Early-Onset Neonatal Sepsis: Inflammatory Biomarkers and MicroRNA as Potential Diagnostic Tools in Preterm Newborns.

Mortality and morbidity of newborns with sepsis can be improved by early and accurate diagnosis and targeted therapy. To evaluate the early molecular events associated with inflammation and infection, we evaluated markers of endothelial activation and injury and circulating plasma miRNAs in preterm newborns with sepsis. The study group consisted of newborns with gestational age ≤ 32 weeks, with culture-positive early-onset neonatal sepsis (sepsis group, N = 8), and as a control group, we enrolled newborns without sepsis (control group, N = 12). Soluble markers of inflammation were measured using Luminex-based multiplex assay. Platelet-free plasma RNA was used to construct the library for miRNA sequencing analysis. Normalized counts were calculated and used to measure differential expression of individual detected miRNAs. We found a significant increase of interleukin 18 (IL-18) in the cord blood of the sepsis group (mean ± SEM, 104.7 ± 30.4 pg/ml vs 52.7 ± 5.6 pg/ml, P = 0.02). In peripheral blood of sepsis group patients, we found a significant increase of VEGF-A compared to controls (196.0 ± 70.5 pg/ml vs 59.6 ± 8.5 pg/ml, P = 0.02). In the cord blood plasma, eight miRNAs had significantly differential expression (P < 0.05), four miRNAs were up-regulated and four miRNAs down-regulated. In peripheral blood plasma, all nine miRNAs with significant differential expression were up-regulated. In conclusion, in early-onset neonatal sepsis, IL-18 and VEGF-A might be considered in diagnostic workup. Early-onset sepsis in preterm newborns is associated with significant changes in the circulating miRNA pattern.

Delivery-Associated Changes in the Levels of Inflammatory Molecules in Newborns.

Inflammation is considered a fundamental process accompanying physiological human birth, also playing a role in perinatal pathologies. The goal of the study was to assess the concentrations of inflammatory molecules with respect to the mode of delivery and dynamics of inflammatory molecules in neonatal samples in the first 48-72 hours of life. The concentrations of inflammatory cytokines were measured using the Luminex®xMAP multi-analyte profiling platform in cord blood and peripheral neonatal blood. Study groups included newborns delivered spontaneously (spontaneous group) and via elective caesarean section (elective group). Cord blood concentrations of interleukin 6 (IL-6) and procalcitonin were significantly higher (P < 0.0001) in the spontaneous group compared to the elective group. Neonatal blood concentrations of tumour necrosis factor (TNF) from the elective group were significantly higher com-pared to the spontaneous group (P = 0.0077). The concentrations of procalcitonin and TNF significantly increased within the first 48 to 72 hours following either mode of delivery. IL-6 and IL-18 were significantly higher in neonatal compared to umbilical cord blood in the elective group only, while the increase in the spontaneous group did not reach statistical significance. The concentrations of IL-1α, IL-1ß, IL-17A and IL-22 did not show significant differen-ces between the spontaneous and elective groups as well as between umbilical cord and neonatal blood. Our findings show physiological differences in the levels of inflammatory molecules following spontaneous vaginal delivery and elective caesarean section. The results can be used as baseline values for the research of various pathologies in newborns.

An international cohort study of autosomal dominant tubulointerstitial kidney disease due to REN mutations identifies distinct clinical subtypes.

There have been few clinical or scientific reports of autosomal dominant tubulointerstitial kidney disease due to REN mutations (ADTKD-REN), limiting characterization. To further study this, we formed an international cohort characterizing 111 individuals from 30 families with both clinical and laboratory findings. Sixty-nine individuals had a REN mutation in the signal peptide region (signal group), 27 in the prosegment (prosegment group), and 15 in the mature renin peptide (mature group). Signal group patients were most severely affected, presenting at a mean age of 19.7 years, with the prosegment group presenting at 22.4 years, and the mature group at 37 years. Anemia was present in childhood in 91% in the signal group, 69% prosegment, and none of the mature group. REN signal peptide mutations reduced hydrophobicity of the signal peptide, which is necessary for recognition and translocation across the endoplasmic reticulum, leading to aberrant delivery of preprorenin into the cytoplasm. REN mutations in the prosegment led to deposition of prorenin and renin in the endoplasmic reticulum-Golgi intermediate compartment and decreased prorenin secretion. Mutations in mature renin led to deposition of the mutant prorenin in the endoplasmic reticulum, similar to patients with ADTKD-UMOD, with a rate of progression to end stage kidney disease (63.6 years) that was significantly slower vs. the signal (53.1 years) and prosegment groups (50.8 years) (significant hazard ratio 0.367). Thus, clinical and laboratory studies revealed subtypes of ADTKD-REN that are pathophysiologically, diagnostically, and clinically distinct.

