Hyper-glycemia and insulinemia induce morphological changes and modulate secretion of S100B, S100A8, amyloid ß 1-40 and amyloid ß 1-42, in a model of human dopaminergic neurons.

Glucose metabolism in neuronal tissue declines during neurodegenerative disorders in a progressive, region-specific, and disease-specific manner. Studies have shown that extracellular hyper-glycemia affects the functioning of adenosine triphosphate (ATP) sensitive potassium channels located in neurons and astrocytes. Also, hyper-insulinemia contributes to the formation and progression of Alzheimer's disease (AD) via competition with amyloid ß (Aß) for insulin-degrading enzyme. Aß disruption is phosphatidylinositol 3-kinase pathway dependent, and increased circulatory insulin concentrations lead to Aß accumulation. In 2008, based on assessment of brain glucose utilization disorders and insulin signaling disruptions, it was proposed that AD could be called "type III diabetes". Proteins from the S100 family are actively secreted during metabolic and oxidative stress, but their role in neuronal cells has yet to be clarified. However, it has been demonstrated that they act in a dose-dependent manner, which may be crucial in the modulation of glucose and insulin metabolism in the brain. The goal of this paper was to elucidate the association between high glucose and insulin concentrations with extra- and intracellular S100B and S100A8 proteins levels as well as the correlation with toxic (Aß42) and physiologic (Aß40) forms of Aß. Medium and high glucose concentrations mimicking pre-diabetic and diabetic state, caused statistically significant discharge of S100b and S100A8 protein to the extracellular compartment. Similar effect was observed after 50 nM insulin incubation. Furthermore, the correlation coefficient patterns between those proteins shows similar associations which highlights possible effective and modulating role of S100 family in the metabolic disturbances occurring in neuropathological disorders.

Crucial players in Alzheimer's disease and diabetes mellitus: Friends or foes?

Alzheimer's disease (AD) and diabetes mellitus, especially type 2 (T2DM), are very common and widespread diseases in contemporary societies, and their incidence is steadily on the increase. T2DM is a multiple metabolic disorder, with several mechanisms including hyperglycaemia, insulin resistance, insulin receptor and insulin growth factor disturbances, glucose toxicity, formation of advanced glycation end products (AGEs) and the activity of their receptors. AD is the most common form of dementia, characterized by the accumulation of extracellular beta amyloid peptide aggregates and intracellular hyper-phosphorylated tau proteins, which are thought to drive and/or accelerate inflammatory and oxidative stress processes leading to neurodegeneration. The aim of this paper is to provide a comprehensive review of the evidence linking T2DM to the onset and development of AD and highlight the unknown or poorly studied "nooks and crannies" of this interesting relationship, hence providing an opportunity to stimulate new ideas for the analysis of comorbidities between AD and DM. Despite, indication of possible biomarkers of early diagnosis of T2DM and AD, this review is also an attempt to answer the question as to whether the crucial factors in the development of both conditions support the link between DM and AD.

Liquid Storage of Hematopoietic Stem Cells Versus Proliferative Potential Colony-Forming Unit Granulocyte-Monocytes: Validation of Cell Processing.

BACKGROUND: The material for transplantation must be of the highest quality. As far as we know, short-term storage is one of the crucial points of stem cell banking. According to the quality assurance system in a stem cell bank, each step of cell processing must be validated. The aim of this study was to assess the influence of short-term storage conditions into a clonogenic assay. METHODS: Material was collected from mobilized peripheral blood by means of leukapheresis from 15 patients. Samples were stored at 4°C and 20°C; samples were evaluated on the day of leukapheresis and after 24 hours and after 48 hours of storage. The number of colony-forming unit granulocyte-monocyte (CFU-GM) precursors was analyzed with the use of in vitro culture. The material was evaluated before freezing and after thawing. RESULTS: The average number of CFU-GM precursors in the material stored at 4°C before freezing on the day of collection was 84/10(5) nuclear cells (nc) and after 24 hours and 48 hours of storage was, respectively, 62/10(5) nc (P = .011719) and 36/10(5) nc (P = .02088). The average of the CFU-GM precursors in material stored at 20°C after 24 hours and 48 hours of storage amounted to 33/10(5) nc (P = .004439) and 2/10(5) nc (P = .00346), respectively. CONCLUSIONS: In our study, the number of colonies of CFU-GM after 24 hours and 48 hours of storage, both at 4°C and 20°C, was significantly reduced compared with the number of colonies on the day of collection. Significantly greater numbers of CFU-GM precursors were observed in the material stored before freezing at 4°C in comparison with the material stored at 20°C.

