Intracellular multiplication of Legionella pneumophila in HL-60 cells functionally differentiated in response to 22-oxacalcitriol.

The agent 1,25-dihydroxyvitamin D3 (D3) induces the differentiation of HL-60 human leukemia cells into functional monocyte-like cells that can support the intracellular multiplication of Legionella pneumophila. 22-Oxacalcitriol (OCT), a synthetic analogue of D3, exhibits greater differentiation-inducing activity than D3 in WEHI-3 mouse leukemia cells and has been suggested to be clinically more useful because of its lower hypercalcemic activity. The abilities of OCT and D3 to induce the functional differentiation of human leukemia HL-60 cells have now been investigated. OCT induced the differentiation of HL-60 cells into monocyte-like cells to a similar extent as D3. Thus, both OCT and D3 increased (1) the surface expression of CD11b, CD11c, CD14, and CD35; (2) nonspecific esterase staining; and (3) phagocytic activity toward fluorescent beads. HL-60 cells differentiated in response to OCT also supported the intracellular multiplication of L. pneumophila. Activation of both OCT- and D3-treated HL-60 cells with human recombinant interferon-gamma (IFN-gamma) for 24 h before infection markedly inhibited L. pneumophila multiplication. IFN-gamma activation enhanced superoxide anion generation by D3-treated HL-60 cells but not by OCT-treated HL-60 cells, suggesting that the inhibition of L. pneumophila multiplication in IFN-gamma-activated cells is independent of superoxide generation. Finally, D3, but not OCT, markedly stimulated the formation of osteoclast-like multinucleated cells from mouse bone marrow cells, consistent with the lower hypercalcemic activity of OCT.

Protein-bound polysaccharide PSK inhibits tumor invasiveness by down-regulation of TGF-beta1 and MMPs.

Transforming growth factor beta1 (TGF-beta1) and matrix metalloproteinases (MMPs) produced by tumor cells play important roles in tumor invasion. PSK, a protein-bound polysaccharide, is widely used in Japan as an immunopotentiating biological response modifier for cancer patients. In this study, we focused on the effects of PSK on invasiveness, TGF-beta1 production, and MMPs expression in two human tumor cell lines, pancreatic cancer cell line (NOR-P1) and gastric cancer cell line (MK-1P3). PSK significantly decreased the invasiveness of both cell lines through Matrigel-coated filters but did not affect cell viability, proliferation, or adhesion. Decreased invasion was associated with the inhibition of TGF-beta1, MMP-2, and MMP-9 at both mRNA and protein levels as assessed by reverse transcriptase-polymerase chain reaction, gelatin zymography, and enzyme-linked immunosorbent assay. Antibody against TGF-beta1 neutralized the MMP activities of both cell lines. PSK also suppressed the expression of urokinase plasminogen activator (uPA) and uPA receptor but did not change plasminogen activator inhibitor-1 (PAI-1) expression. Western blot analysis showed that PSK reduced uPA protein expression but not PAI-1 expression in the both cell lines. These results indicate that PSK suppresses tumor cell invasiveness through down-regulation of several invasion-related factors including TGF-beta1, uPA, MMP-2, and MMP-9.

Anionic properties of beta-lactam-enhancing factor on methicillin-resistant Staphylococcus aureus.

We found a Factor (factor T) in aged mixtures of tungstate and phosphate which greatly enhanced the antibacterial effects of beta-lactams upon methicillin-resistant Staphylococcus aureus (MRSA). Factor T suppressed penicillinase production and the amount of penicillin-binding protein-2' in the membrane fraction, thus sensitizing MRSA strains to beta-lactams. In addition, Factor T caused a metachromatic reaction and prolonged the blood coagulation time, indicating that it is a heparin-like polyanion. Since Factor T becomes ineffective in the presence of a polycation, a charge interaction may play an important role in the enhancing effect. One possibility is that Factor T non-specifically inhibits several anion-sensitive enzymes. Factor T inhibited several nucleotide-interacting enzymes, but not most serum enzymes.

Coexistence of acute monoblastic leukemia and adult T-cell leukemia: possible association with HTLV-I infection in both cases?

