Fuch's Endothelial Corneal Dystrophy in Cataract Patients Is Associated with Elevated Levels of Inflammatory Chemokines, but Not Growth Factors, in the Aqueous Humor.

The study investigated a profile of chemokines and growth factors in the aqueous humor (AH) of eyes with Fuch's endothelial corneal dystrophy (FECD) and cataracts in comparison with cataract patients as a control group. A total of 52 AH samples (26 FECD + cataract and 26 cataract/control) were collected before cataract surgery. None of the patients had any clinically apparent inflammation at the time of AH collection. The AH levels of MCP-1 (CCL2), MIP-1α (CCL3), MIP-1ß(CCL4), RANTES (CCL5), eotaxin (CCL11), IP-10 (CXCL10), FGF basic, G-CSF, GM-CSF, PDGF-bb, and VEGF were compared between the groups. The analyses were performed using the Bio-Plex 200 System from Bio-Rad. Among the studied parameters, the AH levels of RANTES, eotaxin, and IP-10 significantly increased in the FECD + cataract eyes, compared with the cataract controls (p < 0.05). Elevated levels of the RANTES, Eotaxin, and IP-10 indicate more intense inflammation in the eyes of patients in the FECD + cataract group. Moreover, these factors exhibit potential as predictive biomarkers for early detection of FECD in cataract patients. The discovery of elevated concentrations of biochemical markers in a patient, who has not yet received a clinical diagnosis, may suggest the need for heightened observation of the other eye to monitor the potential development of FECD.

Cytokine Profiling in Cerebrospinal Fluid of Patients with Newly Diagnosed Relapsing-Remitting Multiple Sclerosis (RRMS): Associations between Inflammatory Biomarkers and Disease Activity.

Cytokines regulate immune responses and are crucial to MS pathogenesis. This study evaluated pro-inflammatory and anti-inflammatory cytokine concentrations in the CSF of de novo diagnosed RRMS patients compared to healthy controls. We assessed cytokine levels in the CSF of 118 de novo diagnosed RRMS patients and 112 controls, analyzing relationships with time from symptom onset to diagnosis, MRI lesions, and serum vitamin D levels. Elevated levels of IL-2, IL-4, IL-6, IL-13, FGF-basic, and GM-CSF, and lower levels of IL-1ß, IL-1RA, IL-5, IL-7, IL-9, IL-10, IL-12p70, IL-15, G-CSF, PDGF-bb, and VEGF were observed in RRMS patients compared to controls. IL-2, IL-4, IL-12p70, PDGF, G-CSF, GM-CSF, and FGF-basic levels increased over time, while IL-10 decreased. IL-1ß, IL-1RA, IL-6, TNF-α, and PDGF-bb levels negatively correlated with serum vitamin D. TNF-α levels positively correlated with post-contrast-enhancing brain lesions. IL-15 levels negatively correlated with T2 and Gd(+) lesions in C-spine MRI, while TNF-α, PDGF-bb, and FGF-basic correlated positively with T2 lesions in C-spine MRI. IL-6 levels positively correlated with post-contrast-enhancing lesions in Th-spine MRI. Distinct cytokine profiles in the CSF of de novo diagnosed MS patients provide insights into MS pathogenesis and guide immunomodulatory therapy strategies.

A Prognostic Activity of Glutaredoxin 1 Protein (Grx1) in Colon Cancer.

Glutaredoxin 1 (Grx1) is an essential enzyme that regulates redox signal transduction and repairs protein oxidation by reversing S-glutathionylation, an oxidative modification of protein cysteine residues. Grx1 removes glutathione from proteins to restore their reduced state (protein-SH) and regulate protein-SSG levels in redox signaling networks. Thus, it can exert an influence on the development of cancer. To further investigate this problem, we performed an analysis of Grx1 expression in colon adenocarcinoma samples from the Polish population of patients with primary colon adenocarcinoma (stages I and II of colon cancer) and those with regional lymph node metastasis (stage III of colon cancer). Our study revealed a significant correlation between the expression of Grx1 protein through immunohistochemical analysis and various clinical characteristics of patients, such as histological grade, depth of invasion, angioinvasion, staging, regional lymph node invasion, and PCNA expression. It was found that almost 88% of patients with stage I had high levels of Grx1 expression, while only 1% of patients with stage III exhibited high levels of Grx1 protein expression. Furthermore, the study discovered that high levels of Grx1 expression were present in samples of colon mucosa without any pathological changes. These results were supported by in vitro analysis conducted on colorectal cancer cell lines that corresponded to stages I, II, and III of colorectal cancer, using qRT-PCR and Western blot.

