Yoichi Hirakata

Deletions of p15 and/or p16 genes as a poor-prognosis factor in adult T-cell leukemia.

PURPOSE To determine the frequency of the deletions of p15/p16 genes in adult T-cell leukemia (ATL) cells and to evaluate their value in the diagnosis of clinical subtypes of ATL patients and the prediction of their clinical outcome. MATERIALS AND METHODS Peripheral-blood samples from 114 patients with ATL were examined by Southern blot analysis. In five chronic-type patients who showed disease progression to acute type, serial samples also were examined. RESULTS Among 114 patients, 28 (24.6%) showed the deletions of p15 and/or p16 genes. The results were well correlated with the clinical subtypes. Patients with deleted p15 and/or p16 genes had significantly shorter survival times than the patients in whom both genes were preserved (P < .0001). A similar decline in survival time was observed in the analyses within the same subtypes. In multivariate analysis using the Cox proportional hazard model, the deletions of p15 and/or p16 genes emerged as an independent prognostic indicator. Moreover, three of the five chronic-type patients who progressed to acute type lost the p16 gene alone or both the p15 and p16 genes at their exacerbation phase. CONCLUSION The results suggest the following: (1) that the deletions of p15 and/or p16 genes play a key role in the progression of ATL; and (2) that these deletions are reliable prognostic factors that predict shortened survival times.

Antimicrobial activity of superoxidized water

We tested the antimicrobial activity of superoxidized water against methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, Escherichia coli, Pseudomonas aeruginosa and Burkholderia cepacia. The number of bacteria was reduced below detection limit following incubation in superoxidized water for 10 s. The bactericidal activity of superoxidized water was similar to that of 80% ethanol, but superior to that of 0.1% chlorhexidine and 0.02% povidone iodine. We conclude that superoxidized water is a low cost but powerful disinfectant.

Clinical and bacteriological characteristics of IMP-type metallo-β-lactamase-producing Pseudomonas aeruginosa

IMP-type metallo-beta-lactamase-producing bacteria have recently emerged worldwide. We conducted a case-control study in which 69 inpatients harboring bla(IMP)-positive Pseudomonas aeruginosa and 247 control subjects with bla(IMP)-negative pathogens were investigated. Prolonged hospitalization, antineoplastic chemotherapy, corticosteroid therapy (P=.001), and indwelling urinary catheters (P=.04) were risk factors for isolation of bla(IMP)-positive pathogens. The predominant source was urine (P=.001). The duration of antibiotic treatment and the total dose (including of carbapenems) were significantly greater among case patients than among control subjects (P<.01). bla(IMP)-positive P. aeruginosa isolates were more frequently resistant to multiple drugs (P=.001) and caused more infections (P=.001) than bla(IMP)-negative pathogens. There were no significant differences in bacteriological outcome (P=.94); however, infection-related death was more frequent among case patients than among control subjects (P=.023). These results suggest that precautionary measures against the spread of bla(IMP)-positive isolates are needed, because, for most of such pathogens, no antibiotic is potent enough to be used as a single agent in treatment of infection.

Immunokinetics in severe pneumonia due to influenza virus and bacteria coinfection in mice

Coinfections of bacteria and influenza are a major cause of excessive mortality during influenza epidemics. However, the mechanism of the synergy between influenza virus and bacteria are poorly understood. In this study, mice were inoculated with influenza virus, followed 2 days later by inoculation with Streptococcus pneumoniae. The kinetics of viral titres, bacterial numbers and the immune response (cytokine and chemokine production) were also analysed. Short-term survival correlated with pathological changes in the lungs of infected mice. Influenza virus or S. pneumoniae infection alone induced moderate pneumonia; however, severe bronchopneumonia with massive haemorrhage in coinfected mice, which caused death of these mice ∼2 days after inoculation with S. pneumoniae, was noted. Intrapulmonary levels of inflammatory cytokines/chemokines, type‐1 T‐helper cell cytokines and Toll-like receptors, and the related mitogen-activated protein kinase signalling molecules (phosphorylated extracellular signal-regulated kinase ‐1 and ‐2, p38 and c‐Jun N‐terminal kinase), were increased in coinfected mice. These results suggest that immune mediators, including cytokines and chemokines, through Toll-like receptors/mitogen-activated protein kinase pathways, play important roles in the pathology of coinfection caused by influenza virus and Streptococcus pneumoniae.

