Hydrodynamics-based gene delivery of naked DNA encoding fetal liver kinase-1 gene effectively suppresses the growth of pre-existing tumors.

Antiangiogenic gene therapy is a promising strategy for cancer treatment, which generally requires highly efficient delivery systems. To date, success of this strategy has depended almost exclusively on the delivery of high titers of viral vectors, which can result in effective transgene expression. However, their cytotoxicity and immunogenicity are a major concern for clinical applications. Recent advances in delivery efficiency of naked DNA could potentially meet the requirement for both high transgene expression and minimal side effects. To investigate whether naked DNA can be used for antiangiogenic cancer therapy, an expression plasmid was generated that encodes a soluble form of fetal liver kinase-1 (Flk-1) gene, a receptor for vascular endothelial growth factor (VEGF). Hydrodynamic injection of this plasmid resulted in close to 0.1 mg/ml of soluble Flk-1 protein in mouse serum and blocked VEGF-driven angiogenesis in matrigel in vivo. The same delivery significantly suppressed the growth of two different pre-existing subcutaneous tumors, Renca renal cell carcinoma and 3LL lung carcinoma. CD31 immunohistochemistry revealed that the tumor-associated angiogenesis was also highly attenuated in soluble Flk-1-treated mice. Thus, expression of genes by hydrodynamics-based gene delivery of naked DNA appears to be a promising approach for antiangiogenic cancer gene therapy.

Cytokine production by macrophages in association with phagocytosis of etoposide-treated P388 cells in vitro and in vivo.

Chemotherapy and radiotherapy are performed for cancer patients with the hope that dying cancer cells are safely scavenged by phagocytic cells such as macrophages. In this study, we examined cytokine production by macrophages during and after the phagocytosis of etoposide-treated P388 cells in vitro and in vivo. Etoposide caused apoptosis as early as 5 h after treatment, as assessed as to the exposure of phosphatidylserine, increase in membrane permeability and DNA ladder formation. Phagocytosis by phorbol myristate acetate (PMA)-treated THP-1 cells occurred marginally when P388 cells were treated with etoposide for 10 h, while it occurred significantly with P388 cells treated for 24 h, as evidenced by flow cytometry and confocal microscopy. PMA-treated THP-1 cells produced pro-inflammatory cytokines, such as interleukin (IL)-1alpha, IL-8 and macrophage migration inhibitory factor (MIF), but not anti-inflammatory cytokines among those tested at the mRNA level during and after the phagocytosis of apoptotic cells. IL-8 and MIF were also produced at the protein level, and the IL-8 production was dependent on cell-to-cell contact when the plasma membranes of apoptotic cells were intact enough not to leak one of the cytoplasmic enzymes, lactate dehydrogenase. In addition, etoposide-treated P388 cells induced neutrophil infiltration as well as MIP-2 production upon injection into the peritoneal cavity of either normal mice or mice with sterile peritonitis. When macrophages ingesting and/or binding apoptotic P388 cells were isolated from the mice with sterile peritonitis using a cell sorter, they were found to produce MIP-2 upon culture.

A new enzymatic assay for selectively measuring conjugated bilirubin concentration in serum with use of bilirubin oxidase.

A new enzymatic assay for selectively measuring conjugated bilirubin concentration in serum with use of bilirubin oxidase (BOD) has been developed. At pH 5.5 BOD can oxidize only conjugated bilirubin in the presence of reagents such as sodium fluoride and N-acetylcysteine which can decrease BOD reactivity to unconjugated bilirubin and bilirubin covalently bound to albumin (delta bilirubin). The resulting decrease in absorbance at 450 nm is linearly related to the concentration of conjugated bilirubin in serum. The BOD in this new assay was confirmed to oxidize conjugated bilirubin, and neither unconjugated nor delta bilirubin, based on both its reactivity to unconjugated bilirubin and HPLC results. This assay was found to give satisfactory results, such as in terms of the range of measurement, the reproducibility of the results, the lack of interference with coexisting substances in serum and the stability of the reagent solutions, in practical applications. The serum conjugated bilirubin concentrations determined using this assay correlate well with those determined by the HPLC analysis. This assay can be used for accurate monitoring of changes in the conjugated bilirubin concentration in patient sera. These findings suggest that the conjugated bilirubin assay is useful for fractional determination of bilirubin in icteric sera.

Characteristics of coronary heart disease in Japanese taxi drivers as determined by coronary angiographic analyses.