A 64 year-old Japanese man who developed acute monoblastic leukemia during the course of adult T-cell leukemia/lymphoma (ATL) was studied. Leukemic cells in the peripheral blood and bone marrow were monoblasts positive for alpha-naphthol butyrate esterase (alpha-NBE) staining, CD11c and CD36 antigens, whereas tumor cells in the pleural effusion were ATL cells positive for CD2, CD4, CD25, CD29 and CD45RA antigens. These two malignant cells had different chromosomal abnormalities. Monoclonal integration of human T-cell leukemia virus type I (HTLV-I) proviral DNA and T-cell receptor C beta gene (TCR C beta) rearrangement were detected in the ATL cells, but not in the leukemic monoblasts. By polymerase chain reaction (PCR) in the peripheral blood mononuclear cells (CD11c+ 98%, CD2+ 4%, CD20+ 0%) not containing ATL cells, the presence of the gag region of HTLV-I was confirmed. These facts indicate that a double positive T cell (CD29+, CD45RA+) was possibly the target cell for HTLV-I infection and that HTLV-I was not directly related to the oncogenesis of the monocyte lineage in the present case, even if it did infect the monocytes. However, there is still an outside possibility that HTLV-I induced acute monoblastic leukemia indirectly.

Characterization of humoral and cellular immunity in the central nervous system of HAM/TSP.

In HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP) immunopathological processes in the central nervous system (CNS) have not been clarified. We compared the humoral and cellular immunity within the CNS and in the systemic circulation of 24 patients with HAM/TSP (8 men and 16 women) to 6 asymptomatic HTLV-I carriers, 7 patients with active multiple sclerosis, 6 patients with acute viral encephalitis, and 39 patients with other non-inflammatory neurological diseases. Significant differences were observed between the HAM/TSP patients and one or more of the control groups: HAM/TSP cerebrospinal fluids (CSF) exhibited higher levels of IgG, IgG index, de novo IgG synthesis rate, and beta 2-microglobulin, and also a predominance of CD8+ cells that expressed CD11a and CD45RO but lacked CD28 antigens. Results in the 6 patients with acute viral encephalitis suggested that the CD8+ population in the CSF which is positive for CD28 and CD45RO is important for the elimination of virus from infected CNS tissues. Therefore, potentially cytotoxic T cells of a unique CD8+CD11a+CD45RO+CD28- phenotype may play a key role in the CNS pathogenesis of HAM/TSP.

Necrotizing fasciitis caused by Vibrio vulnificus differs from that caused by streptococcal infection.

We reviewed the clinical record of all patients admitted to Saga Medical School Hospital during the most recent 10 years and found that 17 (0.03%) were diagnosed as having necrotizing fasciitis. Bacteriological examination demonstrated that Vibrio vulnificus was the pathogen responsible in five patients (29%). The disease caused by V. vulnificus occurred in the warmer half of the year. All of the patients had underlying chronic liver dysfunction, and three of them had previously consumed raw seafood. In these patients, the predominant skin lesions were oedema and subcutaneous bleeding, such as ecchymosis and purpura, while superficial necrosis was not recognized. Three patients died of systemic complications. By contrast, all of the five patients with necrotizing fasciitis caused by Streptococcus pyogenes had the disorder in winter, and only one of them had chronic liver dysfunction. In skin lesions, subcutaneous bleeding was rare but necrosis was seen often. Despite the high incidence of systemic complications, no patients with streptococcal necrotizing fasciitis died. These findings suggest that the clinical features of necrotizing fasciitis caused by V. vulnificus are different from those of necrotizing fasciitis caused by classical pathogens, and that the two should be differentiated as early as possible to improve the prognosis.

Tumoricidal effect of human PBMC following stimulation with OK-432 and its application for locoregional immunotherapy in head and neck cancer patients.

Both cell-mediated and cytokine-mediated antitumor activities were induced in peripheral blood mononuclear cells (PBMC) in short-term culture with streptococcal preparation, OK-432. Kinetic analysis of OK-432-activated killer activity (OKAK) showed that it reached a plateau level much faster (by 48 h of culture) than that detected in PBMC stimulated with recombinant interleukin 2 (rIL-2) (lymphokine-activated killer: LAK). We also found that the tumor growth inhibitory factor (TGIF) activity was produced in the culture supernatant (CSN) of the OK-432-activated PBMC (OK-MC) and the activity synchronously increased with augmentation of OKAK activity. The TGIF activity was rarely found in the CSN of rIL-2-stimulated PBMC. The TGIF activity detected in CSN of OK-MC was further characterized as derived from a cytokine different from interferon gamma (IFN gamma), tumor necrosis factor (TNF), or lymphotoxin (LT) by a neutralization test using monoclonal antibodies to these cytokines. These 48-h-cultured-OK-MC were adoptively transferred (adoptive immunotherapy: AIT) into 19 head and neck cancer patients either alone or in combination with chemotherapy and/or radiation therapy, and their therapeutic effects were examined. AIT was performed by intra-arterial or intratumoral administration of OK-MC. There were no significant side effects observed in this treatment. In these patients, approximately 1-10 x 10(7) cells were transferred into the tumor burden. Of the 19 patients, 17 had primary cancer, and in 6 (6/17;35%) of them complete remission (CR) of the tumor was obtained. Partial remission (PR) was attained in 9 of the 17 patients (9/17; 53%), giving the overall response rate of 88%.(ABSTRACT TRUNCATED AT 250 WORDS)