Analysis of Cytokine and Chemokine Level in Tear Film in Keratoconus Patients before and after Corneal Cross-Linking (CXL) Treatment.

Keratoconus (KC) is a degenerative corneal disorder whose aetiology remains unknown. The aim of our study was to analyse the expressions of cytokines and chemokines in KC patients before and after specified time intervals after corneal cross-linking (CXL) treatment to better understand the molecular mechanisms occurring before and after CXL in KC patients process of corneal regeneration.; Tear samples were gathered from 52 participants immediately after the CXL procedure and during the 12-month follow-up period. All patients underwent a detailed ophthalmological examination and tear samples were collected before and after CXL at regular intervals: 1 day before and after the surgery, at the day 7 visit, and at 1, 3, 6, 9, and 12 months after CXL. The control group consisted of 20 healthy people. 10 patients were women (50%) and 10 were men (50%). The mean age was 30 ± 3 years of age. Tear analysis was performed using the Bio-Plex 3D Suspension Array System. Corneal topography parameters measured by Scheimpflug Camera included: keratometry values (Ks, Kf), PI-Apex, PI-Thinnest, Cylinder.; All the 12 months post-op values of the KC patients' topographic measurements were significantly lower than the pre-op. As for the tear cytokine levels comparison between the patient and control groups, cytokine levels of TNF-α, IL-6, and CXCL-10, among others, were detected in lower amounts in the KC group. The pre-op level of IL-6 exhibited a significant increase the day after CXL, whereas comparing the day after the procedure to 12 months after CXL, this showed a significant decrease. Both TNF-α and IL-1 showed a significant decrease compared to the day before and after CXL. We observed significantly higher levels of IL-1ß, IL-10, IFN-γ and TNF-α in moderate and severe keratoconus than in mild keratoconus (p < 0.05). We also demonstrated a statistically significant positive correlation between both pre-op and 12 months after CXL TNF-α, IFN-γ, IL-6 and Ks and Kf values (p < 0.05, r > 0); Alterations of inflammatory mediators in tear fluid after CXL link with topographic changes and may contribute to the development and progression of KC.

Glutathione Reductase Expression and Its Prognostic Significance in Colon Cancer.

Maintaining a balanced redox state within cells is crucial for the sustenance of life. The process involves continuous cytosolic disulfide reduction reactions to restore oxidized proteins to their reduced thiol forms. There are two main cellular antioxidant pathways-the thioredoxin (Trx) and glutathione (GSH)/glutaredoxin (Grx) systems. In the GSH/Grx system, glutathione reductase (GR; GSR) catalyses the reduction of GSH disulfide (GSSG) to its sulfhydryl form (GSH), which can then further reduce oxidized Grxs. GR is an essential enzyme that helps in maintaining the supply of reduced glutathione-GSH, which is a significant reducing thiol found in most cells and known for its antioxidant properties. Therefore, it can have a significant impact on cancer development. To investigate this further, we performed an immunohistochemical analysis of GR protein expression in colon adenocarcinoma samples collected from patients with primary colon adenocarcinoma (stage I and II) and patients with metastasis to regional lymph nodes (stage III). The results of our study revealed a significant relationship between the immunohistochemical expression of GR and tumour histological grade, depth of invasion, regional lymph node involvement, staging, and PCNA immunohistochemical expression. It was found that 95% of patients with stage I had low levels of GR expression, whereas 89% of patients with stage III had high levels of immunohistochemical expression. A high level of expression was also detected in the patients with stage II of the disease, where almost 63% were characterized by a high expression of GR. The Western blot method revealed that the highest level of expression was found in the LS 174T cell line, which corresponds to stage II. The results of our study indicate that the immunohistochemical expression of GR may act as an independent prognostic factor associated with colon adenocarcinoma patients' prognosis.

