On the Boundary of Exploratory Genomics and Translation in Sequential Glioblastoma.

OMICS methods brought significant advancements to the understanding of tumor cell biology, which transformed the treatment and prognosis of several cancers. Clinical practice and outcomes, however, changed significantly less in the case of glioblastoma (GBM). In this study, we aimed to assess the utility of whole exome (WES) sequencing in the clinical setting. Ten pairs of formalin-fixed, paraffin-embedded (FFPE) GBM specimens were obtained at onset (GBM-P) and at recurrence (GBM-R). Histopathological and molecular features of all samples supported the diagnosis of GBM based on WHO CNS5. WES data were filtered, applying a strict and custom-made pipeline, and occurrence of oncogenic and likely oncogenic variants in GBM-P, GBM-R or both were identified by using the VarSeq program version 2.5.0 (Golden Helix, Inc.). Characteristics and recurrence of the variants were analyzed in our own cohort and were also compared to those available in the COSMIC database. The lists of oncogenic and likely oncogenic variants corresponded to those identified in other studies. The average number of these variants were 4 and 5 out of all detected 24 and 34 variants in GBM-P and GBM-R samples, respectively. On average, one shared oncogenic/likely oncogenic variant was found in the pairs. We assessed the identified variants' therapeutic significance, also taking into consideration the guidelines by the Association for Molecular Pathology (AMP). Our data support that a thorough WES analysis is suitable for identifying oncogenic and likely oncogenic variants in an individual clinical sample or a small cohort of FFPE glioma specimens, which concur with those of comprehensive research studies. Such analyses also allow us to monitor molecular dynamics of sequential GBM. In addition, careful evaluation of data according to the AMP guideline reveal that though therapeutic applicability of the variants is generally limited in the clinic, such information may be valuable in selected cases, and can support innovative preclinical and clinical trials.

Use of blood products during the first months of COVID-19 pandemic period: A single center report.

The novel coronavirus (SARS-CoV-2) outbreak exerted a serious effect on healthcare. Between 1st of January and May 31, 2020 due to the special regulations in Hungary, the number of reported COVID-19 infections were relatively low (3876 cases). The inpatient and outpatient care and the blood supply were significantly affected by the implemented regulations. The aim of this study was to evaluate the use of blood products amid the first five months of the pandemic situation. This investigation has observed a significant reduction of hospitalizations (37.35%). Analyzing individually the included units, pre-transfusion hemoglobin concentrations of transfused patients presented slight modifications, which were not statistically significant. The special regulations resulted major changes in the frequency of diagnoses at admissions in case of the Department of Surgery, while in case of the other specialities (Division of Hematology and Department of Anesthesiology and Intensive Therapy), there were no major changes compared to pre-pandemic period. Considering each department separately, transfused red blood cell concentrates (RBC) per patient, and the proportion of transfused patients did not change significantly. However, the combination of these modifications resulted in the significant decrease in RBC transfusions (p < 0.0001) compared to the pre-pandemic baseline. With regard to platelet and fresh frozen plasma (FFP), their usage was significantly reduced (44.40% platelet concentrates and 34.27% FFP). Our results indicate that the pandemic had an important effect on the blood product usage at the included departments by introducing different patient care policies and the temporary deferral of the elective surgical interventions. Despite the challenging circumstances of blood collection and blood product supply, the hospitalized patients received adequate care.

Urinary actin, as a potential marker of sepsis-related acute kidney injury: A pilot study.

