Development and validation of the mastocytosis activity score.

BACKGROUND: Mastocytosis is a heterogeneous disease characterized by a clonal expansion of mast cells in various organs. The vast majority of patients suffer from signs and symptoms caused by mediator release from mast cells. Although the disease burden is high, there is currently no specific and validated instrument to measure and monitor signs and symptoms in patients with mastocytosis. OBJECTIVE: To develop and validate a disease-specific tool to measure and monitor the activity of signs and symptoms in patients with mastocytosis, the Mastocytosis Activity Score (MAS). METHODS: Nineteen potential MAS items were developed in a combined approach consisting of semi-structured patient interviews, expert input and literature research. Item selection was performed by impact analysis with 76 patients followed by a review for face validity. The resulting MAS was tested for validity, reliability and influence factors. In parallel, a US American English version of the MAS was developed. RESULTS: A total of 68 mastocytosis patients took part in the MAS validation study. The final 9-item MAS was found to have a three-domain structure ("skin," "gastrointestinal tract" and "other"), a valid total score and an excellent test-retest reliability. Multiple regression analysis revealed that disease duration, age or gender is not a significant determinant of the MAS results. CONCLUSIONS: The MAS is a disease-specific, valid and reliable patient-reported outcome measure for adult patients with cutaneous and indolent systemic mastocytosis. It may serve as a valuable tool to measure and monitor mastocytosis activity, both, in clinical trials and in routine care.

0.7 Structure and zero bias anomaly in ballistic hole quantum wires.

We study the anomalous conductance plateau around G=0.7(2e2/h) and the zero bias anomaly in ballistic hole quantum wires with respect to in-plane magnetic fields applied parallel B parallel and perpendicular B perpendicular to the quantum wire. As seen in electron quantum wires, the magnetic fields shift the 0.7 structure down to G=0.5(2e2/h) and simultaneously quench the zero bias anomaly. However, these effects are strongly dependent on the orientation of the magnetic field, owing to the highly anisotropic effective Landé g-factor g* in hole quantum wires. Our results highlight the fundamental role that spin plays in both the 0.7 structure and zero bias anomaly.

Caspase-2 is required for cell death induced by cytoskeletal disruption.

Caspase-2 is one of the most conserved caspases, yet its biological function remains a matter of controversy. In the present article we analysed mouse embryonic fibroblasts (MEFs) from caspase-2 knockout mice for their sensitivity to various apoptosis inducing agents. We found that cell death induced by drugs that disrupt cytoskeleton is significantly inhibited in Casp2(-/-) MEFs. These drugs included zoledronic acid, vincristine, cytochalasin D and paclitaxel. We demonstrate that MEFs lacking Casp2 show clonogenic survival following drug treatment, whereas all Casp2(+/+) MEFs die, indicating that caspase-2 is required for apoptosis induced by cytoskeletal disruption. We further found that caspase-2 mediates apoptosis via Piddosome, Bid and Bax activation, and cytochrome c release. In the absence of caspase-2, Bid and Bax activation, and cytochrome c release are significantly delayed following drug treatment. Our data provide strong support for a context-dependent function of caspase-2 in apoptosis.

Zeeman splitting in ballistic hole quantum wires.

We have studied the Zeeman splitting in ballistic hole quantum wires formed in a (311)A quantum well by surface gate confinement. Transport measurements clearly show lifting of the spin degeneracy and crossings of the subbands when an in-plane magnetic field B is applied parallel to the wire. When B is oriented perpendicular to the wire, no spin splitting is discernible up to B = 8.8 T. The observed large Zeeman splitting anisotropy in our hole quantum wires demonstrates the importance of quantum confinement for spin splitting in nanostructures with strong spin-orbit coupling.

Fatal hemolytic transfusion reaction due to anti-Ku in a Knull patient.

A fatal transfusion reaction due to anti-Ku in a Knull (Ko) patient is reported. The patient was transfused with 34 units of incompatible RBCs during 44 days of hospitalization. Apart from the first transfusion, all subsequent transfusions failed to raise the patient's Hb. No serum antibody was identified until he was transferred to another hospital for dialysis. A compatibility test demonstrated a weak antibody and autocontrol reacting at room temperature by a manual polybrene method. The antibody was considered to be a "cold agglutinin." A blood sample was sent to a reference laboratory where the patient was found to be Knull and the antibody was identified as anti-Ku.

