Assessment of EN-RAGE, sRAGE, and its isoforms: cRAGE, esRAGE in obese patients treated by moderate caloric restriction combined with physical activity conducted in hospital condition.

BACKGROUND: AGEs, their receptor (RAGE), and the extracellular newly identified receptor for AGEs product-binding protein (EN-RAGE) are implicated in the pathogenesis of inflammation. AIM: We analyzed serum EN-RAGE, soluble RAGE (sRAGE), and their isoforms: endogenous secretory - esRAGE and cleaved - cRAGE concentrations in lean controls (n = 74) and in patients with obesity (n = 71) treated for three weeks with moderate calorie restriction (CR) combined with physical activity in a hospital condition. METHODS: Using the ELISA method, serum sRAGE, esRAGE, and EN-RAGE were measured before and after CR. RESULTS: The serum level of sRAGE and esRAGE in patients with obesity was lower than that in non-obese individuals, contrary to cRAGE. EN-RAGE concentration was about three times higher in obese patients. Gradually, a rise in BMI resulted in sRAGE, esRAGE reduction, and EN-RAGE increase. The sRAGE concentration was sex-dependent, indicating a higher value in lean men. A moderate negative correlation was observed between BMI and all RAGE isoforms, whereas EN-RAGE displays a positive correlation. CR resulted in an expected decrease in anthropometric, metabolic, and proinflammatory parameters and EN-RAGE, but no RAGE isoforms. The ratio EN-RAGE/sRAGE was higher in obese humans than in control and was not modified by CR. CONCLUSION: Obesity decreases sRAGE and esRAGE and increases EN-RAGE concentration. Moderate CR and physical activity by decreasing inflammation reduces EN-RAGE but is insufficient to increase sRAGE and esRAGE to the extent observed in lean patients. EN-RAGE instead of sRAGE could be helpful to indicate a better outcome of moderate dietary intervention in obese subjects.

Effect of cellular senescence on the response of human peritoneal mesothelial cells to TGF-ß.

Transforming growth factor ß (TGF-ß) is implicated in both mesothelial-to-mesenchymal transition (MMT) and cellular senescence of human peritoneal mesothelial cells (HPMCs). We previously showed that senescent HPMCs could spontaneously acquire some phenotypic features of MMT, which in young HPMCs were induced by TGF-ß. Here, we used electron microscopy, as well as global gene and protein profiling to assess in detail how exposure to TGF-ß impacts on young and senescent HPMCs in vitro. We found that TGF-ß induced structural changes consistent with MMT in young, but not in senescent HPMCs. Of all genes and proteins identified reliably in HPMCs across all treatments and states, 4,656 targets represented overlapping genes and proteins. Following exposure to TGF-ß, 137 proteins and 46 transcripts were significantly changed in young cells, compared to 225 proteins and only 2 transcripts in senescent cells. Identified differences between young and senescent HPMCs were related predominantly to wound healing, integrin-mediated signalling, production of proteases and extracellular matrix components, and cytoskeleton structure. Thus, the response of senescent HPMCs to TGF-ß differs or is less pronounced compared to young cells. As a result, the character and magnitude of the postulated contribution of HPMCs to TGF-ß-induced peritoneal remodelling may change with cell senescence.

Novel Aspects of the Immune Response Involved in the Peritoneal Damage in Chronic Kidney Disease Patients under Dialysis.

Chronic kidney disease (CKD) incidence is growing worldwide, with a significant percentage of CKD patients reaching end-stage renal disease (ESRD) and requiring kidney replacement therapies (KRT). Peritoneal dialysis (PD) is a convenient KRT presenting benefices as home therapy. In PD patients, the peritoneum is chronically exposed to PD fluids containing supraphysiologic concentrations of glucose or other osmotic agents, leading to the activation of cellular and molecular processes of damage, including inflammation and fibrosis. Importantly, peritonitis episodes enhance peritoneum inflammation status and accelerate peritoneal injury. Here, we review the role of immune cells in the damage of the peritoneal membrane (PM) by repeated exposure to PD fluids during KRT as well as by bacterial or viral infections. We also discuss the anti-inflammatory properties of current clinical treatments of CKD patients in KRT and their potential effect on preserving PM integrity. Finally, given the current importance of coronavirus disease 2019 (COVID-19) disease, we also analyze here the implications of this disease in CKD and KRT.

Effect of the one-day fasting on cortisol and DHEA daily rhythm regarding sex, chronotype, and age among obese adults.