Development of In-Browser Simulators for Medical Education: Introduction of a Novel Software Toolchain.

BACKGROUND: Simulators used in teaching are interactive applications comprising a mathematical model of the system under study and a graphical user interface (GUI) that allows the user to control the model inputs and visualize the model results in an intuitive and educational way. Well-designed simulators promote active learning, enhance problem-solving skills, and encourage collaboration and small group discussion. However, creating simulators for teaching purposes is a challenging process that requires many contributors including educators, modelers, graphic designers, and programmers. The availability of a toolchain of user-friendly software tools for building simulators can facilitate this complex task. OBJECTIVE: This paper aimed to describe an open-source software toolchain termed Bodylight.js that facilitates the creation of browser-based client-side simulators for teaching purposes, which are platform independent, do not require any installation, and can work offline. The toolchain interconnects state-of-the-art modeling tools with current Web technologies and is designed to be resilient to future changes in the software ecosystem. METHODS: We used several open-source Web technologies, namely, WebAssembly and JavaScript, combined with the power of the Modelica modeling language and deployed them on the internet with interactive animations built using Adobe Animate. RESULTS: Models are implemented in the Modelica language using either OpenModelica or Dassault Systèmes Dymola and exported to a standardized Functional Mock-up Unit (FMU) to ensure future compatibility. The C code from the FMU is further compiled to WebAssembly using Emscripten. Industry-standard Adobe Animate is used to create interactive animations. A new tool called Bodylight.js Composer was developed for the toolchain that enables one to create the final simulator by composing the GUI using animations, plots, and control elements in a drag-and-drop style and binding them to the model variables. The resulting simulators are stand-alone HyperText Markup Language files including JavaScript and WebAssembly. Several simulators for physiology education were created using the Bodylight.js toolchain and have been received with general acclaim by teachers and students alike, thus validating our approach. The Nephron, Circulation, and Pressure-Volume Loop simulators are presented in this paper. Bodylight.js is licensed under General Public License 3.0 and is free for anyone to use. CONCLUSIONS: Bodylight.js enables us to effectively develop teaching simulators. Armed with this technology, we intend to focus on the development of new simulators and interactive textbooks for medical education. Bodylight.js usage is not limited to developing simulators for medical education and can facilitate the development of simulators for teaching complex topics in a variety of different fields.

Noninvasive Immunohistochemical Diagnosis and Novel MUC1 Mutations Causing Autosomal Dominant Tubulointerstitial Kidney Disease.

BACKGROUND: Autosomal dominant tubulointerstitial kidney disease caused by mucin-1 gene (MUC1) mutations (ADTKD-MUC1) is characterized by progressive kidney failure. Genetic evaluation for ADTKD-MUC1 specifically tests for a cytosine duplication that creates a unique frameshift protein (MUC1fs). Our goal was to develop immunohistochemical methods to detect the MUC1fs created by the cytosine duplication and, possibly, by other similar frameshift mutations and to identify novel MUC1 mutations in individuals with positive immunohistochemical staining for the MUC1fs protein. METHODS: We performed MUC1fs immunostaining on urinary cell smears and various tissues from ADTKD-MUC1-positive and -negative controls as well as in individuals from 37 ADTKD families that were negative for mutations in known ADTKD genes. We used novel analytic methods to identify MUC1 frameshift mutations. RESULTS: After technique refinement, the sensitivity and specificity for MUC1fs immunostaining of urinary cell smears were 94.2% and 88.6%, respectively. Further genetic testing on 17 families with positive MUC1fs immunostaining revealed six families with five novel MUC1 frameshift mutations that all predict production of the identical MUC1fs protein. CONCLUSIONS: We developed a noninvasive immunohistochemical method to detect MUC1fs that, after further validation, may be useful in the future for diagnostic testing. Production of the MUC1fs protein may be central to the pathogenesis of ADTKD-MUC1.