The Effect of Methyl-Derivatives of Flavanone on MCP-1, MIP-1ß, RANTES, and Eotaxin Release by Activated RAW264.7 Macrophages.

Chemokines, also known as chemotactic cytokines, stimulate the migration of immune cells. These molecules play a key role in the pathogenesis of inflammation leading to atherosclerosis, neurodegenerative disorders, rheumatoid arthritis, insulin-resistant diabetes, and cancer. Moreover, they take part in inflammatory bowel disease (IBD). The main objective of our research was to determine the activity of methyl-derivatives of flavanone, namely, 2'-methylflavanone (5B), 3'-methylflavanone (6B), 4'-methylflavanone (7B), and 6-methylflavanone (8B), on the releasing of selected cytokines by RAW264.7 macrophages activated by LPS. We determined the concentration of chemokines belonging to the CC chemokine family, namely, MCP-1, MIP-1ß, RANTES, and eotaxin, using the Bio-Plex Magnetic Luminex Assay and the Bio-PlexTM 200 System. Among the tested compounds, only 5B and 6B had the strongest effect on inhibiting the examined chemokines' release by macrophages. Therefore, 5B and 6B appear to be potentially useful in the prevention of diseases associated with the inflammatory process.

Effects of Donepezil on the Musculoskeletal System in Female Rats.

The extension of human life makes it more and more important to prevent and treat diseases of the elderly, including Alzheimer's disease (AD) and osteoporosis. Little is known about the effects of drugs used in the treatment of AD on the musculoskeletal system. The aim of the present study was to investigate the effects of donepezil, an acetylcholinesterase inhibitor, on the musculoskeletal system in rats with normal and reduced estrogen levels. The study was carried out on four groups of mature female rats: non-ovariectomized (NOVX) control rats, NOVX rats treated with donepezil, ovariectomized (OVX) control rats and OVX rats treated with donepezil. Donepezil (1 mg/kg p.o.) was administered for four weeks, starting one week after the ovariectomy. The serum concentrations of CTX-I, osteocalcin and other biochemical parameters, bone mass, density, mineralization, histomorphometric parameters and mechanical properties, and skeletal muscle mass and strength were examined. Estrogen deficiency increased bone resorption and formation and worsened cancellous bone mechanical properties and histomorphometric parameters. In NOVX rats, donepezil decreased bone volume to tissue volume ratio in the distal femoral metaphysis, increased the serum phosphorus concentration and tended to decrease skeletal muscle strength. No significant bone effects of donepezil were observed in OVX rats. The results of the present study indicate slightly unfavorable effects of donepezil on the musculoskeletal system in rats with normal estrogen levels.

Level of Secretion and the Role of the Nerve Growth Factor in Patients with Keratoconus before and after Collagen Fibre Cross-Linking Surgery.

BACKGROUND: The aim of this study was to analyse the concentration of the nerve growth factor (NGF-ß) in patients with keratoconus (KC) who are undergoing collagen fibre cross-linking (CXL) surgery in order to better understand the pathogenesis of this disease and observe the molecular changes occurring after the procedure. Among many cytokines, ß-NGF seems to play an important role in the healing processes of corneal damage. Therefore, its role in the regenerative process after CXL treatment may affect the course of treatment and its final results. Tear samples from 52 patients were collected in this prospective study. Additionally, the patients also had a number of tests performed, including corneal topography using optical coherence tomography. Flat (K 1), steep (K 2), cylindrical (CYL), and central corneal thickness (CCT) keratometry were assessed. The tear samples were collected, and other tests were performed before the CXL procedure and afterwards, during the 12-month follow-up period. The NGF concentration was measured using the Bio-Plex Magnetic Luminex Assay. Lower levels of NGF-ß were detected in the KC patients than in the control group (p < 0.001). The day after the procedure, the NGF-ß level was significantly lower (on average by 2.3 pg/mL) (p = 0.037) than before the procedure, after which, the level of the reagent increases, but only in the group with the advanced cone, one month after CXL it was significantly higher (p = 0.047). Regarding the correlation of NGF with topographic measurements, the following were found: NGF-ß correlates significantly (p < 0.05) and positively (r > 0) with K1 before the CXL procedure; NGF-ß correlates significantly (p < 0.05) and positively (r > 0) with K1 one month after CXL; NGF-ß correlates significantly (p < 0.05) and positively (r > 0) with CYL nine months after CXL; and, after twelve months, NGF-ß correlates significantly (p < 0.05) and positively (r > 0) with K2 and K1. Corneal sensitivity did not statistically and significantly correlate with the level of NGF-ß secretion. Our study suggests that NGF may be crucial in the development and progression of KC as well as in the repair mechanisms after CXL surgery. Further research is needed on the role of NGF and other inflammatory biomarkers for rapid diagnosis and selection of targeted therapy in patients with keratoconus.

