bluuebunny/crossref_metadata_2025 huggingface dataset:
Inducible expression of BCR/ABL using human CD34 regulatory elements results in a megakaryocytic myeloproliferative syndrome
Authors: Huettner Claudia S. , Koschmieder Steffen , Iwasaki Hiromi , Iwasaki-Arai Junko , Radomska Hanna S. , Akashi Koichi , Tenen Daniel G.
Published: July 2003

The BCR/ABL fusion protein is found in more than 90% of patients with chronic myeloid leukemia (CML) as well as in a subset of patients with acute B-cell leukemia. We have previously described a transgenic model for an inducible and reversible acute B-cell leukemia caused by p210 BCR/ABL. Here, we describe a new model of an inducible BCR/ABL disease by directing the expression of the oncogene to megakaryocytic progenitor cells within the murine bone marrow using the tetracycline-responsive expression system under the control of human CD34 regulatory elements. The predominant feature was the development of a chronic thrombocytosis. The condition progressed with the development of splenomegaly accompanied by lymphadenopathy in some mice. Affected animals demonstrated a dramatic increase in the number of megakaryocytes in the bone marrow and the spleen. Immunohistochemistry demonstrated that the reporter gene was expressed in hematopoietic stem cells (HSCs), common myeloid progenitor (CMP) cells, as well as in megakaryocytic/erythroid progenitor cells (MEPs). Although these mice did not display the increase in granulopoiesis commonly found in chronic myeloid leukemia (CML), the phenotype closely resembles a myeloproliferative disorder affecting the megakaryocytic lineage observed in some patients with the BCR/ABL P210 translocation.
Signaling but not trafficking function of HIV-1 protein Nef is essential for Nef-induced defects in human intrathymic T-cell development
Authors: Stove Veronique , Naessens Evelien , Stove Christophe , Swigut Tomek , Plum Jean , Verhasselt Bruno
Published: July 2003

The HIV-1 gene nef is important for progression toward AIDS and cellular depletion of the infected thymus. Expression of the Nef protein alone impairs human thymopoiesis. Here, we performed a structure-function analysis of the Nef protein by comparing the effect on T-cell development of different nef alleles, either wild type or defective for selected functions, expressed by human thymocytes. We show that Nef-mediated impaired thymopoiesis is not due to altered surface marker trafficking, nor dependent on oligomerization of Nef. By contrast, mutations in the myristoylation site and in signaling sites of Nef, ie, sites important for interaction with phosphofurin acidic cluster sorting protein-1 (PACS-1), Src homology domain 3 (SH3) domains, and p21-activated kinase 2 (PAK2), were found to be critical for its effect on T-cell development. These results point to sites in Nef to target therapeutically for restoration of thymopoiesis in HIV infection.
Control of thrombus embolization and fibronectin internalization by integrin αIIbβ3 engagement of the fibrinogen γ chain
Authors: Ni Heyu , Papalia Jessie M. , Degen Jay L. , Wagner Denisa D.
Published: July 2003

Fibrin(ogen) deficiency (Fg-/-) was shown previously to be compatible with rapid thrombus growth within injured arterioles, but platelet fibronectin content was increased and newly formed thrombi were unstable. To further define the role of fibrin(ogen) in thrombus formation and stabilization, platelet biology was examined in mice expressing a form of fibrinogen that clots normally but lacks the γ chain C-terminal binding site for αIIbβ3 (FgγΔ5). Thrombus growth within the arterioles of FgγΔ5 mice appeared faster than in wild-type mice despite a far greater emboli formation. Unlike Fg-/- mice, the emboli were relatively small and released from the top of thrombi, rather than by fracture at the vessel wall. The fibronectin content in FgγΔ5 platelets was also dramatically increased through a β3 integrin-dependent mechanism. The following has been concluded: (1) Fibrin formation contributes to, but is not sufficient for, the stabilization of arterial thrombi. Platelet receptor engagement of the C-terminal of the Fg γ chain contributes to the stable incorporation of platelets into thrombi. (2) Alternative ligands to fibrinogen can support efficient thrombus growth. (3) Fibrinogen is internalized through αIIbβ3 engagement of the fibrinogen γ chain element, and this interaction secondarily controls the fibronectin content of platelets. (Blood. 2003;102: 3609-3614)
Thrombopoietin stimulates Hoxb4 expression: an explanation for the favorable effects of TPO on hematopoietic stem cells
Authors: Kirito Keita , Fox Norma , Kaushansky Kenneth
Published: July 2003

Thrombopoietin (TPO), the primary regulator of platelet production, also plays an important role in hematopoietic stem cell (HSC) biology. In previous studies we demonstrated that the self-renewal and expansion of HSCs is 10 to 20 times less robust in tpo–/– mice than in controls. To explore the molecular basis of this effect, we postulated that Hoxb4 might mediate at least part of the TPO effect on these cells. We first analyzed the effects of TPO on Hoxb4 expression in primitive hematopoietic cell lines; TPO increased expression of the gene 2- to 3-fold in EML and UT-7/TPO cells. We also compared Hoxb4 levels in a candidate HSC population derived from tpo–/– and control mice; Hoxb4 expression was 2- to 5-fold lower in null HSCs. Of the numerous signal transduction molecules induced by TPO, we found that p38 mitogen-activated protein kinase (MAPK) was responsible for the TPO-induced Hoxb4 elevation. We also demonstrated that upstream stimulating factor 1 (USF-1), a transcription factor previously shown to regulate Hoxb4 expression, is also induced by TPO in a p38-dependent manner. Together, these data provide a molecular pathway by which a growth factor can modulate a transcription factor and thereby help direct a critical developmental process.
Inhibition of protein geranylgeranylation induces apoptosis in myeloma plasma cells by reducing Mcl-1 protein levels
Authors: van de Donk Niels W. C. J. , Kamphuis Marloes M. J. , van Kessel Berris , Lokhorst Henk M. , Bloem Andries C.
Published: July 2003

