The liver and blood cells are responsible for creatine kinase clearance in blood Circulation: A retrospective study among different human diseases.

BACKGROUND: Muscle damage leads to increased serum creatine kinase (CK) levels in diseases such as acute myocardial infarction. Still, many individuals have abnormal serum CK activities lacking muscle-related diagnoses. The current study hypothesized that failed or overactivated CK clearance by non-muscle organs/tissues might be responsible for increased or decreased CK activities in blood. METHODS: We analyzing 37,081 independent CK test results in 36 human diseases during the past 5 y. RESULTS: We found that 33 out of 36 diseases were associated with decreased median CK activities compared to healthy controls. Besides muscle damage-related conditions, the highest mean CK activities were observed in hepatitis and cirrhosis. In contrast, 6 blood cell-related illnesses had the lowest mean CK values. ROC analysis showed that CK activities were the best biomarkers (AUC: 0.80-0.94) for the 6 blood-related diseases, especially myeloproliferative disorders. The principal component analysis revealed that the same category of diseases, such as liver-, blood -, kidney-, cancers, and vascular-related diseases, had clustered CK distributions. CONCLUSIONS: We proposed that the liver and blood cells were mainly responsible for CK clearance in blood circulation based on overall results. The testable mechanisms were presented and discussed.

Prolyl carboxypeptidase activity in the circulation and its correlation with body weight and adipose tissue in lean and obese subjects.

BACKGROUND: Prolyl carboxypeptidase (PRCP) is involved in the regulation of body weight, likely by hydrolysing alpha-melanocyte-stimulating hormone and apelin in the hypothalamus and in the periphery. A link between PRCP protein concentrations in plasma and metabolic disorders has been reported. In this study, we investigated the distribution of circulating PRCP activity and assessed its relation with body weight and adipose tissue in obese patients and patients who significantly lost weight. METHODS: PRCP activity was measured using reversed-phase high-performance liquid chromatography in different isolated blood fractions and primary human cells to investigate the distribution of circulating PRCP. PRCP activity was measured in serum of individuals (n = 75) categorized based on their body mass index (BMI < 25.0; 25.0-29.9; 30.0-39.9; ≥ 40.0 kg/m2) and the diagnosis of metabolic syndrome. Differences in serum PRCP activity were determined before and six months after weight loss, either by diet (n = 45) or by bariatric surgery (n = 24). Potential correlations between serum PRCP activity and several metabolic and biochemical parameters were assessed. Additionally, plasma PRCP concentrations were quantified using a sensitive ELISA in the bariatric surgery group. RESULTS: White blood cells and plasma contributed the most to circulating PRCP activity. Serum PRCP activity in lean subjects was 0.83 ± 0.04 U/L and increased significantly with a rising BMI (p<0.001) and decreased upon weight loss (diet, p<0.05; bariatric surgery, p<0.001). The serum PRCP activity alteration reflected body weight changes and was found to be positively correlated with several metabolic parameters, including: total, abdominal and visceral adipose tissue. Plasma PRCP concentration was found to be significantly correlated to serum PRCP activity (0.865; p<0.001). Additionally, a significant decrease (p<0.001) in plasma PRCP protein concentration (mean ± SD) before (18.2 ± 3.7 ng/mL) and 6 months after bariatric surgery (15.7 ± 2.7 ng/mL) was found. CONCLUSION: Our novel findings demonstrate that white blood cells and plasma contributed the most to circulating PRCP activity. Additionally, we have shown that there were significant correlations between serum PRCP activity and various metabolic parameters, and that plasma PRCP concentration was significantly correlated to serum PRCP activity. These novel findings on PRCP activity in serum support further investigation of its in vivo role and involvement in several metabolic diseases.

Altered Aconitase 2 Activity in Huntington's Disease Peripheral Blood Cells and Mouse Model Striatum.