Several epidemiological studies have shown that the prevalence of ischemic heart disease is higher in occupational drivers than in people with other occupations. Although occupation categories can be surrogate measures for coronary risk factors, the relationships between taxi driving and severity of coronary heart disease (CHD) has not been investigated. Even more important, the contribution of risk factors to the severity of CHD in taxi drivers remains unclear. Our study tested the hypothesis that taxi driving could be associated with the severity of CHD. We also examined the relation between this occupation and risk factors and social lifestyle. We analyzed the coronary angiograms of 57 consecutive male taxi driver patients and compared them with those of 215 age-adjusted male non-taxi-driver patients. The number of diseased vessels and risk factors were compared between two groups. The prevalence of myocardial infarction and multi-vessel disease was higher in the taxi-driver patients than in the non-taxi-driver patients. The taxi-driver patients had higher prevalence of body mass index (BMI), diabetes, and smoking, higher levels of low-density lipoprotein cholesterol (LDL-C), and lower levels of apolipoprotein AI (ApoAI). Multiple logistic regression analysis showed that multi-vessel disease was associated with BMI and diabetes mellitus in taxi-driver patients. The taxi-driver patients were characterized by more extensive coronary atherosclerosis associated with higher prevalence of diabetes mellitus and obesity. These characteristics may be explained by in part their working environment.

[A study of methicillin-resistant Staphylococcus aureus (MRSA) infection in the urological field].

Clinical backgrounds of 35 patients with urogenital infection, from whom methicillin-resistant Staphylococcus aureus (MRSA) was isolated, were analyzed. Susceptibilities of these MRSA strains to various antimicrobial agents were also measured. Out of 35 MRSA strains, 29, 4 and 2 were isolated from urine, pus and sputum specimens, respectively, showing a definitely high isolation rate from urine. As for underlying diseases, 22 patients (62.8%), 11 (31.4%) and one each had a malignant tumor of the urinary tract or genital organ, prostatic hypertrophy, urolithiasis and vesicoureteral reflux, respectively. Patients aged at over 60 years numbered 20 (57%), and 32 patients (91.4%) were treated with some antimicrobial agent at the time of MRSA isolation. Out of 35 strains, 17 were isolated after total cystectomy with urinary diversion or transurethral surgery. As for the state of MRSA infection, 9 and 26 patients had single and polymicrobial infections, respectively, but none of patients had serious symptoms definitely thought to be caused by MRSA. On evaluation of susceptibilities of MRSA to various antimicrobial agents, the MRSA strains were found to be sensitive to minocycline, netilmicin and ofloxacin. From these results, MRSA strains isolated from patients treated in the field of urology were thought to rarely cause serious infectious symptoms, especially true for those isolated from the urine.

[Non-destructive and rapid identification of bacteria using near-infrared spectroscopy].

Near-infrared (NIR) spectroscopy has recently come to be applied extensively in agricultural, food and chemical industries, and pharmaceutical science. We have been attempting to expand this method in the field of medical science. For example, we tried to use NIR spectroscopy for determination of bacteria. As the first step of this attempt, we differentiated between Escherichia coli and Staphylococcus aureus using NIR spectroscopy. This method could still further differentiate Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA). Using those results as reference, the true name of bacteria from unknown bacteria was given. Not only untreated bacteria, but also we differentiated untreated MSSA, MSSA cultured in sub MIC concentration of ABPC and heat-killed MSSA. This identification method is sensitive to the bacterial concentration. In the future, the some new idea of a new direction of research from the result of plots of weights from two different bacteria will appear.

[Studies on phage type, beta-lactamase activity and sensitivity of multiple drug-resistant Staphylococcus aureus isolated from clinical specimens to cefmetazole].

