Andrzej Szkaradkiewicz

Proinflammatory cytokines and IL-10 in inflammatory bowel disease and colorectal cancer patients

IntroductionThe aim of the study was to describe the levels of circulating monocyte/macrophage pro-inflammatory cytokines (TNF-α, IL-1β IL-6, and IL-8) and an anti-inflammatory cytokine (IL-10) in inflammatory bowel disease (IBD) and colorectal cancer (CRC) patients and healthy controls.Materials and MethodsThe study was conducted on 15 healthy individuals, 20 patients with ulcerative colitis (UC), 12 with Crohn’s disease (CD), and 15 with CRC (Dukes’ stage B). Blood serum cytokine levels were measured by ELISA.ResultsThe patients with UC had significantly higher levels of the pro-inflammatory cytokines and of circulating IL-10 than the healthy controls. The patients with CD and CRC had the same specific pattern of serum cytokines of significantly elevated levels of the pro-inflammatory cytokines, but the IL-10 levels were within the range found in the healthy individuals.ConclusionsThus our results demonstrate that both IBD and CRC are linked with an intensified production of a wide array of monocyte/macrophage pro-inflammatory cytokines which is not accompanied by elevated levels of circulating IL-10, except for its insufficiently inhibitory elevation in UC patients.

Natural Killer Cell Cytotoxicity and Immunosuppressive Cytokines (IL-10, TGF-β1) in Patients with Gastric Cancer

Cytotoxic activity of NK cells was estimated as related to IL-10 and TGF-β1 serum levels and Helicobacter pylori infection in gastric cancer patients. Moreover, we sought to determine whether human gastric adenocarcinoma cells in vitro release IL-10, TGF-β1 or factor(s) affecting NK cytotoxicity. The studies were conducted on 42 patients with gastric cancer (14 with I-II stage—group 1; 28 with III-IV stage—group 2) and on 20 healthy volunteers. The cytotoxicity was tested on NK cells isolated from peripheral blood. IL-10 and TGF-β1 levels were determined by ELISA. H. pylori was detected in cultures of gastric mucosa biopsies and in direct preparations. In 71.4% patients of group 1 NK cytotoxicity and IL-10 serum levels remained within a normal range while in 68% patients of group 2 a marked decrease was noted in cytotoxic function of NK cells, accompanied by increased levels of IL-10 in serum. In turn, in most patients of either group, independently of NK cytotoxicity and stage grouping in the patients, elevated serum levels of TGF-β1 were detected. Presence of H. pylori infection manifested no relationship with NK cytotoxicity, IL-10, or the TGF-β1 serum levels. In cultures of tumour cells presence of IL-10 and TGF-β1 was demonstrated. Nevertheless, supernatants of the cultures did not change cytotoxic activity of NK cells. Development of gastric carcinoma is accompanied by markedly decreased cytotoxic function of NK cells and by elevated IL-10 and TGF-β1 serum levels. Gastric carcinoma cells may release IL-10, the suppressive activity of which may in a secondary manner decrease NK cytotoxicity.

Bacillus oleronius and Demodex mite infestation in patients with chronic blepharitis

To better recognize the pathogenicity of ocular Demodex mites, we analysed Bacillus oleronius infection in patients with Demodex-related chronic blepharitis. The studies were conducted on 68 adult patients, in whom ophthalmological and parasitological tests permitted the distinction of a group of 38 patients with a diagnosis of Demodex-related chronic blepharitis (group 1, including a subgroup 1a with moderate blepharitis and a subgroup 1b with severe blepharitis) and a group of 30 healthy individuals (group 2). In every person studied six eyelashes were epilated from each eye and the number of Demodex per eyelash was scored. In parallel, bacterial culture and isolation allowed their phenotypic and molecular identification. The drug sensitivity of the isolates was tested using E-tests. Intensity of Demodex infestation showed no significant differences between subgroups 1a and 1b. From the epilated eyelashes 23 bacterial isolates were obtained, identified as being B. oleronius. All the studied strains were sensitive to ciprofloxacin, doxycycline and gentamicin. The Demodex mite represents an independent aetiopathogenetic factor in blepharitis. In parallel, the parasite may act as a carrier of B. oleronius bacteria, which most probably function as a co-pathogen in the development of severe forms of blepharitis.

