Elevated level of lysophosphatidic acid among patients with HNF1B mutations and its role in RCAD syndrome: a multiomic study.

INTRODUCTION: Patients with hepatocyte nuclear factor-1 beta (HNF1B) mutations present a variable phenotype with two main symptoms: maturity onset diabetes of the young (MODY) and polycystic kidney disease (PKD). OBJECTIVES: Identification of serum metabolites specific for HNF1Bmut and evaluation of their role in disease pathogenesis. METHODS: We recruited patients with HNF1Bmut (N = 10), HNF1Amut (N = 10), PKD: non-dialyzed and dialyzed (N = 8 and N = 13); and healthy controls (N = 12). Serum fingerprinting was performed by LC-QTOF-MS. Selected metabolite was validated by ELISA (enzyme-linked immunosorbent assay) measurements and then biologically connected with HNF1B by in silico analysis. HepG2 were stimulated with lysophosphatidic acid (LPA) and HNF1B gene was knocked down (kd) by small interfering RNA. Transcriptomic analysis with microarrays and western blot measurements were performed. RESULTS: Serum levels of six metabolites including: arachidonic acid, hydroxyeicosatetraenoic acid, linoleamide and three LPA (18:1, 18:2 and 20:4), had AUC (the area under the curve) > 0.9 (HNF1Bmut vs comparative groups). The increased level of LPA was confirmed by ELISA measurements. In HepG2HNF1Bkd cells LPA stimulation lead to downregulation of many pathways associated with cell cycle, lipid metabolism, and upregulation of steroid hormone metabolism and Wnt signaling. Also, increased intracellular protein level of autotaxin was detected in the cells. GSK-3alpha/beta protein level and its phosphorylated ratio were differentially affected by LPA stimulation in HNF1Bkd and control cells. CONCLUSIONS: LPA is elevated in sera of patients with HNF1Bmut. LPA contributes to the pathogenesis of HNF1B-MODY by affecting Wnt/GSK-3 signaling.

Interfamilial clinical variability in four Polish families with cranioectodermal dysplasia and identical compound heterozygous variants in WDR35.

Cranioectodermal dysplasia (CED) is a rare autosomal recessive disorder primarily characterized by craniofacial, skeletal, and ectodermal abnormalities. CED is a chondrodysplasia, which is part of a spectrum of clinically and genetically heterogeneous diseases that result from disruptions in cilia. Pathogenic variants in genes encoding components of the ciliary transport machinery are known to cause CED. Intra- and interfamilial clinical variability has been reported in a few CED studies and the findings of this study align with these observations. Here, we report on five CED patients from four Polish families with identical compound heterozygous variants [c.1922T>G p.(Leu641Ter) and c.2522A>T; p.(Asp841Val)] in WDR35. The frequent occurrence of both identified changes in Polish CED families suggests that these variants may be founder mutations. Clinical evaluation of the CED patients revealed interfamilial clinical variability among the patients. This includes differences in skeletal and ectodermal features as well as variability in development, progression, and severity of renal and liver insufficiency. This is the first report showing significant interfamilial clinical variability in a series of CED patients from unrelated families with identical compound heterozygous variants in WDR35. Our findings strongly indicate that other genetic and non-genetic factors may modulate the progression and expression of the patients' phenotypes.

A Taurolidine-Citrate-Heparin Lock Solution Effectively Eradicates Pathogens From the Catheter Biofilm in Hemodialysis Patients.