Endothelial Microvesicles and Soluble Markers of Endothelial Injury in Critically Ill Newborns.

Neonatal systemic inflammatory response and multiple organ dysfunction syndrome are the main postnatal insults influencing mortality and morbidity. Critically ill newborns with high predicted mortality are supported by extracorporeal membrane oxygenation (ECMO). Biomarkers of inflammatory response and endothelial injury can be used for early diagnosis and treatment of critical neonatal situations. The aim of our study was to explore plasma proteins and endothelial microvesicles as markers of inflammation and endothelial activation in newborns on ECMO and to compare them with healthy neonates. Thirteen newborns on ECMO and 13 healthy newborns were included in the study. Plasma soluble biomarkers were measured using multiplex immunoassay based on Luminex® xMAP multianalyte profiling platform. The total microvesicle count and plasma level of surface antigen-specific microvesicles were determined by flow cytometry. The plasma concentration of cell-derived microvesicles was measured using annexin-V labeling, and the endothelial origin of microvesicles was determined using lineage-specific antigen labeling of endothelial cell/microvesicle markers (endoglin/CD105, PECAM1/CD31, VEGFR2/CD309, and MadCAM1). Inflammatory markers (procalcitonin, IL-1ß, IL-6, and IL-22) were increased in the ECMO group (P < 0.01). The assessment of endothelial markers showed higher concentrations of endocan and angiopoietin-2 (P < 0.01) in the ECMO group while VEGF in the ECMO group was significantly lower (P < 0.01). In the ECMO group, the concentration of annexin-V-positive microvesicles (total microvesicles) and endothelial microvesicles expressing mucosal vascular addressin cell adhesion molecule 1 (MadCAM1) was increased (P = 0.05). In summary, we found increased concentrations of soluble inflammatory and endothelial markers in the plasma of critically ill newborns with multiple organ dysfunction. Increased plasma concentrations of microvesicles may reflect the activation or damage of blood cells and vasculature including endothelial cells. The measurement of cell membrane-derived microvesicles may be added to the panel of established inflammatory markers in order to increase the sensitivity and specificity of the diagnostic process in critically ill newborns.

Mouse models of mantle cell lymphoma, complex changes in gene expression and phenotype of engrafted MCL cells: implications for preclinical research.

Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma (NHL) associated with poor prognosis. Animal models of MCL are scarce. We established and characterized various in vivo models of metastatic human MCL by tail vein injection of either primary cells isolated from patients with MCL or established MCL cell lines (Jeko-1, Mino, Rec-1, Hbl-2, and Granta-519) into immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. MCL infiltration was assessed with immunohistochemistry (tissues) and flow cytometry (peripheral blood). Engraftment of primary MCL cells was observed in 7 out of 12 patient samples. The pattern of engraftment of primary MCL cells varied from isolated involvement of the spleen to multiorgan infiltration. On the other hand, tumor engraftment was achieved in all five MCL cell lines used and lymphoma involvement of murine bone marrow, spleen, liver, and brain was observed. Overall survival of xenografted mice ranged from 22 ± 1 to 54 ± 3 days depending on the cell line used. Subsequently, we compared the gene expression profile (GEP) and phenotype of the engrafted MCL cells compared with the original in vitro growing cell lines (controls). We demonstrated that engrafted MCL cells displayed complex changes of GEP, protein expression, and sensitivity to cytotoxic agents when compared with controls. We further demonstrated that our MCL mouse models could be used to test the therapeutic activity of systemic chemotherapy, monoclonal antibodies, or angiogenesis inhibitors. The characterization of MCL murine models is likely to aid in improving our knowledge in the disease biology and to assist scientists in the preclinical and clinical development of novel agents in relapsed/refractory MCL patients.

Rivaroxaban in combination with low-dose aspirin is associated with a reduction in pro-inflammatory and pro-thrombotic circulating vesicle signatures in patients with cardiovascular disease.

