of the surface-attached M protein. Degradation of high-molecular-weight kininogen and upregulation of the bradykinin-1 receptor in inflamed tissues indicated activation of the contact system in 11 patients. Analyses of release of the vasoactive heparin-binding protein (HBP) showed increased levels in the infected as compared with the noninfected areas. The results suggest that group A streptococci induce contact activation and HBP release during skin infection, which likely contribute to the symptoms seen in erysipelas: fever, pain, erythema, and edema.

[Prognostic value of leukocytic migratory activity indices in patients with erysipelas].

Peripheral blood leukocytic migratory activity (LMA) was studied in patients with primary or recurrent erysipelas. A screening cell migration test (SCMT) was used in vitro and it established the prognostic value of MAL parameters at week 1 after the onset of erysipelas. It has been shown that a rapid transition of LMA from the phase of acceleration to that of inhibition characterizes the formation of an adequate immune response, corresponds to the good course of the disease, and has a low likelihood of recurrences. The probability of a recurrence is much higher than that when LMA tends to transit from suppression to acceleration and when LMA parameters are constant in the phase of suppression or acceleration. No transition of LMA to the phase of suppression in early convalescence suggests that the formation of an immune response to streptococcus is delayed.

[T-cell responsiveness to specific group A streptococcus antigens in patients with recurrent erysipelas].

Peripheral blood leukocytic migratory activity (LMA) was studied in 51 patients with recurrent erysipelas versus 63 patients with primary erysipelas. To reveal LMA, the authors employed in vitro a screening cell migration test as an indicator of the cooperation of T and B lymphocytes and macrophages, by stimulating with polysaccharide A, surface proteins, L-antigen, hyaluronidase, streptolysine-O, and a complete set of Grasse S. pyogenes. In patients with recurrent erysipelas, undulating LMA changes were found in the course of the disease in response to the stimulation with partial specific S. pyogenes antigens. There were differences in the time course of LMA changes, when stimulated with specific surface streptococcus antigens and with components of streptococcus with toxic activity. Significant LMA differences were found in relation to the pattern of a local process: active LMA changes from acceleration to suppression in the erythematous-hemorrhagic form and hyperergic reactions of LMA acceleration in the bullous-hemorrhagic one.

[T-cell responsiveness to specific group A streptococcus antigens in patients with primary erysipelas].

Peripheral blood leukocytic migratory activity (LMA) was studied in 63 patients with primary erysipelas. To reveal LMA, a screening cell migration test (SCMT) was used as an indicator of the cooperation of T- and B-lymphocytes and macrophages in the stimulation with polysaccharide A, surface proteins, L-antigen, hyaluronidase, streptolysin O, a complete S. pyogenes antigen complex after Grasse. The prognostic value of MAL parameters was established at week 1 after the onset of erysipelas. A rapid transition of LMA from the phase of acceleration to that of inhibition was shown to characterize the formation of an adequate response, to correspond to the good course of the disease, and to be followed by the low likelihood of recurrences. The probability of a subsequent recurrence is much higher than that when LMA tends to transit from suppression to acceleration and when LMA parameters are constant in the phase of suppression or acceleration. No transition of LMA to the phase of suppression in early convalescence suggests that the formation of an immune response to streptococcus is delayed.

Regulating effects of porcine interleukin-6 gene and CpG motifs on immune responses to porcine trivalent vaccines in mice.

In order to develop novel immunoadjuvants to boost immune response of conventional vaccines, experiments were conducted to investigate the regulating effects of porcine interleukin-6 gene and CpG motifs as the molecular adjuvants on immune responses of mice that were co-inoculated with trivalent vaccines against Swine fever, the Pasteurellosis and Erysipelas suis. Synthetic oligodeoxynuleotides containing CpG motifs were ligated into pUC18, forming recombinant pUC18-CpG plasmid. Eukaryotic plasmid expressing porcine interleukin-6 (VPIL-6) were also constructed as molecular adjuvants in an attempt to enhance levels of immune responses of mice co-administered with the trivalent vaccines in this paper. The cellular and humoral immune responses of mice were systematically analysed, and the experimental results were observed that the number of white blood cells, monocytes, granuloytes and lymphocytes significantly increased, respectively, in the mice immunized with VPIL-6, compared with those of the control; the IgG content and titre of specific antibodies to the trivalent vaccine mounted remarkably in the sera from the VPIL-6 vaccinated mice; the proliferation of lymphocytes and induced IL-2 activities were significantly increased in the vaccinated groups. The above-mentioned immune responses of mice co-inoculated with pUC18-CpG plasmid were significantly stronger than those of co-inoculated with pUC18 plasmid, suggesting that the immunostimulatory effect of oligodeoxynuleotides CpG is closely connected with the number of CpG motifs. These results suggest that the porcine IL-6 gene and CpG motifs could be employed as effective immunoadjuvants to elevate immunity to conventional vaccines.

