Fatal tracheal aspergillosis during rituximab combined chemotherapy for diffuse large B-cell lymphoma that developed after lung transplantation.

Invasive tracheal aspergillosis (ITA) is an infection that is unique to patients who have undergone lung transplantation (LT). Although the activity of this disease often appears on imaging, we encountered a case of ITA that became exacerbated, despite few computed tomography (CT) findings, during rituximab combined chemotherapy for diffuse large B-cell lymphoma. ITA developed during immunosuppressive therapy after LT. Because CT findings may show false-negative results, bronchoscopy is recommended for such cases.

Risk factors for acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation: retrospective analysis of 73 patients who received cyclosporin A.

Cyclosporin A (CsA) has been used most widely as an immunosuppressive agent for preventing graft-versus-host disease (GVHD). To explore the risk factors including CsA blood levels for grades II-IV acute GVHD, we retrospectively analyzed the data of patients who underwent allogeneic hematopoietic stem cell transplantation in our hospital between March 1989 and July 2001. Seventy-three patients (47 males and 26 females) received CsA and short-term methotrexate for GVHD prophylaxis. CsA 1.5 mg/kg was administered as a 3-h infusion twice daily from day 1 until the patient recovered from the toxic gastrointestinal complication. Methotrexate was given at a dose of 15 mg/m(2) on day 1 and 10 mg/m(2) on days 3, 6 and 11. Grades II-IV acute GVHD occurred in 18 patients (24.7%). Multivariate Cox regression analysis revealed that higher C(5) (the whole-blood CsA concentration at 5 h after the start of infusion) before the onset of acute GVHD reduced the onset of grades II-IV acute GVHD with a hazard ratio of 0.994 (95% confidence interval 0.989-0.999) for every increase of 1 ng/ml. Our data indicate that inadequate exposures of CsA can be a vital risk for developing acute GVHD. From our results, we consider that precise monitoring of CsA concentrations and adjustment of CsA dose using the concentration may be effective to prevent the onset of severe acute GVHD. To confirm this finding, further prospective study will be needed.

Expression of coxsackievirus and adenovirus receptor in hearts of rats with experimental autoimmune myocarditis.

The expression of coxsackievirus and adenovirus receptor (CAR) was dominant in the brains and hearts of mice until the newborn phase. There is no detailed information concerning the relation between the expression of CAR and development of hearts. It is also uncertain whether CAR is able to be induced in adult hearts after cardiac injury. We demonstrated that CAR was abundant in the hearts of newborn rats but was barely detectable in the hearts of adult rats. The expression of CAR in rat hearts with experimental autoimmune myocarditis, which was induced by immunization of purified cardiac myosin, was serially investigated. Active myocarditis was observed from day 15 after immunization. By immunohistochemistry, cardiomyocytes were strongly stained for CAR antibody from days 24 to 42. CAR mRNA was also detected from days 18 to 30 by using reverse transcription-polymerase chain reaction. In the next experiment, the induction of CAR on isolated cardiomyocytes was investigated. CAR was barely detectable in cultured cardiomyocytes by Western blot analysis after isolation. This molecule gradually appeared along with the creation of clusters and beating of cardiomyocytes. Furthermore, the induction of CAR in cultured cardiomyocytes increased after supplement with conditioned medium of rat splenocytes activated by concanavalin A. In conclusion, rat CAR is expressed strongly in the hearts of newborn rats and is suppressed in those of adult rats. The expression of CAR is enhanced during the active phase of experimental autoimmune myocarditis and is induced by inflammatory mediators. CAR may play a role in cell-to-cell contact and adhesion of cardiomyocytes.

Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions.

We characterized the fluorescence resonance energy transfer (FRET) from pyrene (donor) to perylene (acceptor) for nucleic acid assays under homogeneous solution conditions. We used the hybridization between a target 32 mer and its complementary two sequential 16 mer deoxyribonucleotides whose neighboring terminals were each respectively labeled with a pyrene and a perylene residue. A transfer efficiency of approximately 100% was attained upon the hybridization when observing perylene fluorescence at 459 nm with 347-nm excitation of a pyrene absorption peak. The Förster distance between two dye residues was 22.3 A (the orientation factor of 2/3). We could change the distance between the residues by inserting various numbers of nucleotides into the center of the target, thus creating a gap between the dye residues on a hybrid. Assuming that the number of inserted nucleo-tides is proportional to the distance between the dye residues, the energy transfer efficiency versus number of inserted nucleotides strictly obeyed the Förster theory. The mean inter-nucleotide distance of the single-stranded portion was estimated to be 2.1 A. Comparison between the fluorescent properties of a pyrene-perylene pair with those of a widely used fluorescein-rhodamine pair showed that the pyrene-perylene FRET is suitable for hybridization assays.