HMG-CoA reductase is the rate-limiting enzyme of the mevalonate pathway leading to the formation of cholesterol and isoprenoids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). The inhibition of HMG-CoA reductase by lovastatin induced apoptosis in plasma cell lines and tumor cells from patients with multiple myeloma. Here we show that cotreatment with mevalonate or geranylgeranyl moieties, but not farnesyl groups, rescued myeloma cells from lovastatin-induced apoptosis. In addition, the inhibition of geranylgeranylation by specific inhibition of geranylgeranyl transferase I (GGTase I) induced the apoptosis of myeloma cells. Apoptosis triggered by the inhibition of geranylgeranylation was associated with reduction of Mcl-1 protein expression, collapse of the mitochondrial transmembrane potential, expression of the mitochondrial membrane protein 7A6, cytochrome c release from mitochondria into the cytosol, and stimulation of caspase-3 activity. These results imply that protein geranylgeranylation is critical for regulating myeloma tumor cell survival, possibly through regulating Mcl-1 expression. Our results show that pharmacologic agents such as lovastatin or GGTase inhibitors may be useful in the treatment of multiple myeloma.
Fanconi anemia type C and p53 cooperate in apoptosis and tumorigenesis
Authors: Freie Brian , Li Xiaxin , Ciccone Samantha L. M. , Nawa Kathy , Cooper Scott , Vogelweid Catherine , Schantz Laurel , Haneline Laura S. , Orazi Attilio , Broxmeyer Hal E. , Lee Suk-Hee , Clapp D. Wade
Published: July 2003

Fanconi anemia (FA) is a recessive genomic instability syndrome characterized by developmental defects, progressive bone marrow failure, and cancer. FA is genetically heterogeneous, however; the proteins encoded by different FA loci interact functionally with each other and with the BRCA1, BRCA2, and ATM gene products. Although patients with FA are highly predisposed to the development of myeloid leukemia and solid tumors, the alterations in biochemical pathways responsible for the progression of tumorigenesis in these patients remain unknown. FA cells are hypersensitive to a range of genotoxic and cellular stresses that activate signaling pathways mediating apoptosis. Here we show that ionizing radiation (IR) induces modestly elevated levels of p53 in cells from FA type C (Fancc) mutant mice and that inactivation of Trp53 rescues tumor necrosis factor α-induced apoptosis in myeloid cells from Fancc-/- mice. Further, whereas Fancc-/- mice failed to form hematopoietic or solid malignancies, mice mutant at both Fancc and Trp53 developed tumors more rapidly than mice mutant at Trp53 alone. This shortened latency was associated with the appearance of tumor types that are found in patients with FA but not in mice mutant at Trp53 only. Collectively, these data demonstrate that p53 and Fancc interact functionally to regulate apoptosis and tumorigenesis in Fancc-deficient cells. (Blood. 2003;102:4146-4152)
Bcl2 retards G1/S cell cycle transition by regulating intracellular ROS
Authors: Deng Xingming , Gao Fengqin , May W. Stratford
Published: July 2003

Bcl2's antiapoptotic function is regulated by phosphorylation. Bcl2 also regulates cell cycle progression, but the molecular mechanism is unclear. Bcl2 is functionally expressed in mitochondria where it can act as an antioxidant that may regulate intracellular reactive oxygen species (ROS). Since ROS have been reported to act as second messengers in cell signaling, we tested whether Bcl2 phosphorylation regulates ROS and cell cycle progression. G1 → S transition and ROS levels were measured in cells expressing either the gain of function phosphomimetic Bcl2 mutants S70E and T69E/S70E/S87E (EEE) or the nonphosphorylatable and survival-deficient mutants S70A and T69A/S70A/S87A (AAA). Expression of S70E and EEE but not the A-containing Bcl2 mutants retards G1 → S transition by 35% to 50% and significantly slows cell growth in association with reduced levels of intracellular ROS. In addition to expression of the phosphomimetic Bcl2 mutants, either interleukin-3 withdrawal or treatment of cells with the antioxidant pyrrolidine dithiocarbamate (PDTC) also reduces intracellular ROS levels in association with up-regulation of p27 and accumulation of cells in G0/G1. Retardation of G1 → S transition can be overridden by directly adding H2O2 to the cells in a mechanism that involves down-regulation of p27 and activation of Cdk2. Thus, Bcl2 may regulate G1 → S transition by a novel signaling mechanism that couples regulation of intracellular ROS with p27 and Cdk2. Furthermore, phosphorylation of Bcl2 may functionally link its antiapoptotic, cell cycle retardation, and antioxidant properties.
Primitive erythropoiesis is regulated by Smad-dependent signaling in postgastrulation mesoderm
Authors: Schmerer Matthew , Evans Todd
Published: July 2003

The bone morphogenetic proteins (BMPs) are required for the development of ventral mesoderm, which contributes to the ventral blood island and primitive (yolk sac stage) hematopoiesis. Primitive erythropoiesis is defective when BMP signaling is blocked during gastrulation of Xenopus embryos. This phenotype might be attributed to changes in mesoderm patterning leading indirectly to altered erythropoiesis. We developed an inducible system in order to block BMP signaling in a controlled fashion at later time points in development. For this purpose, an inhibitory Smad, xSmad6, was fused to the estrogen receptor ligand-binding domain. We show that ER-xSmad6 is inactive when expressed in developing embryos, but its activity is induced by estradiol. When induced early in development, ER-xSmad6 causes a dorsalized phenotype, equivalent to overexpression of native xSmad6. When ER-xSmad6 is induced after gastrulation, there is a specific defect in primitive erythropoiesis without any apparent effect on axial patterning. Our results identify an embryonic signal that is Smad-dependent, is required for maintaining expression of GATA-1, and functions within mesoderm and not the overlying ectoderm. Thus, BMP signaling is necessary both during mesoderm patterning and also following early specification events for proper regulation of the primitive erythroid lineage.
Inhibition of HIV-1 infection by a CCR5-binding cyclophilin from Toxoplasma gondii
Authors: Golding Hana , Aliberti Julio , King Lisa R. , Manischewitz Jody , Andersen John , Valenzuela Jesus , Landau Nathaniel R. , Sher Alan
Published: July 2003

