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T regulatory (Treg) cells play a critical role in the maintenance of self-tolerance, as well as in inhibition of inflammation and exaggerated immune response against exogenous antigens. They develop in the thymus (tTreg cells) but also may be generated at the peripheral tissues, including tumor microenvironment (pTreg cells), or induced in vitro in the presence of transforming growth factor (TGF)-β (iTreg cells). Since tTreg cells constitute a minor fraction of peripheral blood lymphocytes in physiological conditions, an alternative way to obtain high number of functional Treg cells for therapeutic purposes is their generation in vitro from conventional T cells. In our studies, we compared effectiveness of several pharmacological agents with suggested immunomodulatory effects on Treg development (rapamycin, prednisolone, inosine pranobex, glatiramer acetate, sodium butyrate, and atorvastatin) to optimize Treg-inducing protocols. All but one (atorvastatin) immunomodulators augmented induction of polyclonal Treg cells in cultures. They were effective both in increasing the number of CD4+CD25highFoxp3high cells and Foxp3 expression. Rapamycin and prednisolone were found the most effective. Both drugs prolonged also phenotypic stability of Treg cells and induced fully active Treg cells in a functional assay. In the assay, prednisolone appeared superior versus rapamycin. The results, on the one hand, may be helpful in planning optimal protocols for generation of Treg cells for clinical application and, on the other hand, shed some light on mechanisms of the immunomodulatory activity of some tested agents observed in vivo.

Exaggerated oxidative stress (OS) is usually considered as a disturbance in regular function of an organism. The excessive levels of OS mediators may lead to major damage within the organism's cells and tissues. Therefore, the OS-associated biomarkers may be considered as new diagnostic tools of various diseases. In nephrology, researchers are looking for alternative methods replacing the renal biopsy in patients with suspicion of chronic kidney disease (CKD). Currently, CKD is a frequent health problem in world population, which can lead to progressive loss of kidney function and eventually to end-stage renal disease. The course of CKD depends on the primary disease. It is assumed that one of the factors influencing the course of CKD might be OS. In the current work, we review whether monitoring the OS-associated biomarkers in nephrology patients can support the decision-making process regarding diagnosis, prognostication and treatment initiation.

Cyclin C was cloned as a growth-promoting G1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumour suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an 'orphan' CDK19 kinase, as well as CDK8 and CDK3. These cyclin-C-CDK complexes phosphorylate the Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin-C-knockout mice. Cyclin C ablation or heterozygosity collaborates with other oncogenic lesions and accelerates development of T-cell acute lymphoblastic leukaemia (T-ALL). Furthermore, the cyclin C encoding gene CCNC is heterozygously deleted in a significant fraction of human T-ALLs, and these tumours express reduced cyclin C levels. We also describe point mutations in human T-ALL that render cyclin-C-CDK unable to phosphorylate ICN1. Hence, tumour cells may develop different strategies to evade inhibition by cyclin C.

Interleukin 12 (IL-12) seemed to represent the ideal candidate for tumor immunotherapy, due to its ability to activate both innate (NK cells) and adaptive (cytotoxic T lymphocytes) immunities. However, despite encouraging results in animal models, very modest antitumor effects of IL-12 in early clinical trials, often accompanied by unacceptable levels of adverse events, markedly dampened hopes of the successful use of this cytokine in cancer patients. Recently, several clinical studies have been initiated in which IL-12 is applied as an adjuvant in cancer vaccines, in gene therapy including locoregional injections of IL-12 plasmid and in the form of tumor-targeting immunocytokines (IL-12 fused to monoclonal antibodies). The near future will show whether this renewed interest in the use of IL-12 in oncology will result in meaningful therapeutic effects in a select group of cancer patients.

Published epidemiologic data on the administration rates of enteral/parenteral home nutrition is very limited. The aim of this first nationwide study was to assess the availability of pediatric home enteral nutrition (HEN) services in Poland. The questionnaire was sent to all regional centers providing pediatric HEN services in Poland (n = 14). The analysis included the number of pediatric patients who received HEN in 2010, their demographic characteristics and geographical distribution. Furthermore, the distributions of indications and methods of enteral nutrition administration were analyzed, along with the reasons of withdrawal from the HEN program. The number and fraction of children receiving HEN increased in 2010, from 433 (11.34 per 1 million inhabitants) on January 1st to 525 (13.75) on December 31st. Marked differences were observed in geographical distribution of this parameter, from zero to up to 30 pediatric patients per 1 million inhabitants. Median age of patients was 6 years (range: 9 months–18 years). In most cases, HEN was prescribed due to neurological disorders (n = 337, 64.2%), and administered by means of gastrostomy (n = 450, 85.71%). This study revealed the dynamic development of pediatric HEN services in Poland but also documented their potential regional shortages.

