Wojciech Warchoł

An Efficient 3D Cell Culture Method on Biomimetic Nanostructured Grids

Current techniques of in vitro cell cultures are able to mimic the in vivo environment only to a limited extent, as they enable cells to grow only in two dimensions. Therefore cell culture approaches should rely on scaffolds that provide support comparable to the extracellular matrix. Here we demonstrate the advantages of novel nanostructured three-dimensional grids fabricated using electro-spinning technique, as scaffolds for cultures of neoplastic cells. The results of the study show that the fibers allow for a dynamic growth of HeLa cells, which form multi-layer structures of symmetrical and spherical character. This indicates that the applied scaffolds are nontoxic and allow proper flow of oxygen, nutrients, and growth factors. In addition, grids have been proven to be useful in in situ examination of cells ultrastructure.

Antibodies to hepatitis B virus surface antigen and survival of hemodialysis patients – a prospective study

ABSTRACT Background: Antibodies to hepatitis B virus (HBV) surface antigen (anti-HBs) may develop in response to HBV vaccination or infection. We investigated whether anti-HBs are an independent predictor of survival in hemodialysis (HD) patients. Methods: A 6-year prospective study was conducted in 532 HD patients. Survival analyses were performed using the Kaplan-Meier method and the Cox proportional hazard model. Results: In HBV non-infected patients, age (P = 0.005), coronary artery disease (P = 0.002), and non-response to HBV vaccine (P = 0.008) were the independent risk factors of all-cause mortality. In HBV infected patients, the only independent predictor of all-cause mortality was coronary artery disease (P = 0.002). Conclusion: The ability to produce the protective anti-HBs titer in response to HBV vaccine is a positive predictor of survival in HBV non-infected HD patients.

Photodiagnosis and photodynamic therapy of endometriotic epithelial cells using 5-aminolevulinic acid and steroids

BACKGROUND The photodynamic diagnosis and therapy represent relatively new methods used, i.a., in the detection of some preneoplastic and neoplastic conditions. They are based on selective accumulation of photosensitizers in the altered cells, which can be identified by fluorescence of the sensitizers and, using light of an appropriate wavelength, can be eliminated. Currently, investigations continue on application of the methods in diagnosis and therapy of endometriosis, one of the most prevalent causes of a reduced fertility in women. METHODS In this study protoporphyrin IX, a photosensitizer derived from 5-aminolevulinic acid, was used to locate and destroy endometrial epithelium. Material for the investigations involved primary epithelial cells, isolated from 15 normal endometria and 15 ovarian endometriotic epithelia. Taking into account the cyclical hormonal alterations, which affect endometrial cells in individual phases of the menstrual cycle, experiments were conducted on accumulation of the photosensitizer and photodestruction of the cells preceded by their hormonal stimulation (17β-estradiol and progesterone). RESULTS AND CONCLUSION It was found that following 48 h stimulation with 17β-estradiol and/or progesterone a significantly augmented synthesis of protoporphyrin IX can be obtained in cells of endometrial epithelium as compared to the normal epithelium. Moreover, the endometriotic epithelial cells were most effectively eliminated following 48 h prestimulation with progesteron alone. The obtained result permits to assume that photodynamic diagnosis and photodynamic therapy of endometrial epithelium should be performed in the secretory phase of endometrium in order to optimise their results.

Antibodies to HBV surface antigen in relation to interferon-λ3 in hemodialysis patients.