The activation of murine dendritic cells by Toxoplasma gondii has recently been shown to depend on a parasite protein that signals through the chemokine receptor CCR5. Here we demonstrate that this molecule, cyclophilin-18 (C-18), is an inhibitor of HIV-1 cell fusion and infection with cell-free virus. T gondii C-18 efficiently blocked syncytium formation between human T cells and effector cells expressing R5 but not X4 envelopes. Neither human nor Plasmodium falciparum cyclophilins possess such inhibitory activity. Importantly, C-18 protected peripheral blood leukocytes from infection with multiple HIV-1 R5 primary isolates from several clades. C-18 bound directly to human CCR5, and this interaction was partially competed by the β-chemokine macrophage inflammatory protein 1β (MIP-1β) and by HIV-1 R5 gp120. In contrast to several other antagonists of HIV coreceptor function, C-18 mediated inhibition did not induce β-chemokines or cause CCR5 downmodulation, suggesting direct blocking of envelope binding to the receptor. These data support the further development of C-18 derivatives as HIV-1 inhibitors for preventing HIV-1 transmission and for postexposure prophylaxis.
The murine platelet and plasma factor V pools are biosynthetically distinct and sufficient for minimal hemostasis
Authors: Sun Hongmin , Yang Tony L. , Yang Angela , Wang Xixi , Ginsburg David
Published: July 2003

Coagulation factor V (FV) is a central regulator of the coagulation cascade. Circulating FV is found in plasma and within platelet α granules. The specific functions of these distinct FV pools are uncertain. We now report the generation of transgenic mice with FV gene expression restricted to either the liver or megakaryocyte/platelet lineage using bacterial artificial chromosome (BAC) constructs. Six of 6 independent albumin BAC transgenes rescue the neonatal lethal hemorrhage of FV deficiency. Rescued mice all exhibit liver-specific Fv expression at levels ranging from 6% to 46% of the endogenous Fv gene, with no detectable FV activity within the platelet pool. One of the 3 Pf4 BAC transgenes available for analysis also rescues the lethal FV null phenotype, with FV activity restricted to only the platelet pool (approximately 3% of the wild-type FV level). FV-null mice rescued by either the albumin or Pf4 BAC exhibit nearly normal tail bleeding times. These results demonstrate that Fv expression in either the platelet or plasma FV pool is sufficient for basal hemostasis. In addition, these findings indicate that the murine platelet and plasma FV pools are biosynthetically distinct, in contrast to a previous report demonstrating a plasma origin for platelet FV in humans.
Identification of in vitro growth conditions for c-Kit–negative hematopoietic stem cells
Authors: Klarmann Kim , Ortiz Mariaestela , Davies Meghan , Keller Jonathan R.
Published: July 2003

Our laboratory recently identified a quiescent class of pluripotent hematopoietic stem cells (PHSCs) that are lineage negative (Linneg), lack c-Kit, and are able to give rise to c-Kit–positive (c-Kitpos) PHSCs in vivo. This population fails to proliferate in vitro but has delayed reconstituting activity in vivo. In this study, we purified these cells to enrich for the PHSCs and we identified in vitro conditions capable of supporting their maturation. The c-Kit–negative (c-Kitneg) cells exhibited differential expression of Sca-1, CD34, CD43, CD45, and Thy 1.2. We purified the cells based on Sca-1, as it is expressed on active PHSCs. We detected pre–colony-forming unit spleen (pre–CFU-s) activity in both the Sca-1neg and Sca-1pos populations, indicating the presence of primitive PHSCs in both populations. However, our in vitro studies suggest that the Sca-1pos population is enriched for PHSCs. The in vitro systems that support the growth of these dormant cells include a modified long-term marrow culture and various stromal cell lines. In modified long-term bone marrow cultures, c-Kitneg cells gave rise to c-Kitpos PHSCs, with long-term reconstitution activity in vivo. Thus we have established an in vitro system to examine PHSC maturation that will allow us to study the mediators of the c-Kitneg to c-Kitpos transition.
Ifosfamide impairs the allostimulatory capacity of human dendritic cells by intracellular glutathione depletion
Authors: Kuppner Maria C. , Scharner Anabel , Milani Valeria , von Hesler Christoph , Tschöp Katharina E. , Heinz Oksana , Issels Rolf D.
Published: July 2003