Catheter-related bacteremia (CRB) is a typical complication of hemodialysis catheter use. Catheter lumen colonization by pathogens is regarded as a direct cause of CRB. Once settled, the catheter biofilm increases the risk of developing infection, thus necessitating insertion replacement and antibiotic treatment. The study assessed the self-sufficient efficacy of taurolidine-citrate-heparin lock solution in eradicating catheter biofilm bacteria and keeping it sterile in patients on hemodialysis. Twenty-nine chronic patients on hemodialysis with tunneled and nontunneled catheters locked with a heparin filling (the mean time of heparin lock use -30.1 ± 2.0 days) and subsequently converted to a taurolidine-citrate-heparin filling were included. Peripheral vein and catheter lumen blood cultures were obtained before the filling change and after taurolidine-citrate-heparin lock use (mean time 33.8 ± 7.6 days). Twenty-four participants with tunneled and nontunneled catheters locked with taurolidine-citrate-heparin filling served as the control group. During the heparin-locking period, CRB was diagnosed in 3 cases (only nontunneled catheters). The catheter blood cultures findings were positive in 23 patients (10 temporary and 13 permanent catheters), whereas both the catheter and peripheral vein blood cultures were sterile in 3 of 29 subjects (only permanent catheters). Irrespective of catheter type (tunneled or nontunneled), repeated culture revealed no pathogens in any of the 23 patients with initial positive catheter blood culture, after the use of taurolidine-citrate-heparin filling. No positive blood culture was noted in the control group. The taurolidine-citrate-heparin lock solution effectively eradicated pathogens from nontunneled and tunneled catheter biofilms and helped to maintain catheter lumen sterility.

[The influence of side effects related to cinacalcet therapy on compliance in hemodialysis patients suffering from secondary hyperparathyroidism]. / Wplyw wystepowania dzialan niepozadanych na przestrzeganie zalecen terapeutycznych przez chorych przewlekle hemodializowanych leczonych cynakalcetem z powodu wtórnej nadczynnosci przytarczyc.

INTRODUCTION: Secondary hyperparathyroidism (sHPT) in hemodialysis patients often requires an introduction of calcimimetic (cinacalcet) therapy. This medication effectively reduces parathormon (iPTH) serum concentration and modifies calcium phosphorus score, however its oral administration often implicates the occurrence of side effects which may worsen adherence to this medication. The aim of the study was to asses the influence of cinacalcet therapy side effects on patients adherence to the regiment prescription in hemodialysis patients suffering from secondary hyperparathyroidism MATERIAL AND METHODS: Fifty six participants (male 26 and 30 female) in age of 36-75 years, on maintenance hemodialysis 3 times a week, mean session time 4 hours and 25 minutes, treated with orally administered cinacalcet due to sHPT, were involved in the study. The initial dose 30 mg was modified once a visit (an increase or a decrease of 30 mg, if necessary) according to serum iPTH--cinacalcet maximal mean dose was 86 mg. During the treatment any presence of cinacalcet side effect and its intensity was noted. Patients'adherence to medication basing on medication intake were calculated and over 70% intake of cinacalcet doses was regarded as the good adherence. RESULTS: Twenty eight of 56 participants treated with cinacalcet were excluded mainly due to non-compliance. In the cohort, a total number of 70 cases of side effects were noted. In patients who resigned of the treatment the occurrence of cinacalcet adverse events (AE) was not higher than in participants continuing therapy (p > 0.05). Additionally in this group sever AE due to hypocalcaemia i.e. paresthesia, weakness, was rarely reported. In patients with good adherence side effects were presented with similar incidence in comparison to non-compliance patients. CONCLUSION: The incidence of cinacalcet adverse events was regarded as the factor which had an insignificant influence on patients'adherence to this medication. Therefore, it is essential to monitor patients'compliance to decrease non-adherence episodes.

Age-dependent changes of serum soluble CD30 concentration in children.

CD30 was originally described as a marker on Reed-Sternberg cells in Hodgkin lymphoma. The extracellular portion of CD30 is proteolytically cleaved from CD30+ cells, to produce a soluble form of the molecule (sCD30) detectable in serum. Measurement of sCD30 concentration in serum has been suggested to be a potential tool in monitoring of inflammatory status in variety of diseases. Several investigators reported the relevance for sCD30 as a predictive marker for allograft rejection following organ transplantation. The aim of the study was to verify whether sCD30 serum concentrations may be affected by an age in healthy children. Heparinized venous blood was taken from 78 healthy children. For the analysis of sCD30 levels, the commercially available sCD30 ELISA was used. The sCD30 was detected in all serum samples and concentrations ranged from 6.75 to 68.07ng/mL. The statistical analysis of all individuals showed that sCD30 concentration was significantly age depended (r=-0.618, p<0.0001). When sCD30 concentrations were analyzed in regard to gender, no significant differences were identified in age subgroups.