Inhibition of IgE-mediated mast cell activation by the paired Ig-like receptor PIR-B.

The potential of the paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) types for modifying an IgE antibody-mediated allergic response was evaluated in mouse bone marrow-derived mast cells. Although mast cells produced both PIR-A and PIR-B, PIR-B was found to be preferentially expressed on the cell surface, where it was constitutively tyrosine phosphorylated and associated with intracellular SHP-1 protein tyrosine phosphatase. PIR-B coligation with the IgE receptor (FcepsilonRI) inhibited IgE-mediated mast cell activation and release of serotonin. Surprisingly, the inhibitory activity of PIR-B was unimpaired in SHP-1-deficient mast cells. A third functional tyrosine-based inhibitory motif, one that fails to bind the SHP-1, SHP-2, and SHIP phosphatases, was identified in parallel studies of FcepsilonRI-bearing rat basophilic leukemia (RBL) cells transfected with constructs having mutations in the PIR-B cytoplasmic region. These results define the preferential expression of the PIR-B molecules on mast cells and an inhibitory potential that can be mediated via a SHP-1-independent pathway.

Cellular zinc fluxes and the regulation of apoptosis/gene-directed cell death.

The maintenance of discrete subcellular pools of zinc (Zn) is critical for the functional and structural integrity of cells. Among the important biological processes influenced by Zn is apoptosis, a process that is important in cellular homeostasis (an important cellular homeostatic process). It has also been identified as a major mechanism contributing to cell death in response to toxins and in disease, offering hope that novel therapies that target apoptotic pathways may be developed. Because Zn levels in the body can be increased in a relatively nontoxic manner, it may be possible to prevent or ameliorate degenerative disorders that are associated with high rates of apoptotic cell death. This review begins with brief introductions that address, first, the cellular biology of Zn, especially the critical labile Zn pools, and, second, the phenomenon of apoptosis. We then review the evidence relating Zn to apoptosis and address three major hypotheses: (1) that a specific pool or pools of intracellular labile Zn regulates apoptosis; (2) that systemic changes in Zn levels in the body, due to dietary factors, altered physiological states or disease, can influence cell susceptibility to apoptosis, and (3) that this altered susceptibility to apoptosis contributes to pathophysiological changes in the body. Other key issues are the identity of the molecular targets of Zn in the apoptotic cascade, the types of cells and tissues most susceptible to Zn-regulated apoptosis, the role of Zn as a coordinate regulator of mitosis and apoptosis and the apparent release of tightly bound intracellular pools of Zn during the later stages of apoptosis. This review concludes with a section highlighting areas of priority for future studies.

Involvement of intracellular labile zinc in suppression of DEVD-caspase activity in human neuroblastoma cells.

Age-related tissue Zn deficiency may contribute to neuronal and glial cell death by apoptosis in Alzheimer's dementia. To investigate this, we studied the effects of increasing or decreasing the levels of intracellular labile Zn on apoptosis of human neuroblastoma BE(2)-C cells in vitro. BE(2)-C cells were primed for 18 h with butyrate (1 mM) before addition of staurosporine (1 microM), an effector enzyme of apoptosis, for a further 3 h to induce DEVD-caspase activity. An increase in intracellular Zn using Zn ionophore pyrithione suppressed DEVD-caspase activity, while a decrease in intracellular Zn induced by Zn chelator TPEN mimicked staurosporine by activating DEVD-caspase in butyrate-primed cells. The distribution of intracellular Zn in the cells was demonstrated with the UV-excitable Zn-specific fluorophore Zinquin. Confocal images showed distinct cytoplasmic and cytoskeletal fluorescence. We propose that Zn decreases the level of apoptosis in neuronal cells exposed to toxins, possibly by stabilizing their cytoskeleton.

Constitutive tyrosine phosphorylation of the inhibitory paired Ig-like receptor PIR-B.