Introduction: Physiological and biochemical processes in the human body occur in a specific order and show rhythmic variability. Time dependence characterizes the secretion of cortisol and dehydroepiandrosterone (DHEA). One-day fasting implies alternating fasting days and eating days. The study aimed to determine how 24-h fasting affects the daily rhythm of cortisol and DHEA levels in obese people while taking into account gender and chronotype. Methods: Forty-nine obese patients (BMI 32.2-67.1 kg/m2; 25 women and 24 men) underwent a 3-week hospital-controlled calorie restriction diet to reduce body weight. During hospitalization, patients fasted for 1 day, during which only water could be consumed. Samples of whole mixed unstimulated saliva were collected at 2-3-h intervals over a 64-h period and analyzed for cortisol and DHEA by immunoassays. The individual chronotypes were assessed by the morning and evening questionnaire, according to Horne and Östberg. Three components of daily rhythm were evaluated: amplitude, acrophase, and the so-called MESOR. Results: Cortisol rhythm showed differences in amplitude (p = 0.0127) and acrophase (p = 0.0005). The amplitude on the fasting day was 11% higher (p = 0.224) than the day after. The acrophase advanced on the day of fasting, 48 min earlier than the day before (p = 0.0064), and by 39 min to the day after fasting (p = 0.0005). In the rhythm of DHEA, differences were found in the MESOR (p = 0.0381). The MESOR on the fasting day increased. Discussion: Our results obtained during 64 consecutive hours of saliva sampling suggest that one-day fasting may affect three components of cortisol and DHEA daily rhythm. Additionally, no differences were found in the daily rhythm between the morning and evening chronotypes and between females and males. Although aging did not influence daily cortisol rhythm, DHEA amplitude, MESOR, and acrophase changed with age. To the best of our knowledge, this is the first presentation of changes in DHEA rhythm during one-day fasting.

Angiogenic Role of Mesothelium-Derived Chemokine CXCL1 During Unfavorable Peritoneal Tissue Remodeling in Patients Receiving Peritoneal Dialysis as Renal Replacement Therapy.

Peritoneal dialysis (PD) is a valuable 'home treatment' option, even more so during the ongoing Coronavirus pandemic. However, the long-term use of PD is limited by unfavourable tissue remodelling in the peritoneal membrane, which is associated with inflammation-induced angiogenesis. This appears to be driven primarily through vascular endothelial growth factor (VEGF), while the involvement of other angiogenic signaling pathways is still poorly understood. Here, we have identified the crucial contribution of mesothelial cell-derived angiogenic CXC chemokine ligand 1 (CXCL1) to peritoneal angiogenesis in PD. CXCL1 expression and peritoneal microvessel density were analysed in biopsies obtained by the International Peritoneal Biobank (NCT01893710 at www.clinicaltrials.gov), comparing 13 children with end-stage kidney disease before initiating PD to 43 children on chronic PD. The angiogenic potential of mesothelial cell-derived CXCL1 was assessed in vitro by measuring endothelial tube formation of human microvascular endothelial cells (HMECs) treated with conditioned medium from human peritoneal mesothelial cells (HPMCs) stimulated to release CXCL1 by treatment with either recombinant IL-17 or PD effluent. We found that the capillary density in the human peritoneum correlated with local CXCL1 expression. Both CXCL1 expression and microvessel density were higher in PD patients than in the age-matched patients prior to initiation of PD. Exposure of HMECs to recombinant CXCL1 or conditioned medium from IL-17-stimulated HPMCs resulted in increased endothelial tube formation, while selective inhibition of mesothelial CXCL1 production by specific antibodies or through silencing of relevant transcription factors abolished the proangiogenic effect of HPMC-conditioned medium. In conclusion, peritoneal mesothelium-derived CXCL1 promotes endothelial tube formation in vitro and associates with peritoneal microvessel density in uremic patients undergoing PD, thus providing novel targets for therapeutic intervention to prolong PD therapy.

Predictors of Early-Recurrence Atrial Fibrillation after Catheter Ablation in Women and Men with Abnormal Body Weight.