Ifosfamide, a clinically potent chemotherapeutic agent, causes the depletion of intracellular glutathione (GSH) levels in various cell types. GSH is the major intracellular reductant against oxidative stress. 4-Hydroxyifosfamide (4-OH-IF), the activated form of ifosfamide, depletes GSH levels in T cells and natural killer (NK) cells; this is accompanied by a decrease in T-cell and NK-cell function. Here we demonstrate for the first time that human monocyte-derived dendritic cells (DCs) express higher constitutive levels of GSH and are less sensitive to 4-OH-IF-induced GSH depletion than T cells and NK cells. Treatment of DCs with 4-OH-IF significantly reduced their ability to stimulate allogeneic T-cell proliferation and interferon-γ (IFN-γ) production. Ifosfamide also decreased DC interleukin-12p70 (IL-12p70) production after stimulation with lipopolysaccharide (LPS) and IFN-γ. The decrease in allostimulatory capacity and in IFN-γ and IL-12 production correlated with a decrease in intracellular GSH in the DCs. The responses could be restored by reconstituting DC GSH levels with glutathione monoethyl ester (GSH-OEt). 4-OH-IF had no inhibitory effect on the ability of DCs to present exogenously added tyrosinase peptide to tyrosinase-specific cytotoxic T lymphocytes (CTLs). These studies suggest that in cancer patients treated with ifosfamide, protection strategies based on glutathione reconstitution may enhance DC function. (Blood. 2003;102: 3668-3674)
Hsp27 inhibits release of mitochondrial protein Smac in multiple myeloma cells and confers dexamethasone resistance
Authors: Chauhan Dharminder , Li Guilan , Hideshima Teru , Podar Klaus , Mitsiades Constantine , Mitsiades Nicholas , Catley Laurence , Tai Yu Tzu , Hayashi Toshiaki , Shringarpure Reshma , Burger Renate , Munshi Nikhil , Ohtake Yasuyuki , Saxena Satya , Anderson Kenneth C.
Published: July 2003

Smac, second mitochondria-derived activator of caspases, promotes apoptosis via activation of caspases. Heat shock protein 27 (Hsp27) negatively regulates another mitochondrial protein, cytochrome c, during apoptosis; however, the role of Hsp27 in modulating Smac release is unknown. Here we show that Hsp27 is overexpressed in both dexamethasone (Dex)-resistant multiple myeloma (MM) cell lines (MM.1R, U266, RPMI-8226) and primary patient cells. Blocking Hsp27 by an antisense (AS) strategy restores the apoptotic response to Dex in Dex-resistant MM cells by triggering the release of mitochondrial protein Smac, followed by activation of caspase-9 and caspase-3. Moreover, AS-Hsp27 overcomes interleukin-6 (IL-6)-mediated protection against Dex-induced apoptosis. These data demonstrate that Hsp27 inhibits the release of Smac, and thereby confers Dex resistance in MM cells.
Activation-induced cytidine deaminase in chronic lymphocytic leukemia B cells: expression as multiple forms in a dynamic, variably sized fraction of the clone
Authors: Albesiano Emilia , Messmer Bradley T. , Damle Rajendra N. , Allen Steven L. , Rai Kanti R. , Chiorazzi Nicholas
Published: July 2003

The degree of somatic mutation of immunoglobulin variable (Ig V) region genes is an important prognostic indicator of clinical course and outcome in B-cell chronic lymphocytic leukemia (B-CLL), although the reason for this association remains unclear. Furthermore, some B-CLL cells continue to acquire Ig V gene mutations after the transforming event. Because activation-induced cytidine deaminase (AID) is an essential component of the canonical somatic hypermutation process in healthy B cells, its expression in B-CLL is potentially relevant to the disease. We detected full-length AID transcripts and 3 splice variants by conventional reverse transcription polymerase chain reaction (RT-PCR) in approximately 40% of the cases examined. More sensitive real-time quantitative PCR detected AID transcripts in virtually all B-CLL samples tested, although the range of transcript levels was very large between different cases and varied within individual cases over time. Limiting dilution assays revealed that AID expression was restricted to a small fraction of the leukemic cells in the blood. However, this small fraction is not unique in its ability to express AID, because in vitro stimulation of B-CLL cells with appropriate stimuli significantly increased the fraction of AID-expressing cells. These data suggest that AID-mediated DNA alterations may occur in a variably sized, minor subset of B-CLL cells at any given time.
Risks and outcomes of idiopathic pneumonia syndrome after nonmyeloablative and conventional conditioning regimens for allogeneic hematopoietic stem cell transplantation
Authors: Fukuda Takahiro , Hackman Robert C. , Guthrie Katherine A. , Sandmaier Brenda M. , Boeckh Michael , Maris Michael B. , Maloney David G. , Deeg H. Joachim , Martin Paul J. , Storb Rainer F. , Madtes David K.
Published: July 2003

Idiopathic pneumonia syndrome (IPS) is a significant noninfectious complication of hematopoietic stem cell transplantation (HSCT). We compared the incidences and outcomes of IPS among patients who underwent allogeneic HSCT after nonmyeloablative (n = 183) compared with conventional (n = 917) conditioning between December 1997 and December 2001. Patients given nonmyeloablative conditioning were older than those given conventional conditioning (median ages, 53 vs 41 years; P = .001). The cumulative incidence of IPS was significantly lower at 120 days after nonmyeloablative conditioning than conventional conditioning (2.2% vs 8.4%; P = .003). In addition, greater patient age (older than 40 years), diagnosis of acute leukemia or myelodys-plastic syndrome, and severe acute graft-versus-host disease were associated with significantly increased risks for IPS. Among older patients (older than 40 years) given conventional conditioning, high-dose total body irradiation (TBI) was associated with an increased risk for IPS than were non-TBI–based regimens (16% vs 5.8%; P = .001). IPS occurred early after transplantation, progressed rapidly, and was associated with a high mortality rate (75%) despite aggressive support. Initiation of mechanical ventilation and the presence of renal insufficiency at IPS onset were associated with increased risks for death after IPS. These findings support the concept that lung damage from the conditioning regimen plays a crucial role in the development of IPS after HSCT.
Nonneutralizing IgM and IgG antibodies to von Willebrand factor–cleaving protease (ADAMTS-13) in a patient with thrombotic thrombocytopenic purpura
Authors: Scheiflinger Friedrich , Knöbl Paul , Trattner Bettina , Plaimauer Barbara , Mohr Gabriele , Dockal Michael , Dorner Friedrich , Rieger Manfred
Published: July 2003

