Determination of patulin in fruit juice and dried fruit samples by in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry.

A simple and sensitive method for the determination of patulin in fruit juice and dried fruit samples was developed using a fully automated method consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC-MS). Patulin was separated within 5 min by high-performance liquid chromatography using a Synergi MAX-RP 80A column and water/acetonitrile (80/20, v/v) as the mobile phase. Electrospray ionization conditions in the negative ion mode were optimized for MS detection of patulin. The pseudo-molecular ion [M-H](-) was used to detect patulin in selected ion monitoring (SIM) mode. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 microL of sample using a Carboxen 1006 PLOT capillary column as an extraction device. The extracted patulin was readily desorbed from the capillary by passage of the mobile phase, and no carry-over was observed. Using the in-tube SPME LC-MS with SIM method, good linearity of the calibration curve (r=0.9996) was obtained in the concentration range of 0.5-20 ng/mL using (13)C(3)-patulin as an internal standard, and the detection limit (S/N=3) of patulin was 23.5 pg/mL. The in-tube SPME method showed >83-fold higher sensitivity than the direct injection method (10 microL injection volume). The within-day and between-day precision (relative standard deviations) were below 0.8% and 5.0% (n=6), respectively. This method was applied successfully for the analysis of fruit juice and dried fruit samples without interference peaks. The recoveries of patulin spiked into apple juice were >92%, and the relative standard deviations were <4.5%. Patulin was detected at ng/mL levels in various commercial apple juice samples.

Epstein-Barr virus-associated lymphoproliferative disorder after unrelated bone marrow transplantation in a young child with Wiskott-Aldrich syndrome.

We report a case of a 16-month-old Wiskott-Aldrich syndrome (WAS) patient with a WASP gene mutation who received human leukocyte antigen (HLA)-matched, unrelated allogeneic bone marrow transplantation (BMT) followed by an Epstein-Barr virus-associated lymphoproliferative disorder (EB-LPD), diagnosed by clinical findings, polymerase chain reaction detection of the EB virus genome, and spontaneous lymphocyte proliferation of donor cell origin. EB-LPD is one of frequent lethal complications in HLA-mismatched or unrelated BMT in this syndrome. Adoptive immunotherapy with donor leukocyte transfusion, including appropriate numbers of CD3-positive T cells, was effective for the EB-LPD, achieving almost complete recovery 1 year later without any findings of graft-versus-host disease.

Detection of the PGP9.5 and tyrosine hydroxylase mRNAs for minimal residual neuroblastoma cells in bone marrow and peripheral blood.

The "touchdown" polymerase chain reaction (PCR) technique has been applied to analyze expression of the neuron-specific protein, PGP9.5, and tyrosine hydroxylase (TH) genes for detection of minimal residual neuroblastoma cells in bone marrow and peripheral blood. PGP9.5 and TH gene products were not detected in any normal samples (n = 72) examined. However, in patients more than 1 year of age with stage III and IV neuroblastoma PGP9.5 mRNA was detected in six of seven bone marrow samples and in four of eight peripheral blood samples, and TH mRNA in four of seven and three of eight, respectively. The detection sensitivity was up to 10(-6) to 10(-7) micrograms of total cellular RNA for PGP9.5 and 10(-4) micrograms for TH. Among forty bone marrow specimens from nineteen patients with neuroblastoma both PGP9.5 and TH mRNAs were detected in six, and only PGP9.5 mRNA was detected in ten. Since detection of PGP9.5 and TH gene transcripts by the "touchdown" PCR was highly specific and sensitive, it might be most informative at present to carry out both PGP9.5 and TH mRNA assays for minimal residual neuroblastoma cells in blood and bone marrow.

IL-2 receptor gamma chain expression on CD34 positive hematopoietic progenitor cells from bone marrow and cord blood.