Banking of Hematopoietic Stem Cells: Influence of Storage Time on Their Quality Parameters.

BACKGROUND: Banking of hematopoietic stem cells (HSCs) is a rapidly growing part of the transplant field. The essence of the banking process is to maintain the optimal quality parameters throughout the storage period, allowing successful transplantation. METHODS: Our laboratory research was carried out on 126 HSC samples that were collected by means of leukapheresis from patients with lymphoproliferative diseases. The samples were frozen in a controlled rate and stored up to 76 months in containers in vapor phase of liquid nitrogen. The evaluation was performed after thawing the probes. Viability of nuclear cells was assessed after incubation in Trypan blue, CD34+ phenotype cells were determined by means of cytometry with the use of 7-aminoactinomycin D (7-AAD), and an analysis of the proliferative potential of granulocyte-monocyte precursors was performed. For comparative statistical analysis, the material was divided into 3 groups according to storage time: A: <1 month (n = 45); B: 1-12 months (n = 50); C: >12 months (n = 31). RESULTS: In the examined groups, similar median values were observed of nuclear cell viability (A, 86%; B, 87%; and C, 83%) and CD34+ cells (95%, 94.5%, and 95.8%, respectively). A gradual, nonsignificant, reduction in the median of granulocyte-monocyte precursors was found: 68 × 10(4)/kg of body weight (kg bw), 48.5 × 10(4)/kg bw, and 47 × 10(4)/kg bw, respectively. Statistical analysis with the use of the Kruskal-Wallis test showed a P value of >.05 for all variables. CONCLUSIONS: There were no significant differences in the viability of nuclear cells, CD34+ cells, and proliferative potential granulocyte-monocyte precursors between groups. Storage for up to 76 months does not change the essential quality parameters, and HSCs could be qualified for distribution.

Analysis of the effect of cryoprotectant medium composition to viability of autologous hematopoietic cells collected by leukapheresis.

OBJECTIVES: Cryopreservation of hematopoietic stem cells intended for autologous transplantation is a crucial element of the banking process. Although cryopreservation techniques are well known, improvement is needed. This study was designed to optimize cryopreservation to improve the quantitative and qualitative parameters of hematopoietic stem cells in the material intended for transplantation. We used available opportunities to provide the best quantitative and qualitative parameters of hematopoietic stem cell transplants processed in a closed system. MATERIAL AND METHODS: Two hundred forty-eight products of hematopoietic stem cells collected by leukapheresis from patients with lymphoproliferative disorders create the basis of this report. The material was frozen in a controlled-rate freezer and stored in containers in the vapor phase of LN2 (-160°C). The composition of a cryoprotectant medium was modified. For freezing, 192 probes were used with a cryoprotective medium containing 20% dimethyl sulfoxide (DMSO) and enriched RPMI 1640. For 56 samples, we used 20% DMSO in autologous plasma harvested during leukapheresis. Products of hematopoietic stem cells and cryoprotectant medium were combined in a 1:1 ratio. The final number of nuclear cells did not exceed 2 × 10(8)/mL. Analysis was performed after thawing the probes. Viability of nuclear cells has been assessed using the microscopic technique after incubation in Trypan blue and the CD34+ cells by flow cytometry using the 7-aminoactynomycin D. A statistical analysis has been conducted using the Statistica program (StatSoft, Cracow, Poland). RESULTS AND CONCLUSIONS: The results show that the application of autologous plasma is linked with higher viability of nuclear cells and CD34+ cells. Moreover, statistical analysis of the nuclear cells and CD34+ cells viability differs significantly between groups frozen using RPMI 1640 and autologous plasma (P < .05). To assess the viability of CD34+, cells frozen using RPMI 1640 results showed a large span of at 16.4% to 99.1% living cells.

