Rapid skin anaesthesia using high velocity lignocaine particles: a prospective placebo controlled trial.

BACKGROUND: Local anaesthetic creams (EMLA and Ametop) are used widely to provide pain free intravenous cannulation. However, they take a minimum of 45 minutes to become effective. AIMS: To evaluate a prototype device, dermal Powderject lidocaine (DPL), that delivers high velocity lignocaine particles into the skin. METHODS: A total of 132 children (aged 4-12 years) were randomised to receive either a sham delivery or a delivery of DPL on the skin at the antecubital fossa, or back of hand. Pain of intravenous cannulation was assessed four minutes later using self reporting behaviours and blinded observation with standard pain assessment tools. The trial was designed to measure both efficacy of skin anaesthesia and potential skin damage with increasing driving pressure of the device (30 or 40 bar), and different lignocaine particle sizes (<38 micrometer or 38-53 micrometer) in a block randomised fashion. RESULTS: A total of 128 patients were evaluable. There was a trend towards improved anaesthesia at higher device pressures at the antecubital fossa with both self reporting and blinded observation. Acceptable analgesia was achieved in 90% of patients for high pressure at both particle sizes compared to 60% and 40% for the sham device using self reporting measures. The observed differences using the blinded observer were similar: 90% v 20% (40 bar and small particles v sham), and 80% v 40% (40 bar and large particles v sham). At the back of hand the differences between active and sham devices were not significant. The device was well tolerated and not associated with pain on deployment. One patient had mild petechiae and oedema after deployment (Draize score of 3). CONCLUSIONS: This prototype device appears to provide significant skin anaesthesia at the antecubital fossa, but not at the back of hand. The device is not painful to use and causes only minor short term skin changes.

6-formylpterin intracellularly generates hydrogen peroxide and restores the impaired bactericidal activity of human neutrophils.

The effects of 6-formylpterin on the impaired bactericidal activity of human neutrophils were examined ex vivo. When neutrophils isolated from fresh blood were incubated with 6-formylpterin, the intracellular production of hydrogen peroxide (H(2)O(2)) occurred. The H(2)O(2) generation by 6-formylpterin in neutrophils occurred in the presence of diphenyleneiodonium (DPI), an inhibitor of NADPH-oxidase. When neutrophils were incubated with DPI, the killing rate of catalase-positive bacteria, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), significantly decreased. This impaired bactericidal activity of the DPI-treated neutrophils was a mimic for chronic granulomatous disease (CGD). However, the killing rate of the DPI-treated neutrophils against E. coli and S. aureus significantly increased when 6-formylpterin was administered. Since 6-formylpterin intracellularly generates H(2)O(2) independent from the NADPH-oxidase, it was considered to improve the impaired bactericidal activity of the DPI-treated neutrophils. The use of 6-formylpterin may serve as an option of therapy for CGD.

Establishment and characterization of a new human myeloma cell line, KYdelta-1, producing the delta/kappa type immunoglobulin.

We describe the establishment and characterization of a new multiple myeloma (MM) cell line, KYdelta-1, which expressed delta/kappa type immunoglobulin (Ig). The patient was a 65-year-old woman with MM, who presented extramedullary dissemination, lymphadenopathy and short survival. The KYdelta-1 cell line was derived from the pleural fluid obtained in the terminal phase of the disease. The cells expressed delta/kappa Ig in the cytoplasm, and CD10, CD29, CD33, CD38, CD44, CD54, and HLA-DR antigens on the cell surface. Chromosomal analysis revealed two independent translocations, t(3;14)(p21;q32) and t(3;11)(p21;q13), which were confirmed by fluorescence in situ hybridization using chromosome painting probes. Reverse transcriptase-mediated polymerase chain reaction (PCR) and Northern blot analyses demonstrated overexpression of the CCND1 gene, suggesting alteration of the BCL1-CCND1 locus. We thus performed long-distance inverse PCR using nested primers for the Calpha constant region of immunoglobulin heavy chain gene (IGH) and obtained a clone that encompassed the 11q13/IGH fusion. Nucleotide sequencing determined that the fusion occurred at the Salpha2 switch region and at the centromeric side of the major translocation cluster of BCL1. The other IGH allele consisted of a VDJ complex that was adjacent to the Cdelta constant gene, indicating that a class switch-like mechanism from the C(mu) to Cdelta was involved in the production of the Ig delta heavy chain. Point mutations within the P53 and N-RAS genes were presumably related to the rapidly progressive disease in this particular MM patient.