Overexpression and Role of HHLA2, a Novel Immune Checkpoint, in Colorectal Cancer.

The study aimed to investigate correlations between HHLA2 levels and parameters, including microsatellite instability (MSI) status, CD8+ cells, and histopathological features: budding, tumor-infiltrating lymphocytes (TILs), TNM scale, grading, cytokines, chemokines, and cell signaling moleculesin colorectal cancer (CRC). Furthermore, the immune infiltration landscape and HHLA2-related pathways in colorectal cancer using available online datasets were analyzed. The study included 167 patients diagnosed with CRC. Expression of HHLA2 was detected by immunohistochemistry method (IHC) and enzyme-linked immunosorbent assay (ELISA). The IHC was used to evaluate the MSI and CD8+ status. The budding and TILs were measured using a light microscope. The concentrations of cytokines, chemokines, and cell signaling molecules were measured to analyze the data by the Bio-Plex Pro Human cytokine screening panel, 48 cytokine assay, and principal component analysis (PCA). Geneset enrichment analysis (GSEA) was conducted to identify HHLA2-related pathways. The biological function of HHLA2 was predicted by Gene Ontology (GO). Analysis of the immune infiltration landscape of HHLA2 in colorectal cancer was made by the web-based tool Camoip. High HHLA2 expression was detected in CRC tumor tissues compared to the adjacent noncancerous tissues. The percentage of HHLA2-positive tumors was 97%. GSEA and GO showed that HHLA2 upregulation correlated with cancer-related pathways and several biological functions. Tumor-infiltrating lymphocytes score correlated positively with IHC HHLA2 expression level percentage. There was a negative correlation between HHLA2, anti-tumor cytokines and pro-tumor growth factors. This study provides a valuable insight into the role of HHLA2 in CRC. We reveal the role of HHLA2 expression as well as a stimulatory and inhibitory immune checkpoint in colorectal cancer. Further research may verify the therapeutic values of the HHLA2-KIR3DL3/TMIGD2 pathway in colorectal cancer.

The Phenolic Profile and Anti-Inflammatory Effect of Ethanolic Extract of Polish Propolis on Activated Human Gingival Fibroblasts-1 Cell Line.

Propolis, owing to its antibacterial and anti-inflammatory properties, acts as a cariostatic agent, capable of preventing the accumulation of dental plaque and inhibiting inflammation. The anti-inflammatory properties of propolis are attributed to caffeic acid phenethyl ester (CAPE), which is present in European propolis. The objective of the conducted study was to assess the anti-inflammatory effects of the Polish ethanolic extract of propolis (EEP) and isolated CAPE on stimulated with LPS and IFN-α, as well as the combination of LPS and IFN-α. The cytotoxicity of the tested compounds was determined using the MTT assay. The concentrations of specific cytokines released by the HGF-1 cell line following treatment with EEP (25-50 µg/mL) or CAPE (25-50 µg/mL) were assessed in the culture supernatant. In the tested concentrations, both CAPE and EEP did not exert cytotoxic effects. Our results demonstrate that CAPE reduces TNF-α and IL-6 in contrast to EEP. Propolis seems effective in stimulating HGF-1 to release IL-6 and IL-8. A statistically significant difference was observed for IL-8 in HGF-1 stimulated by LPS+IFN-α and treated EEP at a concentration of 50 µg/mL (p = 0.021201). Moreover, we observed that CAPE demonstrates a stronger interaction with IL-8 compared to EEP, especially when CAPE was administered at a concentration of 50 µg/mL after LPS + IFN-α stimulation (p = 0.0005). Analysis of the phenolic profile performed by high-performance liquid chromatography allowed identification and quantification in the EEP sample of six phenolic acids, five flavonoids, and one aromatic ester-CAPE. Propolis and its compound-CAPE-exhibit immunomodulatory properties that influence the inflammatory process. Further studies may contribute to explaining the immunomodulatory action of EEP and CAPE and bring comprehensive conclusions.