INTRODUCTION: A major complication of sepsis is the development of acute kidney injury (AKI). Recently, it was shown that intracellular actin released from damaged tissues appears in the urine of patients with multiple organ dysfunction syndrome. Our aims were to measure urinary actin (u-actin) concentrations of septic and control patients and to test if u-actin levels could predict AKI and mortality. METHODS: Blood and urine samples were collected from septic and sepsis-related AKI patients at three time points (T1-3): T1: within 24 hours after admission; T2: second day morning; T3: third day morning of follow-up. Patients with malignancies needing palliative care, end-stage renal disease or kidney transplantation were excluded. Serum and u-actin levels were determined by quantitative Western blot. Patients were categorized by the Sepsis-3 and KDIGO AKI classifications. RESULTS: In our study, 17 septic, 43 sepsis-induced AKI and 24 control patients were enrolled. U-actin levels were higher in septic patients compared with controls during follow-up (p<0.001). At T1, the septic and sepsis-related AKI groups also showed differences (p<0.001), yet this increase was not statistically significant at T2 and T3. We also detected significantly elevated u-actin concentrations in AKI-2 and AKI-3 septic patients compared with AKI-1 septic patients (p<0.05) at T1 and T3, along with a significant increase in AKI-2 septic patients compared with AKI-1 septic patients at T2 (p<0.01). This tendency remained the same when referring u-actin to urine creatinine. Parameters of first-day septic patient samples could discriminate AKI from non-AKI state (AUC ROC, p<0.001): u-actin: 0.876; se-creatinine: 0.875. Derived cut-off value for u-actin was 2.63 µg/L (sensitivity: 86.0%, specificity: 82.4%). CONCLUSION: U-actin may be a complementary diagnostic biomarker to se-creatinine in sepsis-related AKI while higher u-actin levels also seem to reflect the severity of AKI. Further investigations may elucidate the importance of u-actin release in sepsis-related AKI.

O-GlcNAcylation in early stages of chronic lymphocytic leukemia: Protocol development for flow cytometry.

BACKGROUND: Recent studies proved that metabolic changes in malignant disorders have an impact on protein glycosylation, however, only a few attempts have been made so far to use O-GlcNAc analysis as a prognostic tool. Glucose metabolism is reported to be altered in hematological malignancies thus, we hypothesized that monitoring intracellular O-GlcNAc levels in Rai stage 0-I (Binet A) CLL patients could give deeper insights regarding subtle metabolic changes of progression which are not completely detected by the routine follow-up procedures. OBJECTIVE: In this proof of concept study we established a flow cytometric detection method for the assessment of O-GlcNAcylation as a possible prognostic marker in CLL malignancy which was supported by fluorescence microscopy. METHODS: Healthy volunteers and CLL patients were recruited for this study. Lymphocytes were isolated, fixed and permeabilised by various methods to find the optimal experimental condition for O-GlcNAc detection by flow cytometry. O-GlcNAc levels were measured and compared to lymphocyte count and various blood parameters including plasma glucose level. RESULTS: The protocol we developed includes red blood cell lysis, formalin fixation, 0.1% Tween 20 permeabilisation and employs standardized cell number per sample and unstained controls. We have found significant correlation between O-GlcNAc levels and WBC (R2= 0.8535, p< 0.0029) and lymphocyte count (R2= 0.9225, p< 0.0006) in CLL patients. Interestingly, there was no such correlation in healthy individuals (R2= 0.05664 for O-GlcNAc vs WBC and R2= 0.04379 for O-GlcNAc vs lymphocytes). CONCLUSION: Analyzing O-GlcNAc changes in malignant disorders, specifically in malignant hematologic diseases such as CLL, could be a useful tool to monitor the progression of the disease.

Early occurrence of pseudocysts in acute pancreatitis - A multicenter international cohort analysis of 2275 cases.

BACKGROUND: Pseudocysts being the most frequent local complications of acute pancreatitis (AP) have substantial effect on the disease course, hospitalization and quality of life of the patient. Our study aimed to understand the effects of pre-existing (OLD-P) and newly developed (NEW-P) pseudocysts on AP. METHODS: Data were extracted from the Acute Pancreatitis Registry organized by the Hungarian Pancreatic Study Group (HPSG). 2275 of 2461 patients had uploaded information concerning pancreatic morphology assessed by imaging technique. Patients were divided into "no pseudocyst" (NO-P) group, "old pseudocyst" (OLD-P) group, or "newly developed pseudocyst" (NEW-P) groups. RESULTS: The median time of new pseudocyst development was nine days from hospital admission and eleven days from the beginning of the abdominal pain. More NEW-P cases were severe (15.9% vs 4.7% in the NO-P group p < 0.001), with longer length of hospitalization (LoH) (median: 14 days versus 8 days, p < 0.001), and were associated with several changed laboratory parameters. OLD-P was associated with male gender (72.2% vs. 56.1%, p = 0.0014), alcoholic etiology (35.2% vs. 19.8% in the NO-P group), longer hospitalization (median: 10 days, p < 0.001), a previous episode of AP (p < 0.001), pre-existing diagnosis of chronic pancreatitis (CP) (p < 0.001), current smoking (p < 0.001), and increased alcohol consumption (unit/week) (p = 0.014). CONCLUSION: Most of the new pseudocysts develop within two weeks. Newly developing pseudocysts are associated with a more severe disease course and increased length of hospitalization. Pre-existing pseudocysts are associated with higher alcohol consumption and smoking. Because CP is more frequently associated with a pre-existing pseudocyst, these patients need closer attention after AP.