The IL-2 receptor (IL-2R) gamma chain is shared among receptors for IL-4, IL-7, IL-9 and IL-15 as well as IL-2. In order to clarify the functional role of these cytokines interacting with the common gamma chain in human early hematopoiesis, we studied expression of the IL-2R gamma chain on purified CD34 positive cells from bone marrow and cord blood. Broad populations of bone marrow mononuclear cells were all found to express the IL-2R gamma chain. CD34 positive cells were purified by CD34 monoclonal antibodies and immunomagnetic beads as representative hematopoietic progenitor cells. It was established that only 38 +/- 10% of CD34 positive bone marrow cells (n = 5) and 35 +/- 12% of CD34 positive cord blood cells (n = 11) expressed the IL-2R gamma chain. CD34(+) IL-2R gamma chain(+) and CD34(+) IL-2R gamma chain(-) cells fractionated by cell sorting were subjected to clonogenic assays that showed granulocyte-macrophage colony-forming cells (CFU-GM) were present evenly in both fractions, whereas erythroid burst-forming cells (BFU-E) were enriched in the CD34(+) IL-2R gamma chain(-) fraction approximately two- to six-fold as compared with CD34(+) IL-2R gamma chain(+) fraction. Such clonogenic features did not differ between the bone marrow and cord blood cases. These results indicate that CD34(+) IL-2R gamma chain(-) cells contain immature cells already committed to the erythroid lineage.

Isolation and characterization of two monoclonal antibodies that recognize different epitopes of the human c-kit receptor.

After immunizing mice with a human megakaryoblastic leukemia cell line, M-MOK, we obtained two monoclonal antibodies which recognize the human c-kit receptor. The monoclonal antibodies, designated MTK1 and MTK2, were found to specifically recognize Balb/3T3 cells transfected with human c-kit cDNA and not parent Balb/3T3 cells while showing different immunological, biochemical and biological behaviors. Both allowed visualization of the 140 kDa c-kit protein by Western blot analysis, but MTK1 detected only positive band with non-reducing conditions for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MTK1 partially inhibited the stem cell factor (SCF) induced proliferation of M-MOK cells, whereas, MTK2 was without effect. MTK1 also inhibited the bone marrow derived colony forming unit granulocyte/macrophage (CFU-GM) formed by granulocyte-macrophage colony stimulating factor (GM-CSF) and SCF. Not only anti-CD34 antibodies (HPCA-1) but also MTK1 could be shown to concentrate bone marrow CFU-GM and burst forming unit erythroid (BFU-E) effectively. The presently described monoclonal antibodies may therefore be useful for functional analysis of the ligand binding domain of the human c-kit receptor, as well as for further classification of hematopoietic stem cells in addition to the CD34 positive cells.

Cell surface c-kit receptors in human leukemia cell lines and pediatric leukemia: selective preservation of c-kit expression on megakaryoblastic cell lines during adaptation to in vitro culture.

We produced a monoclonal antibody MTK1 which recognized c-kit protein. Using MTK1, 31 leukemia cell lines and 76 leukemia blasts from pediatric patients were analyzed for expression of the c-kit receptor by flow cytometry. The c-kit receptor was detectable on four of four cell lines assigned to the megakaryo/erythromegakaryoblastic lineage and on one of seven cell lines of myeloid lineage. C-kit expression was not seen on any of 20 cell lines of erythroid and lymphoid lineages. Furthermore, c-kit was expressed on 16 of 24 nonlymphoid blasts without platelet surface antigens (67%) and on six of eight non-lymphoid blasts with platelet surface antigens (75%), but was not detectable on 44 lymphoid blasts from pediatric leukemia patients. In these cases CD34 was expressed on 26 of 32 myeloid blasts (81%) and on 27 of 44 lymphoid blasts (61%). The findings indicate a dominant expression of the c-kit receptor on established cell lines assigned to the megakaryo/erythromegakaryoblastic lineage, though a high percentage of leukemic myeloblasts also expressed the c-kit receptor on their surface.

Establishment and characterization of a novel human immature megakaryoblastic leukemia cell line, M-MOK, dependent on fibroblasts for its viability.