Current practice of management of bacteremic sepsis: a study in a tertiary care teaching hospital in Japan.

OBJECTIVE: To investigate how patients with bacteremic sepsis are managed in a tertiary care teaching hospital. PATIENTS AND METHODS: Prospective observational study on patients with bacteremic sepsis. Clinical and microbiological characteristics of bacteremic sepsis were analyzed in relation to prognosis. Severity of the illness was quantitatively analyzed by the APACHE (Acute Physiology, Age, Chronic Health Evaluation) III scoring system. Also investigated was how closely physicians paid attention to acute physiological alterations in patients. RESULTS: The 28-day mortalities in fifty hemodynamically stable patients and in twenty-three septic shock patients were 26% and 52%, respectively (p=0.028). Gram-positive organisms accounted for 54% of all organisms, with the mortality and incidence of septic shock being the same as with Gram-negative infections. The mean APACHE III score was 42.9 in survivors, and 76.5 in non-survivors (p < 0.001). Although serum levels of C-reactive protein and acute physiology score (APS) was significantly higher in non-survivors than in survivors, the correlation with APACHE III score was more prominent in APS. The number of vital signs recorded was 1.67 in physicians and 3.6 in nurses (p < 0.001). CONCLUSIONS: The present study proved that the APACHE III score accurately discriminates between survivors and non-survivors of patients with sepsis. By addressing the need for an objective evaluation of severity of illness, it strongly recommends that physicians should be made aware of physiologically defined sepsis and that they should pay closer attention to patients' physiological alterations to identify the development of sepsis in critically ill patients.

[A trial of infection control measure by clinical microbiology laboratory].

In 1998, we developed a Total Infection Control System in Saga Medical School Hospital, and would like to introduce it for the practical use. This system was named "Dr. FLEMING" (Flexible Microbiological Test & Information System for the New Generation) and is expected to help physicians by providing highly valuable test results and useful information. For example, bacterial identification and drug susceptibility test can be completed within 4-6 hrs after bacterial colony is isolated, and the test report the contains full-colored pictures to enhance understanding. In addition, we have made an information center for infectious disease, where physicians can have access to various data bases outside our hospital. Furthermore, we offer many kinds of useful information to physicians working at other medical facilities to assist their clinical practice of infectious diseases.

[Hospital infection with methicillin-resistant Staphylococcus aureus (MRSA) in Saga Medical School Hospital, a rapid increase in coagulase type-VIII strains].

Hospital infection with MRSA has increased in Saga Medical School Hospital. The causative MRSA consisted predominantly of coagulase type-II strain before 1989, but after 1990, coagulase type-VII MRSA increased rapidly. This type-VII strain has marked multiple drug-resistance, and the pattern of drug sensitivity of MRSA in this hospital was different from that of MRSA detected in other facilities, which are clinically serious problems, therefore, we conducted an etiological study of the background of the increase in MRSA infection in our hospital. The results of the study are summarized as follows: 1) The proportions of MRSA (on strain from one patient) to all types of S. aureus detected in the hospital were 26% for 1986, 23% for 1988, 37% for 1989, 30% for 1990 and 60% for 1991. The proportion increased greatly in 1991. 2) Coagulase type VII-MRSA was first detected only in 5 patients in 1989, then it tended to spread, and this type (probably derived from the same strain) accounted for 47% of MRSA infection in patients examined in 1991. 3) The study of the drug sensitivity pattern and etiological survey of the infection showed that coagulase type VII-MRSA prevalent in the hospital consisted of two types: CLDM, and EM-sensitive, IPM/CS, and MINO-resistant and TSST-1 non-producing and enterotoxin non-producing type, and CLDM, and EM-sensitive, IPM/CS, and MINO-resistant and TSST-1 non-producing type with enterotoxin serotype A. 4) Coagulase type VII-MRSA (Probably derived from the same strain) was detected in physicians and nurses working in affected wards and also in the patients's room.(ABSTRACT TRUNCATED AT 250 WORDS)

[Isolation of penicillin-resistant Streptococcus pneumoniae in Saga Medical School Hospital].