Involvement of the Catecholamine Pathway in Glioblastoma Development.

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system (CNS). The standard of care improves the overall survival of patients only by a few months. Explorations of new therapeutic targets related to molecular properties of the tumor are under way. Even though neurotransmitters and their receptors normally function as mediators of interneuronal communication, growing data suggest that these molecules are also involved in modulating the development and growth of GBM by acting on neuronal and glioblastoma stem cells. In our previous DNA CpG methylation studies, gene ontology analyses revealed the involvement of the monoamine pathway in sequential GBM. In this follow-up study, we quantitated the expression levels of four selected catecholamine pathway markers (alpha 1D adrenergic receptor-ADRA1D; adrenergic beta receptor kinase 1 or G protein-coupled receptor kinase 2-ADRBK1/GRK2; dopamine receptor D2-DRD2; and synaptic vesicle monoamine transporter-SLC18A2) by immunohistochemistry, and compared the histological scores with the methylation levels within the promoters + genes of these markers in 21 pairs of sequential GBM and in controls. Subsequently, we also determined the promoter and gene methylation levels of the same markers in an independent database cohort of sequential GBM pairs. These analyses revealed partial inverse correlations between the catecholamine protein expression and promoter + gene methylation levels, when the tumor and control samples were compared. However, we found no differences in the promoter + gene methylation levels of these markers in either our own or in the database primary-recurrent GBM pairs, despite the higher protein expression of all markers in the primary samples. This observation suggests that regulation of catecholamine expression is only partially related to CpG methylation within the promoter + gene regions, and additional mechanisms may also influence the expression of these markers in progressive GBM. These analyses underscore the involvement of certain catecholamine pathway markers in GBM development and suggest that these molecules mediating or modulating tumor growth merit further exploration.

DNA CpG methylation in sequential glioblastoma specimens.

PURPOSE: Glioblastoma is the most aggressive form of brain tumors. A better understanding of the molecular mechanisms leading to its evolution is essential for the development of treatments more effective than the available modalities. Here, we aim to identify molecular drivers of glioblastoma development and recurrence by analyzing DNA CpG methylation patterns in sequential samples. METHODS: DNA was isolated from 22 pairs of primary and recurrent formalin-fixed, paraffin-embedded glioblastoma specimens, and subjected to reduced representation bisulfite sequencing. Bioinformatic analyses were conducted to identify differentially methylated sites and pathways, and biostatistics was used to test correlations among clinical and pathological parameters. RESULTS: Differentially methylated pathways likely involved in primary tumor development included those of neuronal differentiation, myelination, metabolic processes, synapse organization and endothelial cell proliferation, while pathways differentially active during glioblastoma recurrence involved those associated with cell processes and differentiation, immune response, Wnt regulation and catecholamine secretion and transport. CONCLUSION: DNA CpG methylation analyses in sequential clinical specimens revealed hypomethylation in certain pathways such as neuronal tissue development and angiogenesis likely involved in early tumor development and growth, while suggested altered regulation in catecholamine secretion and transport, Wnt expression and immune response contributing to glioblastoma recurrence. These pathways merit further investigations and may represent novel therapeutic targets.

[Health policy experiences of publicly financed laboratory services in Hungary with health insurance data analysis]. / A közfinanszírozott laboratóriumi szolgáltatások egészségpolitikai tapasztalatai Magyarországon egészségbiztosítási adatok elemzésével.