The CD14+CD16+ monocytes in erysipelas are expanded and show reduced cytokine production.

In human peripheral blood the classical CD14(++)DR(+) monocytes and the pro-inflammatory CD14(+)CD16(+)DR(++) monocytes can be distinguished. In erysipelas we found strongly increased numbers of CD14(+)CD16(+) monocytes on the day of diagnosis (day 1) in 11 patients with an average of 150.5+/-76.0 cells/microl, while 1 patient had low levels (35 cells/microl, control donors 48.8+/-19.8 cells/microl). The classical monocytes were only moderately elevated in the erysipelas patients (factor 1.7 as compared to controls). Patients exhibited increased body temperature, erythrocyte sedimentation rate and increased serum levels for C-reactive protein (CRP), IL-6 and macrophage-colony-stimulating factor. Among these, body temperature and CRP showed a significant correlation to the numbers of CD14(+)CD16(+) monocytes. In 4 of 4 patients with high levels of CD14(+)CD16(+) monocytes, these levels returned to that seen in controls by day 5 of antibiotic therapy. Determination of intracellular TNF was performed by three-color immunofluorescence and flow cytometry after ex vivo stimulation with lipoteichoic acid, a typical constituent of streptococci. Here, patient CD14(+)DR(++) pro-inflammatory monocytes showed a twofold lower level of intracellular TNF. By contrast, expression of TNF was unaltered in the classical CD14(++) monocytes. These data show that in erysipelas the pro-inflammatory CD14(+)CD16(+)DR(++) monocytes are substantially expanded and selectively tolerant to stimulation by streptococcal products.

Batch potency testing of inactivated erysipelas vaccines by ELISA--development, validation and implementation.

Inactivated erysipelas vaccines are widely used to protect pigs against erysipelas disease caused by the bacterium Erysipelothrix (E.) rhusiopathiae. Quality control tests for this vaccine are laid down in the European Pharmacopoeia (Ph.Eur.) Monograph No. 64. A laboratory animal model using a vaccination-challenge procedure is currently required as batch potency test. More than 10 years ago we initiated the first studies to develop an alternative ELISA potency model to replace this regulatory challenge test in mice. A short retrospective outline of the various steps from the development of the method until implementation into the regulatory requirements is described.

Potency testing of inactivated erysipelas vaccines by ELISA--influence of the adjuvant on antibody development.

The humoral immune response after vaccination may be greatly influenced by the type of adjuvant used. In this study we investigated the influence of three different adjuvants on the serological response in laboratory mice after immunisation with commercial erysipelas vaccines.

Streptococcal DNase B is immunologically identical to superantigen SpeF but involves separate domains.

The previous suggestion that streptococcal superantigen SpeF might be identical to DNase B was confirmed in this study. Polyclonal SpeF-specific antisera were able to inhibit depolymerization of methyl-green DNA by DNase B. However, T-cell mitogenicity and nuclease activity appear to involve separate immune epitopes on SpeF, since sera with the capacity to neutralize the mitogenic activity of SpeF did not always inhibit the DNase activity.

[Immune disorders in patients with purulent necrotic complications of erysipelas]. / Immunnye narusheniia u bol'nykh s gnoino-nekroticheskimi oslozhneniiami rozhistogo vospaleniia.

There were examined 106 patients with purulent-necrotic complications (PNC) of erysipelas and 102 patients without PNC. Significant disorders of the immune status were revealed: the reduction of T-lymphocytes quantity, their subpopulational content dysbalance, the rise of level of a middle- and small-molecular immune complexes. In patients with PNC the immunocorrection conduction is expedient.