AIM To investigate circulating IFN-λ3 and IFNL3 polymorphisms in hemodialysis (HD) patients differing in HBV surface antigen antibody (anti-HBs) production. METHODS The study included 106 HBV-vaccinated HD patients (88 developed anti-HBs) and 36 HBV-infected HD subjects (27 developed anti-HBs). Plasma IFN-λ3 (enzyme-linked immunosorbent assay) and rs12979860 (C>T) and rs8099917 (T>G) in IFNL3 (high-resolution melting curve analysis) were analyzed with regard to the association with anti-HBs production in response to HBV vaccination or infection. The results were adjusted for gender, age, cause of renal disease, dialysis vintage, dialysis modality, IFN-λ3, and 25(OH)D as appropriate. RESULTS HBV vaccine responders had higher circulating IFN-λ3 (ng/L) than non-responders (120, 36-233 vs. 53, 33-109, P<0.000001). Patients who generated anti-HBs after HBV infection also had higher circulating IFN-λ3 levels than those who did not (133, 35-215 vs. 71, 9-229, P=0.043). The IFN-λ3 concentration correlated with the anti-HBs titer in vaccinated (r=0.614, P<0.000001) and infected patients (r=0.589, P=0.0002). Plasma IFN-λ3 was the only significant indicator of responsiveness to HBV vaccination (adjusted P=0.018) and remained the only significant associate for the development of post-infection anti-HBs (adjusted P=0.049). A plasmaIFN-λ3 level of 85.5ng/L was thecut-off value for theprognosis of an anti-HBs titer below vs. equal to or over 10IU/L in the entire group of HD patients (ROC sensitivity 68.7%, specificity 85.2%, and AUC 0.827). Significant associations were not found between IFN-λ3 and IFNL3 rs12979860. Subjects treated with low flux HD that harbored the TT genotype in rs8099917 showed higher IFN-λ3 levels than patients bearing the G allele in rs8099917 (139, 68-233 vs. 103, 9-208, P=0.049). CONCLUSION In HD patients, circulating IFN-λ3 strongly correlates with anti-HBs production after HBV vaccination and infection. IFNL3 rs8099917 polymorphisms seem to be associated with IFN-λ3 plasma levels in HD subjects.

Associations of the calcium-sensing receptor gene CASR rs7652589 SNP with nephrolithiasis and secondary hyperparathyroidism in haemodialysis patients.

Nephrolithiasis, secondary hyperparathyroidism (sHPT), and cardiovascular complications are associated with disturbances in Ca handling and contribute to morbidity/mortality during haemodialysis (HD). Calcimimetics, activators of the calcium-sensing receptor (CaSR), provide an effective means of reducing parathyroid hormone (PTH) secretion in sHPT. Polymorphism in CaSR gene (CASR) influences Ca-related parameters, however it was not shown in HD patients for CASR rs7652589. The minor allele at this polymorphism modifies the binding sites of transcription factors and CaSR expression. We hypothesized that CASR rs7652589 variants may also influence CaSR in end stage renal disease (ESRD). We aimed to determine the associations of rs7652589 with nephrolithiasis-related ESRD, Ca, P, ALP, PTH, response to treatment with cinacalcet, prevalence of coronary artery disease, and all-cause/cardiovascular mortality in HD patients (n = 1162). Healthy individuals (n = 918) were controls. This study shows that the A allele of rs7652589 is a risk allele for nephrolithiasis-related ESRD. The AA genotype is associated with more severe sHPT (higher Ca and PTH concentrations). The A allele is associated with reduced CaSR transcript level in peripheral blood mononuclear cells. According to computational analysis, potential binding sites for GLI3, AHR and TP53 are removed by the A allele, whereas binding sites for SOX18 and TP63 are created.

Contractile properties of motor units and expression of myosin heavy chain isoforms in rat fast-type muscle after volitional weight-lifting training.