Identification of heme axial ligands of cytochrome c' from Alcaligenes sp. N.C.I.B. 11015.

The spectral properties of both ferric and ferrous cytochromes c' from Alcaligenes sp. N.C.I.B. 11015 are reported. The EPR spectra at 77 K and the electronic, resonance Raman, CD and MCD spectra at room temperature have been compared with those of the other cytochromes c' and various hemoproteins. In the ferrous form, all the spectral results at physiological pH strongly indicated that the heme iron(II) is in a high-spin state. In the ferric form, the EPR and electronic absorption spectra were markedly dependent upon pH. EPR and electronic spectral results suggested that the ground state of heme iron(III) at physiological pH consists of a quantum mechanical admixture of an intermediate-spin and a high-spin state. Under highly alkaline conditions, identification of the axial ligands of heme iron(III) was attempted by crystal field analysis of the low-spin EPR g values. Upon the addition of sodium dodecyl sulfate to ferric and ferrous cytochrome c', the low-spin type spectra were induced. The heme environment of this low-spin species is also discussed.

Transcriptional activation of the alternative oxidase gene of the fungus Magnaporthe grisea by a respiratory-inhibiting fungicide and hydrogen peroxide.

Alternative oxidase (AOX) is dramatically induced when the fungus Magnaporthe grisea is incubated with the fungicide SSF-126, which interacts with the cytochrome bc1 complex in the electron transport system of mitochondria. A full-length cDNA for the alternative oxidase gene (AOX) was obtained, and the deduced amino acid sequence revealed marked similarity to other AOXs, but lacks two cysteine residues at corresponding sites which are conserved in plant AOXs and play essential roles in the post-translational regulation. Northern blot experiments showed that treatment of M. grisea cells with SSF-126 induces accumulation of AOX mRNA in a dose-dependent manner, and the level was correlated with the activity of alternative respiration. H2O2 also induced the accumulation of the transcript with a short half-life (<15 min). Nuclear run-on experiments showed that the AOX gene was transcribed constitutively in unstimulated cells. Cycloheximide did not change the basal level of transcription, but induced the accumulation of the transcript, indicating that active degradation of the transcript occurs by factor(s) sensitive to cycloheximide. On the other hand, SSF-126 enhanced the transcriptional activity of AOX gene threefold compared to that of control cells, and H2O2 was also potent for enhancement of the transcription. From these results, it is concluded that the respiratory inhibitor-dependent activation of the transcription is a primary determinant for the induction of alternative respiration in M. grisea. Because we have previously shown that SSF-126 treatment of M. grisea mitochondria induced the generation of superoxide, active oxygen species are thought to be signal mediators to activate AOX gene transcription in M. grisea.

Erythroid involvement in CD36 deficiency.

OBJECTIVE: The CD36 molecule is expressed in platelets, monocytes, erythroblasts, and other different tissues. The two types of platelet CD36 deficiency, types I and II, are associated with the absence and presence of CD36 on monocytes, respectively. To clarify the involvement of the erythroid lineage in CD36 deficiency, we investigated the phenotype and RNA expression of CD36. MATERIALS AND METHODS: CD36 expression was examined in 296 patients with several cardiovascular diseases in our outpatient clinic. There were 12 patients with type I deficiency and 16 with type II CD36 deficiency. A bone marrow sample was examined in five type I and four type II patients. Expression of CD36 mRNA was examined in burst-forming unit-erythroid (BFU-E). The sequences of reverse transcriptase polymerase chain reaction (RT-PCR) products of the CD36 mRNA from monocytes were examined. RESULTS: As expected, CD36 was deficient in erythroblasts from all five patients with type I deficiency. CD36 was present in erythroblasts from three of the four with type II deficiency, suggesting that their abnormality is restricted to platelets (type IIa). CD36 was unexpectedly absent from erythroblasts of a single type II patient (type IIb). CD36-specific mRNA was identified in BFU-E from each of two normals, six type I, and six type II patients, including type IIb. The sequences of RT-PCR products of the CD36 mRNA in a patient with type IIa and another with type IIb showed homozygous wild alleles. CONCLUSION: The findings provide evidence for further heterogeneity among CD36-deficient individuals and the existence of a basic principle mechanism of type II, such as glycosylation abnormality.

Hematopoietic cytokine-dependent differentiation to eosinophils and neutrophils in a newly established acute promyelocytic leukemia cell line with t(15;17).