BACKGROUND: Despite secondary prevention with aspirin, patients with stable cardiovascular disease (CVD) remain at elevated long-term risk of major cardiovascular events (MACE). The COMPASS double-blind randomized clinical trial demonstrated that aspirin plus low-dose Rivaroxaban (COMPASS regime) significantly decreased the incidence of MACE by 24% compared with aspirin alone. However, the mechanisms underlying these potential synergistic/non-antithrombotic effects remain elusive. Extracellular vesicles (EVs) are crucial messengers regulating a myriad of biological/pathological processes and are highly implicated in CVD. We hypothesised that circulating EV profiles reflect the cardioprotective properties of the COMPASS regime. PATIENTS/METHODS: A cohort of stable CVD patients (n=40) who participated in the COMPASS trial and were previously randomised to receive aspirin, were prospectively recruited and assigned a revised regimen of open-label aspirin plus Rivaroxaban. Blood samples were obtained at baseline (aspirin only) and 6-months follow-up. Plasma EV concentration, size and origin were analysed by NTA and flow cytometry. EVs were enriched by ultracentrifugation for proteomic analysis. RESULTS: The COMPASS regime fundamentally altered small (<200nm) and large (200-1000nm) EV concentration and size, compared to aspirin alone. Crucially, levels of platelet-derived, and myeloperoxidase-positive EVs became significantly decreased at follow-up. Comparative proteomic characterisation further revealed a significant decrease in highly pro-inflammatory protein expression at follow-up. CONCLUSIONS: The observed changes in EV subpopulations, together with the differential protein expression profiles, suggest amelioration of an underlying pro-inflammatory and pro-thrombotic state upon dual therapy, which may be of clinical relevance towards understanding the fundamental mechanism underlying the reported superior cardiovascular outcomes associated with this antithrombotic regimen.

A Novel Monoallelic ALG5 Variant Causing Late-Onset ADPKD and Tubulointerstitial Fibrosis.

Introduction: Monoallelic variants in the ALG5 gene encoding asparagine-linked glycosylation protein 5 homolog (ALG5) have been recently shown to disrupt polycystin-1 (PC1) maturation and trafficking via underglycosylation, causing an autosomal dominant polycystic kidney disease-like (ADPKD-like) phenotype and interstitial fibrosis. In this report, we present clinical, genetic, histopathologic, and protein structure and functional correlates of a new ALG5 variant, p.R79W, that we identified in 2 distant genetically related Irish families displaying an atypical late-onset ADPKD phenotype combined with tubulointerstitial damage. Methods: Whole exome and targeted sequencing were used for segregation analysis of available relatives. This was followed by immunohistochemistry examinations of kidney biopsies, and targeted (UMOD, MUC1) and untargeted plasma proteome and N-glycomic studies. Results: We identified a monoallelic ALG5 variant [GRCh37 (NM_013338.5): g.37569565G>A, c.235C>T; p.R79W] that cosegregates in 23 individuals, of whom 18 were clinically affected. We detected abnormal localization of ALG5 in the Golgi apparatus of renal tubular cells in patients' kidney specimens. Further, we detected the pathological accumulation of uromodulin, an N-glycosylated glycosylphosphatidylinositol (GPI)-anchored protein, in the endoplasmic reticulum (ER), but not mucin-1, an O- and N-glycosylated protein. Biochemical investigation revealed decreased plasma and urinary uromodulin levels in clinically affected individuals. Proteomic and glycoproteomic profiling revealed the dysregulation of chronic kidney disease (CKD)-associated proteins. Conclusion: ALG5 dysfunction adversely affects maturation and trafficking of N-glycosylated and GPI anchored protein uromodulin, leading to structural and functional changes in the kidney. Our findings confirm ALG5 as a cause of late-onset ADPKD and provide additional insight into the molecular mechanisms of ADPKD-ALG5.

Dominant renin gene mutations associated with early-onset hyperuricemia, anemia, and chronic kidney failure.