Microbial contamination of peripheral blood and bone marrow hematopoietic cell products and environmental contamination in a stem cell bank: a single-center report.

Hematopoietic stem cells (HSC) derived from peripheral blood (PB) and bone marrow (BM) are frequently used for autologous and allogenic transplantations. Establishing quality control at appropriate steps of the stem cell preparation process is crucial for a successful transplantation. Microbial contamination of haematopoietic stem cells is rare but could cause a potentially mortal complication of a stem cells transplantation. We investigated the microbiological contamination of PB (291 donations) and BM (39 donations) products. Microbial cultures of 330 donations between January 2012 and June 2013 were retrospectively analyzed after the collection and preparation steps. The microbiological analysis was performed with an automated system. Hematopoietic stem cells were processed in a closed system. Additionally, in this report the environment of the working areas of stem cell preparation was monitored. We analyzed microbial contamination of the air in a class I laminar air flow clean bench at the time of preparation and in the laboratory once per month. We reported 9 (2.73%) contaminated HSC products. The most frequent bacteria isolated from PB and BM products were Bacillus species. Coagulase-negative staphylococci and Micrococcus species were the most frequent micro-organisms detected in the air microbial control. Microbial control results are necessary for the safety of hematopoietic stem cell products transplantation. Microbial control of hematopoietic stem cell products enables an early contamination detection and allows for knowledgeable decision making concerning either discarding the contaminated product or introducing an efficient antibiotic therapy. Each step of cell processing may cause a bacterial contamination. A minimum of manipulation steps is crucial for increasing the microbial purity of the transplant material. Also, the air contamination control is essential to ensure the highest quality standards of HSC products preparation.

Three-dimensional positioning of B chromosomes in fibroblast nuclei of the red fox and the chinese raccoon dog.

Great progress has been achieved over the last years in studies on chromosome arrangement in mammalian cell nuclei. Growing evidence indicates that the genome's spatial organization is of functional relevance. So far, no attention has been paid to the nuclear organization of B chromosomes (Bs). In this study we have examined nuclear positioning of Bs in 2 species from the Canidae family--the red fox and the Chinese raccoon dog. Using 2D and 3D fluorescence in situ hybridization and 2 gene-specific probes (C-KIT and PDGFRA), we analyzed the location of Bs in fibroblast nuclei. We found that small Bs of the red fox occupied mostly the interior of the nucleus, while medium-sized Bs of the Chinese raccoon dog were observed in the peripheral area of the nucleus as well as in intermediate and interior locations. The more uniform distribution of B chromosomes in the Chinese raccoon dog may be the result of differences in their size, since 3 morphological types of Bs are distinguished in this species. Our results indicate that 3D positioning of B chromosomes in fibroblast nuclei of the 2 canid species is in agreement with the chromosome size-dependent theory.