We recently established an acute promyelocytic leukemia (APL) cell line (HT93) that has the capacity to differentiate into neutrophils and eosinophils in response to all-trans retinoic acid (ATRA) and human hematopoietic cytokines. The cells had a myeloblastic morphology, were positive for surface CD33, CD34, and CD56, and showed the following karyotypes: 46, XY, t(1;12)(q25;p13), 2q+, t(4;6)(q12;q13), and t(15;17)(q22;q11). When the cells were cultured with ATRA, they showed nuclear segmentation and developed secondary granules consisting in part of neutrophils and eosinophils. In the presence of ATRA and granulocyte colony-stimulating factor (G-CSF), the cells showed polymorphonuclear neutrophil differentiation accompanied by expression of surface CD11b, CD15, CD10, positive activity for neutrophil alkaline phosphatase (NAP), and NAP mRNA expression. In cultures with ATRA and granulocyte-macrophage colony-stimulating factor (GM-CSF), IL (interleukin)-3, or IL-5, HT93 showed remarkable eosinophil maturation at day 8 as determined by luxol fast blue staining, in addition to expression of eosinophil peroxidase and major basic protein. These results indicate that HT93 is an APL cell line with the ability to differentiate into neutrophils and eosinophils, and that these lineages are dependent on the CSF added. HT 93 should prove to be a useful model in analyzing the effects of hematopoietic cytokines on proliferation, differentiation, and maturation of hematopoietic progenitors.

Profile of cell cycle in hematopoietic malignancy by DNA/RNA quantitation using 7AAD/PY.

Using 7-amino-actinomycin-D/pyronin Y (7AAD/PY), we analyzed the surface phenotypes and cell cycle of 22 hematopoietic cell lines based on their cellular DNA/RNA content. Populations of G1a, G1b, S, and G2M, the DNA index (DI), and the RNA index of S phase (SRI) were calculated by means of DNA/RNA dot plots. Two new parameters were extracted from the cell-cycle profiles: the nucleic acid index of S phase (NI) and the coefficient of variations in the RNA at S phase (SVC). DNA/RNA dot plots of cell lines revealed four characteristic profiles of the cell cycle, defined with the calculated NI and SCV. These were type 0 (small NI, large SCV), type I (small NI, small SCV), type II (large NI, small SCV), and type III (large NI, large SCV). Type O included four stem cell lines: one t(1;19) leukemia, two Ph1+ acute lymphocytic leukemia (ALL), and one biphenotypic crisis of chronic granulocytic leukemia (CGL). Type I included five ALL cell lines: three T-ALL and two common B-ALL. Type II contained 10 myeloid cell lines: five AML and five myeloid crisis of CGL. Type III contained three relatively immature lymphoma cell lines: two Burkitt's lymphoma and one follicular center lymphoma. Calculated NI/SCV (%) were as follows: type 0, 2.27 +/- 0.19/16.7 +/- 3.7; type I, 2.20 +/- 0.30/11.1 +/- 0.7; type II, 3.64 +/- 0.52/11.8 +/- 1.0; and type III, 3.60 +/- 0.53/17.5 +/- 1.9. Cell-cycle analysis of blasts using 7AAD/PY combined with surface phenotyping may yield important information for classifying hematopoietic malignancy within 2 hours of patient admission.

Mutants of the phytopathogenic fungus magnaporthe grisea deficient in alternative, cyanide-resistant, respiration

The phytopathogenic fungus Magnaporthe grisea has a cyanide-resistant respiratory pathway. The fungicide SSF-126 ((E)-2-methoxyimino-N-methyl-2-(2-phenoxyphenyl) acetamide) blocks the cytochrome electron transport of M. grisea and induces the alternative respiratory pathway. Twelve mutants of M. grisea more susceptible to SSF-126 than wild type were identified after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Five mutants retained a reduced alternative respiration activity, and seven mutants lacked alternative pathway activity. A monoclonal antibody against the maize alternative oxidase cross-reacted against a 40-kDa mitochondrial protein of M. grisea, indicating that the 40-kDa protein is an alternative oxidase. Immunoblot analysis indicated that the seven completely deficient mutants grouped into two classes: four mutants produced the 40-kDa proteins while the other three mutants failed to produce the functional protein. Copyright 1997 Academic Press. Copyright 1997 Academic Press

Expression of eosinophil peroxidase in the immature basophil cell line KU812-F.