Acquired thrombotic thrombocytopenic purpura (TTP) has been linked to severe deficiency of ADAMTS-13 activity caused by autoantibodies inhibitory to ADAMTS-13. We report data on a patient with confirmed TTP who had severely reduced ADAMTS-13 activity but showed no ADAMTS-13 inhibition in a widely used fluid phase activity assay. With a newly developed enzyme-linked immunosorbent assay, using immobilized recombinant ADAMTS-13, we found high titers of IgM and IgG antibodies that bound to ADAMTS-13, but did not neutralize protease activity. These autoantibodies probably influenced the half-life of ADAMTS-13 or its binding to the endothelial cell surface, thereby compromising ADAMTS-13 activity in vivo. Given that ADAMTS-13 may interact physiologically with various receptors or ligands, the occurrence, distribution, and the epitope mapping of nonneutralizing antibodies will be an important area for future research.
Persistent γ-globin expression in adult transgenic mice is mediated by HPFH-2, HPFH-3, and HPFH-6 breakpoint sequences
Authors: Katsantoni Eleni Z. , Langeveld An , Wai Albert W. K. , Drabek Dubravka , Grosveld Frank , Anagnou Nicholas P. , Strouboulis John
Published: July 2003

Deletions at the 3′ end of the human β-globin locus are associated with the hereditary persistence of fetal hemoglobin (HPFH) in adults, potentially through the juxtaposition of enhancer elements in the vicinity of the fetal γ-globin genes. We have tested how sequences at the HPFH-2, HPFH-3, and HPFH-6 breakpoints, which act as enhancers in vitro, affect the silencing of a locus control region Aγ (LCRAγ) transgene in the adult stage of mice. We found persistent Aγ expression in the adult blood of most of the multicopy HPFH-2, HPFH-3, or HPFH-6 lines, in contrast to the control LCRAγ lines which were silenced. Cre-mediated generation of single copy lines showed persistent γ gene expression maintained in some of the HPFH-2 and HPFH-6 lines, but not in any of the HPFH-3 or LCRAγ lines. In the HPFH-2 and HPFH-6 lines, persistent γ gene expression correlated with euchromatic transgene integrations. Thus, our observations provide support for a model whereby HPFH conditions arise from the juxtaposition of enhancers as well as permissive chromatin subdomains in the vicinity of the γ-globin genes.
Differences in output from corticosteroid inhalers used with a volumatic spacer
Authors: Miller MR , Bright P
Published: July 2003

Corticosteroid inhaler therapy using a spacer device is commonly used as an important part of asthma management. Increasingly, generic corticosteroid inhalers are being used with spacer devices. We have therefore tested whether these generic inhalers yield equivalence in dose when compared to the established inhalers. We measured the in vitro output, discharged into a Volumatic spacer from beclomethasone dipropionate inhalers (250 micrograms.puff-1) made by three manufacturers, Allen & Hanburys, 3M and Baker Norton. The output from 20 of each type of inhaler was sampled, in random order, by a computer driven pump system. Beclomethasone was absorbed onto a coded filter, which was analysed independently for drug content. The output per puff differed significantly between the inhalers of each manufacturer, with a 36% difference between the highest output from the Allen & Hanburys device and the lowest output device. We conclude that there are important differences in output from these inhalers when used with a spacer, and that substitution of one device with another will not necessarily give equivalent therapy to the patient.
Aerosol derived airway morphometry in healthy subjects
Authors: Brand P , Rieger C , Beinert T , Heyder J
Published: July 2003

Monodisperse aerosol particles can be used to non-invasively probe intrapulmonary airspace dimensions. In this study, the aerosol-derived airway morphometry technique was used to study airspace dimensions in 79 healthy subjects, in order to assess reference data for the future clinical application of aerosol-derived airway morphometry, and to investigate the effect of lung inflation, anthropometric, and lung function parameters on aerosol-derived airway morphometry. Intrapulmonary airspace dimensions were assessed by measuring the deposition of monodisperse, hydrophobic submicron aerosol particles during breathholding. Additionally, measurements of spirometric and body plethysmographic lung function were performed. Airspace dimensions were in good agreement with morphometric lung data. Airspace dimensions increased with increasing lung inflation. Interindividual variation of airspace dimensions was lowest in the lung periphery, at high levels of lung inflation, and when the volumetric lung depth was normalized to the end-inspiratory lung volume. Analysis of variance showed an increase of airspace dimensions with age. The results of this study indicate that aerosol-derived airway morphometry is dependent on the level of lung inflation and the age of the subject. These results suggest that in contrast to conventional lung function techniques, aerosol-derived airway morphometry might be a powerful tool for the detection of small changes in peripheral airway geometry.
Potential role of Clara cell protein, an endogenous phospholipase A2 inhibitor, in acute lung injury
Authors: Jorens PG , Sibille Y , Goulding NJ , van Overveld FJ , Herman AG , Bossaert L , De Backer WA , Lauwerys R , Flower RJ , Bernard A
Published: July 2003