Huntington's disease (HD) is caused by an unstable cytosine adenine guanine (CAG) trinucleotide repeat expansion encoding a polyglutamine tract in the huntingtin protein. Previously, we identified several up- and down-regulated protein molecules in the striatum of the Hdh(CAG)150 knock-in mice at 16 months of age, a mouse model which is modeling the early human HD stage. Among those molecules, aconitase 2 (Aco2) located in the mitochondrial matrix is involved in the energy generation and susceptible to increased oxidative stress that would lead to inactivation of Aco2 activity. In this study, we demonstrate decreased Aco2 protein level and activity in the brain of both Hdh(CAG)150 and R6/2 mice. Aco2 activity was decreased in striatum of Hdh(CAG)150 mice at 16 months of age as well as R6/2 mice at 7 to 13 weeks of age. Aco2 activity in the striatum of R6/2 mice could be restored by the anti-oxidant, N-acetyl-l-cysteine, supporting that decreased Aco2 activity in HD is probably caused by increased oxidative damage. Decreased Aco2 activity was further found in the peripheral blood mononuclear cells (PBMC) of both HD patients and pre-symptomatic HD mutation (PreHD) carriers, while the decreased Aco2 protein level of PBMC was only present in HD patients. Aco2 activity correlated significantly with motor score, independence scale, and functional capacity of the Unified Huntington's Disease Rating Scale as well as disease duration. Our study provides a potential biomarker to assess the disease status of HD patients and PreHD carriers.

Characterization of E-NTPDase (EC 3.6.1.5) activity in hepatic lymphocytes: A different activity profile from peripheral lymphocytes.

The activity of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) was characterized in hepatic lymphocytes (HL) of rats. For this purpose, a specific method for the isolation of lymphocytes from hepatic tissue was developed. Subsequently, E-NTPDase activity of rat HL was compared with that of rat peripheral lymphocytes. The HL showed high cell count and viability. Also, the characterization test revealed that the optimal E-NTPDase activities were attained at 37°C and pH 8.0 in the presence of Ca2+ . In addition, in the presence of specific E-NTPDase inhibitors (20mM sodium azide and 0.3mM suramin), there were significant inhibitions in nucleotide hydrolysis. However, there was no significant change in adenosine triphosphate (ATP) or adenosine diphosphate (ADP) hydrolysis in the presence of inhibitors of other E-ATPase (0.1mM Ouabain, 0.5mM orthovanadate, and 1mM, 5mM, and 10mM sodium azide). Furthermore, the kinetic behavior of the enzyme in HL showed apparent Km of 134.90 ± 0.03µM and 214.40 ± 0.06µM as well as Vmax of 345.0 ± 28.32 and 242.0 ± 27.55 Æžmol Pi/min/mg of protein for ATP and ADP, respectively. The Chevillard plot revealed that ATP and ADP were hydrolyzed at the same active site of the enzyme. Our results suggest that the degradation of extracellular nucleotides in HL may have been primarily accomplished by E-NTPDase. The higher E-NTPDase activity observed in HL may be attributed to the important physiological functions of ATP and ADP in HL. SIGNIFICANCE OF THE STUDY: Extracellular purine nucleotides are able to interact with specific receptors and trigger a number of important physiological functions in cells. This interaction is controlled by ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), enzyme that present their catalytic site at the extracellular space and degrades nucleotides. This purinergic signaling has important functions in peripheral lymphocytes and may represent an important new therapeutic target for the treatment of immunological diseases. However, there is dearth of information on the involvement of E-NTPDase in liver lymphocytes. The liver is an important organ, which performs both metabolic and toxicological roles in living organism, and hepatic lymphocytes may play crucial action in the regulation of immune responses in the liver tissue. Furthermore, various chronic diseases such as cirrhosis may be treated with novel pharmacotherapy by targeting the modulation of hepatic lymphocytes. Thus, the significance of this study is to evaluate the activity of E-NTPDase in liver lymphocyte and compare its activity with the peripheral lymphocytes.

Modulation of nuclear PI-PLCbeta1 during cell differentiation.