A recent nationwide increase in beta-lactams-resistant Streptococcus pneumoniae has attracted a great deal of attention. We studied the drug sensitivity of S. pneumoniae isolated from various clinical specimens in Saga Medical School Hospital between April 1988 and December 1991. To determine the drug sensitivity of the strains, we used a micro-dilution method and determined the MIC. Drug resistance was evaluated using MIC of ampicillin (ABPC) as a reference MIC, and the results were roughly classified into the following three groups: sensitive (< or = 0.1 microgram/ml), moderately resistant (0.2-3.13 micrograms/ml) and highly resistant (> or = 6.25 micrograms/ml). The isolation frequency was calculated on the basis of one strain from one patient. No strain of S. pneumoniae with high resistance against ABPC was found in 1988 (94 strains of S. pneumoniae were isolated) and 1990 (115 strains isolated), but one such strain (0.8%) was found among 129 strains isolated in 1989, and 2 such strains (2.4%) among 84 strains isolated in 1991. Moderately resistant strains were isolated at the frequencies of 12.8%, 15.5%, 22.6%, and 21.4% respectively, in 1988, 1989, 1990, and 1991. A sum of the frequencies of "moderately resistant" and "highly resistant" (2.4%) strains was 23.8% in 1991. The frequency of resistant strains is increasing and the intensity of resistance is also being elevated.

[Clinical bacteriological analysis of Vibrio vulnificus infection--a report of five case].

Clinical features in Vibrio infection are generally represented by gastrointestinal involvements such as food poisoning, and its prognosis is usually good. However, Vibrio vulnificus infection not uncommonly causes serious problems including sepsis, necrotizing fasciitis of the extremities, and other conditions, sometimes resulting in fatal outcome. In the present study, we analyzed clinical microbiological aspects of five cases with V. vulnificus infection. All the strains of V. vulnificus isolated in five patients are oxidase-positive Gram negative rods presenting comma-like configuration, which were yielded on TCBS agar forming green colonies; they were grayish-white in color and viscous in texture on 5% sheep blood agar, identification of bacteria were done using VITEK AMS (BioMérieux). Piperacillin and third-generation cephalosporins were found to have bactericidal activities against these strains. All five cases we experienced have primary ailments, and three cases out of the five had taken perishable sea-food before showing disease symptoms. V. vulnificus has two infection channels; one is external wound and the other is oral intake. The latter is said that it may become serious. This has a rather short period from the starting the symptoms to death, and there is high death rate. For life-saving, it is inevitably necessary to dose an effective antibacterial medicine in the early stage. If we suspect this bacteria in the test laboratory, it is important to report this to the clinical doctor. In other words, this is one of the bacteria that needs prompt treatment and further microbiology testing.

[Susceptibility of clinical isolates to aztreonam].

In vitro antibacterial activities of 9 antibiotics including aztreonam (AZT) against clinically isolated Gram-negative bacteria were determined using MIC-2000 plus system. Bacteria were isolated from clinical materials in Saga Medical School during a period from May 1987 to March 1988. Summarized results were as follows: 1. AZT showed excellent antibacterial activities against Escherichia coli, Klebsiella pneumoniae, Proteus sp. and Haemophilus influenzae, and MIC80 values of AZT against these organisms were lower than 0.20 microgram/ml. 2. Antibacterial activities of AZT were superior to cephem antibiotics compared against Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii and Serratia marcescens. 3. The MIC50 and MIC80 of AZT against Pseudomonas aeruginosa were 12.5 micrograms/ml and 25 micrograms/ml, respectively. 4. AZT did not show any antibacterial activity against Acinetobacter sp. and Xanthomonas maltophilia.

[The new generation of laboratory automation systems].

Laboratory automation is essential to release laboratory technicians from simple routine work, allowing them to make use their time for more skilled tasks. In 1998, we developed a Total Infection Control System called "Dr. Fleming", and would like to introduce it into practical use. This systems is expected to help physicians by providing highly valuable test results and useful information.

[A trial approach to the ideal control-management and systematization of clinical laboratory].

