The first nationwide surveillance of bacterial respiratory pathogens conducted by the Japanese Society of Chemotherapy. Part 1: a general view of antibacterial susceptibility.

The Japanese Society of Chemotherapy (JSC) conducted the first nationwide surveillance of bacterial respiratory pathogens during the period from January to August 2006. With the cooperation of 32 medical institutions throughout Japan, a total of 924 strains belonging to seven clinically relevant bacterial species were collected from adult patients with well-diagnosed respiratory tract infections (RTIs). Antimicrobial susceptibility testing of the 887 evaluable strains (205 Staphylococcus aureus, 200 Streptococcus pneumoniae, 9 Streptococcus pyogenes, 165 Haemophilus influenzae, 91 Moraxella catarrhalis, 74 Klebsiella pneumoniae, and 143 Pseudomonas aeruginosa) to 42 antibacterial agents was conducted at the Central Laboratory of the Research Center for Anti-infective Drugs of the Kitasato Institute, according to recommendations issued by the Clinical and Laboratory Standards Institute (CLSI). The antibacterial agents employed were 25 beta-lactams, three aminoglycosides, four macrolides (including one azalide and one ketolide), one lincosamide, one tetracycline, two glycopeptides, five fluoroquinolones, and one oxazolidinone. The incidence of methicillin-resistant S. aureus (MRSA) was 63.4%, and the incidences of penicillin-intermediately resistant S. pneumoniae (PISP) and penicillin-resistant S. pneumoniae (PRSP) were 35.0% and 4.0%, respectively. Among H. influenzae, 21.2% of the strains were found to be beta-lactamase-nonproducing ampicillin (ABPC)-intermediately resistant (BLNAI), 29.1% to be beta-lactamase-nonproducing ABPC-resistant (BLNAR), and 4.8% to be beta-lactamaseproducing ABPC-resistant (BLPAR) strains. The incidence of extended-spectrum beta-lactamase-producing K. pneumoniae was 2.7% (2 of 74 strains). Three (2.1%) of the 143 P. aeruginosa strains were found to be metallo-beta-lactamaseproducing, including 1 (0.7%) multidrug-resistant strain. Through the nationwide surveillance, we obtained fundamental antimicrobial susceptibility data of clinically relevant bacterial pathogens in adult RTI to various antibacterial agents. These data will be a useful reference for future periodic surveillance studies, as well as for investigations to control antimicrobial-resistant pathogens.

Cytomorphologic features characteristic of tumor stages of thymomas.

The cytologic findings of the tumor cells characteristic of the stages of thymomas were investigated to assess the invasiveness of the tumors. Forty-six patients with thymoma who underwent extensive thymectomy without pre-operative corticosteroid therapy were included in this study. The histologic subtypes included 18 round/oval, 20 mixed, and 8 spindle type. The stages of thymoma classified according to Masaoka's clinicopathological classification included 16 stage I, 20 stage II, 6 stage III, 2 stage IVa, and 2 stage IVb, and myasthenia gravis was recognized in 5 patients. Cytologic findings were retrospectively analyzed in the Papanicolaou-stained stamp smears obtained from the cut surfaces of thymoma specimens. Morphometry of the epithelial tumor cells using Cosmozone-1A was performed to evaluate the validity of our cytologic categories. Compared with the cytologic findings of stage I or II thymomas, those of epithelial tumor cells in stage III or IV more frequently showed necrotic background (50.0%-stage III or IV vs 11.1%-stage I or II, p=0.006), large clusters of epithelial tumor cells (70.0% vs 36.1%, p=0.055), marked nuclear enlargement (90.0% vs 52.7%, p=0.033), marked anisokaryosis (100% vs 52.7%, p=0.006), marked nuclear polymorphism (40.0% vs 5.5%, p=0.004), hyperchromasia (50.0% vs 11.4%, p=0.007) and prominent nucleoli (50.0% vs 16.6%, p=0.028) whereas no significant correlation was observed between cytologic findings and tumor volume. Morphometric studies of thymoma tumor cells revealed that the nuclear size (mean values, 78.8 microm(3)-stage III or IV vs 58.2 microm(3)-stage I or II), the coefficient of variation of the nuclear size (0.326 vs 0.282), and the nuclear rotundity (0.849 vs 0.858) differed significantly between the two categories (p<0.05). Our findings demonstrated that there were significant differences between the cytologic findings of epithelial tumor cells of stage I or II thymomas and those of stage III or IV thymomas, and that the cytologic findings of thymoma tumor cells appear to be useful for distinguishing between non-invasive and invasive thymomas.