Cytokine response in patients with chronic infections caused by Staphylococcus aureus strains and diversification of their Agr system classes

This study aimed to describe the levels of circulating cytokine levels produced by Th lymphocytes (IFN-γ, IL-4, IL-10, IL-17A), as well as the levels of cytokines produced by monocytes/macrophages (TNF-α, IL-1β, IL-12), in patients with chronic infections caused by Staphylococcus aureus strains, particularly in the context of the diversification of their Agr system classes. The studies were conducted on adult patients, including 50 patients with chronic suppurative dermatitis, 40 patients with chronic infections of the upper respiratory tract and 25 healthy individuals (control group). Blood serum cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA). S. aureus was detected in cultures of suppurative dermal exudates or of pharyngeal smears. Classes of Agr systems in the S. aureus strains were identified using polymerase chain reaction (PCR). In both groups of patients, on average, levels of IFN-γ were doubled, while levels of IL-17A were increased by 2.5-fold, which, however, was not accompanied by increased levels of TNF-α or IL-12. The data indicate that the development of S. aureus infection among the studied patients was linked to an impoverished cytokine response of monocytes/macrophages, while that induced by the pathogen lymphocytes Th17/Th1 may be responsible for promotion of the chronic inflammatory response. In parallel, no quantitative or qualitative differences were disclosed between cytokine responses manifested by subgroups of patients infected with S. aureus strains belonging to class IV Agr, as compared to patients infected with strains of classes I to III Agr. Nevertheless, in the patients, strains belonging to class IV Agr prevailed, which points to the preferential relationship between the class and the pathogenicity of S. aureus.

Inflammatory bowel disease — is there something new in the immunological background?

In the present paper we correlate clinical data, as well as histopathological, immunohistochemical and molecular biology methods, with the occurrence of both forms of inflammatory bowel disease (IBD) i.e. ulcerative colitis and Crohns disease. We found that patients with a history of Epstein-Barr virus (EBV) or cytomegalovirus (CMV) infections, as well as steroid treatment, had increased susceptibility to the development of IBD. The diagnosis of IBD was confirmed by histopathology. Previous infections by EBV and CMV, as well as M. tuberculosis, were proved by PCR-based techniques and in situ hybridization. We found PCR-proved latent viral infections in 30-50% of the IBD patients we studied. However, we were unable to prove the presence of viral antigens by immunohistochemistry for EBV or CMV. We found positive correlations between the presence of anti-CMV IgG, as well as PCR-positive results for M. tuberculosis with an ulcerative colitis diagnosis. Additionally, up to 80% of IBD patients used steroids, which was found to be correlated with a diagnosis of Crohns disease. Our data may support the theory that IBD could be related to previous viral infections and the use of steroids.

The Participation of p53 and bcl-2 Proteins in Gastric Carcinomas Associated with Helicobacterpylori and/or Epstein-Barr Virus (EBV).

In the presented studies p53 and bcl-2 proteins expression were evaluated in samples of gastric carcinomas in patients with Helicobacter pylori or EBV or without H. pylori/EBV infection. The studies were conducted on 64 adult patients with gastric adenocarcinomas: 16 patients with H. pylori (cagA+)-positivity (group 1), 14 with EBV-positive tumours (group 2), 12 with H. pylori/EBV-positive tumours (group 3) and 22 patients with H. pylori/EBV-negative tumours (group 4). H. pylori presence in gastric tumour specimens was detected using Giemsa staining and bacterial culture technique. Moreover, cagA gene was detected using PCR. EBV infection was detected based on EBER presence in the tissue by RNA in situ hybridization. Expressions of p53 and bcl-2 proteins were analysed using immunohistochemistry. Expression of p53 was noted in 14 (84%) patients from group 1, 8 (57%) patients from group 2, 7 (58%) patients from group 3, and 19 (86%) patients from group 4, whereas expression of bcl-2 was noted in 12 (75%) patients from group 1, in 10 (71%) patients from group 2, 9 (75%) patients from group 3, and 6 (27%) patients from group 4. The obtained results allow the conclusion, that H. pylori (cagA+)-associated development of the gastric adenocarcinoma is determined by abnormalities in the p53 protein function and overexpression of anti-apoptotic bcl-2 protein, whereas EBV-associated adenocarcinomas seem to be related with apoptosis resistance associated with bcl-2 overexpression.

Evaluation of antimicrobial resistance of Helicobacter pylori in the last 15 years in West Poland

Increasing resistance to drugs represents a serious problem in treatment of infections with Helicobacter pylori, providing cause of frequent therapeutic failures. Present study aimed at analysis of changes in resistance of H. pylori to antibiotics in West Poland within the recent 15 years. 108 strains of H. pylori were analysed, isolated from gastric mucosa of adult patients. Group 1 involved 66 strains isolated in years of 1998/1999. Group 2 comprised 42 isolates obtained in years of 2013/2014. Susceptibility to amoxicillin (AMX), clarithromycin (CL), tetracycline (TC) and metronidazole (MTZ) was determined by E-test (AB Biodisc). All strains on both studied groups were susceptible to AMX. In group 1 all strains proved to be susceptible to TC, while 9% and 36% of tested strains were resistant to CL and MTZ, respectively. By contrast, in group 2, 31% and 83% of strains were resistant to CL and MTZ, respectively. In parallel, 14% strains were found to be resistant to TC (according to EUCAST interpretations). In West Poland, within recent 15 years a dramatic increase was noted in H. pylori strains resistant to metronidazole. In parallel, a significant increase was noted in proportion of strains resistant to clarithromycin.