A novel fibroblast-dependent human immature megakaryoblastic leukemia cell line (M-MOK) was established from the bone marrow of a girl with acute megakaryoblastic leukemia, and its growth was determined to be completely dependent on the presence of human embryonic lung-derived fibroblasts, HEL-O. Adhesive interaction between M-MOK and HEL-O was crucial for viability; once HEL-O was removed from the culture, mortality was total within a few days. On HEL-O cells, M-MOK could be passaged for more than 2 years. With regard to surface marker profile, the established cells were positive for CD11a, CD13, CD18, CD33, CD34, CD41b, CD42b, CD54, and c-kit antigens, but negative for HLA class II antigen and glycophorin. Histochemically, the cells were negative for myeloperoxidase, nonspecific esterase, and naphthol ASD chloroacetate esterase staining. Electron-microscope examination revealed the cells to be negative for platelet peroxidase (PPO). After induction of differentiation by a phorbol ester, however, the cells were demonstrated to be positive for PPO with a morphological change to megakaryocytes. From these results, M-MOK was considered to represent an immature cell line of megakaryocyte lineage. Studies of the mechanisms sustaining the HEL-O-dependent continuous in vitro growth of M-MOK cells revealed the following results: (1) M-MOK could grow even when separated from HEL-O by a nucleopore membrane; (2) conditioned medium (CM) from HEL-O supported the growth of M-MOK for more than 1 month without feeder cells; (3) the growth of M-MOK on HEL-O or CM supplement was nearly entirely inhibited by anti-GM-CSF (1 microgram/mL); (4) GM-CSF mRNA was detected in HEL-O cells; and (5) HEL-O was found to secrete GM-CSF into the culture medium. Taken together, the growth of M-MOK might therefore be driven by a soluble factor, that is, GM-CSF secreted from HEL-O cells. The presence of HEL-O, however, inhibited anti-GM-CSF-induced M-MOK death. Co-culture of M-MOK and HEL-O cells thus offers a useful experimental model for analysis of interactions between hematopoietic stem cells and stromal cells.

bcr/abl mRNA in leukemic blasts of an unusual patient with acute lymphoblastic leukemia followed after 5-year remission by chronic myelogenous leukemia in blast crisis.

A 13-year-old boy without any previous illness was diagnosed as suffering from acute lymphoblastic leukemia (ALL). After a period of apparent complete remission until 17 years of age, the presence of Ph1 positive cells in bone marrow was demonstrated by karyotype analysis. This finding suggested chronic myelogenous leukemia (CML) because of the absence of blastic changes in bone marrow but mild leukocytosis with basophilia at that time. Six months later he had a relapse (blast crisis) with the appearance of peroxidase negative lymphoid blasts and myeloid surface markers. To make differential diagnosis, leukemia blasts at onset and relapse were examined for rearrangement of immunoglobulin JH gene and bcr/abl fusion mRNA, and were found to have the same JH gene rearrangement pattern and the same bcr/abl mRNA of bcr exon 2/abl exon 2. These results indicate an unusual case of CML which appeared in blast crisis at onset, followed by a long-term remission.

[Quantitative evaluation of 111In-antimyosin Fab myocardial imaging in acute myocardial infarction patients].