Diagnostic accuracy of intraoperative imprint cytology in ovarian epithelial tumors.

OBJECTIVE: To evaluate imprint cytology (IC) as the intraoperative pathological consultations for ovarian epithelial tumors (OET). METHOD: We reviewed ICs obtained from 354 consecutive surgical specimens of OET. Cytological specimens were classified into five categories. Final pathological diagnoses were made according to the WHO classification. We performed logistic regression analysis, calculated the limits among benign, borderline, and malignant lesions, and analyzed the diagnostic accuracy. We also made receiver operating characteristic (ROC) curves regarding IC. RESULTS: The accuracies to differentiate benign and malignant lesions were 87.1 and 83.6%, respectively. In contrast, that of borderline lesions was 30.0%. The areas under ROC curves to diagnose benign, and malignant lesions were 0.888 (P<0.05) and 0.951 (P<0.05), respectively, that meant IC was significantly useful for diagnosis of malignancy. CONCLUSIONS: IC applied to OET was proved to be practically useful in establishing an intraoperative diagnosis by ROC curves.

Overexpression of bcl-2 in transient abnormal myelopoiesis associated with Down syndrome.

Transient abnormal myelopoiesis (TAM) is a haematological complication found in Down syndrome. To determine the mechanisms of sustained proliferation of TAM cells, we studied the expression of apoptosis-related proteins, such as bcl-2, Fas (APO-1/CD95) and p-53, in peripheral blood cells from a new-born infant with Down syndrome and TAM. Using flow cytometry, peripheral blood mononuclear cells (PBMCs), consisting mostly of blast cells, showed marked expression of bcl-2 protein but not of Fas or p-53 products. DNA gel electrophoresis of PBMCs, cultured in the absence of serum factors, revealed no marked fragmentation. Our findings suggest that bcl-2 overexpression may be associated with prolonged cell survival of TAM cells.

Establishment of a myelodysplastic syndrome (MDS)/secondary AML-derived T lymphoid cell line K2-MDS.

We have established a T lymphoid cell line, K2-MDS, from the peripheral blood mononuclear cells (PBMC) of a patient with acute myeloblastic leukemia (AML) transformed myelodysplastic syndrome (MDS). K2-MDS cells are positive for the expression of CD4, CD5, CD13, CD25, CD71, CD95, HLA-DR and cytoplasmic CD3. Southern blotting analysis shows T cell receptor (TCR) beta chain genes rearrangements, whereas immunoglobulin heavy chain (IgH) genes are not rearranged. Further, the patient PBMC contains TCR beta chain genes rearrangements in the same manner as K2-MDS cells. The data indicate that K2-MDS is a T lymphoid cell line derived from a myelodysplastic clone in the patient PBMC. This new MDS-derived cell line K2-MDS may be a useful in vitro model for studies on the pathogenetic mechanisms leading to MDS.

Importance of positive peritoneal lavage cytology findings in the stage grouping of gastric cancer.

Although peritoneal lavage cytology is widely performed during surgery for gastric cancer and the results have been reported to be one of the accurate prognostic factors, the cancer stage is determined independent of the results of lavage cytology according to the First English Edition of Japanese Classification of Gastric Carcinoma. In this study we demonstrated the validity of lavage cytology for accurately staging gastric cancer. Between 1988 and 1996, peritoneal lavage cytology was performed in 347 patients with resectable gastric cancer. Among them, cytology was positive in 29 cases (8.4%). The survival rate of the cytology-positive patients in each stage was worse than that of all patients in the same stage. The prognosis of patients with positive cytology findings and serosa-exposed gastric cancer was significantly worse than that of negative cytology findings and serosa-exposed gastric cancer, and similar to that of negative cytology findings and serosa-infiltrating gastric cancer. Our data indicated that positive cytology findings thus indicated a poor prognosis, and the prognostic difference between positive and negative cytology findings was approximately a one-stage difference in the Japanese stage grouping. Based on our findings, the results of peritoneal lavage cytology should thus be included in the factors for staging gastric cancer.