It is now recognized that epithelial cells lining airways and alveoli are capable of releasing various mediators, which have the potential to modulate local inflammatory reactions. The amount of the 16 kDa Clara cell protein (CC16), an inhibitor of phospholipase A2 activity produced by pulmonary epithelial cells, was measured by means of a sensitive immunoassay in the unconcentrated bronchoalveolar lavage fluid (BALF) of 13 control subjects, and in patients with acute lung injury (14 with the full-blown adult respiratory distress syndrome (ARDS); 21 after standard cardiopulmonary bypass surgery, a known risk factor for ARDS). The level of CC16 was compared with other markers of inflammation with a wide range of molecular weights: albumin (nephelometry); total protein (spectrophotometry); beta 2-microglobulin (latex immunoassay); cystatin C (latex immunoassay); alpha 1-antitrypsin (immunoradiometry), and lipocortin-1 (enzyme-linked immunosorbent assay (ELISA)). The Clara cell protein (CC16) was detectable in all BALF, and significantly higher levels of this protein were observed in BALF from patients with acute lung injury. Changes in BALF Clara cell protein levels differed from those of alpha 2-macroglobulin and the natural phospholipase inhibitor lipocortin-1. Alpha 2-macroglobulin levels were not significantly enhanced in patients at risk for ARDS, but were increased in patients with ARDS; whereas, lipocortin 1 levels were not elevated in either group. Pretreatment of patients at risk for ARDS with high dose methylprednisolone did not alter the amount of Clara cell protein recovered in BALF. The mean CC16 level in BALF from patients with ARDS who died was significantly lower than from those who survived. The data presented in this study suggest that pulmonary epithelial cells secrete a natural anti-inflammatory protein during acute lung injury, which might have a protective and immunosuppressive role.
Protein 1 (Clara cell protein) serum levels in lung cancer patients receiving chemotherapy
Authors: Nomori H , Horio H , Kobayashi R , Morinaga S , Hirabayashi Y
Published: July 2003

As many antineoplastic drugs can cause injury to alveolar pneumocytes, Clara cells (nonciliated, nonmucous epithelial cells of the bronchioles) may also be damaged by such drugs. Protein 1 (P1) is an alpha-microprotein secreted by Clara cells. The effect of antineoplastic drugs on Clara cells is examined by the measurement of serum levels of P1 in patients with lung cancer receiving chemotherapy. Serum levels of P1 were studied in 36 patients with lung cancer, before chemotherapy and 5-7, 10-12 and 14-18 days after chemotherapy. One hundred and eight healthy subjects, matched by sex and age, acted as controls. There was no significant difference in P1 serum levels between patients with lung cancer and healthy controls. P1 serum levels decreased significantly 5-7 and 10-12 days after chemotherapy, recovering thereafter. We conclude that P1 serum levels do not differ between lung cancer patients and healthy controls, and that antineoplastic drugs inhibit the synthesis or secretion of P1 by Clara cells in the early period after the administration of medication.
Plasma levels of soluble tumour necrosis factor receptors are increased in coal miners with pneumoconiosis
Authors: Schins RP , Borm PJ
Published: July 2003

Among other cytokines, tumour necrosis factor (TNF)-alpha is considered to play a key role in the development of mineral dust related fibrosis. Previously, we showed that ex-vivo release of TNF by peripheral blood monocytes is a marker for progression of coal workers' pneumoconiosis (CWP). Since soluble TNF receptors (sTNF-Rs) are believed to play an important regulatory role in systemic effects of TNF, we measured plasma levels of sTNF-R55 and sTNF-R75 in coal miners with (n = 28) or without (n = 76) CWP and in nonexposed controls (n = 29). sTNF-R75 levels were significantly increased in miners with CWP (2.09 +/- 0.44 ng.mL-1) versus the nonexposed controls (1.86 +/- 0.23 ng.mL-1). Neither sTNF-R55 nor sTNF-R75 were related to exposure, stage of pneumoconiosis, smoking, or (spontaneous or ex-vivo induced) monocyte TNF-release. sTNF-R55 was increased in subjects with medication (especially those using cardiovascular drugs); upon exclusion of these subjects, sTNF-R55 was found also to be significantly increased in CWP. In conclusion, bearing in mind a confounding effect of medication, soluble TNF receptors are elevated in plasma of retired miners with coal workers' pneumoconiosis. These observations further support the important role of TNF-mediated pathways in the pathogenesis of mineral dust related fibrosis.
Nitrogen dioxide exposure impairs the frequency of the mucociliary activity in healthy subjects
Authors: Helleday R , Huberman D , Blomberg A , Stjernberg N , Sandstrom T
Published: July 2003

In previous studies, we have reported bronchoalveolar lavage (BAL) findings following single and repeated exposures to NO2. Here, we present the first data on measurements of the frequency of the mucociliary activity in healthy subjects following exposure to NO2. Twenty four healthy nonsmoking volunteers underwent fibreoptic bronchoscopy, during which mucociliary activity was measured using an in vivo method based on a two-way fibreoptic system, to obtain baseline data. Two weeks later, the subjects were reinvestigated following NO2 exposure. Eight volunteers were investigated 45 min after a 20 min exposure to 1.5 ppm NO2 (Group A); eight subjects 45 min after a 20 min exposure to 3.5 ppm NO2 (Group B); and eight subjects 24 h after a 4 h exposure to 3.5 ppm NO2 (Group C). No activity was detected in Group A or B 45 min after exposure, in contrast to the easily identifiable mucociliary activity waves at the reference bronchoscopy. In group C, a significant elevation of the frequency of the mucociliary activity waves was seen at 24 h. It is concluded that short-term exposure to NO2 in man produces a significant reduction in the mucociliary activity 45 min after exposure. The effect ceases within 24 h. Further studies are needed to evaluate the effects of repeated exposures with NO2 on mucociliary activity in man.
Use of pulse transit time as a measure of inspiratory effort in patients with obstructive sleep apnoea
Authors: Pitson DJ , Sandell A , van den Hout R , Stradling
Published: July 2003