Dynamic resistance training increases the force and speed of muscle contraction, but little is known about modifications to the contractile properties of the main physiological types of motor units (MUs) that contribute to these muscle adaptations. Although the contractile profile of MU muscle fibers is tightly coupled to myosin heavy chain (MyHC) protein expression, it is not well understood if MyHC transition is a prerequisite for modifications to the contractile characteristics of MUs. In this study, we examined MU contractile properties, the mRNA expression of MyHC, parvalbumin, and sarcoendoplasmic reticulum Ca2+ pump isoforms, as well as the MyHC protein content after 5 wk of volitional progressive weight-lifting training in the medial gastrocnemius muscle in rats. The training had no effect on MyHC profiling or Ca2+-handling protein gene expression. Maximum force increased in slow (by 49%) and fast (by 21%) MUs. Within fast MUs, the maximum force increased in most fatigue-resistant and intermediate but not most fatigable MUs. Twitch contraction time was shortened in slow and fast fatigue-resistant MUs. Twitch half-relaxation was shortened in fast most fatigue-resistant and intermediate MUs. The force-frequency curve shifted rightward in fast fatigue-resistant MUs. Fast fatigable MUs fatigued less within the initial 15 s while fast fatigue-resistant units increased the ability to potentiate the force within the first minute of the standard fatigue test. In conclusion, at the early stage of resistance training, modifications to the contractile characteristics of MUs appear in the absence of MyHC transition and the upregulation of Ca2+-handling genes.

Polymorphisms of Vitamin D Signaling Pathway Genes and Calcium-Sensing Receptor Gene in respect to Survival of Hemodialysis Patients: A Prospective Observational Study

We evaluated in the 7-year prospective study whether variants in vitamin D pathway genes and calcium-sensing receptor gene (CASR) are determinants of mortality in hemodialysis (HD) patients (n = 532). HRM analysis was used for GC rs2298849, GC rs1155563, RXRA rs10776909, RXRA rs10881578, and CASR rs7652589 genotyping. GC rs7041, RXRA rs749759, VDR rs2228570, and VDR rs1544410 were genotyped using PCR-RFLP analysis. The minor allele in GC rs2298849 was associated with all-cause mortality in univariate analysis (HR 1.330, 95% CI 1.046–1.692, P = 0.020). Bearers of the minor allele in GC rs2298849 demonstrated higher infection/neoplasm mortality than major allele homozygotes also in multivariate analysis (HR 2.116, 95% CI 1.096–4.087, P = 0.026). Cardiovascular mortality was associated with major homozygosity (CC) in VDR rs2228570 (HR 1.896, 95% CI 1.163–3.091, P = 0.010). CC genotype patients were more often dyslipidemic than TT genotype subjects (46.1% versus 31.9%, P = 0.047). Dyslipidemics showed higher frequency of rs1544410_rs2228570 haplotype AC than nondyslipidemics (26 versus 18%, P corr = 0.005), whereas TT genotype patients were at lower risk of dyslipidemia compared with CC/CT genotype patients (HR 0.59, 95% CI 0.37–0.96, P = 0.04). In conclusion, GC rs2298849 and VDR rs2228570 SNPs are associated with survival on HD. VDR-related cardiovascular mortality may occur due to connections of rs2228570 with dyslipidemia.

The role of HE4 in differentiating benign and malignant endometrial pathology.

OBJECTIVES The incidence of endometrial cancer is constantly growing. More aggressive types of endometrial cancer as well as the incidence in younger women is being observed. More than 80% of cases is diagnosed in early stages due to early symptoms like abnormal bleeding. The remaining 20% of asymptomatic cases of endometrial cancer as well as the cases of false negative histopathological diagnoses are mostly the incidences of serous endometrial cancer and are a true diagnostic and therapeutic challenge. This was the reason of our study in which we proposed investigation of HE4 levels as a complementary diagnostic method in management and diagnosing of EC. MATERIAL AND METHODS Serum HE4 level was measured in 92 patients with abnormal vaginal bleeding. Based on histhology after curretage the study group was divided into the benign and malignant endometrial pathology groups. Statistical analysis was performed using Mann-Whitney test RESULTS The difference of serum HE4 level between benign endometrial pathology and cancer was significant (p = 0.000) and the cut-off for identification of patients with endometrial cancer was 58.08 pmol/l. There was a significant difference between G2 and G3 endometrial cancer, and G1 and G3. (p = 0,4 and p = 0,008 respectively) Patients who needed lymphadenectomy had significantly higher HE4 level than those who had no indications for this procedure (p = 0,001). CONCLUSIONS HE4 is a useful biomarker in diagnosing endometrial cancer. HE4 is associated with high grade endometrial cancer. It can also serve as an useful preoperative counseling tool to identify patients, who may require pelvic and paraaortic lymphadenectomy.