Thiol compounds rescue growth inhibition by retinoic acid on HTLV-I (+) T lymphocytes; possible mechanism of retinoic-acid-induced growth inhibition of adult T-cell leukemia cells.

We demonstrated significant growth inhibition by retinoic acid (RA) of HTLV-I (+) T-cell lines (ATL-2 and HUT102), but not HTLV-I (-) T-cell lines (MOLT-4 and Jurkat). We hypothesized that the mechanism of growth inhibition by RA depends on an imbalance in redox potential. To examine the effect of exogenous thiol compounds for the growth of HTLV-I (+) T-cell lines by RA, HTLV-I (+) T-cell lines were cultured with several thiol compounds (thioredoxin, L-cystine, and GSH), following addition of 13-cis RA or ATRA, respectively, in cultured with thiol free medium. Unexpectedly, thiol compounds alone did not restore growth inhibition of HTLV-I (+) T-cell lines. However, when those cells were preincubated with thiol compounds for 24 hours, no growth inhibition by 13-cis RA or ATRA was observed. These results suggest that thiol compounds are associated strongly with sensitivity to RA of HTLV-I (+) T cells, but not of HTLV-I (-) T cells and that thiol compounds serve an important role on HTLV-I (+) T cells.

[Anesthetic management of a patient with Duchenne's muscular dystrophy undergoing radical repair for tetralogy of Fallot].

This report describes a 13-month-old-girl with Duchenne's muscular dystrophy (DMD) who had radical repair for tetralogy of Fallot safely. Patients with DMD are considered to be at risk of malignant hyperthermia (MH). Drugs for induction and maintenance were chosen from a list of agents rarely associated with MH. To wash out the inhalation anesthetics from the equipment, oxygen was circulated continuously for 24 hours. Dantrolene sodium was kept readily available in case of MH occurrence. Differential diagnosis during surgery is difficult in term of the episodes of MH and complications of cardiac surgery, as cardiac surgery is also associated with tachycardia, tachyarrhythmias, metabolic asidosis and red colored urine, which are frequently accompanied by MH. Although increased levels of CK, GOT, LDH and myoglobin strongly support the diagnosis of MH, such evidence can only be confirmed after operation. Fortunately, these factors recovered to the normal range without treatment by dantrolene sodium. During the cardiac surgery, treatment of MH may be delayed due to its late confirmation.

Soluble intercellular adhesion molecule-1 levels of patients with acute myeloid leukemia after allogeneic bone marrow transplantation.

Concentrations of serum-soluble intercellular adhesion molecule-1 (ICAM-1) were measured for 10 patients with or without chronic graft versus host disease after allogeneic bone marrow transplantation. Levels of soluble ICAM-1 were higher among patients with chronic graft versus host disease than among those without it, a statistically significant difference. The results indicated that measurement of serum-soluble ICAM-1 is useful for prediction of chronic graft versus host disease.

[Inhibition of proliferation by retinoic acid on adult T cell leukemia cells].

We observed the effects of the retinoic acid (13-cis retinoic acid; 13-cis RA, and all-trans retinoic acid; ATRA) for the cell growth and the expression of CD 25 on peripheral blood mononuclear cells (PBMC) from 17 patients with adult T cell leukemia (ATL). Fourteen had acute type, 1 had chronic type, and 2 had smoldering type of ATL. We divided those patient into 3 groups (hyper-sensitive, sensitive and resistant group) by determined with reduction rate of [3H]-thymidine incorporation obtained before and after treatment with 13 -cis RA or ATRA respectively. Growth inhibition was not observed in normal PBMC by 13 -cis RA or ATRA. However, no down-regulation of CD 25 expression was observed on PBMC in all patients and normal individuals after treatment with 13-cis RA or ATRA. In the aspect of growth inhibition on PBMC in ATL patients, we tried to clarify the mechanism of the phenomenon. In agarose gel electrophoresis, extracted genomic DNA from retinoic acid treated PBMC in hyper-sensitive and sensitive ATL patients showed multimer DNA fragmentation pattern. On the other hand, genomic DNA from PBMC after treatment with retinoic acid in resistant ATL patients and normal individuals showed high molecular DNA pattern without fragmentation. Taken together, it is suggested that retinoic acid could induce growth inhibition of PBMC in some ATL patients resulting in DNA fragmentation, apoptosis. We deeply consider that retinoic acid may be an useful agent for ATL patients in clinical aspect.