Quantitative evaluation of 111In-antimyosin Fab myocardial (InAM) imaging was performed in 15 patients with acute myocardial infarction to evaluate which organ is the most appropriate for the control of myocardial accumulation, to compare the quantitative method with the conventional visual method, and to study which clinical indices correlate with the InAM quantitative evaluations. InAM images demonstrated the myocardium with 31 +/- 6 mean counts/pixel, lung with 14 +/- 4, upper mediastinum with 20 +/- 5, middle mediastinum with 26 +/- 5, and liver with 75 +/- 10. We considered the lung to be the most appropriate control organ for quantitative evaluations of InAM imaging, because it could be separated from the myocardium, and the measurement range was narrow. The InAM uptake index [IUI = (myocardial counts-lung counts)/lung counts] was calculated as the index of myocardial accumulation. Visual evaluations of myocardial accumulation on InAM images were classified into three grades. The IUI of grade 1 (slight) was 0.98 +/- 0.19, grade 2 (moderate) was 1.34 +/- 0.38, and grade 3 (severe) was 1.97 +/- 0.19. Visual grading was nearly in accordance with the IUI, although it was difficult to distinguish visually between grades 1 and 2. Measurement of wall motion by left ventriculography showed that reduced wall motion was associated with an IUI of 1.01 +/- 0.18 and dyskinesis with an IUI of 1.92 +/- 0.16, showing IUI can indicate regional myocardial damage. However, IUI was not correlated with indices of the overall left ventricular function, such as ejection fraction, cardiac index, and peak creatine kinase level.

Allogeneic bone marrow transplantation for malignant hematologic disorders in children.

In the present study we carried out allogeneic bone marrow transplantation (BMT) in 14 leukemia children with high risk prognostic factors. Six patients with acute nonlymphocytic leukemia (ANLL), four with acute lymphocytic leukemia (ALL), two with chronic myelogenous leukemia (CML), and two with myelodysplastic syndrome (MDS). Among these patients, six with ANLL, two with ALL, one with CML and one with MDS were alive in complete remission 8 to 58 months post-BMT. Four patients died of relapse (one with ALL, and one with MDS), and chronic GVHD (one with ALL and one with CML). In six patients recombinant granulocyte colony stimulating factor (rG-CSF) was used to shorten the period of granulocytopenia. The mean time of recovery to granulocyte count of 500/mm3 was 13.2 days in the rG-CSF+ group, being 15.9 days faster than that in the rG-CSF- group. In light of these results, allogeneic BMT is shown to be a choice of treatment for leukemia children with high risk prognostic factors and rG-CSF may be an effective reagent to prevent infectious episodes in BMT.

Mass screening for neuroblastoma in Miyagi Prefecture.

In Japan, aiming at early and preclinical detection of neuroblastoma in infancy a mass screening program for the tumor has been implemented nationwide using urinary tests for catecholamine metabolites, vanillylmandelic acid (VMA) and homovanillic acid (HVA) (Sawada 1990; Sawada et al. 1991). In this report, the results obtained from the screening program in Miyagi Prefecture for the last 6 years are described. The detection rate of neuroblastoma by mass screening was 1:8,377 among 125,652 infants tested in Miyagi Prefecture. All but one patients survived after removal of the primary tumor and none or minimal chemotherapy.

[The influence of findings of coronary artery on myocardial salvage in acute myocardial infarction].

201Tl stress myocardial scintigraphy was performed in convalescent patients with acute myocardial infarction, to evaluate the influence of stenosis and collateral circulation of coronary artery in acute phase, on myocardial salvage in chronic phase. In 14 cases of unsuccessful coronary revascularization (complete occlusion), a complete defect of thallium imaging in chronic phase was seen in only one case of four cases with good collateral circulation, while eight of 10 cases with poor collateral circulation. In 16 cases of poor collateral circulation, six cases showed a complete defect, although the target vessel had improved to less than 75% of stenosis. However, in cases of good collateral circulation, no case showed a complete defect when the target vessel had improved to less than 75% of stenosis. The myocardial salvage is quite possible (p less than 0.05), when the coronary angiography in acute phase showed the forward flow (99% or 90% of stenosis) before coronary revascularization and/or good collateral circulation (Rentrop 2 degrees or 3 degrees).

[Acute myocardial infarction: comparison of results of Tl-201, Tc-99m pyrophosphate and In-111 antimyosin Fab imagings].