In Vitro Anti-Inflammatory Activity of Methyl Derivatives of Flavanone.

Inflammation plays an important role in the immune defense against injury and infection agents. However, the inflammatory chronic process may lead to neurodegenerative diseases, atherosclerosis, inflammatory bowel diseases, or cancer. Flavanones present in citrus fruits exhibit biological activities, including anti-oxidative and anti-inflammatory properties. The beneficial effects of flavanones have been found based on in vitro cell cultures and animal studies. A suitable in vitro model for studying the inflammatory process are macrophages (RAW264.7 cell line) because, after stimulation using lipopolysaccharide (LPS), they release inflammatory cytokines involved in the immune response. We determined the nitrite concentration in the macrophage cell culture and detected ROS using chemiluminescence. Additionally, we measured the production of selected cytokines using the Bio-Plex Magnetic Luminex Assay and the Bio-PlexTM 200 System. For the first time, we have shown that methyl derivatives of flavanone inhibit NO and chemiluminescence generated via LPS-stimulated macrophages. Moreover, the tested compounds at 1-20 µM dose-dependently modulate proinflammatory cytokine production (IL-1ß, IL-6, IL-12p40, IL-12p70, and TNF-α) in stimulated RAW264.7 cells. The 2'-methylflavanone (5B) and the 3'-methylflavanone (6B) possess the strongest anti-inflammatory activity among all the tested flavanone derivatives. These compounds reduce the concentration of IL-6, IL-12p40, and IL12p70 compared to the core flavanone structure. Moreover, 2'-methylflavanone reduces TNF-α, and 3'-methylflavanone reduces IL-1ß secreted by RAW264.7 cells.

Allergy to Der p 23 influences the cytokine profile in patients with allergic asthma - a preliminary study.

OBJECTIVE: Der p 23 is a major allergen of Dermatophagoides pteronyssinus, which could contribute to allergic asthma. The study compared the cytokine profile (Il-1beta, Il-4, Il-5, Il-6, Il-13, Il-17, TNF-alpha) in patients with allergic asthma, with confirmed allergy to D. pteronyssinus and with the presence or absence of allergy to Der p 23. METHODS: Among 173 included patients, the following combinations were analyzed: profile A - Der p 1 (+), Der p 2 (+), and Der p 23 (-) observed in 38 (22%) patients; profile B - Der p 1 (+), Der p 2 (+), and Der p 23 (+) in 87 (50.3%) patients; and profile C - Der p 1 (-), Der p 2 (-), and Der p 23 (+) in 15 (8.7%) patients. RESULTS: The mean concentration of Il-1beta was significantly lower in profile A than in profiles B and C: 10.51 ± 5.22 (pg/ml) vs. 21.92 ± 11.34 vs. 23.1 ± 8.56 (A vs. B for p = 0.03 and A vs. C for p = 0.019). Similar trends were observed for Il-5: 38.5 ± 10.45 (pg/ml) vs. 94.8 ± 54.11 vs. 103.61 ± 34.9 (A vs. B for p = 0.008 and A vs. C for p = 0.001). CONCLUSION: The higher Il-1 and Il-5 activities observed in profiles B and C with Der p 23 (+) could be responsible for the more effective acceleration of allergic inflammation than in profile A with Der p 23.

The effect of ethanolic extract of Brazilian green propolis and artepillin C on aFGF-1, Eselectin, and CD40L secreted by human gingival fibroblasts.

