Nemanja Zdravkovic

Galectin-3 Deficiency Accelerates High-Fat Diet Induced Obesity and Amplifies Inflammation in Adipose Tissue and Pancreatic Islets

Obesity-induced diabetes is associated with low-grade inflammation in adipose tissue and macrophage infiltration of islets. We show that ablation of galectin-3 (Gal-3), a galactoside-binding lectin, accelerates high-fat diet–induced obesity and diabetes. Obese LGALS3−/− mice have increased body weight, amount of total visceral adipose tissue (VAT), fasting blood glucose and insulin levels, homeostasis model assessment of insulin resistance, and markers of systemic inflammation compared with diet-matched wild-type (WT) animals. VAT of obese LGALS3−/− mice exhibited increased incidence of type 1 T and NKT lymphocytes and proinflammatory CD11c+CD11b+ macrophages and decreased CD4+CD25+FoxP3+ regulatory T cells and M2 macrophages. Pronounced mononuclear cell infiltrate, increased expression of NLRP3 inflammasome and interleukin-1β (IL-1β) in macrophages, and increased accumulation of advanced glycation end products (AGEs) and receptor for AGE (RAGE) expression were present in pancreatic islets of obese LGALS3−/− animals accompanied with elevated phosphorylated nuclear factor-κB (NF-κB) p65 and mature caspase-1 protein expression in pancreatic tissue and VAT. In vitro stimulation of LGALS3−/− peritoneal macrophages with lipopolysaccharide (LPS) and saturated fatty acid palmitate caused increased caspase-1–dependent IL-1β production and increased phosphorylation of NF-κB p65 compared with WT cells. Transfection of LGALS3−/− macrophages with NLRP3 small interfering RNA attenuated IL-1β production in response to palmitate and LPS plus palmitate. Obtained results suggest important protective roles for Gal-3 in obesity-induced inflammation and diabetes.

Regulatory T cells and ST2 signaling control diabetes induction with multiple low doses of streptozotocin.

Several peripheral mechanisms appear to be operational in limiting autoimmune damage of the islets of Langerhans and organ-specific T cell-mediated autoimmunity in general. These include cyclophosphamide sensitive T regulatory cells (Treg cells) and Th2 derived cytokine downregulation. We used the model of multiple low doses of streptozotocin (MLD-STZ) induced diabetes in susceptible C57BL/6 mice and resistant BALB/c mice to study these regulatory mechanisms. We show that low dose cyclophosphamide (CY) sensitive CD4(+)CD25(+)FoxP3(+) Treg cell-dependent mechanisms can be demonstrated in C57Bl/6 mice susceptible to MLD-STZ diabetes induction. CY pretreatment decreased Foxp3(+) cell count, glycemia, glycosuria and insulitis. In contrast, CY did not overcome resistance to diabetes induction in BALB/c mice. However, in BALB/c mice, deletion of ST2, an orphan member of the IL-1R family responsible for Th2 cell signaling leads to enhanced susceptibility to diabetes induction as evaluated by level of glycemia and glycosuria, number of infiltrating cells and beta cell loss. RT-PCR analysis of mRNA transcripts of diabetogenic cytokines revealed that the expression of TNF-alpha, and IFN-gamma was significantly enhanced in pancreatic lymph nodes by day 10 after diabetes induction in ST2-deficient mice in comparison with wild type BALB/c mice while IL-17 was detected only in ST2(-/-) mice by day 21. Our results are compatible with the notion that Treg cells are involved in MLD-STZ diabetes in susceptible mice and demonstrate that ST2-mediated signaling may also be involved in limiting Th1/Th17-mediated autoimmune pathology in diabetes resistant strain.

Intravenous immunoglobulins exposed to heme (heme IVIG) are more efficient than IVIG in attenuating autoimmune diabetes

Intravenous immunoglobulins (IVIG) are known to have a therapeutic effect in some autoimmune diseases. We examined the effect of IVIG and heme-exposed IVIG on the development of immune mediated diabetes induced in C57BL/6 mice by multiple low doses of streptozotocin. IVIG were used in a dose of 200mg/kg daily for 15 days. Treatment with IVIG resulted in significant attenuation of diabetes induction as evaluated by glycemia, glycosuria and HbA1c level. Interestingly, heme-exposed IVIG had a still stronger antidiabetogenic effect. Serum levels of proinflammatory cytokines TNF-α, IFN-γ and IL17 were lower in IVIG treated animals when compared with controls, while IL10 level was higher. The number of CD4(+)Foxp3(+) cells was higher in pancreatic lymph nodes of heme-exposed IVIG treated mice. Our results show that IVIG may downregulate diabetes induction possibly by favouring induction of T regulatory cells and suggest enhanced effect upon heme-binding to IVIG.