Clinical laboratory examinations have continued to expand with the increase in demand for medical care. However, recent high-technology, medical care has made introduction of a new system of quality control in the clinical laboratory indispensable. First, regarding personnel (clinical laboratory technicians), reducing staff hours led to a drop in the employment of newcomers and subsequent aging of the staff. Next, introducing a system to save labor is extremely expensive, making the cost effectiveness very poor and consequently making it difficult to introduce such a system in many laboratories. This symposium was initiated to assist the search for ways in which the present clinical laboratory can survive despite rising personnel expenses and reagent costs, while reducing medical expenses. And at the same time developing a guide to establishing our ideal for the clinical laboratory of the 21st century.

[The introduction of automation and systematization in the department of microbiology].

The Department of Laboratory Medicine is considered to be the transmission subdivision of medical information. However, most of the information is provided from the subdivisions of clinical chemistry, hematology and immunology. Moreover, the information is slowly transferred from the subdivision of microbiology. The Department of Laboratory Medicine at Saga Medical School developed a flexible microbiological test & information system in 1998, called Dr. Fleming. However, many methods of operation, effects, and problems are associated with the standardization and introduction of such a system. Therefore, the ideas of leaders in the field of microbiology and infectious disease will be discussed before establishing this system.

[Laboratory examination of contact factors].

The methods of measurement, clinical evaluation and application as molecular marker of contact factors are described. It is important to scrutinize the measurement methods and normal control of contact factors regularly in the institution. A slightly abnormal value and repeated measurement of APTT become valuable, which may reveal asymptomatic hereditary contact factor deficiencies. There is more thrombosis symptoms than bleeding in that cases. Acquired contact factors deficiency is recognized in disseminated intravascular coagulation or liver cirrhosis. Although the cases of the clinical application of molecular marker with contact factors is not enough, the diagnostic and therapeutical trials in hypercoagulopathy with sepsis are starting. There are many unknown clinical functions about contact factors, and careful assessment will be necessary in the previously reported cases of hereditary contact factors deficiency.

[Centralization of all medical laboratory data by laser disk filing system].

In recent years, the medical laboratory system is being widely applied. We describe the medical laboratory system of our Saga Medical School Hospital, including the electrocardiogram (ECG) and electroencephalogram (EEG) laser disk data filing systems. The main computer system of FUJITSU M-730/4 (main memory of host computer 13 MB) handles a large amount of data, in the fields of chemistry, hematology, serology, microbiology, blood banking and histology laboratory. All 17 chemistry and hematology automatic analyzers are linked through communication lines to the main computer. The important tasks of this system are data processing, storing of information in the database, data reporting and quality control statistics. This laboratory system is also connected to the total hospital information system of FUJITSU M-760, main memory 48 MB. For pattern recognition, ECG and EEG laser disk data filing systems have been constructed. The main purpose of these filing systems is mass storage of analog data signals in laser disk, computer assisted analysis and data communication. ECG and EEG analog data are converted into digital form by the analog-to-digital converter, and then transmitted over hospital telephone lines to the central computer system for analysis. The computer assisted statements are then sent back to the ECG terminals at the nurse station. As necessary, after the physician reads over the ECG, statements are printed in the final reports. These optical reporting systems are also linked to the total hospital information system. One of the main tasks in the laboratory is the control of the seemingly endless paper work.(ABSTRACT TRUNCATED AT 250 WORDS)

A variant translocation (11;22) (p15;q11) in a patient with chronic myelocytic leukemia.

Chromosome analyses were carried out on bone marrow specimens of a 55 year-old female patient with CML. As a result, a chromosome aberration, i.e. the translocation between chromosome 22 and 11, considered to be the 3rd case ever reported. However, chromosome 9 of bone marrow cells and chromosome analysis of the peripheral lymphocytes, were normal.

The current concepts of "normal values" and "clinical reference values" in the clinical laboratory (trials for determining clinical reference values of the Tokai University Hospital).

The purposes of this commentary are 1) to summarize the problems and ambiguities that cause many clinical pathologists to discard the term "normal values"; 2) to describe and define two terms, 'normal values" and "clinical reference values", which are becoming important for clinical interpretation of laboratory data; 3) to describe a new method for estimating clinical reference ranges of blood chemistry laboratory tests performed on selected Tokai University Hospital patients; and 4) to provide a bibliography of articles that have focused attention on the conceptual problems in this field.