Comparative in vitro exoenzyme-suppressing activities of azithromycin and other macrolide antibiotics against Pseudomonas aeruginosa.

The inhibitory effects of azithromycin (AZM), a new 15-membered macrolide antibiotic, on the production of exotoxin A, total protease, elastase, and phospholipase C by Pseudomonas aeruginosa were determined, and the virulence-suppressing effects of AZM were compared with those of erythromycin (EM), roxithromycin (RXM), and rokitamycin (RKM). The effect of exposure of P. aeruginosa PA103 or B16 in cultures to sub-MICs of these macrolide antibiotics on the production of exoenzymes was determined. AZM suppressed the in vitro production of extracellular and intracellular exotoxin A by P. aeruginosa PA103 more than did EM, even at a concentration of only 2 micrograms/ml. At concentrations of between 4 and 32 micrograms/ml, AZM also inhibited total protease, elastase, and phospholipase C production by P. aeruginosa B16 more than did EM, RXM, and RKM. AZM was effective in suppressing exotoxin A and total protease production through 24 h of incubation in the presence of drug at sub-MICs, but it had no significant effect on either the growth of P. aeruginosa or its total protein production. Moreover, at a concentration of 4 micrograms/ml, AZM suppressed exoenzyme production by other strains of P. aeruginosa more than did EM. These findings indicate that AZM, EM, RXM, and RKM each has an inhibitory effect on exoenzyme production separate from the antimicrobial effect and that, of these macrolides, AZM has the strongest virulence-suppressing effect.

Potential effects of erythromycin on host defense systems and virulence of Pseudomonas aeruginosa.

We evaluated several potential effects of erythromycin (EM) on host defense systems and the virulence of Pseudomonas aeruginosa. Peritoneal macrophages obtained from mice given 250 mg of EM per kg of body weight for 7 days by the intraperitoneal, intravenous, subcutaneous, or oral route produced significantly greater amounts of thymocyte-activating factors. These data suggest that EM enhances the in vivo production of cytokines, such as interleukins 1 and 6. Treatment of P. aeruginosa D4 with subinhibitory concentrations of EM enhanced the association of bacteria with murine Kupffer cells in vitro and increased bacterial clearance from the blood in mice. EM suppressed the in vitro production of exotoxin A, total protease, elastase, and phospholipase C by P. aeruginosa D4; exotoxin A production by P. aeruginosa PA-103; and total protease production by P. aeruginosa B16 and PAO1 in a generally dose-dependent manner. These data demonstrate that EM produces various effects in addition to its direct antimicrobial activity, suggesting that it has potential as an immunomodulator or bacterial virulence-suppressing agent against P. aeruginosa and other infections.

Comparison between Wako‐WB003 and Fungitec G tests for detection of (1→3)‐β‐D‐glucan in systemic mycosis