Elevated levels of soluble ICAM-1 in serum of patients with acute myeloid leukemia undergoing bone marrow transplantation.

Serum soluble ICAM-1 concentrations were measured in 10 patients with or without chronic graft-vs.-host disease (GVHD) after allogeneic bone marrow transplantation. The serum soluble ICAM-1 levels in the patients with chronic GVHD were significantly higher than that in the patients without chronic GVHD. The data indicated that serum soluble ICAM-1 is a useful parameter for predicting chronic GVHD.

Inhibition of growth and induction of apoptosis by all-trans retinoic acid in lymphoid cell lines transfected with the PML/RAR alpha fusion gene.

The interaction of an exogenous PML/RAR alpha fusion gene associated with acute promyelocytic leukaemia, with all-trans retinoic acid (ATRA) was examined in two lymphoid cell lines. L1210 and MOLT-4 cells were transfected with PML/RAR alpha cDNA in the expression vector pGD and stable transformants (L1210PML/RAR alpha and MOLT-4PML/RAR alpha) were selected with G418. ATRA inhibited the growth of these stable transformants, as assessed by [3H]thymidine incorporation, in a dose-dependent manner, but had no effect on the growth of control cells stably transformed with neomycin resistant gene alone. ATRA also induced apoptosis, as assessed by fragmentation of genomic DNA, in L121OPML/RAR alpha and MOLT-4PML/RAR alpha cells but not in control cells. The exogenous PML/RAR alpha fusion gene therefore probably mediates the effects of ATRA on cell growth and apoptosis in these cell lines.

13-cis retinoic acid inhibits growth of adult T cell leukemia cells and causes apoptosis; possible new indication for retinoid therapy.

We evaluated the effects of 13-cis retinoic acid (13-cis RA) on the growth of peripheral blood mononuclear cells (PBMC) obtained from 12 patients with adult T cell leukemia (ATL). In general, 13-cis RA potently inhibited the growth of PBMC from ATL patients. However, the sensitivity of the cells to 13-cis RA-induced growth inhibition varied among the patients. The ATL patients fell into three groups (hypersensitive, sensitive and resistant to 13-cis RA) according to the percent reduction of 3H-thymidine incorporation before and after treatment with 13-cis RA. Agarose gel electrophoresis of total genomic DNA from a patient sensitive to 13-cis RA provided evidence of the DNA fragmentation indicative of apoptosis. The ability of 13-cis RA to induce apoptosis in PBMC from ATL patients suggests that retinoids may be useful in the treatment of ATL.

Detection of minimal residual disease using clonospecific primers for CDRIII in patients with acute B lymphocytic leukemia with or without Philadelphia chromosome: possibility of clinical application as a tool for improving prognosis.

We attempted to identify the minimal residual leukemic clone as related to the clinical course in patients with acute B lymphocytic leukemia (B-ALL). DNA was extracted from stored bone marrow slides, and the third complementarity determining region (CDRIII) was amplified by polymerase chain reaction (PCR) using primers with consensus sequences for VH and JH. After amplification of the CDRIII band, the DNA fragment of CDRIII was inserted into the cloning vector PUC118. After cloning, the DNA sequences for CDRIII were determined. Clonospecific DNA sequences in CDRIII were selected, and clonospecific primers for each patient were synthesized. Using the clonospecific primers, we carried out second-round PCR to detect minimal residual disease (MRD) during several stages of the clinical course. Basically, the sensitivity of detection for MRD was between 10(-4) and 10(-5) cells. Even when leukemic cells were not detected in the morphologic study, with this detection system, the MRD was identified as an amplified CDRIII band stained with ethidium bromide on agarose gel. After bone marrow transplantation (BMT), MRD was detected for at least 4 months. In this article, we discuss the difference in sensitivity of detection for MRD between the BCR-ABL fusion gene and CDRIII in Philadelphia chromosome-positive (Ph+) B-ALL, as well as the possible clinical application of this method to predict relapse and prognosis.