Our study aimed to select factors that affect the rate of early recurrence (up to 3 months) of atrial fibrillation (AF) (ERAF) following pulmonary veins isolation (PVI) in obese women and men. The study comprised 114 patients: 54 women (age: 63.8 ± 6.3, BMI 31 ± 4 kg/m2), and 60 men (age: 60.7 ± 6.7; BMI 31 ± 3 kg/m2) with paroxysmal, persistent and long-standing persistent AF. They had been scheduled to undergo cryoballoon (men n = 30; women n = 30) and radiofrequency (RF) ablation (men n = 30; women n = 24) using the CARTO-mapping. The blood was collected at baseline and 24 h after ablation. The rate of ERAF was comparable after cryoballoon and RF ablation and constituted 18% in women and 22% in men. Almost 70 parameters were selected to perform univariate and multivariate analysis and to create a multivariate logistic regression (MLR) model of ERAF in the obese men and women. The MLR analysis was performed by forward stepwise logistic regression with three variables. It was only possible to create the MLR model for the group of obese men. It revealed a poor predictive value with an unsatisfactory sensitivity of 31%. Men with ERAF: smokers (OR 39.25, 95% CI 1.050-1467.8, p = 0.0021), with a higher ST2 elevation (OR 1.68, 95% CI 1.115-2.536, p = 0.0021) who received dihydropyridine calcium channel blockers (OR 0.042, 95% CI 0.002-1.071, p = 0.0021) less frequently. Our results indicate a complex pathogenesis of ERAF dependent on the patients' gender.

Control of neutrophil influx during peritonitis by transcriptional cross-regulation of chemokine CXCL1 by IL-17 and IFN-γ.

Neutrophil infiltration is a hallmark of peritoneal inflammation, but mechanisms regulating neutrophil recruitment in patients with peritoneal dialysis (PD)-related peritonitis are not fully defined. We examined 104 samples of PD effluent collected during acute peritonitis for correspondence between a broad range of soluble parameters and neutrophil counts. We observed an association between peritoneal IL-17 and neutrophil levels. This relationship was evident in effluent samples with low but not high IFN-γ levels, suggesting a differential effect of IFN-γ concentration on neutrophil infiltration. Surprisingly, there was no association of neutrophil numbers with the level of CXCL1, a key IL-17-induced neutrophil chemoattractant. We investigated therefore the production of CXCL1 by human peritoneal mesothelial cells (HPMCs) under in vitro conditions mimicking clinical peritonitis. Stimulation of HPMCs with IL-17 increased CXCL1 production through induction of transcription factor SP1 and activation of the SP1-binding region of the CXCL1 promoter. These effects were amplified by TNFα. In contrast, IFN-γ dose-dependently suppressed IL-17-induced SP1 activation and CXCL1 production through a transcriptional mechanism involving STAT1. The SP1-mediated induction of CXCL1 was also observed in HPMCs exposed to PD effluent collected during peritonitis and containing IL-17 and TNFα, but not IFN-γ. Supplementation of the effluent with IFN-γ led to a dose-dependent activation of STAT1 and a resultant inhibition of SP1-induced CXCL1 expression. Transmesothelial migration of neutrophils in vitro increased upon stimulation of HPMCs with IL-17 and was reduced by IFN-γ. In addition, HPMCs were capable of binding CXCL1 at their apical cell surface. These observations indicate that changes in relative peritoneal concentrations of IL-17 and IFN-γ can differently engage SP1-STAT1, impacting on mesothelial cell transcription of CXCL1, whose release and binding to HPMC surface may determine optimal neutrophil recruitment and retention during peritonitis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The intensity of joint pain in relation to changes in serum TNFα during therapy with anti-TNFα inhibitors.

INTRODUCTION: Tumor necrosis factor-alpha (TNFα) inhibitors have significantly improved the outcomes of treatment for rheumatoid arthritis (RA). In the present study, we aimed to determine whether serum levels of TNFα during therapy with TNFα inhibitors do really reflect the disease activity and correspond to the intensity of pain experienced. MATERIALS AND METHODS: Thirty RA patients were examined before and after 12 weeks of routine therapy with TNFα inhibitors. Serum levels of TNFα were measured with a high-sensitivity immunoassay and related to patients' clinical and biochemical status. Disease activity was assessed by the modified disease activity score (DAS28). RESULTS: A median relative change in TNFα was 13%. The patients were stratified according to whether the relative change in serum TNFα after therapy was above or below this median value. The patients from both subgroups did not differ in baseline characteristics and response to therapy. However, the patients in whom serum TNFα increased after therapy above the median value had more tender joints after treatment than patients from the other group. Consequently, the number of tender joints after the treatment correlated with absolute TNFα concentrations at this time (r = 0.37; p = 0.049) and the magnitude of changes in serum TNFα correlated with a change in the number of tender joints (r = - 0.48; p = 0.008). CONCLUSIONS: Circulating TNFα levels did not decrease in RA patients treated with TNFα inhibitors, despite clinical and biochemical improvement. It is possible, that circulating TNFα is responsible for the persistence of joint pain in this group of patients.