PI-PLCbeta1 plays an important role in cell differentiation, and particularly in myogenesis, osteogenesis and hematopoiesis. Indeed, the increase of PI-PLCbeta1, along with Cyclin D3, has been detected in C2C12 mouse myoblasts induced to differentiate, as well as in human cells obtained from myotonic dystrophy. Also in the case of osteogenic differentiation there is a specific induction of PI-PLCbeta1, but in this case the role of PI-PLCbeta1 seems to be independent from Cyclin D3, so that a different mechanism could be involved. As for the hematopoietic system, PI-PLCbeta1 has a peculiar behavior: it increases during myeloid differentiation and decreases during erythroid differentiation, thus confirming the role of PI-PLCbeta1 as a modulator of hematopoiesis.

Urokinase type plasminogen activator mediates Interleukin-17-induced peripheral blood mesenchymal stem cell motility and transendothelial migration.

Mesenchymal stem cells (MSCs) have the potential to migrate toward damaged tissues increasing tissue regeneration. Interleukin-17 (IL-17) is a proinflammatory cytokine with pleiotropic effects associated with many inflammatory diseases. Although IL-17 can modulate MSC functions, its capacity to regulate MSC migration is not well elucidated so far. Here, we studied the role of IL-17 on peripheral blood (PB) derived MSC migration and transmigration across endothelial cells. IL-17 increased PB-MSC migration in a wound healing assay as well as cell mobilization from collagen gel. Concomitantly IL-17 induced the expression of urokinase type plasminogen activator (uPA) without affecting matrix metalloproteinase expression. The incremented uPA expression mediated the capacity of IL-17 to enhance PB-MSC migration in a ERK1,2 MAPK dependent way. Also, IL-17 induced PB-MSC migration alongside with changes in cell polarization and uPA localization in cell protrusions. Moreover, IL-17 increased PB-MSC adhesion to endothelial cells and transendothelial migration, as well as increased the capacity of PB-MSC adhesion to fibronectin, in an uPA-dependent fashion. Therefore, our data suggested that IL-17 may act as chemotropic factor for PB-MSCs by incrementing cell motility and uPA expression during inflammation development.

Potentials of the circulating pruritogenic mediator lysophosphatidic acid in development of allergic skin inflammation in mice: role of blood cell-associated lysophospholipase D activity of autotaxin.

Itching and infiltration of immune cells are important hallmarks of atopic dermatitis (AD). Although various studies have focused on peripheral mediator-mediated mechanisms, systemic mediator-mediated mechanisms are also important in the pathogenesis and development of AD. Herein, we found that intradermal injection of lysophosphatidic acid (LPA), a bioactive phospholipid, induces scratching responses by Institute of Cancer Research mice through LPA1 receptor- and opioid µ receptor-mediating mechanisms, indicating its potential as a pruritogen. The circulating level of LPA in Naruto Research Institute Otsuka Atrichia mice, a systemic AD model, with severe scratching was found to be higher than that of control BALB/c mice, probably because of the increased lysophospholipase D activity of autotaxin (ATX) in the blood (mainly membrane associated) rather than in plasma (soluble). Heparan sulfate proteoglycan was shown to be involved in the association of ATX with blood cells. The sequestration of ATX protein on the blood cells by heparan sulfate proteoglycan may accelerate the transport of LPA to the local apical surface of vascular endothelium with LPA receptors, promoting the hyperpermeability of venules and the pathological uptake of immune cells, aggravating lesion progression and itching in Naruto Research Institute Otsuka Atrichia mice.

Identification of inducible nitric oxide synthase in peripheral blood cells as a mediator of myocardial ischemia/reperfusion injury.