Possible involvement of protein kinase C activation in down-regulation of CD3 antigen on adult T cell leukaemia cells.

The role of protein kinase C (PKC) system on CD3 expression on adult T-cell leukaemia (ATL) was examined. The down-regulation of CD3 on ATL cells is reportedly induced by CD3 down-regulating factor (CD3DF) contained in serum and culture supernatants of leukaemia cells from acute type ATL patients. After we cultured normal PBMC with a PKC inhibitor, H-7, CD3DF activity for PBMC was reduced significantly. Culture with H-7 of HTLV-1 transformed T cells, ATL-2 cells whose CD3 expression had been decreased, led to enhancement of CD3 expression in a time-dependent manner. These findings suggest that CD3DF may play an important role as a PKC system activator, resulting in CD3 down-regulation.

CD3 down-regulating factor in sera and culture supernatants of leukaemic cells from patients with adult T cell leukaemia.

Immunological abnormality of T lymphocytes in patients with adult T cell leukemia (ATL) is characterized by abnormal expression of the 55 kD chain of the receptor for interleukin 2 (IL-2R/p55) (Tac), and the down-regulation of CD3 expression. Using serum and culture supernatants of leukaemic cells from ATL patients (Group A) whose CD3 expression was down-regulated and those (Group B) whose CD3 was not low, the possible mechanism of CD3 down-regulation on ATL cells was discussed. When PBMC from normal individuals were cultured with sera from ATL patients for 24 h, CD3 expression revealed by mean fluorescent intensity (MFI) was down-regulated by sera from ATL patients in Group A (MFI: Pt 1 = 51.6 +/- 4.5, Pt 2 = 48.0 +/- 6.9, control = 96.5 +/- 6.6), not by sera from patients in Group B (MFI: Pt 3 = 105.5 +/- 7.9, Pt 4 = 102.5 +/- 8.3, control = 96.5 +/- 6.6). When normal PBMC were cultured with supernatants of leukaemic cells from ATL patients in Group A, this CD3 down-regulating activity was also detected (MFI: Pt 1 = 78.0 +/- 10.2, Pt 2 = 70.6 +/- 8.7, control = 94.0 +/- 6.6). By using gel-chromatography, the fractionated supernatants from ATL patients in Group A decreased CD3 expression of normal PBMC significantly (MFI: Pt = 22.9 +/- 5.8, Pt 2 = 28.8 +/- 7.4, control = 92.1 +/- 9.6). This CD3 down-regulating activity in fractionated supernatant was not inhibited by any lymphokine antibodies, anti-IL-1 alpha antibody (Ab), anti-IL-1B Ab, anti-IL-2 Ab, anti-IL-3 Ab, anti-IL-4 Ab, anti-IL-6 Ab, anti-TNF-alpha Ab and anti-IFN-gamma Ab. Any known cytokines (IL-1, IL-2, IL-3, IL-4, IL-6, TNF-alpha and IFN-gamma) could not modulate CD3 expression of normal PBMC. These findings suggested that there are novel factor(s) with CD3 down-regulating activity in the serum and culture supernatant of ATL patient and those factor(s) are involved in progression of ATL.

[Studies on pseudo-Chediak-Higashi granules formation in acute promyelocytic leukemia].

Leukemic cells from acute promyelocytic leukemia containing pseudo-Chediak-Higashi (P-CH) granules in a 38-year-woman were studied with ultrastructural and cytochemical techniques to evaluate the origin and nature of the granules. Wright-Giemsa stain revealed giant granules to be azurophilic. Cytochemical stain revealed p-CH granules ot the basic of their peroxidase and glycoprotein content. Electron microscopy revealed numerous giant granules formed by fusion of azurophilic granules these morphological, different type granules were classified into four types, 1) circular granule with homogeneous matrix, 2) circular granule with heterogeneous change by autolysis, 3) Auer body-like granule with crystalline arrangement, 4) vacuolar formation. The results demonstrate that the Auer body-like granule of P-CH granules in leukemic cells is a morphologically variant type of the classical Auer body observed in common acute myeloid leukemia.