Pulse transit time (PTT) is the time taken for the arterial pulse pressure wave to travel from the aortic valve to a peripheral site. For convenience, it is usually measured from the R wave on the electrocardiogram to the pulse wave arrival at the finger. Pulse transit time is inversely proportional to blood pressure, and the falls in blood pressure which occur with inspiration (pulsus paradoxus) correspond to rises (lengthening) in pulse transit time. In awake normal subjects, the size of these inspiratory rises in pulse transit time correlate well with the degree of inspiratory effort. The aim of this study was to investigate whether inspiratory rises in pulse transit time could provide a quantitative measure of inspiratory effort in patients with obstructive sleep apnoea. Eight patients with obstructive sleep apnoea, attending the laboratory for institution of nasal continuous positive airway pressure, took part in the study. Once asleep, airway pressure was varied between optimal treatment level and minimum pressure, to produce a range of inspiratory efforts whilst continuous recordings of oesophageal pressure and pulse transit time were made. There was an excellent correlation between the size of the swings in oesophageal pressure and the size of the swings in pulse transit time (mean r = 0.94). Pulse transit time may, therefore, provide a clinically useful noninvasive and quantitative measure of inspiratory effort in patients with sleep-related breathing disorders.
Cardiovascular changes during acute episodic repetitive hypoxic and hypercapnic breathing in rats
Authors: Bakehe M , Miramand JL , Chambille B , Gaultier C , Escourrou P
Published: July 2003

It has been shown that chronic repetitive ambient hypoxia, simulating pulmonary gas disturbances observed in apnoea, leads to systemic hypertension in rats. However, the relative roles of hypoxia (HO), hypercapnia (HC), gas stress and vigilance on the cardiovascular changes have not yet been demonstrated. The aim of this study was to investigate the acute haemodynamic changes observed during the repetitive inhalation of various gas mixtures in rats for HO alone and HO+HC, and to analyse the effects of vigilance and of the stress of gas administration. We studied 6 unanaesthetized Wistar rats chronically instrumented with an aortic catheter. Nitrogen, nitrogen+CO2 mixtures and compressed air were randomly administered in a Plexiglass chamber for 10 s and then flushed by compressed air for 20 s. Two cycles were repeated every min for 10 to 12 min. The inhaled gas fractions (FI,O2,FI,CO2) were monitored by O2 and CO2 analysers. Blood pressure (BP) was measured by a P23XL transducer. The blood gases were analysed by a 1306 IL meter. In control experiments, with compressed air alone, there were no significant acute changes in heart rate (HR) and BP. During HO there were no changes in HR or BP at FI,O2 values from 0.05-0.14, whilst at FI,O2 values from 0-0.05 systolic blood pressure (SBP) rose significantly (+25.3 +/- 25.7 (SD) mmHg) and HR decreased (-93.8 +/- 124.1 bpm). During HOHC, SBP rose (+35.1 +/- 26.4 mmHg) and HR decreased (-139.3 +/- 75.7 bpm), significantly more than in HO alone. SBP was linearly correlated with Pa,O2 during HO (r = 0.53) and also during HOHC (r = 0.44) and was not directly related to Pa,CO2 which has, nevertheless, an additive effect to HO. SBP rose with each challenge significantly more when the rats were awake than when asleep (behavioural sleep). We conclude that in this acute repetitive inhalation model, the rise in SBP is not related to gas stress or to Pa,CO2 but to a decrease in Pa,O2 and is enhanced by wakefulness.
Aspergillus culture filtrates and sputum sols from patients with pulmonary aspergillosis cause damage to human respiratory ciliated epithelium in vitro
Authors: Amitani R , Murayama T , Nawada R , Lee WJ , Niimi A , Suzuki K , Tanaka E , Kuze F
Published: July 2003

Aspergillus species frequently colonize lower respiratory tracts and lungs with localized underlying conditions (healed tuberculous cavity, cystic fibrosis, bronchiectasis, etc.) even in subjects without systemic predisposing factors. We investigated the in vitro effects of culture filtrates of Aspergillus species and sputum sols from patients with pulmonary aspergillosis on ciliary beat frequency (CBF) and epithelial integrity of human respiratory ciliated epithelium. Culture filtrates of 25 clinically isolated fungi (16 Aspergillus fumigatus, three Aspergillus niger, one Aspergillus flavus, three Candida albicans, and two Cryptococcus neoformans) were obtained by culturing the fungi in Medium-199 at 37 degrees C for 7 days, and five sputum sols were obtained from patients with pulmonary aspergillosis infected by A. fumigatus. During 6 h experiments using a photometric technique, 14 out of 16 A. fumigatus culture filtrates caused progressive and significant reduction in CBF associated with marked epithelial disruption, whilst the culture filtrates of A. niger and A. flavus caused minor epithelial damage without slowing of CBF, and Medium-199 alone (Control) showed neither epithelial damage nor slowing of CBF. All of the sputum sols also caused significant slowing of CBF as well as epithelial disruption. Culture filtrates of C. albicans and Cr. neoformans had no effects on human respiratory epithelium. We conclude that Aspergillus species, especially A. fumigatus release a factor (or factors) which causes damage to respiratory epithelium and slows CBF, and that these factors may contribute to the colonization of the lower respiratory tracts by the Aspergillus species and may possibly contribute to the further proliferation and spread of the lesions in pulmonary aspergillosis.
Rapid drug susceptibility testing of Mycobacterium tuberculosis using conventional solid media
Authors: Schaberg T , Reichert B , Schulin T , Lode H , Mauch H
Published: July 2003