Although the late phase of ischemic preconditioning is known to be mediated by increased inducible nitric oxide synthase (iNOS) activity, controversy persists regarding the role of iNOS in ischemia/reperfusion (I/R) injury and, specifically, whether this protein is protective or detrimental. We hypothesized that iNOS is protective in myocytes but detrimental in inflammatory cells. To test this hypothesis, we created chimeric mice with iNOS-deficient peripheral blood cells by transplanting iNOS knockout (KO) bone marrow in wild-type (WT) mice after lethal irradiation. 2 months later, the mice underwent a 30-min coronary occlusion followed by 24 h of reperfusion. In WT naïve mice (iNOS(+/+) naïve; group I, n = 17), infarct size was 56.9 ± 2.8% of the risk region. In iNOS KO naïve mice with whole-body iNOS deletion (iNOS(-/-) naïve; group II, n = 10), infarct size was comparable to group I (53.4 ± 3.5%). When irradiated WT mice received marrow from WT mice (iNOS(+/+) chimera; group III, n = 10), infarct size was slightly reduced versus group I (44.3 ± 3.2%), indicating that irradiation and/or transplantation slightly decrease I/R injury. However, when WT mice received marrow from iNOS KO mice (iNOS(-/-) chimera; group IV, n = 14), infarct size was profoundly reduced (22.8 ± 2.1%, P < 0.05 vs. group III), indicating that selective deletion of iNOS from peripheral blood cells (with no change in myocardial iNOS content) induces protection against myocardial infarction. Together with our previous work showing the cardioprotective actions of NO donors, iNOS gene therapy, and cardiac-specific overexpression of iNOS, these data support a complex, dual role of iNOS in myocardial infarction (i.e., protective in myocytes but deleterious in blood cells). To our knowledge, this is the first study to identify a critical role of iNOS in peripheral blood cells as a mediator of myocardial I/R injury. The results support heretofore unknown differential actions of iNOS depending on cell source and have important translational implications.

Shear stress-induced activation of Tie2-dependent signaling pathway enhances reendothelialization capacity of early endothelial progenitor cells.

Although endothelial progenitor cells (EPCs) play a pivotal role in the endothelial repair following arterial injury and shear stress has a beneficial effect on EPCs, however, the molecular mechanism underlying the influence of EPCs on the endothelial integrity and the regulation of shear stress on the EPC signaling remained to be studied. Here, we investigated the effects of laminar shear stress on the tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2)-dependent signaling and its relation to in vivo reendothelialization capacity of human early EPCs. The human early EPCs were treated with shear stress. Shear stress in a dose-dependent manner increased angiopoietin-2 (Ang2)-induced migratory, adhesive and proliferatory activities of EPCs. Transplantation of EPCs treated by shear stress facilitated in vivo reendothelialization in nude mouse model of carotid artery injury. In parallel, the phosphorylation of Tie2 and Akt of EPCs in response to shear stress was significantly enhanced. With treatment of Tie2 knockdown or Akt inhibition, shear stress-induced phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) of EPCs was markedly suppressed. After Tie2/PI3K/Akt/eNOS signaling was blocked, the effects of shear stress on in vitro function and in vivo reendothelialization capacity of EPCs were significantly inhibited. The present findings demonstrate for the first time that Tie2/PI3k/Akt/eNOS signaling pathway is, at least in part, involved in the EPCs-mediated reendothelialization after arterial injury. The upregulation of shear stress-induced Tie2-dependent signaling contributes to enhanced in vivo reendothelialization capacity of human EPCs.

Gene expression profile of Ci-VSP in juveniles and adult blood cells of ascidian.

VSP is a transmembrane protein whose cytoplasmic region shows significant similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Notably, VSP exhibits a unique ability to transduce electrical signals into phosphoinositide turnover by coupling a transmembrane voltage sensor domain to the PTEN-like phosphoinositide phosphatase domain. Moreover, VSP gene is known to be widely conserved among deuterostome genomes, though the function of VSP in vivo remains largely unknown. In the present study, the expression pattern of ascidian VSP(Ci-VSP) was examined in embryos and juveniles of a marine invertebrate chordate, Ciona intestinalis. RT-PCR showed that Ci-VSP is expressed at the larval stage and that expression persists in juveniles. Whole mount in situ hybridization showed that Ci-VSP is expressed in cells of the stomach, intestine and blood cells of 2- to 3-week-old juveniles. Moreover, double staining blood cells from 2-month-old adults with Ci-VSP and Ci-PTEN probes showed that Ci-VSP-positive cells are a distinct population, separate from cells expressing Ci-PTEN. These findings suggest that in addition to its previously suggested roles in testis or sperm, Ci-VSP plays a key role in voltage-induced signal transduction in cells of the digestive system and blood.

Circulating osteogenic cells: characterization and relationship to rates of bone loss in postmenopausal women.