Histamine Blood Concentration in Ischemic Heart Disease Patients

The aim of this study was to investigate histamine blood concentration in subjects suffering from different types of ischemic heart diseases during the period of eight days. Our results showed that the histamine blood level was associated with different types of ischemic heart diseases. The blood histamine level in all investigated patients was significantly higher when compared to control subjects (44.87 ± 1.09 ng mL−1), indicating the increase of histamine release in patients suffering from coronary diseases. In patients suffering from ACS-UA and ACS-STEMI, the second day peak of histamine level occurs (90.85 ± 6.34 ng mL−1 and 121.7 ± 6.34 ng mL−1, resp.) probably as the reperfusion event. Furthermore, our data suggest that histamine can be additional parameter of myocardial ischemia along with cardiac specific enzymes and may prove to be an excellent single prognostic marker for multitude of ischemic heart diseases.

Elevated serum level of IL-23 correlates with expression of VEGF in human colorectal carcinoma.

BACKGROUND AND AIMS Interleukin-23 (IL-23) has a role in inflammatory bowel diseases (IBD) as a condition of higher risk of colorectal carcinogenesis. Vascular endothelial growth factor (VEGF) is overexpressed in IBD and colorectal carcinoma. Therefore, we aimed at uncovering the relationship between serum level of IL-23 and expression of VEGF in colorectal cancer (CRC) patients and to establish the relationship between VEGF and p53 and serum levels of IL-23, as well as its possible role in carcinogenesis of colorectal carcinomas. METHODS Levels of IL-23 from serum samples of patients with colorectal carcinoma (n = 40) and healthy control samples (n = 37) were examined for IL-23-Ab using an ELISA assay. We also determined the expression of VEGF and p53 by immunohistochemistry in 59 cases of CRC. RESULTS We found significantly higher serum levels of IL-23 in patients with CRC compared to control subjects (IL-23; mean 189.46 pg/mL vs. mean 34.77 pg/mL, p = 0.033). We also detected higher serum levels of IL-23 in patients with overexpressed VEGF (p = 0.028). Our results also showed that concomitant expression of VEGF and increased serum levels of IL-23 are in positive correlation with histological grade 2 (p <0.05). CONCLUSIONS Our data indicate that serum IL-23 levels are significantly elevated in CRC vs. control patients and are strongly associated with overexpression of VEGF, thus they may play an important role in carcinogenesis of CRC.

NT-proBNP for prognostic and diagnostic evaluation in patients with acute coronary syndromes

BACKGROUND AND AIM N terminal-proB-type natriuretic peptide (NT-proBNP) is synthesised and secreted from the ventricular myocardium. This marker is known to be elevated in patients with acute coronary syndromes (ACS). We evaluated NT-proBNP asa significant diagnostic marker and an important independent predictor of short-term mortality (one month) in patients with ACS. METHODS NT-proBNP and cardiac troponin I (cTI) were assessed in 134 consecutive patients (median age 66 years, 73% male)hospitalised for ACS in a cardiological university department. The patients were classified into ST-elevation ACS (STE-ACS, n = 74) and non-ST-elevation ACS (NSTE-ACS, n = 60) groups based on the ECG findings on admission. Patients with Killip class ≥ II were excluded. RESULTS The serum level of NT-proBNP on admission was significantly higher (p < 0.0005), while there was no difference in cTI serum level in the NSTE-ACS patients compared to STE-ACS patients. There was a significant positive correlation between NT-proBNP and cTI in the NSTE-ACS (r = 0.338, p = 0.008) and STE-ACS (r = 0.441, p < 0.0005) patients. There was a significant difference in NT-proBNP (p < 0.0005) and cTI (p < 0.0005) serum level between ACS patients who died within 30 days or who survived after one month. The increased NT-proBNP level is the strongest predictor of mortality in ACS patients, also NT-proBNP cut-point level of 1,490 pg/mL is a significant independent predictor of mortality. CONCLUSIONS We demonstrated the differences and the correlation in the secretion of NT-proBNP and cTI in patients with STE-ACS vs. NSTE-ACS. Our results provide evidence that NT-proBNP is a significant diagnostic marker and an important independent predictor of short-term mortality in patients with ACS.

Systemic photochemotherapy decreases the expression of IFN-γ, IL-12p40 and IL-23p19 in psoriatic plaques

Psoriasis vulgaris (PV) is a chronic skin disease with unclear pathogenesis. In the present study we investigated the effect of systemic photochemotherapy (PUVA therapy- psoralen and UVA therapy) on the expression of IFN-γ, IL-12p40 and IL-23p19 in lesional psoriatic skin. Fifteen patients with chronic plaque type psoriasis selected to be treated with PUVA therapy were recruited for this study. Expression of IFN-γ, IL-12p40 and IL-23p19 in psoriatic lesions before and after twenty PUVA treatments was established by using immunohistochemistry (IHC). A significant decrease in expression (p < 0.05) of IFN-γ, IL-12p40 and IL-23p19 in epidermis and dermis of psoriatic lesions was observed. The immunosuppressive effect of PUVA therapy presented with decreased expression of biologically active IL-23 (IL-12/IL-23p40 + IL-23p19) as a part of the Th17 pathway, and IFN-γ (Th1 pathway) led, in our patients, to a marked clinical improvement as shown by PASI (before therapy 20.55 and after therapy 3.33).