Evaluation of CGB, GNRH1, MET and KRT19 Genes Expression Profile as a Circulating Tumor Cells Marker in Blood of Cancer Patients

The aim of this study was to evaluate human chorionic gonadotropin beta subunit (CGB), gonadotropin releasing hormone type 1 (GNRH1), hepatocyte growth factor receptor (MET) and keratin 19 (KRT19) genes expression profile as a circulating tumor cells (CTC) marker in blood of cancer patients. Expression level of studied genes was assessed in peripheral blood of 122 patients with different types of cancers and 86 healthy volunteers using reverse transcription real time quantitative polymerase chain reaction (RT-qPCR). The result of the experiments demonstrated that in blood of cancer patients the level of MET transcripts showed positive correlation with KRT19 and negative correlation with GNRH1. In the control group negative correlation between CGB and GNRH1 was documented. What is more the level of CGB, MET and KRT19 expression was significantly higher in blood of cancer patients. Even though the analysis proved that studied genes are expressed in blood of both cancer patients and healthy volunteers, their expression level was highly heterogeneous. In order to interpret the results, the obtained data was log-transformed and fitted to multiplied normal distribution model using the maximal likelihood method. The results of this analysis showed elevated expression of CGB, MET and KRT19 together with extremely high levels of GNRH1 in blood of cancer patients which might indicate the presence of circulating tumor cells and increased risk of metastasis.

Correlations of indoleamine 2,3-dioxygenase, interferon-λ3, and anti-HBs antibodies in hemodialysis patients

BACKGROUND Indoleamine 2,3-dioxygenase (IDO) contributes to maintaining immune homeostasis. Polymorphisms (SNPs) of the IDO encoding gene (IDO1) influence the IDO activity. Interferon (IFN)-λ3 induces IDO expression. We aimed to investigate whether IDO1 variants are associated with anti-HBs production in response to HBV vaccination or infection, interact with IFN-λ3 associated variants of IFNL4, and influence survival of hemodialysis (HD) patients. We also tested circulating IDO concerning IDO1 SNPs and plasma IFN-λ3 and anti-HBs levels. METHODS The study included HD patients who had established status concerning responsiveness to HBV vaccination (n = 1022) or were exposed to HBV (n = 315). Ability to generate anti-HBs was diagnosed if anti-HBs after vaccination or infection exceeded 10 IU/L. Genotyping of IDO1 (rs3739319 A < G, rs9657182 C < T), IFNL4 rs8099917 G < T and IFNL4 rs12979860 C > T polymorphisms was carried out by high-resolution melting curve analysis. Circulating IDO and IFN-λ3 were measured with ELISA in 57 subjects. Survival probability was analyzed using the Kaplan-Meier method. RESULTS IDO1 SNPs did not correlate with the ability to produce anti-HBs after vaccination or infection. Anti-HBs titers, including a frequency of anti-HBs ≥ 1000 IU/l, also did not associate with IDO1 SNPs, but there was an epistatic interaction between rs9657182, rs8099917, and rs12979860 concerning anti-HBs titers (P = 0.028). Significant associations between IDO1 SNPs and circulating IDO were not demonstrated. Anti-HBs titers negatively correlated with plasma IDO (r = -0.358, P = 0.006), and positively with circulating IFN-λ3 (r = 0.498, P = 0.00008). IDO and IFN-λ3 did not correlate. Patients possessing the rs9657182 TT genotype showed higher infection-related mortality, also in multivariate analysis (HR 2.073, 1.221-3.518, P = 0.007). CONCLUSIONS IDO1 rs9657182, IFNL4 rs8099917, and IFNL4 rs12979860 show epistatic interaction concerning anti-HBs titers. Overreacting immune responses to HBsAg occur in patients with lower IDO but simultaneously higher IFN-λ3 levels. The rs9657182 TT genotype associates with infection-related mortality of HD patients.