There is increasing evidence that osteogenic cells are present not only in bone marrow (BM) but also in peripheral blood (PB). Since staining for alkaline phosphatase (AP) identifies osteoprogenitor cells in BM, we sought to further characterize BM versus PB hematopoietic lineage negative (lin-)/AP+ cells and to compare gene expression in PB lin-/AP+ cells from postmenopausal women undergoing rapid versus slow bone loss. PB lin-/AP+ cells were smaller than their BM counterparts, and both were negative for the pan-hematopoietic marker, CD45. BM and PB lin-/AP+ cells were capable of mineralization in vitro. Using whole genome linear amplification followed by quantitative polymerase chain reaction (QPCR) analysis, we found that relative to the BM cells, PB lin-/AP+ cells expressed similar levels of a number of key osteoblast marker genes (runx2, osterix, osteopontin, OPG, periostin), consistent with the PB cells being in the osteoblastic lineage. Importantly, however, compared to the BM cells, PB lin-/AP+ cells expressed lower levels of mRNAs for AP, type I collagen, and for a panel of proliferation markers, but higher levels of osteocalcin, osteonectin, and PTHR1 mRNAs, as well as those for RANKL and ICAM-1, both of which are important in supporting osteoclastogenesis. Using microarray followed by QPCR analysis, we further demonstrated that, compared to postmenopausal women undergoing slow bone loss, PB lin-/AP+ cells from women undergoing rapid bone loss expressed lower levels of mRNAs for hydroxyprostaglandin dehydrogenase, interferon regulator factor 3, Wnt1-induced secreted protein 1, and TGFbeta2, but higher levels of the Smad3 interacting protein, zinc finger DHHC-type containing 4 and col1alpha2. These data thus demonstrate that while PB lin-/AP+ cells express a number of osteoblastic genes and are capable of mineralization, they are a relatively quiescent cell population, both in terms of cell proliferation and matrix synthesis. However, their higher expression of RANKL and ICAM-1 mRNAs as compared to BM lin-/AP+ cells suggests a role for the PB lin-/AP+ cells in regulating osteoclastogenesis that warrants further investigation. Our study also provides "proof-of-concept" for the use of PB lin-/AP+ cells in clinical-investigative studies, and identifies several pathways that could potentially regulate rates of bone loss in postmenopausal women.

Characterization of an atypical gamma-secretase complex from hematopoietic origin.

Gamma-secretase is a widely expressed multisubunit enzyme complex which is involved in the pathogenesis of Alzheimer disease and hematopoietic malignancies through its aberrant processing of the amyloid precursor protein (APP) and Notch1, respectively. While gamma-secretase has been extensively studied, there is a dearth of information surrounding the activity, composition, and function of gamma-secretase expressed in distinct cellular populations. Here we show that endogenous gamma-secretase complexes of hematopoietic origin are distinct from epithelial derived gamma-secretase complexes. Hematopoietic gamma-secretase has reduced activity for APP and Notch1 processing compared to epithelial gamma-secretase. Characterization of the active complexes with small molecule affinity probes reveals that hematopoietic gamma-secretase has an atypical subunit composition with significantly altered subunit stoichiometry. Furthermore, we demonstrate that these discrete complexes exhibit cell-line specific substrate selectivity suggesting a possible mechanism of substrate regulation. These data underscore the need for studying endogenous gamma-secretase to fully understand of the biology of gamma-secretase and its complexity as a molecular target for the development of disease therapeutics.

Dendritic cells express hematopoietic prostaglandin D synthase and function as a source of prostaglandin D2 in the skin.