PIR-A and PIR-B are activating and inhibitory Ig-like receptors on murine B lymphocytes, dendritic cells, and myeloid-lineage cells. The inhibitory function of PIR-B is mediated via its cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, whereas PIR-A pairs with the Fc receptor common gamma chain to form an activating receptor complex. In these studies, we observed constitutive tyrosine phosphorylation of PIR-B molecules on macrophages and B lymphocytes, irrespective of the cell activation status. Splenocyte PIR-B molecules were constitutively associated with the SHP-1 protein tyrosine phosphatase and Lyn protein tyrosine kinase. In Lyn-deficient mice, PIR-B tyrosine phosphorylation was greatly reduced. Unexpectedly, tyrosine phosphorylation of PIR-B was not observed in most myeloid and B cell lines but could be induced by ligation of the PIR molecules. Finally, the phosphorylation status of PIR-B was significantly reduced in MHC class I-deficient mice, although not in mice deficient in TAP1 or MHC class II expression. These findings suggest a physiological inhibitory role for PIR-B that is regulated by endogenous MHC class I-like ligands.

Regulation of caspase activation and apoptosis by cellular zinc fluxes and zinc deprivation: A review.

Non-toxic agents that target intracellular signalling pathways in apoptosis may have potential therapeutic use in many diseases. One such agent is the transition metal Zn, a dietary cytoprotectant and anti-oxidant, which stimulates cell proliferation and suppresses apoptosis. Zn is maintained in discrete subcellular pools that are critical for the functional and structural integrity of cells. The present review initially describes the current state of knowledge on the cellular biology of Zn, especially the critical free or loosely bound (labile) pools of Zn, which are thought to regulate apoptosis. We then review the evidence relating Zn to apoptosis, including studies from our laboratory showing potent synergy between intracellular Zn deficiency and the short chain fatty acid butyrate in induction of caspase activation and the downstream events of apoptosis. Our studies have also reported the suppressive effects of micromolar concentrations of Zn on caspase-3 activation in cell-free models. Other key issues that will be discussed include the identification of the putative molecular targets of Zn and the evidence that systemic changes in labile Zn levels are sufficient to alter susceptibility to apoptosis and lead to physiopathological changes in the human body. Finally, we propose that labile Zn may serve as a coordinate regulator of mitosis and apoptosis to regulate tissue growth.

Biochemical nature and cellular distribution of the paired immunoglobulin-like receptors, PIR-A and PIR-B.

PIR-A and PIR-B, paired immunoglobulin-like receptors encoded, respectively, by multiple Pira genes and a single Pirb gene in mice, are relatives of the human natural killer (NK) and Fc receptors. Monoclonal and polyclonal antibodies produced against a recombinant PIR protein identified cell surface glycoproteins of approximately 85 and approximately 120 kD on B cells, granulocytes, and macrophages. A disulfide-linked homodimer associated with the cell surface PIR molecules was identified as the Fc receptor common gamma (FcRgammac) chain. Whereas PIR-B fibroblast transfectants expressed cell surface molecules of approximately 120 kD, PIR-A transfectants expressed the approximately 85-kD molecules exclusively intracellularly; PIR-A and FcRgammac cotransfectants expressed the PIR-A/ FcRgammac complex on their cell surface. Correspondingly, PIR-B was normally expressed on the cell surface of splenocytes from FcRgammac-/- mice whereas PIR-A was not. Cell surface levels of PIR molecules on myeloid and B lineage cells increased with cellular differentiation and activation. Dendritic cells, monocytes/macrophages, and mast cells expressed the PIR molecules in varying levels, but T cells and NK cells did not. These experiments define the coordinate cellular expression of PIR-B, an inhibitory receptor, and PIR-A, an activating receptor; demonstrate the requirement of FcRgammac chain association for cell surface PIR-A expression; and suggest that the level of FcRgammac chain expression could differentially affect the PIR-A/PIR-B equilibrium in different cell lineages.

Hydrophilic-hydrophobic biodegradable polymers: release characteristics of hydrogen-bonded, ring-containing polymer matrices.

Biodegradable hydrogen-bonded, ring-containing polymers were prepared. These included poly(enolketones) by the controlled oxidation of poly(vinyl alcohol), and poly(amide-amines) and poly(amide-enamine-esters) by the reaction of diketene with diamines. These polymers had both hydrophilic and hydrophobic properties and are potentially matrix materials for the controlled release of drugs.

Human biliary beta-glucuronidase: correlation of its activity with deconjugation of bilirubin in the bile.