Epithelial-to-Mesenchymal Transition and Migration of Human Peritoneal Mesothelial Cells Undergoing Senescence.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs) contributes to fibrotic thickening of the peritoneum that develops in patients on peritoneal dialysis (PD). The process is thought to be largely mediated by transforming growth factor-beta (TGF-ß). As TGF-ß has also been implicated in senescence of HPMCs, we have performed an exploratory study to examine if senescent HPMCs can undergo EMT. METHODS: Omentum-derived HPMCs were rendered senescent by repeated passages in culture. Features of EMT were assessed by immunostaining and quantitative polymerase chain reaction (qPCR) at various stages of the HPMC lifespan and after treatment with or without TGF-ß. The motility of HPMCs was assessed in a scratch wound migration assay. RESULTS: Replicative senescence of HPMCs was associated with a gradual increase in the constitutive expression of EMT markers, including increased production of extracellular matrix proteins. However, senescent HPMCs also retained epithelial cell features such as cytokeratin, calretinin, and E-cadherin and showed decreased, rather than increased, motility. In contrast, exposure to TGF-ß resulted in an up-regulation of mesenchymal markers and down-regulation of epithelial markers. Such effects of TGF-ß occurred both in young and senescent cells, although they were less pronounced in senescence. CONCLUSIONS: Senescence of HPMCs is associated with spontaneous development of several EMT features. At the same time, senescent HPMCs preserve epithelial cell-like characteristics and are less prone to develop a full EMT phenotype in response to TGF-ß. These observations may support the concept of cellular senescence being antagonistically pleiotropic with regard to EMT.

IL-17 in Peritoneal Dialysis-Associated Inflammation and Angiogenesis: Conclusions and Perspectives.

Long-term peritoneal dialysis (PD) is associated with peritoneal membrane remodeling. This includes changes in peritoneal vasculature, which may ultimately lead to inadequate solute and water removal and treatment failure. The potential cause of such alterations is chronic inflammation induced by repeated episodes of infectious peritonitis and/or exposure to bioincompatible PD fluids. While these factors may jeopardize the peritoneal membrane integrity, it is not clear why adverse peritoneal remodeling develops only in some PD patients. Increasing evidence points to the differences that occur between patients in response to the same invading microorganism and/or the differences in the course of inflammatory reaction triggered by different species. Such differences may be related to the involvement of different inflammatory mediators. Here, we discuss the potential role of IL-17 in these processes with emphasis on its impact on peritoneal mesothelial cells and peritoneal vascularity.

Thy-1+/- fibroblast subsets in the human peritoneum.

Fibrotic thickening of the peritoneum develops in patients receiving peritoneal dialysis (PD) for renal failure. For unknown reasons, however, in some patients it progresses to extensive fibrosis that compromises dialysis capacity of the peritoneum. It is increasingly clear that fibroblasts display large heterogeneity not only between but also within tissues. Differential surface expression of thymocyte differentiation antigen 1 (Thy-1) has been shown to identify functionally distinct fibroblast subsets in several organs. Here, we isolated Thy-1+/- subsets of human peritoneal fibroblasts (HPFB) and analyzed them in terms of profibrotic myofibroblast features. In healthy individuals, Thy-1+ cells constituted ~45% of the HPFB population found in the greater omentum but were not detected in the parietal peritoneum. When propagated in culture and compared with Thy-1- cells, omentum-derived Thy-1+ HPFB consistently displayed an increased expression of α-smooth muscle actin, collagen I, and transforming growth factor-ß1. They also showed greater proliferation capacity and enhanced contractile properties. The number of Thy-1+ HPFB increased significantly in PD patients and made up more than 70 and 95% of all HPFB found in the omentum and parietal peritoneum, respectively. These data indicate that the expansion of Thy-1+ fibroblasts may contribute to fibrotic thickening of the peritoneal membrane during PD.

New developments in peritoneal fibroblast biology: implications for inflammation and fibrosis in peritoneal dialysis.