6-formylpterin, a xanthine oxidase inhibitor, intracellularly generates reactive oxygen species involved in apoptosis and cell proliferation.

The chemical property of 6-formylpterin and its biological functions were examined. Polarographic studies revealed that 6-formylpterin reacted with NAD(P)H and consumed oxygen. In contrast, other conjugated pterins, such as biopterin and neopterin, showed no consumption of oxygen. The production analysis using high-performance liquid chromatography documented that 6-formylpterin catalyzes the conversion from NADH to NAD. Electroparamagnetic resonance spin trapping experiments demonstrated that this reaction is accompanied with the generation of reactive oxygen species (ROS), superoxide anion and hydrogen peroxide. When 6-formylpterin was administered to HL-60 cells, intracellular ROS generation was observed and apoptosis was induced. In contrast, other conjugated pterins induced neither intracellular ROS generation nor apoptosis in HL-60 cells. The intracellular ROS generation by 6-formylpterin was observed in other cells, such as PanC-1 cells and Jurkat cells. 6-formylpterin suppressed cell proliferation in PanC-1 cells and inhibited Fas-mediated apoptosis in Jurkat cells. These findings indicate that, among conjugated pterins, 6-formylpterin has the unique property to transfer electron from NAD(P)H to oxygen and that the property brings about intracellular ROS generation, which exerts various biological functions such as induction of apoptosis, suppression of cell proliferation, and inhibition of Fas-mediated apoptosis.

Rapid and prominent up-regulation of high-affinity receptor for immunoglobulin G (Fc gamma RI) by cross-linking of beta 2 integrins on polymorphonuclear leukocytes.

Receptors for the Fc region (FcR) of immunoglobulin (Ig)G play essential roles in effector functions of polymorphonuclear leukocytes (PMNs) including the antibody-mediated clearance of microbes. In contrast to the constitutive expression of the low-affinity receptors for IgG (Fc gamma RII [CD32] and Fc gamma RIII [CD16]), the high-affinity receptor Fc gamma RI (CD64) is barely detectable on unactivated PMNs. CD64 expression is induced in a slow kinetic manner by interferon (IFN)-gamma and granulocyte colony-stimulating factor (G-CSF) after 12 to 24 hours of exposure to these agents. We found that the cross-linking of CD11b as well as of CD18 induced comparable rapid increases in CD64 expression on the surface of PMNs, occurring within 15 minutes of exposure. Cross-linking of neither CD11a nor CD11c induced CD64 expression. In contrast to slow induction by IFN-gamma and G-CSF, the integrin-induced rapid CD64 expression did not require RNA synthesis. Genistein, herbimycin A, and 1,2-bis(o-aminophenoxy)ethan-N,N-N',N'-tetraacetic acid blocked the immediate expression of CD64 in a dose-dependent manner, suggesting that the signal is mediated through calcium mobilization and protein tyrosine kinase(s). Such rapid modulation of the high-affinity Fc gamma RI receptor by integrin cross-linking may reflect the requirement for rapid up-regulation of PMN effector functions, after interaction with endothelial cells, platelets or bacteria.

Caspases mediate tumor necrosis factor-alpha-induced neutrophil apoptosis and downregulation of reactive oxygen production.

Tumor necrosis factor-alpha (TNF-alpha) exerts two separate effects on neutrophils, stimulating effector functions while simultaneously inducing apoptosis. We examined here the involvement of caspases in neutrophil apoptosis and the effect of TNF-alpha-induced apoptosis on reactive oxygen production. Immunoblotting and affinity labeling showed activation of caspase-8, caspase-3, and a caspase with a large subunit of 18 kD (T18) in TNF-alpha-treated neutrophils. Active caspase-6 and -7 were not detectable in this cell type. Caspase-8 activated caspase-3 and T18 in neutrophil cytoplasmic extracts. zVAD-fmk blocked neutrophil apoptosis, in parallel with the inhibition of caspase activation. TNF-alpha-induced caspase activation was accompanied by a decrease in the ability of neutrophils to release superoxide anion. Conversely, TNF-alpha treatment in the presence of zVAD-fmk caused a prolonged augmentation of superoxide release. Granulocyte-macrophage colony-stimulating factor inhibited TNF-alpha-induced caspase activation and apoptosis, while reversing the diminution in superoxide release. These observations not only suggest that a caspase cascade mediates apoptotic events and downregulates oxygen radical production in TNF-alpha-treated neutrophils, but also raise the possibility that suppression of caspase activation with enhanced proinflammatory actions of TNF-alpha may underlie the pathogenesis of inflammatory diseases.