Introduction: In order to provide appropriate prevention, diagnosztics, decision on therapy and monitoring the results of medical treatment, there is an increasing need for laboratory examinations. Aim: The aim of our study is the health-ecnomics analysis of laboratory budget of the Hungarian Health Insurance Fund. Data and method: Data were derived from the financial database of the National Health Insurance Fund Administration. The analysis covered the period of 2002-2018. We analysed the annual budget for laboratory examinations, the number of patients and examinations, the market share of laboratory services providers according to their owner structure from the health insurance curative-preventive budget. Results: The budget available for financing the laboratory examinations (21-22 billion Hungarian forint (Ft)/év) did not change significantly between 2005 and 2015. There was a significant decrease in the number of both patients and examinations between 2006 and 2008. In the latest years, there were 14-15 million cases per year and 180 million examinations per year. The market share of for-profit companies decreased from 29.0% in 2010 to 10.6% in 2018, while the market share of governmental institutions increased from 27.1% in 2010 to 78.7% in 2018. Conclusion: The activity of laboratories was stabilized in the latest years. After the necessary correction of professional regulations and code maintenance, the laboratory budget can be increased towards the mainly public laboratory services providers. Orv Hetil. 2020; 161(12): 468-473.

Hyperglycemia-Induced Aberrant Cell Proliferation; A Metabolic Challenge Mediated by Protein O-GlcNAc Modification.

Chronic hyperglycemia has been associated with an increased prevalence of pathological conditions including cardiovascular disease, cancer, or various disorders of the immune system. In some cases, these associations may be traced back to a common underlying cause, but more often, hyperglycemia and the disturbance in metabolic balance directly facilitate pathological changes in the regular cellular functions. One such cellular function crucial for every living organism is cell cycle regulation/mitotic activity. Although metabolic challenges have long been recognized to influence cell proliferation, the direct impact of diabetes on cell cycle regulatory elements is a relatively uncharted territory. Among other "nutrient sensing" mechanisms, protein O-linked ß-N-acetylglucosamine (O-GlcNAc) modification emerged in recent years as a major contributor to the deleterious effects of hyperglycemia. An increasing amount of evidence suggest that O-GlcNAc may significantly influence the cell cycle and cellular proliferation. In our present review, we summarize the current data available on the direct impact of metabolic changes caused by hyperglycemia in pathological conditions associated with cell cycle disorders. We also review published experimental evidence supporting the hypothesis that O-GlcNAc modification may be one of the missing links between metabolic regulation and cellular proliferation.

Fractalkine Induces Hepcidin Expression of BV-2 Microglia and Causes Iron Accumulation in SH-SY5Y Cells.

Fractalkine (CX3CL1) is a potent inflammatory mediator of the central nervous system, which is expressed by neurons and regulates microglial functions by binding to fractalkine receptor (CX3CR1). It has been demonstrated that neuroinflammation plays an important role in iron accumulation of the brain leading to neuronal cell death. The major regulator of iron homeostasis is the peptide hormone hepcidin. Hepcidin expression is triggered by inflammatory conditions, which may contribute to the neuronal iron accumulation. In the present study, we established a bilaminar co-culture system of differentiated SH-SY5Y cells and BV-2 microglia as a neuronal model to examine the effect of soluble fractalkine on iron homeostasis of microglia and SH-SY5Y cells. We determined the hepcidin expression of fractalkine-treated microglia which showed significant elevation. We examined the relation between increased hepcidin secretion, the known hepcidin regulators and the signalling pathways controlled by fractalkine receptor. Our data revealed that TMPRSS6 and alpha 1-antitrypsin levels decreased due to fractalkine treatment, as well as the activity of NFκB pathway and the tyrosine phosphorylation of STAT5 factor. Moreover, fractalkine-induced hepcidin production of microglia initiated ferroportin internalisation of SH-SY5Y cells, which contributed to iron accumulation of neurons. Our results demonstrate that soluble form of fractalkine regulates hepcidin expression of BV-2 cells through fractalkine-mediated CX3CR1 internalisation. Moreover, fractalkine indirectly contributes to the iron accumulation of SH-SY5Y cells by activating ferroportin internalisation and by triggering the expressions of divalent metal transporter-1, ferritin heavy chain and mitochondrial ferritin.

Elevated urinary orosomucoid excretion as a novel biomarker in Crohn's disease.