Expression of cagA, virB/D Complex and/or vacA Genes in Helicobacter pylori Strains Originating from Patients with Gastric Diseases

In order to better understand pathogenicity of Helicobacter pylori, particularly in the context of its carcinogenic activity, we analysed expression of virulence genes: cagA, virB/D complex (virB4, virB7, virB8, virB9, virB10, virB11, virD4) and vacA in strains of the pathogen originating from persons with gastric diseases. The studies were conducted on 42 strains of H. pylori isolated from patients with histological diagnosis of non-atrophic gastritis—NAG (group 1, including subgroup 1 containing cagA+ isolates and subgroup 2 containing cagA- strains), multifocal atrophic gastritis—MAG (group 2) and gastric adenocarcinoma—GC (group 3). Expression of H. pylori genes was studied using microarray technology. In group 1, in all strains of H. pylori cagA+ (subgroup 1) high expression of the gene as well as of virB/D was disclosed, accompanied by moderate expression of vacA. In strains of subgroup 2 a moderate expression of vacA was detected. All strains in groups 2 and 3 carried cagA gene but they differed in its expression: a high expression was detected in isolates of group 2 and its hyperexpression in strains of group 3 (hypervirulent strains). In both groups high expression of virB/D and vacA was disclosed. Our results indicate that chronic active gastritis may be induced by both cagA+ strains of H. pylori, manifesting high expression of virB/D complex but moderate activity of vacA, and cagA- strains with moderate expression of vacA gene. On the other hand, in progression of gastric pathology and carcinogenesis linked to H. pylori a significant role was played by hypervirulent strains, manifesting a very high expression of cagA and high activity of virB/D and vacA genes.

Selected Factors of Innate Immunity in Healthy Individuals with S. aureus Nasal Carriage

Nasal carriage of Staphylococcus aureus represents a well-defined factor of risk involving community and hospital-acquired infections. Recently a significance of several host factors has been pointed out and, in particular, of immune determinants in nasal S. aureus colonization. Therefore, this study aimed at analysis of manifestation involving manifestation in the nasal secretions of important components of the host innate immunity – human beta-defensin-2 (HBD-2), lysozyme (Ly), and interferon-gamma (IFN-γ) in healthy individuals and in persons with persistent carriage of S. aureus. The studies were conducted in two groups of healthy volunteers, encompassing non-carriers (group 1) or persistent carriers of S. aureus (group 2). Elisa assays were employed to evaluate levels of HBD-2, Ly, and IFN-γ in nasal secretions of the examined donors. In S. aureus carriers a significant variability of HBD-2 levels was detected, corresponding to, respectively, the high (averaging at 1.46 ng/ml) and the low (averaging at 0.13 ng/ml) secretory response of the defensin. The level of Ly in S. aureus carriers averaged at 1.46 μg/ml and it manifested no significant difference as compared to that noted in non-carriers. In turn, concentrations of IFN-γ in nasal secretions in the group of carriers of S. aureus amounted on the average to 81.7 pg/ml and they were 1.3-fold higher that in the group of non-carriers. The obtained results allow to conclude that IFN-γ secretion by the nasal cavity-colonizing S. aureus remains quantitatively insufficient to eliminate the pathogen. Nevertheless, a significant increase in levels of this host factor may be important for restriction of the staphylococcal colonization and protection against development of an invasive infection. In turn, the role of HBD-2 and Ly in inactivation of the colonizing S. aureus remains doubtful.

Cytokine Response in Autovaccine-Treated Patients with Chronic Staphylococcus Aureus Infections

This study aims to describe the levels of circulating cytokines produced by Th lymphocytes (IFN-γ, IL-4, IL-10, IL-17A), as well as the levels of cytokines produced by monocytes/macrophages (TNF-α, IL-1β, IL-12), in patients with chronic Staphylococcus aureus infections before treatment and following completion of autovaccine treatment. The study was carried out on adult individuals, including 25 healthy subjects (group 1, control, not treated), 50 patients with chronic suppurative dermatitis (group 2) and 40 patients with chronic infections of the upper respiratory tract (group 3). Blood serum cytokine levels were measured by enzyme–linked immunosorbent assay (ELISA). S. aureus was detected in cultures of suppurative dermal exudates or of pharyngeal smears. For every individual patient an autovaccine was prepared, containing a suspension of inactivated S. aureus bacteria (1.5 × 108 bacteria/ml) isolated from the patient. The autovaccine was administered subcutaneously for a period exceeding 3 months, for a total of 18 injections. The average level of IFN-γ and IL-17 was 2–2.5 times higher in the infected patients. This was not accompanied by an increase in TNF-α or IL-12 levels. A treatment with autovaccine eradicated S. aureus infection in 42 (84%) patients of group 2 and in 14 (35%) patients of group 3. A significant increase (two-fold) in IL-17A was observed in treated patients. Also, following the treatment with autovaccine, all patients demonstrated a significant increase in the levels of IFN-γ, TNF-α and IL-12. These studies showed for the first time that efficiency of the autovaccine treatment in patients with chronic S. aureus infection depends on an adequate secretory response of TH17 cells.