The expression of co-stimulatory molecules and their relationship to the prognosis of human acute myeloid leukaemia: poor prognosis of B7-2-positive leukaemia.

We examined the expression of co-stimulatory molecules on leukaemic cells of 52 adult patients with acute myeloid leukaemia (AML) (34 men and 18 women) and analysed the relationship between these expressions and the patient's prognosis. B7-1 was not expressed in any of the 23 patients investigated, whereas B7-2 was expressed in 26/52 patients (50.0%). B7-2 was expressed in all AML patients with monocytic morphology (M4 or M5) and in 16/42 cases without monocytic morphology. CD54 was expressed in 28/ 37 patients examined (75.7%), and CD58 was expressed in all of the AML patients except one (M 7). The overall survival of the 26 B7-2-positive leukaemia patients (1-24 months, median survival 11.5 months) was significantly shorter than that of the 26 B7-2-negative leukaemia patients (1-71+ months, median 35.1 months) (P=0.0080). In addition, the B7-2-positive patients exhibited significantly shorter disease-free survival periods compared to the B7-2-negative patients (P=0.021). There was no significant difference in age, sex, haematological data and complete remission rate between the B7-2-positive and B7-2-negative patients. Our results indicated that B7-2 is one of the most crucial factors in the prognosis of adult acute leukaemia and can be expected to have an important role in tumour immunity.

[Successful treatment with donor leukocyte transfusion followed by interferon-alpha in a patient with relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation].

A 44-year-old man with CML in chronic phase was admitted for BMT from an HLA-identical sibling. Ph positive cells were undetectable at 3 and 7 months after BMT but became detectable by cytogenetic analysis of bone marrow aspirates at 12 months after BMT. He was treated with IFN-alpha (6 million units/day, 3 times a week) without apparent effect. Donor leukocyte transfusion (DLT) was performed four times between 20 months and 23 months after BMT, transfusing 3.4 x 10(8) mononuclear cells/kg. However, leukocytosis appeared and the NAP score declined at 25 months after BMT. FISH analysis revealed an increase in bcr-abl positive cells. IFN-alpha was restarted using the same schedule at 26 months after BMT. Three months after restarting IFN-alpha, the leukocyte count fell to the normal range, NAP score increased to a normal level, and bcr-abl positive cells decreased markedly. He has remained in hematological and cytogenetic remission for 20 months, and bcr-abl chimeric mRNA remained undetectable by PCR. These results suggest that CML which does not respond to DLT may be cured by subsequent IFN-alpha therapy, possibly by inducing anti-leukemia immune responses.

Down-regulation of CXCR2 expression on human polymorphonuclear leukocytes by TNF-alpha.

TNF-alpha is implicated in the initiation of cytokine cascades in various inflammatory settings. To assess the interactions of multiple cytokines at the level of inflammatory effector cells, we examined the effects of TNF-alpha on the expression of two IL-8Rs (CXCR1 and CXCR2) on polymorphonuclear leukocytes (PMNs). TNF-alpha decreased the surface expression of CXCR2 in a dose- and time-dependent manner. In contrast, CXCR1 expression was not affected by TNF-alpha. The release of CXCR2 into the supernatant of TNF-alpha-treated PMNs was detected by immunoblotting and immuno-slot-blot analyses, suggesting that the down-regulation of CXCR2 was caused mainly by shedding from the cell surface. The CXCR2 down-regulation was inhibited by PMSF and aprotinin, supporting the hypothesis that the shedding was mediated by serine protease(s). The intracellular Ca2+ mobilization and chemotaxis in response to IL-8 were suppressed by the pretreatment of PMNs with TNF-alpha, indicating that the decrease in CXCR2 was reflected in the decreased functional responses to IL-8. In contrast, the O2- release, which is mediated by CXCR1, was not suppressed by TNF-alpha. The treatment of whole blood with TNF-alpha also caused a significant reduction in CXCR2 and markedly suppressed intracellular Ca2+ mobilization and chemotaxis in response to IL-8, while enhancing the O2- release. These findings suggest that TNF-alpha down-regulates CXCR2 expression on PMNs and modulates IL-8-induced biologic responses, leading to the intravascular retention of PMNs with an enhanced production of reactive oxygen metabolites.

Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis.

Two novel synthetic tetrapeptides, VEID-CHO and DMQD-CHO, could selectively inhibit caspase-6 and caspase-3, respectively. We used these inhibitors to dissect the pathway of caspase activation in Fas-stimulated Jurkat cells and identify the roles of each active caspase in apoptotic processes. Affinity labeling techniques revealed a branched protease cascade in which caspase-8 activates caspase-3 and -7, and caspase-3, in turn, activates caspase-6. Both caspase-6 and -3 have major roles in nuclear apoptosis. Caspase-6 cleaves nuclear mitotic apparatus protein (NuMA) and mediates the shrinkage and fragmentation of nuclei. Caspase-3 cleaves NuMA at sites distinct from caspase-6, and mediates DNA fragmentation and chromatin condensation. It is also involved in extranuclear apoptotic events: cleavage of PAK2, formation of apoptotic bodies, and exposure of phosphatidylserine on the cell surface. In contrast, a caspase(s) distinct from caspase-3 or -6 mediates the disruption of mitochondrial membrane potential (permeability transition) and the shrinkage of cytoplasm. These findings demonstrate that caspases are organized in a protease cascade, and that each activated caspase plays a distinct role(s) in the execution of Fas-induced cell death.

Performance of a digital video camcorder for the Autonomous Biological System experiment onboard Space Station Mir.

A video imaging and recording system was utilized in the Autonomous Biological System experiment onboard the space station Mir. Video image of the mini-ecological system was successfully recorded. The whole system was retrieved to the ground after its operation in orbit for four months. Performance of the video system is summarized here together with technical problems encountered. Defects of pixel had been developed in the imaging device. Cause of these defects could be attributed to its exposure against space radiation. Auto white balance was another function of the camcorder that was deviated from normal range of its performance once in orbit but recovered to normal after a while. Possible use of imaging devices for dosimetry is proposed to record space radiation environment at the site close to the experiment.

Interleukin-12 gene expression in human monocyte-derived macrophages stimulated with Mycobacterium bovis BCG: cytokine regulation and effect of NK cells.

Macrophage-derived interleukin-12 (IL-12) is essential for the activation of a protective immune response against intracellular pathogens. In this study, we examined the regulation of IL-12 mRNA expression by monocyte-derived macrophages (MDM) in response to Mycobacterium bovis BCG stimulation. A reverse transcription-PCR assay detected p40 mRNA of IL-12 at 3 h and showed a peak at 6 to 12 h with a subsequent decline. Semiquantitation of mRNA levels by competitive PCR revealed that pretreatment with gamma interferon (IFN-gamma) amplified the expression approximately 100-fold, while pretreatment with tumor necrosis factor alpha (TNF-alpha) or granulocyte-macrophage colony-stimulating factor augmented this expression about 10-fold. In contrast, pretreatment with IL-10 and IL-4 inhibited IL-12 mRNA expression. These results were further confirmed by measuring the p70 bioactive protein level in each conditioned medium by an enzyme-linked immunosorbent assay. Since IL-12 mRNA expression was weak without cytokine pretreatment and IFN-gamma strongly augmented production, we speculated that IFN-gamma might have a role in BCG stimulation of IL-12 mRNA expression. Unexpectedly, the addition of three different kinds of anti-IFN-gamma antibodies and anti-IFN-gamma receptor antibody and the coaddition of anti-TNF-alpha antibody with anti-IFN-gamma receptor antibody all failed to inhibit IL-12 mRNA expression. However, the MiniMACS method used to remove NK cells from a mononuclear cell suspension inhibited the expression of p40 mRNA but not the expression of mRNA of TNF-alpha or IL-1beta. We concluded that the coexistence of NK cells was essential for the induction of IL-12 in MDM stimulated with BCG rather than through the secretion of IFN-gamma.

Leukotriene B4-activated human endothelial cells promote transendothelial neutrophil migration.

We explored the effect of leukotriene B4 (LTB4) on endothelial cells in LTB4-induced transendothelial migration (TEM) of neutrophils as an in vitro model of neutrophil extravasation. Chemotactic response of human neutrophils to LTB4 was significantly lower than that in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP), whereas the extent of TEM in response to LTB4 was significantly higher than that to fMLP. The study on random migration induced by LTB4 and fMLP also showed similar results, which indicated that LTB4 might affect the human umbilical cord vein endothelial cell (HUVEC) barrier. Neutrophil TEM was induced by pretreatment of HUVEC monolayer with LTB4 but not with fMLP. Treatment of endothelial cells by ONO-4057, a LTB4 receptor antagonist, abolished the effect of LTB4 almost completely whereas neutrophils treated with ONO-4057 could transmigrate through HUVEC treated with LTB4. These findings indicated that LTB4 could induce neutrophil TEM by acting on HUVEC.