INTRODUCTION: Periodontal diseases are among the most common diseases of the oral cavity in the worldwide population. The prevention of gingivitis and periodontitis is based on the removal of bacterial plaque from the teeth with use of toothpaste containing active substances. Noteworthy is the ethanolic extract of Brazilian green propolis (EEP-B), which, due to the high content of artepillin C, has anti-inflammatory, antibacterial, or immunostimulatory effects. Little is known about interactions between EEP-B and gingival fibroblasts within the oral cavity. The purpose of the article is to determine the role of acidic fibroblast growth factor (aFGF-1), E-selectin, and ligand of CD40 (CD40L) secreted by human gingival fibroblasts (HGF-1) in the gingiva. MATERIAL AND METHODS: We performed our experiments on gingival fibroblasts (HGF-1), which are an ideal in vitro model for studying the processes taking place within the gingiva. We incubated cells with EEP-B or artepillin C at the concentrations of 1 µg/ml and 10 µg/ml. The aFGF-1, E-selectin, and CD40L were detected using the Bio-Plex Magnetic Luminex Assay and the Bio-Plex 200 System. RESULTS: Ethanolic extract of Brazilian green propolis and artepillin C increased the levels of aFGF-1 secreted by HGF-1. Moreover, EEP-B decreased the levels of E-selectin in both tested concentrations, which was not proved for artepillin C. No changes in the concentration of CD40L released by HGF-1 were observed. CONCLUSIONS: The obtained results may suggest that EEP-B, due to the mixture of various compounds including flavonoids, accelerates the wound healing effects and has anti-inflammatory activity.

Negligible Effect of Estrogen Deficiency on Development of Skeletal Changes Induced by Type 1 Diabetes in Experimental Rat Models.

Although postmenopausal osteoporosis often occurs concurrently with diabetes, little is known about interactions between estrogen deficiency and hyperglycemia in the skeletal system. In the present study, the effects of estrogen deficiency on the development of biochemical, microstructural, and mechanical changes induced by streptozotocin-induced diabetes mellitus (DM) in the rat skeletal system were investigated. The experiments were carried out on nonovariectomized (NOVX) and ovariectomized (OVX) control and diabetic mature female Wistar rats. Serum levels of bone turnover markers (CTX-I and osteocalcin) and 23 cytokines, bone mass and mineralization, histomorphometric parameters, and mechanical properties of cancellous and compact bone were determined. The results were subjected to two-way ANOVA and principal component analysis (PCA). Estrogen deficiency induced osteoporotic changes, with increased bone resorption and formation, and worsening of microstructure (femoral metaphyseal BV/TV decreased by 13.0%) and mechanical properties of cancellous bone (the maximum load in the proximal tibial metaphysis decreased by 34.2%). DM in both the NOVX and OVX rats decreased bone mass, increased bone resorption and decreased bone formation, and worsened cancellous bone microarchitecture (for example, the femoral metaphyseal BV/TV decreased by 17.3% and 18.1%, respectively, in relation to the NOVX controls) and strength (the maximum load in the proximal tibial metaphysis decreased by 35.4% and 48.1%, respectively, in relation to the NOVX controls). Only in the diabetic rats, profound increases in some cytokine levels were noted. In conclusion, the changes induced by DM in female rats were only slightly intensified by estrogen deficiency. Despite similar effects on bone microstructure and strength, the influence of DM on the skeletal system was based on more profound systemic homeostasis changes than those induced by estrogen deficiency.

Chemokines and Growth Factors Produced by Lymphocytes in the Incompetent Great Saphenous Vein.