To evaluate the extent and characteristics of infarct areas, we performed indium-111 monoclonal antimyosin Fab (InAM), thallium-201 (TL) and Tc-99m pyrophosphate (PYP) imagings in 17 patients with acute myocardial infarction, and tried to find out the mechanism that causes difference of these imagings. In each study, the extent scores as an index of the infarct area were obtained by single photon emission computed tomography (SPECT), and comparisons were made between the results obtained. The overlap between InAM and TL imagings obtained by SPECT was evaluated. Location, severity, extent and patterns of accumulation were compared between InAM and PYP with both planar image and SPECT. The extent scores of InAM correlated well with those of TL (r = 0.73, p < 0.01). However, the overlap of both methods was recognized in 8 of 17 patients, in whom wall thickness of the infarct area as obtained by echocardiography was well preserved. The left ventricular regional asynergy was mild in 6 of these 8 patients. Coronary angiography showed poor or no collateral circulation in these cases. Although there were generally close correlations of the extent scores between InAM and PYP, discrepancy was noted in 2 cases for location; 2 for severity, 5 for extent, and 3 for patterns of accumulation. These differences may be attributed to the timings of imaging, coronary reperfusion and different mechanisms of accumulation. In conclusion, the extent of acute myocardial infarction obtained by InAM correlates well with those obtained by TL and PYP, with some exceptions.

[Myocardial imaging in acute myocardial infarction using beta-methyl-p-(123I)-iodophenylpentadecanoic acid: comparison with 201Tl imaging and wall motion].

Myocardial imaging using beta-methyl-p-(123I)-iodophenylpentadecanoic acid (BMIPP) was performed in 11 patients with acute myocardial infarction. The left ventricular images were divided into 12 segments, and myocardial imagings with BMIPP were compared with coronary angiography (CAG), thallium-201 myocardial scintigraphy (TL) and wall motion obtained by two-dimensional echocardiography (WM). When the culprit lesion was at the proximal point of the left anterior descending artery (LAD), all segments showed depressed uptake. In 3 cases with single vessel disease of the LAD, inferior wall of the basis showed reduced uptake of BMIPP despite the location of the culprit lesion. In cases with discordant uptake between the two tracers, BMIPP frequently showed more severely depressed uptake than TL in the subacute phase, although the uptake of BMIPP correlated with that of TL (tau = 0.82, p less than 0.001). In such cases, the discordance was related to the improvement in WM from the acute phase to the convalescent phase. BMIPP uptake correlated with WM in the subacute phase (tau = 0.50, p less than 0.001). BMIPP showed more severely depressed uptake while WM showed mild asynergy in most cases in which discordance was found between the BMIPP and WM findings. However, there was no correlation between the change in WM from the acute to subacute phases, or the uptakes of BMIPP and TL alone. We concluded that the myocardial condition can be evaluated in detail in acute myocardial infarction by comparing the findings of BMIPP with those of TL and WM.

[Diagnostic utility of 111In-antimyosin Fab scintigraphy in acute myocardial infarction: comparison with 201Tl and 99mTc-pyrophosphate myocardial scintigraphy].

To assess the diagnostic accuracy, extent, and characteristics of 111In-antimyosin Fab scintigraphy (In-AM) in acute myocardial infarction (AMI), we studied In-AM in 17 patients with AMI and compared with In-AM, 99mTc-PYP and 201Tl scintigraphy. Intensity of In-AM uptake was classified into 3 grades. Fourteen of 17 patients (82%) showed positive uptake of In-AM. The locations of infarct area diagnosed by In-AM were in accordance with those by electrocardiography. There was a good correlation between the extent score of In-AM planar and that of SPECT (r = 0.72), In-AM SPECT and Tl SPECT (r = 0.79), In-AM planar and PYP planar (r = 0.92), In-AM SPECT and PYP SPECT (r = 0.76), respectively (p less than 0.01). Thus, In-AM is a useful method for diagnosis of AMI.

[Quantitative evaluation of acute myocardial infarction by In-111 antimyosin Fab myocardial imaging].