The 868 strains of S. aureus were isolated at the Department of Pediatrics, Third Hospital and Aoto Hospital, The Jikei University, School of Medicine and the Kanagawa Prefectural Nursing and Hygienic School Hospital during 6 months from May to October in 1981. From them 66 strains not sensitive to CEZ were selected by a 3-concentration disk method. A total number of 54 strains except for 12 isolated from the same infant patient was examined for their MIC's for 6 drugs, CMZ, CEZ, CTM, CXM, MCIPC and GM. Moreover, phage typing and beta-lactamase activity determination were carried out in them. 1. Antibacterial activity Sixty-six (7.6) of 868 strains were not sensitive to CEZ. The MIC's of CMZ against these resistant strains were between 1.56 and 50 micrograms/ml with a peak between 3.13 and 6.25 micrograms/ml when the 10(5) cells/ml bacterial suspension were inoculated. CMZ was superior in antibacterial activity to CEZ and CXM by about 4 degrees and to CTM by about 3 degrees. MCIPC and GM has higher antibacterial activity against a few strains than CMZ. However, the number of strains with MIC higher than or equal to 50 micrograms/ml was 17 for MCIPC and 40 for GM, but only 2 for CMZ. Thus, the former 2 drugs were far inferior to the latter one. 2. Phage type (1) Nineteen strains (35.2%) had MIC's for CEZ greater than 50 micrograms/ml and CMZ less than 6.25 micrograms/ml. Seventeen of them belonged to the nontypable. (2) Fourteen (25.9%) had MIC's for CEZ greater than 100 micrograms/ml. Of them 9 were allocated to the group III, 3 to the mixed (I + II + III, I + III) group and 2 to the nontypable. (3) Of 20 strains (37.0%) which had MIC's for CEZ greater than 100 micrograms/ml and CMZ less than 6.25 micrograms/ml 5 belonged to the group I, 3 to the group III and 12 to the nontypable. (4) Five strains classified into the group I were all isolated at the Kanagawa Perfectural Nursing and Hygienic School Hospital. (5) Eleven of 15 strains belonging to the group III were isolated at the Third Hospital, The Jikei University, School of Medicine. (6) Seventeen of 21 strains isolated at the Aoto Hospital, The Jikei University, School of Medicine belonged to the nontypable. (7) Phage type was considered to be influenced by difference in areas, infection within hospitals, etc., 3. beta-Lactamase activity beta-Lactamase activity was demonstrated at levels between 0.07 and 3.26 mumol/min/mg in all 54 strains. There was no correlation between MIC for CEZ or CEZ and beta-lactamase activity. It was suggested that beta-lactamase might not contribute to mechanism of resistance of S. aureus to each drug examined.

[Basic aspects of Staphylococcus aureus and MRSA--typing and extracellular toxins and proteins].

Many kinds of staphylococcal typing systems have been developed and tested for epidemiology of Staphylococcus aureus. Coagulase typing and phage typing are more useful and important for this epidemiology. The methods of coagulase typing and phage typing were discussed in this paper. A large number of toxins and proteins of Staphylococcus aureus have been released in the field of infectious environment. Some of these toxins and proteins, working as causative agents of staphylococcal infection, are of interest and discussed here. MRSA shows the same mechanisms of infection as methicillin sensitive Staphylococcus aureus.

Rapid separation of DNA constituents, bases, nucleosides and nucleotides, under the same chromatographic conditions using high-performance liquid chromatography with a reversed-phase column.

Four components of three sets of DNA constituents, bases, deoxyribonucleosides and deoxyribonucleoside 5'-monophosphate, were sufficiently resolved under one set of chromatographic conditions using high-performance liquid chromatography with a reversed-phase column (Zorbax ODS) and the solvent 0.4 M NH4H2PO4 (pH 3.5). The effect of pH and salt concentration in the solvent on the retention of these compounds in the column was thoroughly investigated. Proportionality of peak height to the content, and reproducibility and recovery of the four bases were satisfactory under appropriate conditions and as little as 1 microgram of DNA could be analysed for its base composition by this method.

Production of proinflammatory cytokines by phorbol myristate acetate-treated THP-1 cells and monocyte-derived macrophages after phagocytosis of apoptotic CTLL-2 cells.

Because it is generally believed that apoptosis is not associated with inflammation, we hypothesized that the interaction of phagocytes with apoptotic cells provides a negative or null signal for inflammation. However, we recently found that the interaction led to the production of proinflammatory cytokines but not antiinflammatory cytokines, although the apoptotic cell membranes appeared to be intact. In this study, we examined in detail the relationship among the kinetics of apoptosis, phagocytosis and production of cytokines by macrophages. Among the time points examined, murine CTLL-2 cells became apoptotic in terms of cell size and exposure of phosphatidylserine after 12 h of culture in the absence of IL-2, and at the same time they began to be phagocytosed and lead to proinflammatory cytokine production by PMA-treated THP-1 cells (human macrophages). The phagocytosis of apoptotic cells by macrophages was also confirmed by confocal laser microscopy. The coculturing of human macrophages with murine apoptotic cells led to the production of human proinflammatory cytokines, notably IL-8, at both the mRNA level and the protein level. The coculturing of monocyte-derived macrophages with the apoptotic cells also led to the production of IL-8 protein. Both the phagocytosis and production of the cytokines were suppressed by either phospho-L-serine or RGDS (Arg-Gly-Asp-Ser), but not by RGES (Arg-Gly-Glu-Ser). Thus, the production of proinflammatory cytokines and phagocytosis of apoptotic CTLL-2 cells appear to be closely interrelated.