The role of cytokines in the pathogenesis of chronic venous disease (CVD) remains obscure. It has been postulated that oscillatory flow present in incompetent veins causes proinflammatory changes. Our earlier study confirmed this hypothesis. This study is aimed at assessing chemokines and growth factors (GFs) released by lymphocytes in patients with great saphenous vein (GSV) incompetence. In 34 patients exhibiting reflux in GSV, blood was derived from the cubital vein and from the incompetent saphenofemoral junction. In 12 healthy controls, blood was derived from the cubital vein. Lymphocyte culture with and without stimulation by phytohemagglutinin (PHA) was performed. Eotaxin, interleukin 8 (IL-8), macrophage inflammatory protein 1 A and 1B (MIP-1A and MIP-1B), interferon gamma-induced protein (IP-10), monocyte chemoattractant protein-1 (MCP-1), interleukin 5 (IL-5), fibroblast growth factor (FGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor (VEGF) were assessed in culture supernatants by a Bio-Plex assay. Higher concentrations of eotaxin and G-CSF were revealed in the incompetent GSV, compared with the concentrations in the patients' upper limbs. The concentrations of MIP-1A and MIP-1B were higher in the CVD group while the concentration of VEGF was lower. In the stimulated cultures, the concentration of G-CSF proved higher in the incompetent GSV, as compared with the patients' upper limbs. Between the groups, the concentration of eotaxin was higher in the CVD group, while the IL-5 and MCP-1 concentrations were lower. IL-8, IP-10, FGF, GM-CSF, and PDGF-BB did not reveal any significant differences in concentrations between the samples. These observations suggest that the concentrations of chemokines and GFs are different in the blood of CVD patients. The oscillatory flow present in incompetent veins may play a role in these changes. However, the role of cytokines in CVD requires further study.

10H-1,9-diazaphenothiazine and its 10-derivatives: synthesis, characterisation and biological evaluation as potential anticancer agents.

10H-1,9-diazaphenothiazine was obtained in the sulphurisation reaction of diphenylamine with elemental sulphur and transformed into new 10-substituted derivatives, containing alkyl and dialkylaminoalkyl groups at the thiazine nitrogen atom. The 1,9-diazaphenothiazine ring system was identified with advanced 1H and 13C NMR techniques (COSY, NOESY, HSQC and HMBC) and confirmed by X-ray diffraction analysis of the methyl derivative. The compounds exhibited significant anticancer activities against the human glioblastoma SNB-19, melanoma C-32 and breast cancer MDA-MB-231 cell lines. The most active 1,9-diazaphenothiazines were the derivatives with the propynyl and N, N-diethylaminoethyl groups being more potent than cisplatin. For those two compounds, the expression of H3, TP53, CDKN1A, BCL-2 and BAX genes was detected by the RT-QPCR method. The proteome profiling study showed the most probable compound action on SNB-19 cells through the intrinsic mitochondrial pathway of apoptosis. The 1,9-diazaphenotiazine system seems to be more potent than known isomeric ones (1,6-diaza-, 1,8-diaza-, 2,7-diaza- and 3,6-diazaphenothiazine).

Cytokines Produced by Lymphocytes in the Incompetent Great Saphenous Vein.

The pathogenesis of chronic venous disease (CVD) remains unclear, but lately inflammation is suggested to have an important role in its development. This study is aimed at assessing cytokines released by lymphocytes in patients with great saphenous vein (GSV) incompetence. In 34 patients exhibiting oscillatory flow (reflux) in GSV, blood was derived from the cubital vein and from the incompetent sapheno-femoral junction. In 12 healthy controls, blood was derived from the cubital vein. Lymphocyte culture with and without stimulation by phytohemagglutinin (PHA) was performed. Interleukins (IL) 1ß, 2, 4, 10, 12 (p70), and 17A; interleukin 1 receptor α (IL-1ra); tumor necrosis factor-α (TNF-α); interferon-gamma (IFN-γ); and RANTES were assessed in culture supernatants by the Bio-Plex assay. In both stimulated and unstimulated samples, in the examined group, IL-1ß and IFN-γ had higher concentrations and RANTES had lower concentrations when compared to those in the control group. In the examined group, IL-4 and IL-17A had higher concentrations without stimulation and TNF-α had higher concentrations with stimulation. The GSV samples had higher IL-2, IL-4, IL-12 (p70), and IFN-γ concentrations without stimulation and lower IL-2 and TNF-α concentrations with stimulation when compared to those of the upper limb in the examined group. These observations indicate that the oscillatory flow present in incompetent veins causes changes in the cytokine production by lymphocytes, promoting a proinflammatory profile. However, the relations between immunological cells, cytokines, and the endothelium require more insight.