Total and conjugated bilirubin contents of gall-bladder and hepatic biles before and after 24-h incubation at 37 degrees C and beta-glucuronidase activity of hepatic biles were determined in forty-eight patients divided equally into four groups: no stones or control (C), cholesterol stones (CS), black pigment stones (black PS), and brown pigment stones (brown PS). The percent conjugation of bilirubin is lower in gall-bladder biles and hepatic biles after incubation, particularly in black PS and brown PS, when compared with hepatic biles before incubation. Mean endogenous beta-glucuronidase activities at pH 5.2 were 12.0, 15.5, 44.5 and 147.7 nmol min-1 ml-1 for C, CS, black PS, and Brown PS, respectively, which correlated well with the degree of deconjugation of bilirubin in gall-bladder and hepatic biles and with the rate of deconjugation of hepatic bile incubated at 37 degrees C. Only four biles in brown PS exhibited bacterial enzyme activity. We concluded that though bacterial beta-glucuronidase might be responsible for deconjugation of bilirubin in some patients in brown PS, endogenous biliary beta-glucuronidase could play a key role in the pathogenesis of pigment cholelithiasis.

Human hepatic beta-glucuronidase: an enzyme kinetic study.

beta-Glucuronidase (EC 3.2.1.31) was purified from human liver and its activity was determined by enzyme kinetic method employing phenolphthalein glucuronic acid (PGA) and conjugated bilirubin, primarily bilirubin diglucuronide purified from human bile, as substrates in the absence or presence of D-glucaro-1,4-lactone. The enzyme was capable of acting on both PGA and conjugated bilirubin with Michaelis constants of 0.435 mmol/l at 56 degrees C and 1.02 mmol/l at 37 degrees C, respectively. Both reactions were beta-glucuronidase-specific because both were inhibited by D-glucaro-1,4-lactone in a competitive fashion. Conjugated bilirubin acted as a noncompetitive inhibitor of the enzyme for PGA in a two-substrate system. The study indicates that these two substrates bind at different catalytic sites of the enzyme and, on molar base, conjugated bilirubin had higher affinity for the enzyme and less degree of inhibition by D-glucaro-1,4-lactone than PGA. Whether such catalytic sites are also common for other beta-D-glucuronid ethers and esters remains to be proven.

Determination of urinary beta-glucuronidase activity. Single-point versus enzyme kinetic measuring system.

beta-Glucuronidase activity was determined in dialyzed and undialyzed urine from 50 healthy adult males by single-point and enzyme kinetic measuring systems. The enzyme had a pH optimum of 5.2 and a Michaelis constant of 1.465 +/- 0.206 mmol/l (mean +/- SD) of phenolphthalein glucuronide. Substrate inhibition occurred at a concentration of 0.006 mol/l in one fourth of the urine samples. A significant amount of a competitive inhibitor, D-glucaro-1,4-lactone, was present in one third of the specimens. The activity measured by the single-point determination was always lower than the maximal velocity, particularly in the presence of the competitive inhibitor and substrate inhibition. Such pitfalls could be avoided by the enzyme kinetic method which not only permits the detection of substrate inhibition of the enzyme and the elimination of 2-hour dialysis of urine, but also allows the determination of both the maximal velocity of the enzyme and the quantity of D-glucaro-1,4-lactone in urine.

Characterization and determination of the activity of biliary beta-glucuronidase in rats.

beta-Glucuronidase activity determined in 100 diluted bile samples from 12 rats with bile duct fistula by using phenolphthalein glucuronide as substrate incubated at 56 degrees C and pH 6 was 636 +/- 650 (mean +/- S.D.) modified Sigma units/ml. The enzyme had an optimal pH of 6.0 and was inhibited slightly by cholate by markedly by chenodeoxycholate and deoxycholate. The biliary beta-glucuronidase had, thus, low activity under normal physiologic condition because of the high pH (8.1) and high bile salt content (20 mumoles/ml) of the bile. The enzyme kinetic studies revealed that the direct bilirubin was a competitive inhibitor to phonolphthalein glucuronide for the enzyme. The affinity of the former to the enzyme was 163 times that of the latter. The studies provided a method for measuring the true activity of biliary beta-glucuronidase (Vmax) devoid of interfering factors by measuring the enzyme velocity (v) in the diluted bile with at least five different concentrations of substrate (s). The plotting of (1/v) vs. (1/s) should yield the y intercept or (1/Vmax).