Prostaglandin D2 (PGD2), an arachidonic acid metabolite, has been implicated in allergic responses. A major source of PGD2 in the skin is mast cells that express hematopoietic PGD synthase (H-PGDS). In this study, we show the expression of H-PGDS in human dendritic cells (DCs) and the regulatory mechanisms by which DCs produce PGD2. We detected H-PGDS in epidermal Langerhans cells, dermal DCs, plasmacytoid DCs, and myeloid DCs. Monocyte-derived DCs rapidly secreted PGD2 when stimulated with the calcium ionophore A23187. More importantly, pretreatment of monocyte-derived DCs with PMA (phorbol 12-myrisate 13-acetate) synergistically enhanced the rapid PGD2 secretion induced by A23187, whereas PMA alone did not induce PGD2 secretion. Lipopolysaccharide (LPS) reduced H-PGDS expression, but interferon-gamma followed by LPS induced significant PGD2 production in a delayed time course at 6 hours. This effect was associated with inhibition of LPS-induced H-PGDS reduction. Interestingly, an irritant compound, SDS, also induced a rapid PGD2 release. PGD2 synergistically enhanced CCL22/macrophage-derived chemokine synthesis in interferon-gamma-treated human keratinocytes. In addition, bone marrow-derived DCs from wild-type mice stimulated lymph node cells to produce higher amounts of interleukin-17 than did DCs from mice lacking the H-PGDS gene. Thus, DCs could be an important source of skin PGD2 and may mediate or regulate skin inflammation by releasing PGD2 in response to various stimuli, contributing to the innate and/or acquired immune responses.

Regulation of hematopoiesis by the K63-specific ubiquitin-conjugating enzyme Ubc13.

The ubiquitin-conjugating enzyme Ubc13 mediates lysine-63-specific protein ubiquitination involved in signal transduction by immune receptors; however, the in vivo physiological functions of Ubc13 remain incompletely understood. Using Ubc13 conditional knockout mice, we show that somatic deletion of the Ubc13 gene causes severe loss of multi lineages of immune cells, which is associated with profound atrophy of the thymus and bone marrow, as well as lethality of the mice. Ubc13 has a cell-intrinsic function in mediating hematopoiesis and is essential for the survival and accumulation of hematopoietic stem cells in the bone marrow. Interestingly, loss of Ubc13 results in accumulation of beta-catenin and hyperexpression of Wnt target genes, a condition known to cause impaired hematopoiesis. These results establish Ubc13 as a crucial regulator of hematopoiesis and suggest a role for Ubc13 in the control of Wnt signaling in hematopoietic stem cells.

Elevated human telomerase reverse transcriptase gene expression in blood cells associated with chronic arsenic exposure in Inner Mongolia, China.

BACKGROUND: Arsenic exposure is associated with human cancer. Telomerase-containing human telomerase reverse transcriptase (hTERT) can extend telomeres of chromosomes, delay senescence, and promote cell proliferation leading to tumorigenesis. OBJECTIVE: The goal of this study was to investigate the effects of As on hTERT mRNA expression in humans and in vitro. METHOD: A total of 324 Inner Mongolia residents who have been exposed to As via drinking water participated in this study. Water and toenail samples were collected and analyzed for As. Blood samples were quantified for hTERT mRNA expression using real-time polymerase chain reaction. The hTERT mRNA levels were linked to water and nail As concentrations and skin hyperkeratosis. Human epidermal keratinocytes were treated with arsenite to assess effects on cell viability and hTERT expression in vitro. RESULTS: hTERT mRNA expression levels were significantly associated with As concentrations of water (p<0.0001) and nails (p=0.002) and also associated with severity of skin hyperkeratosis (p<0.05), adjusting for age, sex, smoking, and pesticide use. Females showed a higher slope than males (females: 0.126, p=0.0005; males: 0.079, p=0.017). In addition to water and nail As concentrations, age (p<0.0001) and pesticide use (p=0.025) also showed significant associations with hTERT expression. The hTERT expression levels decreased with age. Tobacco smoking did not affect hTERT expression (p=0.13). hTERT expression was significantly correlated with OGG1 and ERCC1 expression. The in vitro results also showed a dose-response relationship between arsenite concentrations and hTERT expression and reached the peak at 1 microM. CONCLUSIONS: hTERT expression was associated with As exposure in vivo and in vitro. The increased hTERT expression may be a cellular response to genomic insults by As and may also indicate that As may function as a tumor promoter in carcinogenesis in humans.

CD45, CD148, and Lyp/Pep: critical phosphatases regulating Src family kinase signaling networks in immune cells.