Uraemia and long-term peritoneal dialysis (PD) can lead to fibrotic thickening of the peritoneal membrane, which may limit its dialytic function. Peritoneal fibrosis is associated with the appearance of myofibroblasts and expansion of extracellular matrix. The extent of contribution of resident peritoneal fibroblasts to these changes is a matter of debate. Recent studies point to a significant heterogeneity and complexity of the peritoneal fibroblast population. Here, we review recent developments in peritoneal fibroblast biology and summarize the current knowledge on the involvement of peritoneal fibroblasts in peritoneal inflammation and fibrosis.

Activation of nuclear factor of activated T cells 5 in the peritoneal membrane of uremic patients.

Peritoneal inflammation and fibrosis are responses to the uremic milieu and exposure to hyperosmolar dialysis fluids in patients on peritoneal dialysis. Cells respond to high osmolarity via the transcription factor nuclear factor of activated T cells (NFAT5). In the present study, the response of human peritoneal fibroblasts to glucose was analyzed in vitro. Expression levels of NFAT5 and chemokine (C-C motif) ligand (CCL2) mRNA were quantified in peritoneal biopsies of five nonuremic control patients, five uremic patients before PD (pPD), and eight patients on PD (oPD) using real-time PCR. Biopsies from 5 control patients, 25 pPD patients, and 25 oPD patients were investigated using immunohistochemistry to detect the expression of NFAT5, CCL2, NF-κB p50, NF-κB p65, and CD68. High glucose concentrations led to an early, dose-dependent induction of NFAT5 mRNA in human peritoneal fibroblasts. CCL2 mRNA expression was upregulated by high concentrations of glucose after 6 h, but, most notably, a concentration-dependent induction of CCL2 was present after 96 h. In human peritoneal biopsies, NFAT5 mRNA levels were increased in uremic patients compared with nonuremic control patients. No significant difference was found between the pPD group and oPD group. CCL2 mRNA expression was higher in the oPD group. Immunohistochemistry analysis was consistent with the results of mRNA analysis. CD68-positive cells were significantly increased in the oPD group. In conclusion, uremia results in NFAT5 induction, which might promote early changes of the peritoneum. Upregulation of NFAT5 in PD patients is associated with NFκB induction, potentially resulting in the recruitment of macrophages.

Regulation of chemokine CCL5 synthesis in human peritoneal fibroblasts: a key role of IFN-γ.

Peritonitis is characterized by a coordinated influx of various leukocyte subpopulations. The pattern of leukocyte recruitment is controlled by chemokines secreted primarily by peritoneal mesothelial cells and macrophages. We have previously demonstrated that some chemokines may be also produced by human peritoneal fibroblasts (HPFB). Aim of our study was to assess the potential of HPFB in culture to release CCL5, a potent chemoattractant for mononuclear leukocytes. Quiescent HPFB released constitutively no or trace amounts of CCL5. Stimulation of HPFB with IL-1ß and TNF-α resulted in a time- (up to 96 h) and dose-dependent increase in CCL5 expression and release. IFN-γ alone did not induce CCL5 secretion over a wide range of concentrations (0.01-100 U/mL). However, it synergistically amplified the effects of TNF-α and IL-1ß through upregulation of CCL5 mRNA. Moreover, pretreatment of cells with IFN-γ upregulated CD40 receptor, which enabled HPFB to respond to a recombinant ligand of CD40 (CD40L). Exposure of IFN-γ-treated HPFB, but not of control cells, to CD40L resulted in a dose-dependent induction of CCL5. These data demonstrate that HPFB synthesise CCL5 in response to inflammatory mediators present in the inflamed peritoneal cavity. HPFB-derived CCL5 may thus contribute to the intraperitoneal recruitment of mononuclear leukocytes during peritonitis.

The proto-oncogene c-Fos transcriptionally regulates VEGF production during peritoneal inflammation.