Brain tumors are associated with genetic alterations of oncogenes and tumor suppressor genes. Accumulation of reactive oxygen species (ROS) in cells leads to oxidative stress-induced damage, resulting in tumorigenesis. Here, we showed that the nuclear matrix protein nuclear restricted protein in brain (NRP/B) was colocalized and interacted with NF-E2-related factor 2 (Nrf2). During oxidative stress response, NRP/B expression and its interaction with Nrf2 were upregulated in SH-SY5Y cells. Association of NRP/B with Nrf2 was crucial for NAD(P)H:quinone oxidoreductase 1 (NQO1) expression. NRP/B was localized predominantly in the nucleus of normal brain cells, whereas in primary brain tumors NRP/B was almost exclusively contained in the cytoplasm. In addition, unlike wild-type NRP/B, the expression of NRP/B mutants isolated from primary brain tumors was found in the cytoplasm, and these mutants failed to induce Nrf2-dependent NQO1 transcription. Thus, NRP/B mutations and their altered localization resulted in changes in NRP/B function and deregulation of Nrf2-dependent NQO1 activation in brain tumors. This study provides insights into the mechanism by which the NRP/B modulates Nrf2-dependent NQO1 induction in cellular protection against ROS in brain tumors.

Photofrin®-based photodynamic therapy (PDT) has recently been approved for palliative and curative purposes in cancer patients. It has been demonstrated that neutrophils are indispensable for its anti-tumour effectiveness. We decided to evaluate the extent of the anti-tumour effectiveness of PDT combined with administration of granulocyte colony-stimulating factor (G-CSF) as well as the influence of Photofrin®and G-CSF on the myelopoiesis and functional activity of neutrophils in mice. An intensive treatment with G-CSF significantly potentiated anti-tumour effectiveness of Photofrin®-based PDT resulting in a reduction of tumour growth and prolongation of the survival time of mice bearing two different tumours: colon-26 and Lewis lung carcinoma. Moreover, 33% of C-26-bearing mice were completely cured of their tumours after combined therapy and developed a specific and long-lasting immunity. The tumours treated with both agents contained more infiltrating neutrophils and apoptotic cells then tumours treated with either G-CSF or PDT only. Importantly, simultaneous administration of Photofrin®and G-CSF stimulated bone marrow and spleen myelopoiesis that resulted in an increased number of neutrophils demonstrating functional characteristics of activation. Potentiated anti-tumour effects of Photofrin®-based PDT combined with G-CSF observed in two murine tumour models suggest that clinical trials using this tumour therapy protocol would be worth pursuing. © 2000 Cancer Research Campaign

Of a number of factors involved in apoptosis, protease activity may play a crucial role. We show that N-benzyloxycarbonyl-Ile-Glu( O-t-butyl)-Ala-leucinal (PSI), a selective inhibitor of the chymotrypsin-like activity of the proteasome, induces massive apoptosis in murine leukaemia L1210 cells. At 50 nM concentration, PSI induces a block of cytokinesis, while higher concentrations (500 nM) cause S phase block and massive apoptosis. Z-Leu-leucinal, a specific calpain inhibitor, did not induce apoptosis. In contrast to previous reports, TNF-alpha did not enhance apoptosis when combined with PSI. Our results suggest that proteasome inhibitors may be considered as potential anti-neoplastic agents.

We developed a BioMEMS device to study cell- mitochondrial physiological functionalities. The pathogenesis of many diseases including obesity, diabetes, heart failure as well as aging has been linked to functional defects of mitochondria. This is understandable as the mitochondria produces up to 90% of ATP, and plays a critical role in cell signaling and apoptosis. The synthesis of ATP is determined by the electrical potential across the inner mitochondrial membrane (IMM) and by the pH difference due to proton flux across it. Therefore, electrical characterization by E-fields with complementary chemical testing was used here. Mitochondrial ion channels present in the IMM control specific ion fluxes, and maintain ion homeostasis, matrix volume, IMM potential etc and thus serve a central role in cell growth and death related processes. Defects in ion channels (Channelopathies) are being attributed to many diseases like cancer, neurodegeneration, etc. Complete physiological roles of various ion channels and their interactions are still unknown, hindering the development of targeted therapeutic agents. The BioMEMS device was fabricated as an SU-8 based microfluidic system with gold electrodes on SiO2/Si wafers for electromagnetic interrogation. Ion Sensitive Field Effect Transistors (ISFETs) were incorporated for proton studies important in electron transport chain, together with monitoring Na+, K+, Ca++ions for ion channel studies. ISFETs are chemically sensitive MOSFET devices, their threshold voltage is directly proportional to the electrolytic H+ ion variation. These ISFETs (sensitivity ˜55 mV/pH for H+) were further realized as specific ion sensitive CHEMFETs by depositing a poly-HEMA layer sandwiched between the gate and a final specific ion sensitive membrane. Electrodes for dielectric spectroscopy studies of mitochondria were designed as 2- and 4-probe structures for optimized operation over a wide frequency range. In addition, to limit polarization effects (which masks actual impedance for high conductivity solutions at low frequencies), a 4-electrode set-up with unique meshed pickup electrodes (7.5×7.5 μm2 loops with 4 μm wires) was fabricated. An electrical model was developed for the mitochondrial sample, and its frequency response correlated with impedance spectroscopy experiments of sarcolemmal mitochondria. Using the mesh electrode structure, we obtained a reduction of 83.28% in impedance at 200 Hz. COMSOL simulations of selected electrical structures in this sensor were compared with experimental results to better understand the physical system. The simultaneous measurement of membrane potential, ion concentrations and pH would enhance diagnostics and studies of mitochondrial diseases.