Reciprocal regulation of tyrosine phosphorylation by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) is central to normal immune cell function. Disruption of the equilibrium between PTK and PTP activity can result in immunodeficiency, autoimmunity, or malignancy. Src family kinases (SFKs) play a central role in both immune cell function and disease due to their proximal position in numerous signal transduction cascades including those emanating from integrin, T and B-cell antigen receptors, Fc, growth factor, and cytokine receptors. Given that tight regulation of SFKs activity is critical for appropriate responses to stimulation of these various signaling pathways, it is perhaps not surprising that multiple PTPs are involved in their regulation. Here, we focus on the role of three phosphatases, CD45, CD148, and LYP/PEP, which are critical regulators of SFKs in hematopoietic cells. We review our current understanding of their structures, expression, functions in different hematopoietic cell subsets, regulation, and putative roles in disease. Finally, we discuss remaining questions that must be addressed if we are to have a clearer understanding of the coordinated regulation of tyrosine phosphorylation and signaling networks in hematopoietic cells and how they could potentially be manipulated therapeutically in disease.

Serglycin proteoglycan: regulating the storage and activities of hematopoietic proteases.

Serglycin (SG), like all other proteoglycans, consists of a protein "core" to which sulfated and thereby negatively charged polysaccharide chains of glycosaminoglycan type are attached. The recent generation of mice lacking a functional SG gene has revealed a number of biological functions of SG. In particular, it has been shown that SG has a key role in promoting the storage and in regulating the activities of a number of proteases expressed in hematopoietic cell types, most notably various mast cell proteases. In this review, we summarize the recent development in our understanding of the biological function of SG, in particular by focusing on the novel insight provided through analysis of the SG-deficient mouse strain.

Various functions of caspases in hematopoiesis.

The role of cysteine proteases of the caspase family in apoptosis is well defined. Some caspases were initially shown to be involved in cytokine maturation along inflammatory response. In the recent years, several other non apoptotic functions of caspases were identified. In hematopoietic cells, caspases play a role in specific pathways of differentiation (erythropoiesis, differentiation of monocytes into macrophages, formation of proplatelets by megakaryocytes). These enzymes also play a non-apoptotic and complex role in regulating the maturation and proliferation of specific lymphocytes. Lastly, the apoptotic functions of caspases regulate the life span of several but not all blood cell types. The present review summarizes the current knowledge in these different functions. We show that the nature of involved enzymes, the pathways leading to their activation and the specificity of their cellular target proteins varies strongly from a cell type to another. We indicate also that, in most situations, specific Bcl-2-related proteins are involved in the control of caspase activation. Lastly, we discuss the deregulation of these pathways in hematopoietic diseases, including those in which an excess in caspase activation leads to cell death and those in which a default in caspase activation could block cell differentiation.

Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia.

PURPOSE: Gene expression profiling identified receptor tyrosine kinase ROR1, an embryonic protein involved in organogenesis, as a signature gene in B-cell chronic lymphocytic leukemia (B-CLL). To assess the suitability of ROR1 as a cell surface antigen for targeted therapy of B-CLL, we carried out a comprehensive analysis of ROR1 protein expression. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells, sera, and other adult tissues from B-CLL patients and healthy donors were analyzed qualitatively and quantitatively for ROR1 protein expression by flow cytometry, cell surface biotinylation, Western blotting, and ELISA. RESULTS: ROR1 protein is selectively expressed on the surface of B-CLL cells, whereas normal B cells, other normal blood cells, and normal adult tissues do not express cell surface ROR1. Moreover, cell surface expression of ROR1 is uniform and constitutive, i.e., independent of anatomic niches, independent of biological and clinical heterogeneity of B-CLL, independent of B-cell activation, and found at similar levels in all B-CLL samples tested. The antibody binding capacity of B-CLL cell surface ROR1 was determined to be in the range of 10(3) to 10(4) molecules per cell. A portion of B-CLL cell surface ROR1 was actively internalized upon antibody binding. Soluble ROR1 protein was detectable in sera of <25% of B-CLL patients and a similar fraction of healthy donors at concentrations below 200 ng/mL. CONCLUSIONS: The restricted, uniform, and constitutive cell surface expression of ROR1 protein in B-CLL provides a strong incentive for the development of targeted therapeutics such as monoclonal antibodies.