[The influence of short-term magnesium carbonate treatment on calcium-phosphorus balance in dialysis patients]. / Wplyw krótkoterminowego leczenia weglanem magnezu na gospodarke wapniowo-fosforanowa u chorych przewlekle hemodializowanych.

INTRODUCTION: The phosphate-binders presently used in the treatment of calcium-phosphorus disorders in dialysis patients remain a crucial element of cardio-vascular protection. The aim of the study was to assess short-time magnesium carbonate treatment efficacy in hemodialysis patients with hyperphosphatemia. MATERIAL AND METHODS: The study involved 64 participants (32 male and 32 female) aged 29-84 years, with end-stage renal disease, hyperphosphatemia (> 1.78 mmol/l), dialysis 3 times a week, mean session time 4 hours 15 minutes. All the patients were divided into three groups: I--30 patients treated with magnesium carbonate 3 x 1 g; group II--10 patients treated with sevelamer hydrochloride 0.8 g--3 x 2 tabl (3 x 1.6 g); group III--24 patients treated with calcium carbonate 3 x 2 g. Participants were categorized randomly to groups I and II and to group III only patients with decreased serum calcium concentration (< 2.1 mmol/1) were assigned. The doses stayed constant within 12 weeks of therapy. RESULTS: In group treated with magnesium carbonate after 3 months of the treatment the decrease of plasma parathormon (iPTH) from 526.1 +/- 463.3 to 443.2 +/- 223.1 (pg/ml), calcium (Ca) from 2.4 +/- 0.2 to 2.3 +/- 0.1 (mmol/1); the highest reduction of phosphate (P) 2.1 +/- 0.5 to 1.6 +/- 0.4 (mmol/1) and calcium phosphate product (Cax P) from 4.6 +/- 2.3 to 3.5 +/- 1.1 (mmol2/ l2) were observed. In group II, iPTH slightly increased from initial 425.26 +/- 192.5 to 445.6 +/- 222.3 (pg/ml); serum calcium decreased from 2.23 +/- 0.17 to 2.0 +/- 0.2 (mmol/l); phosphates dropped from 2.35 +/- 0.43 to 2.0 +/- 0.3 (mmol/l) and Ca x P index from 5.1 +/- 1.2 to 4.1 +/- 0.7 (mmol2/l2). In group treated with calcium carbonate iPTH decreased from 308.2 +/- 196.6 to 301.9 +/- 188.5 (pg/ml). Calcium, phosphate and Ca x P dropped during the treatment from 2.06 +/- 0.23 to 2.05 +/- 0.2 (mmol/l), 2.17 +/- 0.36 to 1.86 +/- 0.45 (mmol/l) and from 4.7 +/- 0.8 to 3.7 +/- 0.9 (mmol2/l2), respectively. Calcium-phosphorus disorders were normalized to actual guidelines only in participants treated with magnesium carbonate. CONCLUSIONS: Magnesium carbonate seems to be the effective treatment of calcium-phosphorus disorders in hemodialysis patients. However its administration, similarly to other non-calcium phosphate-binders, is limited and dedicated to patients with normal serum calcium concentration.

[The assessment of cynacalcet (Mimpara) accompanied by alfacalcidol treatment efficacy in haemodialysis patients with different secondary hyperparathyroidism severity recognized by iPTH]. / Ocena skutecznosci cynakalcetu (Mimpara) i alfakalcydolu w leczeniu róznych stadiów zaawansowania wtórnej nadczynnosci przytarczyc monitorowanych stezeniami iPTH u chorych przewlekle hemodializowanych.