Through linkage analysis and candidate gene sequencing, we identified three unrelated families with the autosomal-dominant inheritance of early onset anemia, hypouricosuric hyperuricemia, progressive kidney failure, and mutations resulting either in the deletion (p.Leu16del) or the amino acid exchange (p.Leu16Arg) of a single leucine residue in the signal sequence of renin. Both mutations decrease signal sequence hydrophobicity and are predicted by bioinformatic analyses to damage targeting and cotranslational translocation of preprorenin into the endoplasmic reticulum (ER). Transfection and in vitro studies confirmed that both mutations affect ER translocation and processing of nascent preprorenin, resulting either in reduced (p.Leu16del) or abolished (p.Leu16Arg) prorenin and renin biosynthesis and secretion. Expression of renin and other components of the renin-angiotensin system was decreased accordingly in kidney biopsy specimens from affected individuals. Cells stably expressing the p.Leu16del protein showed activated ER stress, unfolded protein response, and reduced growth rate. It is likely that expression of the mutant proteins has a dominant toxic effect gradually reducing the viability of renin-expressing cells. This alters the intrarenal renin-angiotensin system and the juxtaglomerular apparatus functionality and leads to nephron dropout and progressive kidney failure. Our findings provide insight into the functionality of renin-angiotensin system and stress the importance of renin analysis in families and individuals with early onset hyperuricemia, anemia, and progressive kidney failure.

Nutritional hepatic iron overload is not prevented by parenteral hepcidin substitution therapy in mice.

The peptide hormone hepcidin functions as a negative regulator of intestinal Fe absorption and Fe recycling. Since its discovery as a systemic negative regulator of Fe metabolism, hepcidin has attracted enormous interest as a potential drug for the treatment and/or prevention of several forms of Fe overload. We therefore tested whether multiple doses of intraperitoneally administered synthetic renatured hepcidin can prevent hepatic Fe loading in mice concurrently fed an Fe-rich diet, and whether the same treatment affects hepatic Fe stores in mice fed a normal diet. Cohorts of male mice were fed either a normal defined diet (180 parts per million Fe) or an Fe-rich diet (the same diet supplemented with 2 % carbonyl iron for 2 weeks). Concurrently, half of the animals in each diet group received 100 µg of renatured hepcidin intraperitoneally every 12 h, for the same 2-week period. The second half of the animals received PBS only. The renatured synthetic hepcidin demonstrated biological activity by significantly decreasing transferrin saturation, which lasted for up to 24 h after a single hepcidin dose. However, the 14 d intraperitoneal hepcidin therapy did not prevent hepatic Fe overload in mice fed the Fe-rich diet, nor did it affect hepatic Fe stores in mice fed the normal diet. Both hepcidin agonists and antagonists are expected to have broad therapeutic potential. The absence of an effect of biologically active hepcidin on hepatic Fe loading shows the need for thorough future studies on the hepcidin regulation of Fe absorption and tissue distribution.

Exosomes released by imatinib­resistant K562 cells contain specific membrane markers, IFITM3, CD146 and CD36 and increase the survival of imatinib­sensitive cells in the presence of imatinib.

Chronic myeloid leukemia (CML) is a malignant hematopoietic disorder distinguished by the presence of a BCR­ABL1 fused oncogene with constitutive kinase activity. Targeted CML therapy by specific tyrosine kinase inhibitors (TKIs) leads to a marked improvement in the survival of the patients and their quality of life. However, the development of resistance to TKIs remains a critical issue for a subset of patients. The most common cause of resistance are numerous point mutations in the BCR­ABL1 gene, followed by less common mutations and multiple mutation-independent mechanisms. Recently, exosomes, which are extracellular vesicles excreted from normal and tumor cells, have been associated with drug resistance and cancer progression. The aim of the present study was to characterize the exosomes released by imatinib­resistant K562 (K562IR) cells. The K562IR­derived exosomes were internalized by imatinib­sensitive K562 cells, which thereby increased their survival in the presence of 2 µM imatinib. The exosomal cargo was subsequently analyzed to identify resistance­associated markers using a deep label­free quantification proteomic analysis. There were >3,000 exosomal proteins identified of which, 35 were found to be differentially expressed. From this, a total of 3, namely the membrane proteins, interferon­induced transmembrane protein 3, CD146 and CD36, were markedly upregulated in the exosomes derived from the K562IR cells, and exhibited surface localization. The upregulation of these proteins was verified in the K562IR exosomes, and also in the K562IR cells. Using flow cytometric analysis, it was possible to further demonstrate the potential of CD146 as a cell surface marker associated with imatinib resistance in K562 cells. Taken together, these results suggested that exosomes and their respective candidate surface proteins could be potential diagnostic markers of TKI drug resistance in CML therapy.

Spontaneous delivery is associated with increased endothelial activity in cord blood compared to elective cesarean section.