BACKGROUND: Laboratory markers are essential tools in the follow-up of patients with Crohn's disease (CD). Our aim was to investigate urinary concentrations of orosomucoid in relation to the inflammatory activity of CD and to compare it with clinical indices and conventional laboratory parameters. MATERIALS AND METHODS: Blood and urine samples of 86 patients (55 adults and 31 children) with CD and 68 healthy individuals (38 adults and 30 children) as controls were analysed. Patients were categorized according to their clinical scores (Harvey-Bradshaw Index [HBI] or Pediatric Crohn's Disease Activity Index [PCDAI]). Urinary orosomucoid (u-ORM) was determined by automated immune turbidimetric assay, and values were referred to urinary creatinine (u-ORM/u-CREAT, mg/mmol). RESULTS: U-ORM/u-CREAT values were seven times higher in children with active CD (0.50 vs 0.07 mg/mmol, P < 0.001) and two times higher in adults (0.32 vs 0.14 mg/mmol, P = 0.01) compared with patients with inactive disease. U-ORM/u-CREAT showed good correlation with conventional inflammatory markers (hs-CRP, serum ORM; P < 0.01) and activity indices (HBI, P = 0.018; PCDAI, P < 0.001). U-ORM/u-CREAT had similar discriminative performance to hs-CRP and serum ORM in the differentiation of active from inactive paediatric CD patients. CONCLUSIONS: Our findings suggest that u-ORM/u-CREAT might serve as a valuable additional marker in the follow-up of CD patients, especially in children for whom the non-invasive sampling is a further advantage.

Accelerated pre-senile systemic amyloidosis in PACAP knockout mice - a protective role of PACAP in age-related degenerative processes.

Dysregulation of neuropeptides may play an important role in aging-induced impairments. Among them, pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent cytoprotective peptide that provides an endogenous control against a variety of tissue-damaging stimuli. We hypothesized that the progressive decline of PACAP throughout life and the well-known general cytoprotective effects of PACAP lead to age-related pathophysiological changes in PACAP deficiency, supported by the increased vulnerability to various stressors of animals partially or totally lacking PACAP. Using young and aging CD1 PACAP knockout (KO) and wild type (WT) mice, we demonstrated pre-senile amyloidosis in young PACAP KO animals and showed that senile amyloidosis appeared accelerated, more generalized, more severe, and affected more individuals. Histopathology showed age-related systemic amyloidosis with mainly kidney, spleen, liver, skin, thyroid, intestinal, tracheal, and esophageal involvement. Mass spectrometry-based proteomic analysis, reconfirmed with immunohistochemistry, revealed that apolipoprotein-AIV was the main amyloid protein in the deposits together with several accompanying proteins. Although the local amyloidogenic protein expression was disturbed in KO animals, no difference was found in laboratory lipid parameters, suggesting a complex pathway leading to increased age-related degeneration with amyloid deposits in the absence of PACAP. In spite of no marked inflammatory histological changes or blood test parameters, we detected a disturbed cytokine profile that possibly creates a pro-inflammatory milieu favoring amyloid deposition. In summary, here we describe accelerated systemic senile amyloidosis in PACAP gene-deficient mice, which might indicate an early aging phenomenon in this mouse strain. Thus, PACAP KO mice could serve as a model of accelerated aging with human relevance. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Novel automated immune turbidimetric assay for routine urinary cystatin-C determinations.

AIM: There is no commercially available urinary cystatin-C (u-CYSC) test in the market. Therefore, we optimized and validated an automated immune turbidimetric test for u-CYSC measurements and investigated u-CYSC concentrations in acute and chronic diseases which might lead to renal tubular disorders. MATERIALS & METHODS: A particle-enhanced immune turbidimetric assay was adapted and validated on a Cobas 8000/c502 analyzer. Urine samples of different patient groups were also analyzed. RESULTS: Our method showed excellent analytical performance. U-CYSC/u-creatinine (u-CREAT) was higher in sepsis-related acute kidney injury group (p < 0.001) compared with controls and to patients with chronic hypertension and Type 2 diabetes. CONCLUSION: We validated a fast, sensitive, fully automated u-CYSC assay which is ideal for routine use and might be a potential complementary laboratory test to evaluate renal tubular function.

Monitoring urinary orosomucoid in patients undergoing cardiac surgery: A promising novel inflammatory marker.