Radiometric methods for M. tuberculosis drug susceptibility testing yield much faster results than standard techniques; however, these methods require sophisticated equipment and are expensive. We investigated a rapid drug susceptibility testing method for isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide in specimens from 197 patients with pulmonary tuberculosis using a simplified agar-dilution method. Middlebrook 7H11 agar solid medium and microcolony detection were used to test sputum from 64 smear-positive, and from 70 culture-positive but smear-negative patients. Culture-positive material from bronchoscopy, surgical biopsy, pleural fluid or gastric fluid of 63 patients was tested. In 64 smear-positive patients, the median time for final susceptibility results was 11 days (95% confidence interval (95% CI) 10-12 days) compared to 62 days (95% CI 56-66 days) with the standard method. In 133 smear-negative patients, results were available after a median of 35 days (95% CI 32-40 days) in contrast to 72 days (95% CI 62-83 days) with the standard method, regardless of whether or not sputum or other materials were used for primary culture. The rapid method detected all cases of single-drug resistance (n = 20) and multidrug resistance (n = 14) within 13 days (95% CI 9-17 days) in smear-positive patients (n = 8), or within 38 days (95% CI 35-48 days) in smear-negative patients (n = 26). Only one discrepancy was encountered in 985 resistance tests. Moreover, contamination was not observed. Our rapid susceptibility testing method for M. tuberculosis on Middlebrook 7H11 agar is fast, practical and inexpensive. It provides an alternative when more sophisticated techniques are not available or affordable.
Risk factors for death and hospitalization from pneumonia. A prospective study of a general population
Authors: Lange P , Vestbo J , Nyboe J
Published: July 2003

The aim of this study was to identify possible risk indicators for pneumonia leading to death and hospitalization in the general population. We followed 6,158 men and 7,265 women aged 30-70 years, who participated in the Copenhagen City Heart Study, a prospective population study, for 12 years with regard to mortality and hospital admissions for pneumonia. A total of 260 deaths with pneumonia as main or contributory death cause had occurred, and 405 subjects had been admitted to hospital at least once because of pneumonia. Mortality and hospitalization were analysed by multivariate Cox regression models. In addition to increasing age, forced expiratory volume in one second (FEV1) was strongly and consistently related to both pneumonia related mortality and hospitalization. Women with FEV1 < 60% predicted had a relative risk of 5.7 (95% confidence interval: 2.9-11) and 3.6 (2.1-6.4) for death and hospitalization, respectively, when compared with women with FEV1 > or = 100% predicted. Similar, although lower, relative risks were observed in men. Other significant risk indicators for hospitalization were: self-reported asthma (women), mucus hypersecretion (women and men), history of stroke (men) and smoking (women). We conclude that, in addition to age, reduced FEV1 is the most important risk indicator for severe pneumonia.
Antitussive and antibronchoconstriction actions of fenspiride in guinea-pigs
Authors: Laude EA , Bee D , Crambes O , Howard P
Published: July 2003

Fenspiride is a nonsteroidal anti-inflammatory agent, which we have previously shown to have an in vivo antibronchoconstrictor action in guinea pigs. We have currently studied this action using the constrictors Substance P, neurokinin A, citric acid and capsaicin in anaesthetized guinea-pigs. Fenspiride has also been reported to produce a subjective improvement in cough in patients. We have used a conscious guinea-pig model of cough as a more definitive method to study the effect of fenspiride on capsaicin- and citric acid-induced cough. Aerosolized fenspiride (1 mg.mL-1) caused a 58% reversal of capsaicin-induced bronchoconstriction; and i.v. fenspiride (1mg.kg-1) a 45% reversal of citric acid induced bronchoconstriction. Substance P- and neurokinin A-induced bronchoconstriction were unaffected by 1 mg.kg-1 i.v. fenspiride. Aerosolized fenspiride (1, 3 and 10 mg.mL-1) administered for 4 min reduced citric acid (300 mM) induced cough, but 0.1 mg.mL-1 was without effect. Pretreatment with aerosolized fenspiride (10 mg.mL-1) caused a shift in the citric acid dose response curve to the right. For citric acid-induced cough, the duration of action of aerosolized fenspiride (10 mg.mL-1) was found to be 5 and 15 min post-treatment. Aerosolized capsaicin (30 microM) induced cough was also reduced by 3 and 10 mg.mL-1 aerosolized fenspiride, but no significant effect was found with 1 mg.mL-1. We conclude that aerosolized fenspiride reduces capsaicin- and citric acid-induced bronchoconstriction as well as induced cough in guinea-pigs in vivo. Whether a pathway common to both cough and bronchoconstriction is the site of action of fenspiride remains to be established. We postulate that fenspiride, acting as an antitussive and antibronchoconstrictor agent, would be beneficial in the clinical situation for those patients with hyperresponsive airways.
Effect of thromboxane A2 synthetase inhibitor, OKY-046, on sputum in chronic bronchitis and diffuse panbronchiolitis
Authors: Kurashima K , Fujimura M , Hoyano Y , Takemura K , Matsuda T
Published: July 2003

The mechanisms of excessive sputum production are only partially understood. We speculated that a selective thromboxane (Tx) A2 synthetase inhibitor, OKY-046, now used in the treatment of asthma in Japan, could decrease excess sputum production in patients with chronic airways disease. To test this hypothesis, we carried out a double-blind, placebo-controlled study of the effects of OKY-046, administered orally at 400 mg.day-1, on the sputum of patients with chronic bronchitis and patients with diffuse panbronchiolitis. Patients treated with OKY-046 showed a significant decrease (22%) in sputum volume after 1 month, and a 39% decrease after 3 months. Although the rheological properties of the sputum and the concentrations of fucose and immunoglobulin (Ig) A in the sputum remained unchanged, significant decreases were observed in the concentrations of total protein, albumin, sialic acid and phospholipid. Since albumin and fucose are chemical markers of plasma exudation and mucus secretion, respectively, whilst sialic acid and phospholipid are derived both from serum and mucus, our results indicate that this TxA2 synthetase inhibitor reduced sputum volume by inhibiting microvascular leakage in the airway. OKY-046 may, therefore, be of value in the treatment of chronic bronchitis and diffuse panbronchiolitis.