Production of proinflammatory cytokines by resident tissue macrophages after phagocytosis of apoptotic cells.

It is generally believed that apoptosis is not associated with inflammation. However, we have found that phagocytosis of apoptotic cells by PMA-treated THP-1 cells and human monocyte-derived macrophages led to the production of proinflammatory cytokines, notably IL-8. These macrophages were obtained either by PMA treatment or by M-CSF treatment, possibly affecting the cytokine production after phagocytosis of apoptotic cells. In order to exclude the possibility, we employed resident tissue macrophages such as Kupffer cells and alveolar macrophages in this study and examined the production of cytokines after phagocytosis of apoptotic cells. Kupffer cells produced proinflammatory cytokines MIP-2 and TNF-alpha at the mRNA level. The MIP-2 protein was also detected by means of ELISA. Alveolar macrophages also produced the MIP-2 protein after phagocytosis of apoptotic cells. Furthermore, apoptotic thymocytes induced a similar response by these macrophages. These findings do support the notion that macrophages are apt to produce proinflammatory cytokines after phagocytosis of apoptotic cells.

A 38 kDa precursor protein of aqualysin I (a thermophilic subtilisin-type protease) with a C-terminal extended sequence: its purification and in vitro processing.

The precursor of aqualysin I, an extracellular subtilisin-type protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, a protease domain, and a C-terminal extended sequence. In an Escherichia coli expression system for the aqualysin I gene, a 38 kDa precursor protein consisting of the protease domain and the C-terminal extended sequence is accumulated in the membrane fraction and processed to a 28 kDa mature enzyme upon heat treatment at 65 degrees C. The 38 kDa precursor protein is separated as a soluble form from denatured E. coli proteins after heat treatment. Accordingly, purification of the 38 kDa proaqualysin I was performed using chromatography. The purified precursor protein gave a single band on SDS-polyacrylamide gels. The precursor protein exhibited proteolytic activity comparable to that of the mature enzyme. The purified precursor protein was processed to the mature enzyme upon heat treatment. The processing was inhibited by diisopropyl fluorophosphate. The processing rate increased upon either the addition of mature aqualysin I or upon an increase in the concentration of the precursor, suggesting that the cleavage of the C-terminal extended sequence occurs through an intermolecular self-processing mechanism.

Antimicrobial activity of lipoprotein particles containing apolipoprotein Al.

Human plasma in vitro inhibits the growth of coagulase negative staphylococci, S. epidermidis, which may be pathogenic in the immunocompromised host. To determine the antimicrobial components, serum was fractionated by column chromatography, which revealed that elution areas where lipoproteins can be yielded had high antimicrobial activity against S. epidermidis. Therefore, lipoprotein fractions, including very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL), were separated by ultracentrifugation and incubated with S. epidermidis. All 3 lipoprotein fractions suppressed bacterial growth within the first 3 h but VLDL enhanced bacterial growth after 9 h of incubation compared with the control. HDL, however, inhibited bacterial growth throughout 21 h of incubation. To confirm these results, serum from healthy volunteers was separated by ion exchange column chromatography and again by HPLC to purify the antimicrobial fraction. In the protein analysis with gradient polyacrylamide-SDS gel, apolipoprotein Al (apo Al), which is a major apolipoprotein of HDL, was detected in the antimicrobial fraction. Therefore, this fraction was loaded onto an immunoaffinity column coupled with the anti-apo Al monoclonal antibody (Mab). Unbound fraction had no antimicrobial activity, but anti-S. epidermidis activity was recovered from the bound fraction which consisted mainly of apo Al, All and apo C in protein composition. These results indicated that the antimicrobial activity was associated with the apo Al-containing lipoprotein particles (HDL). This property of HDL may directly affect bacterial growth and promote the self-defense mechanisms of normal and immunocompromised individuals.

Interaction of phagocytes with apoptotic cells leads to production of pro-inflammatory cytokines.

It is generally believed that apoptosis is not associated with inflammation. To explore the possibility that the interaction of phagocytes with apoptotic cells provides a negative or null signal for inflammation, we examined the cytokine production by thioglycollate-induced peritoneal exudate cells (PEC) upon interaction with a murine T cell clone (CTLL-2) cultured in the absence of interleukin-2 (IL-2). Coculturing of PEC with apoptotic CTLL-2 cells led to the production of not only anti-inflammatory cytokines but also pro-inflammatory ones, notably MIP-2, at the mRNA level, and neutrophils were accumulated in vivo in response to the culture supernatant. Our findings suggest the possibility that apoptosis may be associated with leukocyte infiltration in vivo in some situations.