Pancreatic secretory trypsin inhibitor acts as an effective inhibitor of cysteine proteinases gingipains from Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis has been implicated as the major pathogen of periodontitis in adults. This organism produces an array of virulence factors, of which cysteine proteinases, referred to as gingipains K and R, are believed to play a crucial role in pathogenicity. The aim of this study was to investigate the susceptibility of gingipains K and R to inhibition by a pancreatic secretory trypsin inhibitor. MATERIAL AND METHODS: Enzyme activities were measured spectrophotometrically using chromogenic turnover substrates. To estimate the value of the association constant (Ka), constant amounts of enzyme were reacted with increasing amounts of inhibitor to reach equilibrium. The Ka was calculated by fitting the experimental data to the given equation. RESULTS: In this study it was shown that gingipains are susceptible to pancreatic Kazal-type trypsin inhibitors (pancreatic secretory trypsin inhibitors). Bovine pancreatic secretory trypsin inhibitor, having an Arg residue at the P1 position of the reactive site, specifically inhibited the activity of the Arg-specific cysteine proteinase gingipain R, whereas porcine inhibitor, possessing a Lys residue at the P1 position, exhibited activity only against the Lys-specific cysteine proteinase gingipain K. The Ka values for the inhibitor-proteinase interaction were 1.6 x 10(6) m(-1) and 2.0 x 10(4) m(-1) for gingipain R and gingipain K, respectively. CONCLUSION: This finding is the first demonstration of the inhibitory potency of the Kazal-type specific trypsin inhibitors against cysteine proteinases. These discoveries open new possibilities for the use of naturally occurring inhibitors, displaying activity across enzyme families, as a model in designing new molecules of therapeutic significance.

Expression of two alternative transcription forms of platelet-derived growth factor-A chain in the normal human kidney and in glomerulonephritis.

PURPOSE: Up to now, a role of platelet-derived growth factor (PDGF)-AA in glomerulonephritis (GN) remains unclear. PDGF-A chain may be produced in two forms, as a result of the alternative splicing. MATERIAL AND METHODS: We examined the expression of this growth factor in the renal tissue of 57 patients with GN and seven normal kidneys (NK). The gene expression of PDGF-A was examined by reverse transcriptase-polymerase chain reaction. Sets of primers allowing distinction between the two forms of transcripts were used. Specificity of the PCR products was confirmed by restriction enzyme analysis and sequencing. The expression of PDGF-AA/AB was also evaluated by immunohistochemistry. RESULTS: Compared to NK, the expression of PDGF-A gene was higher in the renal tissue with GN. This expression was higher in non-proliferative GN (NPGN) than in proliferative forms of GN (PGN) (1.24 +/- 0.34 vs. 0.86 +/- 0.14). In NK, both forms of transcripts (N = 4) or only the short one (N = 3) were found. In 45.5% of patients with NPGN, only the short form could be detected. In contrast, in 68.6% of patients with PGN both or only the longer form of transcripts were found. In NK, a faint staining for PDGF-AA/AB was observed within glomerular capillaries, whereas a statistically significant increase in this protein expression was particularly stated in NPGN. These results suggest that the production of the longer PDGF-A chain variant is associated with glomerular cells' proliferation. However, the higher expression of PDGF-AA/AB protein in NPGN could indicate an essential role of this growth factor in the maintaining the glomerular architecture.

Can von Willebrand factor, platelet-endothelial cell adhesion molecule-1 and thrombomodulin be used as alternative markers of endothelial cell injury in human glomerulonephritis?

PURPOSE: There is growing evidence that endothelial cells (EC) are active participants of an inflammatory process in glomeruli. MATERIAL AND METHODS: We compared the glomerular expression of three EC-coupled molecules, i.e. platelet-endothelial cell adhesion molecule-1 (PECAM-1 or CD31), von Willebrand factor (vWF) and thrombomodulin (TM) in 60 patients with glomerulonephritis (GN) and five normal kidneys (NK). The alkaline phosphatase anti-alkaline phosphatase method was used to examine the expression of these proteins in the biopsy specimens. RESULTS: In NK, the expression of CD31 and vWF comprised the whole glomerular network. In contrast, the expression of TM was much lower and localized mainly to EC at the vascular pole and adjacent areas. In GN, the glomerular staining for CD31 and vWF was significantly reduced. A fall in the expression of both these EC antigens was more pronounced in proliferative forms of GN (PGN) than in non-proliferative GN (NPGN) (CD31: NPGN vs. PGN, p < 0.02; vWF: NPGN vs. PGN, p < 0.05). In addition, a linear relationship between the expression of CD31 and vWF was found in GN (r = 0.8, p < 0.001). Conversely to CD31 and vWF, a marked increase in glomerular reactivity for TM was observed in all the patients with GN (GN: 2.12 +/- 0.32, NK: 0.95 +/- 0.05, p < 0.02). However, the highest expression of TM was found in membranoproliferative GN and lupus GN. CONCLUSIONS: Our results suggest that CD31 and vWF may be used as markers of glomerular EC loss during GN, whereas TM staining seems to reflect EC activation in response to circulating and/or released in situ procoagulant factors.