We report on complex permittivity measurements of single-walled carbon nanotubes (SWNTs) over the microwave frequency range. The SWNT samples contained a mixture of semiconducting and metallic nanotubes and were homogeneously suspended using surfactant (Pluronic, F108). Other samples that were characterized included a Pluronic powder and a carpet of oriented multi-walled nanotubes (MWNT). Shielded open-circuited transmission line technique was used for broadband frequency measurements. Single frequency (resonant) measurements for SWNT samples were carried out by using two specially designed microwave dielectric resonators (DR). The first DR, with an axial cylindrical hole in the dielectric disk, could excite either TE011 or TM011 mode (3.4 GHz and 6 GHz) and was designed for liquid/powder sample characterization. The second DR used was split-post dielectric resonator (12 GHz). At 3.4 GHz, the real and imaginary parts of permittivity for Pluronic only suspended SWNTs were experimentally found to be 3.5 and 0.72, respectively. From our calculations conductivity of SWNT mixture was 1.16×105 (S/m) and for Pluronic it was 1.6587×10−2 (S/m).

Nano-gap metal oxide semiconductor (MOS) capacitors were studied to evaluate their limitations in applications of dielectric spectroscopy in living cells. The purpose was to optimize the design of a transducer to avoid interfacial polarization at the electrodes. Silicon IC technology was selected for designing processes in which we could limit electric double layer impedance by precisely controlling dielectric thickness of the capacitors in the range of 17 to 150 nm. The working capacitance was defined by lateral oxide etching of capacitor structures of various configuration to ensure high perimeter to area ratio. Highly doped n+ polysilicon and n+ implanted Si substrate were acting as capacitor's electrodes. Restrictions known from CMOS circuits regarding oxide leakage current, which depends on geometry and increases with the gate area were taken into account. To allow for testing cells (yeasts), which have larger dimensions than nano structures it was necessary to include cell manipulation using dielectrophoresis (DEP). Entrapment of cells at the electrode perimeter preceded electrical measurements. Our focus in analyses was on the frequency dependence of impedance parameters.

Gallium Nitride (GaN) thin films were successfully grown by electron cyclotron resonance molecular beam epitaxy (ECR-MBE), gas source MBE (GSMBE), and chemical beam epitaxy (CBE). Time of flight mass spectroscopy of recoiled ions (TOF-MSRI) and reflection high energy electron diffraction (RHEED) were used in-situ to determine the surface composition, crystalline structure, and growth mode of GaN thin films deposited by the three MBE methods. The substrate nitridation and the buffer layers were monitored and optimized by TOF-MSRI and RHEED. For GSMBE, the gallium to nitrogen ratio is found to correlate well with ex-situ optical properties. In the case of CBE, carbon incorporation determines the surface morphology, crystalline quality and optical activity of the epilayers.

Proximity rapid thermal diffusion (RTD) using arsenic has been used for doping high aspect ratio trench capacitors which may be used in high density dynamic memories. Nonuniformity of doping has been observed in SEM analyses at the trench comers after preferential etching used for junction delineation. Severe thinning was seen at the bottom comers of the junctions produced at low temperatures for long times (90 sec). This non- uniformity was alleviated at high diffusion temperatures for short time resulting in very uniform junctions. We modeled diffusion processes for trench doping using a process simulator, TSUPREM-IV. Default program data produced large discrepancies with experimental results and a new set of parameters was derived. The stress model in the oxide growth was incorporated in our calculations of the diffusion profiles to simulate formation of the dopant glass and obtain match with the experimental results.