BACKGROUND: Urinary biomarkers might provide non-invasive tool for monitoring of systemic processes. We aimed to investigate the time-course of urinary orosomucoid (u-ORM) excretion after cardiac surgery hypothesizing that u-ORM is an early and sensitive marker of systemic inflammatory activation. METHODS: During a 5-day follow-up study we monitored u-ORM levels in cardiovascular patients who underwent on-pump cardiac surgery (n=38). The patients baseline data were compared to healthy control individuals (n=40). u-ORM was measured by a newly developed automated turbidimetric assay and values were referred to urinary creatinine and expressed as u-ORM/u-CREAT (mg/mmol). RESULTS: The cardiovascular patients showed slightly increased baseline u-ORM excretion compared to healthy controls (0.29 vs 0.08mg/mmol, p<0.001). After cardiac surgery, a rapid 10-fold elevation in u-ORM/u-CREAT levels was found. The values remained high till the 3rd postoperative day, and they then decreased significantly (p<0.01) on the 5th day after surgery. u-ORM/u-CREAT mirrored well the perioperative tendency of hs-CRP levels, but it did not follow the non-decreasing kinetics of serum ORM concentrations during the follow-up. u-ORM/u-CREAT correlated significantly (p<0.001) with inflammatory parameters (hs-CRP, se-ORM, WBC). CONCLUSIONS: We described u-ORM as an early and sensitive marker of inflammatory activation. The rapid elevation of u-ORM/u-CREAT after surgery and its postoperative kinetics could reflect the magnitude of inflammatory response better than serum ORM and similar to hs-CRP. u-ORM measurements might provide a novel non-invasive tool for real-time monitoring of systemic inflammation, however further investigations are required to confirm it.

Urinary orosomucoid: validation of an automated immune turbidimetric test and its possible clinical use.

INTRODUCTION: Besides routine serum markers of inflammatory diseases, the diagnostic potential of selected urinary proteins has not been fully exploited yet. Former studies revealed that urinary orosomucoid (u-ORM) might have complementary information in inflammatory disorders. Our aim was to develop and validate a fully automated method for u-ORM measurements and to evaluate its potential clinical impact on systemic inflammatory diseases. MATERIALS AND METHODS: A particle-enhanced immune turbidimetric assay was validated for a Cobas 8000/c502 analyzer to determine u-ORM levels. Spot urine samples from 72 healthy individuals, 28 patients with Crohn's disease and 30 septic patients were studied. RESULTS: Our assay time was 10 minutes and the detection limit of u-ORM was 0.02 mg/L. The intra- and inter-assay imprecision expressed as CV was less than 5%, and the recovery ranged between 95-103%. Within 10 to 60 years of age, a preliminary reference range for urinary orosomucoid/creatinine ratio (u-ORM/u-CREAT) was found to be 0.08 (0.01-0.24) mg/mmol [median (2.5-97.5 percentiles)]. Compared to controls, a five-fold increase of u-ORM/u-CREAT values in Crohn's disease and approximately a 240-fold increase in sepsis were observed. CONCLUSIONS: We set up a fast, sensitive and precise turbidimetric approach for automated u-ORM determination. Our highly sensitive assay is ideal for routine u-ORM measurements and might be a potential novel laboratory test in the management of systemic inflammatory processes.

Oxidative stress induces transient O-GlcNAc elevation and tau dephosphorylation in SH-SY5Y cells.

O-linked ß-N-acetlyglucosamine or O-GlcNAc modification is a dynamic post-translational modification occurring on the Ser/Thr residues of many intracellular proteins. The chronic imbalance between phosphorylation and O-GlcNAc on tau protein is considered as one of the main hallmarks of Alzheimer's disease. In recent years, many studies also showed that O-GlcNAc levels can elevate upon acute stress and suggested that this might facilitate cell survival. However, many consider chronic stress, including oxidative damage as a major risk factor in the development of the disease. In this study, using the neuronal cell line SH-SY5Y we investigated the dynamic nature of O-GlcNAc after treatment with 0.5 mM H2 O2 for 30 min. to induce oxidative stress. We found that overall O-GlcNAc quickly increased and reached peak level at around 2 hrs post-stress, then returned to baseline levels after about 24 hrs. Interestingly, we also found that tau protein phosphorylation at site S262 showed parallel, whereas at S199 and PHF1 sites showed inverse dynamic to O-Glycosylation. In conclusion, our results show that temporary elevation in O-GlcNAc modification after H2 O2 -induced oxidative stress is detectable in cells of neuronal origin. Furthermore, oxidative stress changes the dynamic balance between O-GlcNAc and phosphorylation on tau proteins.