The role of alert pathogens in bacterial diseases in patients hospitalized at the Department of Pulmonary Diseases and Tuberculosis Public Clinical Hospital No 3 in Zabrze in 2008-2012

BACKGROUND: The current public health problem is the increasing bacterial resistance to antibiotics. Microorganisms isolated from infections are more often non-susceptible to most available drugs. The microorganisms producing resistance mechanisms have been classified as so called alert pathogens. METHODS: We performed a total number of 3810 tests of bronchoalveolar lavage and sputum of patients hospitalized for respiratory diseases at the Department of Pulmonary Diseases and Tuberculosis at Public Clinical Hospital No 3 in Zabrze (Poland). The research was performed in the microbiological laboratory of the Department of Microbiology and Immunology in Zabrze, Medical University of Silesia in Katowice, Poland. The analysis included Gram-positive and Gram-negative alert species strains. RESULTS: In the period of five years, 144 strains of alert microorganisms have been isolated. The percentage of Gramnegative alert pathogens producing ESBL and KPC increased. MRSA, Steptococcus pneumoniae and Streptococcus pyogenes have been found to be the most often present among Gram-positive alert microorganisms. The lowest value of cultured alert pathogens (3.9%) was noted in 2008, whereas the highest (16.5%) in 2011. Gram-positive alert microorganisms showed resistance to macrolides and lincosamides, however, Gram-negative alert microorganisms showed the highest percentage of resistance to penicillins, penicillins with inhibitors and cephalosporins. CONCLUSIONS: Our work has shown that over the period 2008­2012 an increased percentage of Gramnegative and Gram-positive alert microorganisms was observed.

Novel Structurally Related Flavones Augment Cell Death Induced by rhsTRAIL.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) was identified as a powerful activator of apoptosis in tumor cells and one of the most promising candidates for cancer therapy with no toxicity against normal tissues. However, many tumor cells are resistant to TRAIL-induced apoptosis. The aim of this work was to analyze the improvement of the anticancer effect of rhsTRAIL (recombinant human soluble TRAIL) by nine flavones: 5-Hydroxyflavone, 6-Hydroxyflavone, 7-Hydroxyflavone and their new synthetic derivatives 5-acetoxyflavone, 5-butyryloxyflavone, 6-acetoxyflavone, 6-butyryloxyflavone, 7-acetoxyflavone and 7-butyryloxyflavone. We examined the cytotoxic and apoptotic effects of rhsTRAIL enhanced by novel structurally-related flavones on SW480 and SW620 colon cancer cells using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test, the lactate dehydrogenase assay and annexin V-FITC fluorescence staining. We observed a slight difference in the activities of the flavones that was dependent on their chemical structure. Our study indicates that all nine flavones significantly augment cell death by rhsTRAIL (cytotoxicity range 36.8 ± 1.7%-91.4 ± 1.7%; apoptosis increase of 33.0 ± 0.7%-78.5 ± 0.9%). Our study demonstrates the potential use of tested flavones in TRAIL-based anticancer therapy and prevention.

Novel Triazole Hybrids of Betulin: Synthesis and Biological Activity Profile.

Betulin derivatives containing a 1,2,3-triazole ring possess a wide spectrum of biological activities, including antiviral, anticancer, and antibacterial activity. A series of novel triazoles were prepared by the 1,3-dipolar cycloaddition reaction between the alkyne derivatives of betulin and organic azides. The chemical structures of the obtained compounds were defined by ¹H and 13C NMR, IR, and high-resolution mass spectrometry (HR-MS) analysis. The target triazoles were screened for their antiviral activity against DNA and RNA viruses. The cytotoxic activity of the obtained compounds 5a-k and 6a-h was determined using five human cancer cell lines (T47D, MCF-7, SNB-19, Colo-829, and C-32) by a WST-1 assay. The bistriazole 6b displayed a promising IC50 value (0.05 µM) against the human ductal carcinoma T47D (500-fold higher potency than cisplatin). The microdilution method was applied for an evaluation of the antimicrobial activity of all of the compounds. The triazole 5e containing a 3'-deoxythymidine-5'-yl moiety exhibited antibacterial activity against two gram-negative bacteria vz. Klebsiellapneumoniae and Escherichia coli (minimal inhibitory concentration (MIC) range of 0.95-1.95 µM).