Demonstration of functionally distinct human polymorphonuclear leukocyte fractions by simultaneous measurement of phagocytosis and oxygen radical generation.

Phagocytosis and oxygen radical generation by human polymorphonuclear leukocytes (PMNLs) were studied by a two-color flow cytometric analysis, where the red fluorescent product(s) of hydroethidine was used as an indicator of intracellular generation of oxygen radicals and opsonized zymosan (OZ) as an indicator of phagocytosis. Unstimulated cells formed a single population of cells without any significant fluorescence. PMNLs stimulated by OZ exhibited a high red fluorescence. Most PMNLs phagocytosed OZ and generated oxygen radicals when stimulated by zymosan particles opsonized with human AB serum at concentrations of more than 10%, whereas three distinct subpopulations (designated as R1, R2 and R3) appeared when stimulated by zymosan particles opsonized with 3% serum; R1 cells enhanced neither green nor red fluorescence, R2 cells enhanced green fluorescence but not red fluorescence, and R3 cells enhanced both green and red fluorescence. The R2 cells completely disappeared by the addition of Trypan blue. Most of the R3 cells disappeared by the addition of cytochalasin B. These findings on the three fractions were confirmed by the observation under fluorescence microscopy of cells in each fraction obtained by sorting. In conclusion, PMNLs could be separated into three functionally distinct subpopulations when stimulated by zymosan particles opsonized with a suboptimal concentration of serum.

Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8.

The activation of multiple interleukin-1beta converting enzyme-related proteases (caspases) in apoptotic mammalian cells raises questions as to whether the multiple active caspases have distinct roles in apoptotic execution as well as how these proteases are organized in apoptotic signaling pathways. Here we used an affinity-labeling agent, YV(bio)KD-aomk, to investigate the caspases activated during apoptotic cell death. YV(bio)KD-aomk identified six distinct polypeptides corresponding to active caspases in Fas-stimulated Jurkat T cells. On staurosporine treatment, four polypeptides were detected. Competition experiments showed that the labeled caspases have distinct substrate preferences. Stepwise appearance of the labeled caspases in each cell death event was consistent with the view that the activated caspases are organized into protease cascades. Moreover, we found that stepwise activation of caspases similar to that induced by Fas ligation is triggered by exposing non-apoptotic Jurkat cell extracts to caspase-8 (MACH/FLICE/Mch5). Conversely, CrmA protein, a viral suppressor of Fas-induced apoptosis, inhibited the protease activity of caspase-8. Overall, these findings provide evidence that caspase-8, a CrmA-sensitive protease, is responsible for initiating the stepwise activation of multiple caspases in Fas-stimulated cells.

Random migration of polymorphonuclear leukocytes induced by GM-CSF involving a signal transduction pathway different from that of fMLP.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced random migration of human polymorphonuclear leukocytes (PMNs) but not chemotaxis. Chemoattractants such as N-formyl-methionyl-leucyl-phenylalanine (fMLP), leukotriene B4 (LTB4), and interleukin-8 (IL-8) induced both random migration and chemotaxis. Other inflammatory cytokines, including granulocyte colony-stimulating factor (G-CSF), interleukin 1alpha (IL-1alpha), and tumor necrosis factor alpha (TNF-alpha), did not induce either movement. One-minute exposure of PMNs to GM-CSF was sufficient for the induction of random migration, whereas fMLP-induced random migration required continued presence of fMLP. Inhibitors of phosphatidylinositol 3-kinase (PI3-K), protein kinase C (PKC), and protein tyrosine kinase (PTK) had no effect on random migration induced by GM-CSF, whereas fMLP-induced movements were partially inhibited by PTK inhibitors but not by inhibitors of PI3-K inhibitors nor PKC inhibitors. Myosin light chain kinase inhibitors inhibited movements of PMNs induced by both GM-CSF and fMLP. These findings also imply that some aspects of the signal transduction pathway of GM-CSF leading to random migration is different from that of fMLP. Our findings suggest that cell movements are controlled through diverse signal transduction systems.