INTRODUCTION: Calcimimetics are highly efficient drugs in treatment of secondary hyperparathyroidism (sHPT) in patients on haemodialysis. The effect and the dose of cinacalcet may depend on severity of sHPT, and alfacalcidol supplementation helps in the treatment optimization. The study evaluated cinacalcet and alfacalcidol treatment efficacy in haemodialysis patients with different secondary hyperparathyroidism severity recognized by iPTH. MATERIAL AND METHODS: The study group comprised of 82 participants (male 67 and 34 female) in aged from 36 to 75 years, on haemodialysis. All patients were divided into two groups: the study group--40 participants treated with cinacalcet accompanied by alfacalcidol started after 8 months of the study (0.25 to 0.5 microg/day) and the control group--42 patients. The study group comprises of two subgroups: I--moderate sHPT with iPTH 500 to 800 pg/ml and II--severe sHPT with iPTH > 800 pg/ml. The basic phosphate binder treatment throughout the study period in all groups was calcium carbonate. RESULTS: In the subgroup I initial mean iPTH 700 +/- 129 pg/ml was reduced to 550 +/- 61 pg/ml (p < 0.05) in the third month with no need of the Mimpara dose change. No further iPTH decrease up to eighth month of the treatment was observed despite the cinacalcet dose increase to 53 mg (p < 0.05). The alfacalcidol supplementation decreased iPTH to 331 +/-55 pg/ml (p < 0.05) and the cynacalcet dose to 42 mg (p < 0.05). In the II subgroup iPTH was reduced from 1035 +/- 149 pg/ml to 885 +/- 101 pg/ml (p < 0.05) in the third month of the treatment and Mimpara dose changed to 90 mg. Up to eighth month iPTH did not change (790 +/- 92 pg/ml; p > 0.05) despite the cinacalcet dose increase to 122 mg (p < 0.05). The alfacalcidol supplementation induced iPTH reduction to 622 +/- 71 pg/ml (p < 0.05) and the cinacalcet dose to 100 mg (p < 0.05). CONCLUSIONS: Cinacalcet convinced its effectiveness in the iPTH serum concentration control in haemodialysis patients independently of secondary hyperparathyroidism severity and alfacalcidol supplementation enhanced its efficacy. Still in case of the late introduction of Mimpara this drug was recognized as potent however the efficient dose was mandatory multiply.

Serum intact fibroblast growth factor 23 in healthy paediatric population.

INTRODUCTION: It is believed that fibroblast growth factor 23 (FGF23) can become an early biomarker of chronic kidney disease progression. Data on FGF23 age dependency are inconsistent. We present the results of the cross-sectional study concerning FGF23 levels in healthy Polish children. MATERIAL AND METHODS: This study was conducted in 121 children aged 0-18 years. Kidney function and intact FGF23 levels in serum were assessed. Differences between age groups and according to gender were analysed. RESULTS: The difference in FGF23 between age groups and according to gender was statistically insignificant. In the youngest and the oldest group, a trend to higher FGF23 levels was observed. FGF23 level in girls tended to be higher than boys, apart from the age group between 1 and 4 years. There was a negative correlation between eGFR and FGF23 (r = -0.26, p < 0.05) - strong in girls (r = -0.38, p < 0.05), but not in boys. In each age group, we found no significant correlation between eGFR and FGF23. CONCLUSIONS: Our study supports the evidence that the FGF23 level in paediatric population is not age or sex dependent. The results can serve as a reference point under clinical conditions and for other studies on the topic.

Serum microRNA profiles in patients with autosomal dominant polycystic kidney disease show systematic dysregulation partially reversible by hemodialysis.