OBJECTIVES: It is suggested that delivery whether spontaneous or by elective cesarean section is associated with an inflammatory reaction which may be modified by the type of delivery. Inflammatory reactions are associated with endothelial activation. The aim of our study was to assess endothelial biomarkers in cord and neonatal blood following different modes of delivery. STUDY DESIGN: The study group consisted of term healthy newborns after uncomplicated pregnancies and either spontaneous vaginal delivery (n = 39) or elective cesarean section (n = 20). Plasma soluble biomarkers were measured using multiplex magnetic bead immunoassay. The microvesicle count and number of surface antigen-specific microvesicles were determined by flow cytometry. RESULTS: We found significantly increased concentrations of cord blood endothelial markers (sVEGFR1, Endothelin-1 and sVCAM1) and microvesicles (EPCR/CD201+, ICAM1/CD54+ and PECAM1/CD31+) in spontaneous vaginal delivery when compared to elective cesarean section. Irrespective of the delivery mode endothelial markers sVEGFR1, Endocan, Angiopoietin-2, VEGF, and sICAM1, were significantly increased in neonatal compared to cord blood. CONCLUSION: We found increased cord blood concentrations of endothelial markers and microvesicles following spontaneous vaginal delivery, which may reflect the natural activation of endothelial cells during labor. Following the delivery, most of the soluble markers increased, as a possible consequence of activation of neonatal innate immunity and postnatal cardiovascular transition.

Identification of molecular targets for selective elimination of TRAIL-resistant leukemia cells. From spots to in vitro assays using TOP15 charts.

The resistance of malignant cells to chemotherapy calls for the development of novel anti-cancer drugs. TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic cytokine, which selectively induces apoptosis in malignant cells. We derived two TRAIL-resistant HL-60 subclones, HL-60/P1 and HL-60/P2, from a TRAIL-sensitive HL-60 acute promyelocytic leukemia cell line. To identify therapeutically exploitable "weaknesses" of the TRAIL-resistant leukemia cells that could be used as molecular targets for their elimination, we performed proteomic (2-DE) analysis and compared both TRAIL-resistant subclones with the original TRAIL-sensitive HL-60 cells. We identified over 40 differentially expressed proteins. To significantly narrow the lists of candidate proteins, we excluded proteins that are known to be often differentially expressed, regardless of experiment type and tissue (the so-called "TOP15" proteins). Decreased expression of DNA replication and maintenance proteins MCM7 and RPA32 in HL-60/P1 cells, and the marked down-regulation of enzyme adenosine deaminase in HL-60/P2 cells, suggests increased sensitivity of these cells to DNA-interfering drugs, and adenosine and its homologues, respectively. In a series of in vitro assays, we confirmed the increased toxicity of etoposide and cisplatin to TRAIL resistant HL-60/P1 cells, and adenosine and vidarabine to HL-60/P2, compared with TRAIL-sensitive HL-60 cells.

TRAIL-induced apoptosis of HL60 leukemia cells: two distinct phenotypes of acquired TRAIL resistance that are accompanied with resistance to TNFalpha but not to idarubicin and cytarabine.

TNF-related apoptosis-inducing ligand (TRAIL) is a proapoptotic cytokine implicated in cancer cell surveillance. A potential of TRAIL as a cancer-specific therapeutic agent has been proposed, either as a single agent or in combination with chemotherapy. Prolonged exposure of TRAIL-sensitive leukemia cell line, wild-type (WT) HL60 cells to recombinant soluble TRAIL or to cytostatic agents, cytarabine and idarubicin, resulted in the establishment of resistant subclones with distinct phenotypic features. The TRAIL resistant HL60 subclones were characterized by decreased expression of TRAIL and TNFalpha death receptors. These resistant subclones had impaired activation of caspases 8 and 10 in response to TRAIL and TNFalpha, decreased TRAIL-induced nuclear translocation of NFkappaB RelA/p65, and dysregulation of the expression of several apoptosis regulators. Among the TRAIL resistant HL60 subclones we identified two separate phenotypes that differed in the expression of CD14, osteoprotegerin, and several apoptosis regulators. Both these TRAIL resistant HL60 subclones were resistant to TNFalpha, suggesting disruption of the extrinsic apoptotic pathway, but not to cytostatic agents, cytarabine and idarubicin. The concurrently derived HL60 subclones were cytarabine and idarubicin-resistant but remained sensitive to TRAIL-induced apoptosis. We identified distinct pathways for the development of HL60 leukemia cell resistance to apoptosis induction. These findings are relevant for the design of more effective strategies for leukemia therapy.