Although peroxidase activity in basophils can be detected by optical and ultrastructural cytochemistry, its characteristics remain to be determined. We have demonstrated the characteristics of peroxidase activity induced in the immature basophil cell line, KU812-F. Ultrastructurally, peroxidase activity was detected in granules as well as in the perinuclear space and endoplasmic reticulum. Immunocytochemistry revealed that KU812-F cells were stained by anti-eosinophil peroxidase antibodies, and eosinophil peroxidase mRNA, not myeloperoxidase, was detected in the cells using Northern hybridization and reverse transcription-polymerase chain reaction. Eosinophil peroxidase can be one of the molecules shared with eosinophils and basophils. The biological function of eosinophil peroxidase detected in basophils remains uncertain.

Effects of transfection of p210bcr-abl and bcr-v-abl into the factor-dependent human leukemia cell line HSM-911.

In order to clarify the action of the bcr-abl, a growth factor dependent human leukemic cell line (HSM-911) was transfected with p210bcr-abl or bcr-v-abl by electroporation. The cells transfected with bcr-v-abl, but not the cells transfected with p210bcr-abl, became growth factor independent. Some clones of the cells transfected with p210bcr-abl demonstrated cellular maturation (nuclear segmentation, becoming positive for naphthol ASD chloroacetate esterase, the disappearance of CD34 expression and the appearance of glycophorin A and CD10 expression). Moreover, these clones transfected with p210bcr-abl demonstrated apoptosis (increased expression of Fas and DNA ladder formation suggesting apoptotic DNA fragmentation). These findings demonstrated the different actions of p210 bcr-abl and bcr-v-abl, the former of which gave the cells the characteristics of maturation like the cells from chronic myelogenous leukemia, and the latter of which rendered the cells grow autonomously.

The coxsackievirus-adenovirus receptor protein as a cell adhesion molecule in the developing mouse brain.

In an attempt to elucidate the molecular mechanisms underlying neuro-network formation in the developing brain, we analyzed 130 proteolytic cleavage peptides of membrane proteins purified from newborn mouse brains. We describe here the characterization of a membrane protein with an apparent molecular mass of 46 kDa, a member of the immunoglobulin superfamily of which the cDNA sequence was recently reported, encoding the mouse homologue of the human coxsackievirus and adenovirus receptor (mCAR). Western and Northern blot analyses demonstrated the abundant expression of mCAR in the mouse brain, the highest level being observed in the newborn mouse brain, and its expression was detected in embryos as early as at 10. 5 days post-coitus (dpc), but decreased rapidly after birth. On in situ hybridization, mCAR mRNA expression was observed throughout the newborn mouse brain. In primary neurons from the hippocampi of mouse embryos the expression of mCAR was observed throughout the cells including those in growth cones on immunohistochemistry. In order to determine whether or not mCAR is involved in cell adhesion, aggregation assays were carried out. C6 cells transfected with mCAR cDNA aggregated homophilically, which was inhibited by specific antibodies against the extracellular domain of mCAR. In addition to its action as a virus receptor, mCAR may function naturally as an adhesion molecule involved in neuro-network formation in the developing nervous system.

High-dose cytosine arabinoside and etoposide with total body irradiation as a preparatory regimen for allogeneic hematopoietic stem-cell transplantation in patients with acute lymphoblastic leukemia.

One approach to improving the outcome of allogeneic hematopoietic stem-cell transplantation for acute lymphoblastic leukemia (ALL) is to intensify the pretransplant conditioning regimen without increasing toxicity. We used an intensified conditioning regimen consisting of high-dose cytosine arabinoside (3 g/m(2) twice daily i.v. for 3 consecutive days, total six doses), high-dose etoposide (1 g/m(2) once daily i.v. during the first 2 days) and total body irradiation (TBI) (HDACE-TBI) in ALL patients. We retrospectively analyzed 21 patients treated with HDACE-TBI, of whom 18 were in complete remission (CR) and three were in non-CR at transplantation. Although gastrointestinal toxicities were common, critical regimen-related toxicities were not seen in any patients. One patient demonstrated veno-occlusive disease, which could be controlled conservatively. The disease-free survival rate of 18 patients in CR at transplantation was 61%. These results demonstrate that the HDACE-TBI combination regimen is a feasible alternative to other preparatory regimens and does not increase the regimen-related toxicity.

Characterization of nitrite reductase from a denitrifier, Alcaligenes sp. NCIB 11015. A novel copper protein.

A copper-containing dissimilatory nitrite reductase [nitric-oxide: ferricytochrome c oxidoreductase, EC 1.7.2.1] was purified from a denitrifier, Alcaligenes sp. NCIB 11015, by ion-exchange chromatography on CM-cellulose, gel filtration on Sephadex G-150, and adsorption on hydroxyapatite. The preparation was homogeneous by SDS-polyacrylamide gel electrophoretic criteria, and its enzymatic activity increased considerably by freezing (at -20 degrees C) and thawing. The enzyme consists of two subunits with a molecular weight of 37,000, and the isoelectric point and redox potential are 8.4 and +260 mV (pH 7.2), respectively. The EPR spectrum and copper analysis clearly indicated that the enzyme contains two type I copper atoms per molecule but no other types of copper. This is the first blue copper protein that exhibits catalytic activity despite possessing only type I copper.