Timed, sequential administration of paclitaxel improves its cytotoxic effectiveness in a cell culture model.

Paclitaxel (taxol) is a chemotherapeutic agent frequently used in combination with other anti-neoplastic drugs. It is most effective during the M phase of the cell-cycle and tends to cause synchronization in malignant cells lines. In this study, we investigated whether timed, sequential treatment based on the cell-cycle characteristics could be exploited to enhance the cytotoxic effect of paclitaxel. We characterized the cell-cycle properties of a rapidly multiplying cell line (Sp2, mouse myeloma cells) by propidium-iodide DNA staining such as the lengths of various cell cycle phases and population duplication time. Based on this we designed a paclitaxel treatment protocol that comprised a primary and a secondary, timed treatment. We found that the first paclitaxel treatment synchronized the cells at the G2/M phase but releasing the block by stopping the treatment allowed a large number of cells to enter the next cell-cycle by a synchronized manner. The second treatment was most effective during the time when these cells approached the next G2/M phase and was least effective when it occurred after the peak time of this next G2/M phase. Moreover, we found that after mixing Sp2 cells with another, significantly slower multiplying cell type (Jurkat human T-cell leukemia) at an initial ratio of 1:1, the ratio of the two different cell types could be influenced by timed sequential paclitaxel treatment at will. Our results demonstrate that knowledge of the cell-cycle parameters of a specific malignant cell type could improve the effectivity of the chemotherapy. Implementing timed chemotherapeutic treatments could increase the cytotoxicity on the malignant cells but also decrease the side-effects since other, non-malignant cell types will have different cell-cycle characteristic and be out of synch during the treatment.

Low intensity, long term exposure to tobacco smoke inhibits hippocampal neurogenesis in adult mice.

Previous data have shown that high dose of nicotine administration or tobacco smoke exposure can reduce cell formation and the survival rate of adult-born neurons in the dentate gyrus. Here, we subjected adult mice to low intensity cigarette smoke exposure over long time periods. We did a 2×30min/day smoke exposure with two cigarettes per occasion over 1- or 2-months. Subsequently, we carried out a systematic quantitative histopathological analysis to assess the number of newborn neurons in the dentate gyrus. To investigate cell proliferation, the exogenous marker 5-bromo-2'-deoxyuridine (BrdU) was administered on the last experimental day and animals were sacrificed 2h later. To investigate the effect of tobacco smoke on the population of immature neurons, we quantified the number of doublecortin-positive (DCX+) neurons in the same animals. We found that exposing animals to cigarette smoke for 1- or 2-months had no influence on cell proliferation rate, but significantly reduced the number of DCX-positive immature neurons. Our tobacco smoke exposure regimen caused no substantial changes in respiratory functions, but histopathological analysis of the pulmonary tissue revealed a marked perivascular/peribronchial edema formation after 1-month and signs of chronic pulmonary inflammation after 2-months of cigarette smoke exposure. These data demonstrate that even mild exposure to cigarette smoke, without significantly affecting respiratory functions, can have a negative effect on adult-born neurons in the dentate gyrus, when applied over longer time periods. Our data indicate that besides nicotine other factors, such as inflammatory mediators, may also contribute to this effect.

Hepcidin and its potential clinical utility.

A number of pathophysiological conditions are related to iron metabolism disturbances. Some of them are well known, others are newly discovered or special. Hepcidin is a newly identified iron metabolism regulating hormone, which could be a promising biomarker for many disorders. In this review, we provide background information about mammalian iron metabolism, cellular iron trafficking, and the regulation of expression of hepcidin. Beside these molecular biological processes, we summarize the methods that have been used to determine blood and urine hepcidin levels and present those pathological conditions (cancer, inflammation, neurological disorders) when hepcidin measurement may have clinical relevance.