INTRODUCTION: The impact of autosomal dominant polycystic kidney disease (ADPKD) on serum microRNAs (miRNA) is unknown. MATERIAL AND METHODS: For profiling experiment we recruited 30 patients from three equinumerous groups: controls, ADPKD and ADPKD on hemodialysis. From the last group extra samples were collected for in pre-/postdialysis analysis. Additionally, 23 healthy volunteers were used for selected biomarker verification. Real-time PCR arrays were used for quantification of 752 miRNAs. Validation of selected miRNAs was performed in total RNA extracted from the serum and the exosomal fraction in pre-/postdialysis samples. RESULTS: In total, 37 significant circulating miRNAs were found to differ between ADPKD patients and controls. In validation, 3 miRNAs with the highest fold change in comparison of dialyzed vs non-dialyzed patients (miR-532-3p, miR-320b, miR-144-5p) were not significantly altered by hemodialysis and from the top down-regulated ones, miR-27a-3p was significantly lower after dialysis in both total and exosomal fractions, miR-20a-5p was down-regulated in the exosomal fraction and miR-16-5p was unaltered by hemodialysis. MiR-16-5p was selected as the best circulating biomarker of ADPKD. Circulating representatives of the miR-17 family sharing the same seed region (miR-20a-5p, miR-93-5p and miR-106a-5p) showed significantly lower expression among dialyzed vs. non-dialyzed patients and their exosomal fraction dropped after hemodialysis. CONCLUSIONS: The serum miRNAs among ADPKD patients differ substantially depending on the stage of CKD. The exosomal fraction of miRNA was more affected by dialysis than the total one. There was a common pattern of down-regulation for circulating miR-17 family members sharing the same seed region.

Evaluation of receptor expression on immune system cells in the peripheral blood of asthmatic children undergoing food challenges.

BACKGROUND: The role of food allergens in the induction of allergic reactions in the airways is not completely understood. The aim of the present study was to evaluate fluorocytometric assays of the peripheral blood during food challenge tests in children with asthma and food allergy. PATIENTS AND METHODS: 22 children with asthma and concomitant food allergy and 18 children with asthma without food allergy participated in the study. Oral challenge tests were performed using double-blind, placebo-controlled food challenge. Blood samples were collected before and 4 and 24 h after the challenge. CD25 and CD23 antigen expression was determined with monoclonal antibodies using a FACSCalibur flow. RESULTS: The evaluation of the CD25+ T subpopulation and CD19+CD23+ B lymphocytes revealed statistically significant differences between the study group and the control group. In children with asthma and food allergy, the cell pool consisted (on average) of 9 +/- 2.8% of CD3+CD25+ cells before the challenge and of 10.3 +/- 3.8% (mean delta: 1.623; p = 0.01) after the provocation. However, placebo challenge did not significantly change the number of this T-lymphocyte subpopulation (mean delta: -0.121; p > 0.05). The highest increase in the CD25+ T-subpopulation expression was found in patients with respiratory reactions during the positive food challenge (mean delta: 4.065; p < 0.004). CONCLUSIONS: An increase in CD25+ T-lymphocyte and CD23 B-lymphocyte populations after food allergen challenge may indicate their significant role in the pathogenesis of the active phase of the immunoinflammatory process in children with asthma and concomitant food allergy.

[Prehypertension in type 1 diabetic children and adolescents]. / Stan przednadcisnieniowy u dzieci i mlodziezy chorej na cukrzyce typu 1.

BACKGROUND: The elevated blood pressure is one of the most important risk factors of diabetic micro- and macroangiopathy. AIM OF THE STUDY: Evaluation of the prevalence of prehypertension and relationship between prehypertension, metabolic control and chronic complications in children and adolescents with type 1 diabetes mellitus. MATERIALS AND METHODS: 83 patients aged 12.0-18.9 years, with a duration of diabetes 0.5-17.3 years, without evidence of arterial hypertension were recruited. In all patients 24-hour automatic blood pressure monitoring was performed with oscillometric device. The individuals with >40% of systolic and/or diastolic blood pressure >120/80 mmHg were defined as prehypertensive. HbA(1)c was measured by HPLC, plasma lipid levels--by an enzymatic method and urinary albumin excretion rate by chemiluminescent enzyme immunoassay method. Body mass index (BMI) and daily dose of insulin were calculated. Ophthalmoscopic examination and power spectral analysis of heart rate variation were performed. RESULTS: In 30 individuals (36.1%) prehypertension was diagnosed. The prehypertension group had older age (17.5+/-1.1 vs. 15.9+/-2.3 years; p<0.001) and longer duration of the disease (7.3+/-4.7 vs. 4.7+/-3.4 years; p=0.005) as compared with the group with normal blood pressure. There were no significant differences between groups in HbA1c, daily dose of insulin, BMI-SDS, lipids profile, prevalence of microalbuminuria and retinopathy. In the patients with prehypertension the a greater activity of sympathetic activation was observed (LF/HF: 1.00+/-0.06 vs. 0.78+/-0.04, p=0.018). CONCLUSIONS: Prehypertension is frequently recognized in type 1 diabetic children and adolescents. The prevalence of prehypertension is associated with older age, longer duration of diabetes and the shift of the sympatho-vagal balance toward sympathetic activation. There is no relationship between prehypertension and metabolic control or the prevalence of microvascular complications.