For quantitative evaluation of acute myocardial infarction, In-111 antimyosin Fab myocardial imaging (InAM) was performed in 17 patients with myocardial infarction who underwent Tl-201 (TL) and Tc-99m pyrophosphate (PYP) myocardial imaging in acute phase. For calculating the infarct size, voxel counter method was used for analysis in PYP and InAM, and extent and severity score were used on bull's-eye polar map in TL. The most appropriate cut-off level ranged from 65 to 80% by the fundamental experiment using cardiac phantom. The cut-off level of 0.70 (InAM) and 0.65 (PYP) were used for clinical application of voxel counter analysis. The infarct size calculated by InAM and PYP was compared with wall motion abnormality index by echocardiography (WMAI), TL extent score, TL severity score, peak CK and sigma CK. Infarct size by InAM showed the following correlations with other indices. PYP: r = 0.26 (ns), TL extent score: r = 0.72 (p less than 0.01), TL severity score: r = 0.65 (p less than 0.05), WMAI: r = 0.69 (p less than 0.05). The infarct size by PYP did not show any correlations with these indices. Therefore, the infarct size by InAM showed better correlations with TL and WMAI than that of PYP. So InAM was considered superior to PYP for quantitative evaluation of acute myocardial infarction.

[Coronary revascularization evaluated by thallium-201 myocardial scintigraphy: a follow-up study].

To elucidate changes with time in T1-201 scintigraphy after coronary revascularization, T1-201 stress myocardial scintigraphy was performed at least twice during the follow-up period (from one to 12 months) in 58 patients with ischemic heart disease (12 with angina, and 46 with myocardial infarction) who had undergone PTCA or A-C bypass surgery. The perfusion defects were classified in 4 grades, and scintigraphic changes over grade 1 were judged significant. We evaluated; 1) time of scintigraphic improvement after revascularization, 2) presence of reverse redistribution, and 3) assessment of coronary restenosis. Scintigraphic improvement was observed in 21 of 58 patients during a 3- to 12- month follow-up period, 7 of whom improved within one month. Reverse redistribution after coronary revascularization was observed in 8 of the 58 patients (14%), including 6 who showed scintigraphic improvement in 3 to 12 months (2 were not examined). Among 29 patients whose coronary angiogram and Tl-201 scintigram were compared, 11 had angiographic evidence of restenosis and 4 of them showed deterioration of scintigraphic findings (sensitivity 57%, specificity 68%, and accuracy 66%). In conclusion, scintigraphic improvement was observed over various periods (immediately after and up to 12 months) after coronary revascularization. Reverse redistribution appears to be a predictor of good prognosis. Coronary restenosis cannot always be reliably assessed by Tl-201 scintigraphy.

[Evaluation of syndrome X by exercise stress thallium-201 SPECT].

Using exercise stress thallium (Tl)-201 SPECT, we studied 11 patients with Syndrome X who had anginal pain and ischemic ECG change during exercise in spite of angiographically normal coronary artery. In three patients, the initial stress image showed mild hypoperfusion in the area of ST segment depression, but the delayed image showed complete or incomplete redistribution. Eight cases showed normal perfusion. This result suggests that some patients of Syndrome X could be caused by small vessel disease.

[111In-antimyosin scintigraphy on acute myocardial infarction].

Indium-111 antimyosin (InAM) scintigraphy was performed in 17 patients with acute myocardial infarction (on 15 +/- 6 days from the onset). The degree of myocardial uptake was classified into 3 groups. They were ranged from low intensity as in bone marrow to high intensity as in liver. All of 17 cases showed positive myocardial uptake including low intensity. The locations of infarction judged by InAM were in agreement with those by electrocardiography, coronary angiography (CAG), and 99mTc-pyrophosphate scintigraphy (PYP, performed on 5 +/- 2 days from the onset). In 5 cases, the uptake of InAM showed doughnuts or diffuse pattern which was occasionally observed on PYP. These cases showed myocardial uptake of 4th degree of Parkey's classification with doughnuts-like pattern on PYP, and showed involvement of left anterior descending artery on CAG. In some cases, the extent of myocardial uptake on InAM did not agree with those on PYP.