ST2 gene-deletion reveals a role of Foxp3+ regulatory T cells in diabetes modulation in BALB/c mice

BALB/c mice are resistant to diabetes induced by multiple low doses of streptozotocin (MLD-STZ; 5 × 40 mg/kg body weight [b.w.]) regimen in contrast to C57/BL6 mice. The deletion of ST2 gene renders BALB/c mice susceptible to diabetes induction. Cyclophosphamide (CY) in the dose of 175 mg/kg b.w. eliminated CD4+Foxp3+ regulatory T cells (Tregs) and enhanced disease severity in C57/BL6 mice, but it did not overcome resistance to diabetes in BALB/c mice and did not affect diabetes progression in ST2 knock-out (ST2KO) mice. We argued that a lower dose of CY may selectively eliminate Tregs while sparing effector T cells in BALB/c mice. Indeed, only a very low dose of CY (50 mg/kg b.w.) enhanced diabetes severity in ST2KO mice. This treatment eliminated Tregs in pancreatic lymph nodes in ST2KO mice, while markedly increasing the influx of CD8+, CD4+TNF-α+, and CD4+IFN-γ+ effector T cells (Teffs) in pancreata. Also, the aggravation of diabetes was accompanied with increased serum levels of TNF-α, IFN-γ, and IL-17. Taken together, our data suggest that the prevailing Th2 immune response in BALB/c mice may be responsible for the resistance to MLD-STZ diabetes and that ST2 gene deletion reveals the role of highly cyclophosphamide sensitive CD4+Foxp3+ regulatory T cells in the pancreatic lymph nodes in diabetes modulation.

Nitric oxide and tumour necrosis factor alpha in the process of pseudoexfoliation glaucoma

AIM To establish the role of nitric oxide (NO), ascorbic acid and tumour necrosis factor-α (TNF-α) in the pathogenesis of pseudoexfoliation glaucoma (XFG). METHODS Our study included 120 patients who were referred for cataract surgery. All patients were divided into four groups according to clinical findings: XFG, early and late pseudoexfoliation syndrome (XFS), and cataract (without pseudoexfoliation). Serum and aqueous humour levels of the ascorbic acid, NO and TNF-α were measured. The concentrations of the ascorbic acid and NO were measured by an appropriate spectrophotometric method. Enzyme-linked immunosorbent assay (ELISA) was used to determine TNF-α level. RESULTS Aqueous humour concentration of ascorbic acid was significantly lower in patients with late XFS (0.61±0.11 mmol/L) and XFG (0.48±0.15 mmol/L) compared to patients with early XFS (0.9±0.15 mmol/L) and cataract (1.16±0.22 mmol/L), while there was no difference in serum concentration in all examined groups. Aqueous humour concentration of NO was significantly higher in patients with XFG (77.7±11.4 µmol/L) compared to patients with early XFS (50.27±9.34 µmol/L) and cataract (49.77±7.1 µmol/L), while serum concentration was increased in the early stage of XFS (73.26±8.29 µmol/L). Aqueous humour level of proinflammatory cytokine TNF-α was increased in patients with XFS (early 460.04±18.32 pg/mL; late 502.42±53.23 pg/mL) and XFG (510.34±43.07 pg/mL), while there was no difference in serum level in all examined groups of patients. CONCLUSION Reduced ascorbic acid and elevated NO and inflammation related cytokine TNF-α level in aqueous humour of the patients with developed XFG suggest that oxidative stress induces local inflammation.

Tear Film Stability in Patients with Pseudexfoliation

Abstract Pseudoexfoliation syndrome is an age related disorder, characterized by abnormal fibrous fiber production and accumulation in different visceral organs as well as in the eye and periocular tissues. Hystological examination recorded the presence of the pseudoexfoliation in the conjunctiva, and they can disturb the accessory lacrimal gland and goblet cell function. This can explain tear film instability in patients with pseudoexfoliations. In our study, we examined the tear film stability in patients with and without pseudoexfoliation, using Schirmer test and tear break up time test. Our results indicated that patients with pseudoexfoliation had lower values of Schirmer and tear break up time tests than patients without it. Pseudoexfoliation is the main reason for the instability of the tear film, because of its negative impact on the conjucntival goblet cells. In conclusion, ophthalmologists must have these data on their mind in the process of the pseudoexfoliation glaucoma treatment and controlling.