Vascular endothelial growth factor (VEGF) and transforming growth factor-ß1 (TGF-ß1) are key mediators of adverse peritoneal membrane remodeling in peritoneal dialysis eventually leading to ultrafiltration failure. Both are pleiotropic growth factors with cell type-dependent regulation of expression and biological effects. Here we studied regulation of TGF-ß1-induced VEGF expression in human peritoneal mesothelial cells in the absence or presence of proinflammatory stimuli, tumor necrosis factor-α (TNF-α) or interleukin-1ß (IL-1ß). Quiescent human peritoneal mesothelial cells secreted only trace amounts of VEGF. Stimulation with TGF-ß1 resulted in time- and dose-dependent increases in VEGF mRNA expression and protein release. TNF-α and IL-1ß alone had minimal effects but acted in synergy with TGF-ß1. Combined stimulation led to induction of transcription factor c-Fos and activation of the VEGF promoter region with high-affinity binding sites for c-Fos. Inhibition of c-Fos by small interfering RNA interference or by pharmacological blockade with SR-11302 decreased VEGF promoter activity and downregulated its expression and release. Exposure of human peritoneal mesothelial cells to dialysate effluent containing increased levels of TGF-ß1, TNF-α, and IL-1ß obtained during peritonitis resulted in a dose-dependent VEGF induction that was significantly attenuated by SR-11302. Thus, dialysate TGF-ß1, IL-1ß, and TNF-α act through c-Fos to synergistically upregulate VEGF production in peritoneal mesothelium and may represent an important regulatory link between inflammation and angiogenesis in the peritoneal membrane.

Recovery of senescent endothelial cells from injury.

Percutaneous coronary intervention is increasingly performed in elderly patients. Because the procedure is associated with endothelial cell (EC) denudation, we compared recovery of young and old ECs from scratch injuries inflicted in culture. Although senescent ECs displayed markedly reduced potential to proliferate and migrate, they repopulated the wounds as fast as young cells. Morphometric analysis revealed that senescent cells were significantly larger and as a result far fewer senescent cells managed to cover the lesion. Compared with young EC, senescent cells displayed increased expression of senescence-associated ß-galactosidase, nitric oxide synthase (eNOS), and AKT kinase, and secreted increased amounts of growth factors (VEGF, TGF-ß), cytokines (IL-6, IL-8, MCP-1), adhesion molecules (sICAM-1), and matrix proteins (fibronectin). This secretory phenotype rather than the rate of wound closure per se may contribute to unfavorable vascular remodeling in the elderly undergoing coronary catheterization.

Interpretation of elevated serum VEGF concentrations in patients with myocardial infarction.

Serum has been considered an unsuitable medium for measurements of circulating vascular endothelial growth factor (VEGF) since platelets release significant quantities of VEGF during clotting. Nevertheless, the assessment of platelet-derived VEGF may be important in patients with acute coronary syndromes characterized by intraluminal thrombosis. The present study aimed at identifying the factors that impact on the interpretation of serum VEGF concentrations in patients with ST-elevation myocardial infarction (STEMI). VEGF was measured in 106 patients with STEMI and correlated with clinical and angiographic parameters. Serum VEGF levels were significantly higher in patients with STEMI than in healthy controls. Although the average number of platelets did not differ between the groups, the patients with STEMI, but not the controls, exhibited a significant correlation between serum VEGF levels and platelet counts. Stratification of patients according to different criteria revealed that VEGF concentrations were particularly elevated shortly (<3h) after the onset of chest pain in those patients who had occluding thrombi graded as large (3-4) on a TIMI scale. These data demonstrate that high levels of serum VEGF detected early in the course of STEMI may derive from activated platelets and may characterize patients with extensive intracoronary thrombosis.

Relation of salivary antioxidant status and cytokine levels to clinical parameters of oral health in pregnant women with diabetes.

OBJECTIVE: Both pregnancy and diabetes are thought to predispose to the impairment of oral health. As saliva contributes to oral homeostasis, we have characterised its properties and flow rate in pregnant women with or without diabetes. DESIGN: Unstimulated whole mixed saliva was collected from 63 women in the first trimester of pregnancy and analysed for the concentration of selected antioxidants, cytokines, and growth factors. RESULTS: Pregnant women with diabetes were found to have markedly increased indexes of caries activity, plaque formation, gingival and periodontal status, as well as increased salivary antioxidant capacity and pro-inflammatory cytokine levels. These changes were more pronounced in patients with long-term disease and systemic diabetic complications, but only partly correlated with the level of blood glycated haemoglobin. Of the cytokines examined, salivary VEGF and HGF concentrations in diabetic pregnant women correlated in a positive and negative manner, respectively, with the prevalence of caries. Moreover, VEGF levels in this group correlated inversely with the probing depth and clinical attachment levels. All such associations did not occur in healthy individuals. In contrast, the salivary pH and flow rate correlated inversely with several parameters of caries and plaque formation irrespectively of whether the pregnant women were diabetic or not. CONCLUSIONS: Diabetes in pregnant women significantly changes saliva properties, which may contribute to accelerated deterioration of the oral status in this population.