[Empiric antibiotic therapy for treatment of infection in patients with severe neutropenia]. / Empiryczna antybiotykoterapia w leczeniu infekcji u chorych w okresie glebokiej neutropenii.

Infection is the most frequent cause of death in patients with severe neutropenia. Fever and other signs of infection with neutrophil count below 0.5 G/L require an early and rapid treatment--the empiric antibiotic therapy. This treatment comprises various combinations of bactericidal broad-spectrum antibiotics such as ureidopenicillins, cephalosporins, quinolones and aminoglycosides. If defervescence is not attained within 3 days, modification of the treatment scheme should be done. The addition of vancomycin or teicoplanin, antibiotics active against Gram + cocci, and changing of the beta-lactams should be considered. In the case of persistent microbiologically not recognized infection after 7 days of therapy, empiric antimycotic treatment with amphotericin B is indicated. Duration of the empiric antibiotic therapy is dependent on the granulocyte recovery and the resolution of infection.

Incidence of measurement disturbances in the Dental Admission Quantitative Reasoning Test.

This paper describes the incidence of inconsistent person-response patterns in the Quantitative Reasoning Test (QRT), which is part of the Dental Admission Test (DAT) battery. using the Rasch person analysis approach to estimate the likelihood of a response pattern resulting from only two factors (the difficulty of the items and the ability of the person), a statistical profile of inconsistent response patterns was developed for a sample of students taking the DAT. The results of two test dates, April 1987 and October 1988, were included in this study. A random sample of 1,000 persons was drawn from each of these two test administrations. For the Fall 1988 sample, the person analysis was supplemented by a questionnaire designed to check the correspondence between a student's self-reported response behavior and the statistical profile provided by the person analysis. The analysis of the two samples of response patterns indicates a high incidence (50 percent+) of atypical response patterns on the test. This has serious implications for the admissions procedure since many of the atypical response patterns invalidate the standard scores reported for those students.

Gender differences in item performance and predictive validity on the DAT Quantitative Reasoning Test.

This article reports on examination of the performance of males and females on individual items of a graduate admission mathematics test--i.e., the Quantitative Reasoning Test administered as part of the Dental Admission Test--and relates the results to success in dental school. Items that are differentially familiar to males and females were identified and used as independent predictors of success in the first year of dental school. There was no significant difference between male and female performance in dental schools and no significant difference in the predictive validity of items that "favor males" and those that "favor females." Furthermore, the items that "favor females" produce approximately equal raw score distributions for males and females without significant adjustments in the content specifications of the test and thus would not underpredict female performance in dental schools.

Construct and predictive validities of the Perceptual Ability Test.

The construct and predictive validities of the Perceptual Ability Test (PAT) were examined. The results indicate that the PAT is a multidimensional test of spatial abilities, with each of the PAT subtests exhibiting different predictive validity. Furthermore, a linear combination of the PAT subtest scores was found to be more predictive of first-year dental school technique performance than the total PAT score.

Revision of Dental Admission Test standard score scale.

In October 1988 the Dental Admission Testing Program will begin reporting DAT scores on a new standard score scale. This scale will be based on the underlying ability metric rather than on the normal distribution that is the basis of the current-1 to 9 scale. A comparison of the two standard score scales is included, as well as a discussion of the advantages offered by the ability based metric.