Déjà vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins.

After reading many 2-DE-based articles featuring lists of the differentially expressed proteins, one starts experiencing a disturbing déjà vu. The same proteins seem to predominate regardless of the experiment, tissue or species. To quantify the occurrence of individual differentially expressed proteins in 2-DE experiment reports, we compiled the identities of differentially expressed proteins identified in human, mouse, and rat tissues published in three recent volumes of Proteomics and calculated the appearance of the most predominant proteins in the dataset. The most frequently identified protein is a highly abundant glycolytic enzyme enolase 1, differentially expressed in nearly every third experiment on both human and rodent tissues. Heat-shock protein 27 (HSP27) and heat-shock protein 60 (HSP60) were differentially expressed in about 30 percent of human and rodent samples, respectively. Considering protein families as units, keratins and peroxiredoxins are the most frequently identified molecules, with at least one member of the group being differentially expressed in about 40 percent of all experiments. We suggest that the frequent identification of these proteins must be considered in the interpretation of any 2-DE studies. We consider if these commonly observed changes represent common cellular stress responses or are a reflection of the technical limitations of 2-DE.

Reduced erythroid cell and erythropoietin production in response to acute anemia in prion protein-deficient (Prnp-/-) mice.

Cellular prion protein (PrPc) participates in the pathogenesis of prion diseases but its normal function remains unclear. PrPc is expressed on hematopoietic cells, including erythroid precursors. We investigated the role of PrPc in erythropoiesis in vivo with phenylhydrazine-induced acute anemia. Induction of equivalent anemia in wild-type (WT) and Prnp-/- mice resulted in a higher number of circulating reticulocytes, hematocrits and spleen weights in WT mice than in Prnp-/- mice on Days 5 and 7. Examination of bone marrow erythroid precursor cells (Ter119+) on Day 5 revealed no significant differences in the number of these cells between the two types of animals. However, a higher percentage of Ter119+ cells were going through apoptosis in Prnp-/- mice than in WT mice. Plasma erythropoietin (Epo) levels and Epo mRNA in kidneys peaked on Day 3 in response to anemia for both types of animals but rose less in Prnp-/- (5500 pg/ml ) than in WT (18,000 pg/ml) animals. Administration of recombinant human Epo to mice produced an equivalent reticulocyte response in both types of animals suggesting that the potential for erythroid generation is intact in Prnp-/- animals. These observations indicate that PrPc may modulate tissue hypoxia-sensing mechanisms or effect hypoxia target gene expression.

Endothelial cell-derived microvesicles: potential mediators and biomarkers of pathologic processes.

This review focuses on the formation, composition and function of endothelial microvesicles (MV), often called microparticles (MP). MV release is a controlled event and is considered a hallmark of cellular activation or alteration. MV may affect the function of target cells through surface interaction and receptor activation, cellular fusion and the delivery of intravesicular cargo. Endothelial MV are released as a consequence of endothelial activation during inflammation and have been described to affect hemostasis, various aspects of inflammatory reaction, vessel formation, apoptosis and cell survival, endothelial cell differentiation and function. Recent data suggest the potential use of MV in diagnostics, assessment of severity and prediction of outcomes in inflammatory diseases and their utilization as targets, mediators and vectors in therapy.

Expression of angiopoietic factors in normal and type-I diabetes human placenta: a pilot study.

OBJECTIVES: Morphological changes of blood vessel wall have been described in placenta from pregnancies complicated by diabetes mellitus type-I. STUDY DESIGN: We measured mRNA expression of vascular endothelial growth factor (VEGF), angiopoietin 1 and 2 (Ang-1 and Ang-2), their receptors VEGFR-1, VEGFR-2, Tie-2, fibroblast growth factor 2 (FGF-2), and its receptor FGF-2R in placental tissue of diabetes type-I patients, in normal term placenta, and endometrium of non-pregnant women by real time reverse transcriptase PCR. RESULTS: The expression of Ang-2 and VEGFR-1 mRNAs was significantly higher in placenta (P