The effect of IL-2, IL-12 and IL15 on the function of natural killer cells in children suffering from type 1 diabetes mellitus.

INTRODUCTION: The immunologic reaction of pancreatic islets destruction leads to the occurrence of type 1 diabetes mellitus (T1D). The autoreactive lymphocytes play the pivotal role in this process although mechanisms regulating the lymphocyte migration and infiltration of Langerhans islets have not been fully understood yet. The in vitro studies showed natural killer (NK) cells potency to initiate pancreatic islets cell lyses. Many authors postulate that NK cells may be involved in this reaction. AIM OF THE STUDY: The aim of the study was to evaluate the effect of IL-2, IL-12 and IL-15 stimulation on peripheral blood NK cells in children suffering from type 1 diabetes mellitus in comparison to healthy controls. MATERIAL AND METHODS: Fifteen children with type 1 diabetes and 10 healthy adults were examined. NK cells were isolated by the magnetic cell separation system (MACS). For activation, NK cells were cultured with IL-2, IL-12 and IL-15 for 24 hours. The production of IFN-γ and IL-10 by NK cells was measured using commercial ELISA kits. FACS analysis of cell surface antigens--CD16, CD56, NKG2D and CD137 was performed using LSR II flow cytometer. RESULTS: In children with T1D the IFN-γ median concentration in supernatant obtained from NK cells culture was 16.831 ng/ml (inter quartile range 5.566-25.509) and did not statistically differ from median IFN-γ concentration in the control group--14.810 ng/ml (7.022-18.785), p = 0.76. In contrast, the IL-10 median concentration was statistically higher in T1D patients 7.87 pg/ml (1.32-11.37) than in healthy participants--1.41 pg/ml (1.05-4.81), p = 0.01. The median (inter-quartile range) percentage of NK NKG2D(+) was found in 0.42% (0.28-0.76) cells of TID patients versus 0.72% (0.53-1.08) in the controls (p = 0.05). There was no difference between -T1D group and the control group in regard to NK cells expressing CD137 - 6.58% (3.38-12.4) versus 6.85% (2.94-10.8); p = 0.8. CONCLUSIONS: The observed activity of NK cells after in vitro stimulation by IL2, IL-12 and IL15 in children suffering from type 1 diabetes mellitus indicates the tendency for supporting the inhibition of autoimmunological reaction by increased IL10 synthesis and increased number of NK cells with surface NKG2D receptors.

Influence of interleukin IL-2 and IL-12 + IL-18 on surface expression of immunoglobulin-like receptors KIR2DL1, KIR2DL2, and KIR3DL2 in natural killer cells.

Natural killer (NK) cells express killer cell inhibitory receptors (KIRs) that recognize polymorphic class I MHC molecules. In the present study, we analyze the modulatory effect of IL-2 alone or a combination of IL-12 with IL-18 on surface expression of killer cell immunoglobulin-like receptors KIR2DL1, KIR2DL2, and KIR3DL2 in NK cells. Thus, it was found that IL-2 causes a significant increase in the proportion of cells with given studied receptors. Stimulation by a mixture of IL-12 and IL-18 caused significant increase in the fraction of cells with the KIR2DL1 and KIR2DL2, however no significant change in the percentage of cells with KIR3DL2 receptor on their surface was observed. The results of the study show the presence of KIRs on both resting and activated NK cells, this may suggest that KIRs have also an important role in the regulatory processes after activation of this subpopulation of cells.