Effects of freezing on purified nitrite reductase from a denitrifier, Alcaligenes sp. NCIB 11015.

The effects of freezing on Alcaligenes sp. nitrite reductase [nitric-oxide: ferricytochrome c oxidoreductase, EC 1.7.2.1] dissolved in sodium phosphate (pH 7.2) were investigated. The nitrite reductase was gradually activated with time in the frozen state, resulting in an increase in its activity of 2.5-4.5 times. The final freezing temperature influenced the enzyme activation, maximal activation being observed at around -20 degrees C. All the enzymatic activities that the nitrite reductase is known to catalyze were enhanced by freeze-thawing. The activation was followed by neither association-dissociation nor any gross conformational change of the enzyme molecule, but was accompanied by an increase in the fluorescence intensity of 2-p-toluidinonaphthalene-6-sulfonate used as a hydrophobic probe. The results are consistent with the hypothesis that the activation of the NiR is due to a limited conformational change of the enzyme molecule, particularly in the hydrophobic region. The mechanism of the activation of NiR by freeze-thawing is discussed, in comparison with the mechanisms of inactivation by freeze-thawing of many enzymes reported by previous workers.

A novel method for detection and counting of single bacteria in a wide field using an ultra-high-sensitivity TV camera without a microscope.

We describe a novel method for enumeration of bacteria, based on the principle that small, light emitting particles on a flat surface can be easily and rapidly detected and counted using an ultra-high-sensitivity TV camera. To test this method, we obtained TV images of individual cells of a luminous bacterium on a membrane filter without the use of a microscope. The positions of the luminous points in the TV images were almost the same as the positions of the bacterial colonies after growth. Our results show that the single cells can be efficiently detected and counted by our method if they emit light or can be stimulated to emit light.

Rapid detection and counting of single bacteria in a wide field using a photon-counting TV camera.

Using Escherichia coli as a model bacterium, we tested a photon-counting method for enumeration of bacteria. This method is based on the principle that microscopic sized luminous particles in a wide field can be directly detected and counted using a photon-counting TV camera without the use of a microscope. E. coli cells were labeled with peroxidase and luminescence induced by adding a luminol-based reaction mixture. The number of luminous spots in the TV images was in good agreement with the number of bacterial colonies grown from labeled cells. The results show that our method provides a rapid and easy microbial counting system for such purposes as clinical diagnosis, microbial analysis in food, and environmental assessment.

Nucleic acid hybridization accompanied with excimer formation from two pyrene-labeled probes.

We developed a novel nucleic acid hybridization method based on excimer formation. We used two different 16-mer oligonucleotide probes that had a combined continuous-sequence run that was complementary to a target 32-mer. Prior to hybridization, the adjacent terminal ends (i.e. the 3'-terminal of one probe and the 5'-terminal of the other probe) were each labeled with one pyrene residue. When these probes simultaneously hybridized to the target, a 495 nm broad fluorescence band was produced. The intensity of this band increased as the intensity of the pyrene monomer bands decreased, indicating that the 495 nm band was attributed to the pyrene excimer. The excimer fluorescence, easily differentiated from the monomer bands for emission wavelength, opens up a new way to perform homogeneous hybridization assays and in vivo imaging of nucleic acids.

[Treatment of eustachian tube using a remodelled fiberscope conformed to the anatomical variety of its orifice].

The structure of the opening of the Eustachian tube of 702 cases were observed with fiberoptic endoscope, and the relationship between the difficulty in ear douche by Eustachian tube catheterization and the shape of the orifice was discussed. The shape of pharyngeal orifice of the tube was classified into two types. The one which is well-known as ordinary shaped was named as type I, while the other which has an arched upper edge by torus tubarius was termed as type II. Type II orifices were found more frequently (54.3%) than type I. Insertion of a catheter was difficult in type II ones because the tip of catheters was apt to slip down from the tubal elevation. Moreover, anatomical irregularities in the nasopharynx, which is observed quite often, seem to be a factor of difficulties in douching the tube. In case of a failure in ear douches, it is necessary for us to detect its cause, and in case of type II orifice, the way of probing the orifice should be changed. I have treated some patients who were suffered from tubal obstruction with an injection of remedies into the auditory tubes, observing with remodelled Olympus NPF-S3 fiberscope, and have got a good result.