The limulus factor G reacts with (1→3)‐β‐D‐glucan, a major structural component of fungal cell walls. The Fungitec G test is a colorimetric assay that measures the concentration of (1→3)‐β‐D‐glucan and is used as a serodiagnostic test for deep mycosis. Wako‐WB003 is another assay for (1→3)‐β‐D‐glucan that determines the change in turbidity of the gelatin reaction of limulus factor G with (1→3)‐β‐D‐glucan. In five rabbits inoculated intravenously with 1 × 107 CFU of Candida albicans, the concentration of (1→3)‐β‐D‐glucan measured by the fungitec G test increased gradually reaching a peak of 660.9 ± 427.9 pg/ml (mean ± SD) 4 days after inoculation, but to 42.225 ± 41.275 ng/ml on day 6 in the Wako‐WB003 test. In one rabbit challenged intravenously with 5 × 106 CFU of C. albicans, (1→3)‐β‐D‐glucan increased to 101.5 pg/ml on day 4 on the fungitec G test, whereas the level remained below the detection limit of the Wako‐WB003 test throughout the course of the disease. We also detected high concentrations of (1→3)‐β‐D‐glucan in 11 patients with candidemia, 4 with suspected candidemia, 1 with invasive pulmonary aspergillosis, and 12 patients with aspergilloma. The concentration of (1→3)‐β‐D‐glucan measured by the Fungitec G test was > 150, > 1006.8, 312.1, and 55.6 ± 37.4 pg/ml (range, 20.1–138.0 pg/ml), and by the Wako‐WB003 test > 153.000, > 17.70, 153.000 and 2.645 ± 7.248 ng/ml (range, < 25.20 ng/ml) in these patients, respectively. In contrast, the concentration of (1→3)‐β‐D‐glucan in 9 patients with pulmonary cryptococcosis and 6 with superficial candida colonization ranged from < 13.2 and < 15.3 pg/ml in the Fungitec G test and < 0.53 and < 0.12 ng/ml in Wako‐WB003 test. There was a weak relationship between the concentration of (1→3)‐β‐D‐glucan measured by the Fungitec G test and Wako‐WB003 test (r = 0.521). Our results indicate that the sensitivity of the Wako‐WB003 test is lower than that of the Fungitec G test. J. Clin. Lab. Anal. 11:73–77.

Effect of anti-PcrV antibody in a murine chronic airway Pseudomonas aeruginosa infection model

Pseudomonas aeruginosa is one of the most important pathogens in patients with chronic airway conditions, such as cystic fibrosis and diffuse panbronchiolitis. Type III secretion system-mediated virulence factors contribute to the lung damage in chronic P. aeruginosa infection. The effects of the anti-PcrV immunoglobulin (Ig)G, which blocks the type III secretion system, were evaluated in a mouse model of chronic P. aeruginosa infection. On bacteriological examination, anti-PcrV IgG showed no bactericidal effects. On bronchoalveolar lavage fluid (BALF) analysis, total cell number and neutrophil ratios in the anti-PcrV IgG-treated groups were lower than those in the control group. In addition, macrophage inflammatory protein-2, tumour necrosis factor-α, and interleukin-β concentrations in BALF were lower in the anti-PcrV IgG-treated groups when compared with controls. Plasma anti-PcrV IgG titre was elevated after administration of anti-PcrV IgG. Although plasma titre decreased gradually, a significant concentration was maintained during the experimental period. These data suggest that anti-PcrV immunoglobulin G reduces the inflammatory reaction caused by chronic Pseudomonas aeruginosa respiratory infection and may be useful in treating respiratory diseases.

Chemotherapy targeting methylthioadenosine phosphorylase (MTAP) deficiency in adult T cell leukemia (ATL)

Methylthioadenosine phosphorylase (MTAP) is an important enzyme used for the salvage of adenine and methionine. Cells lacking this enzyme are expected to be sensitive to purine synthesis inhibitors and/or methionine starvation. We reported previously that the MTAP gene is deleted in adult T cell leukemia (ATL) cells. In the present study, we expanded our series and used a real-time quantitative PCR assay for accurate diagnosis of the deletion and nine of 65 primary ATL samples (13.8%) were MTAP negative. In spite of this low incidence, ATL cells showed significantly higher sensitivity to L-alanosine, an inhibitor of de novo adenosine monophosphate (AMP) synthesis, than normal lymphocytes, suggesting that the MTAP gene is inactivated not only by deletion but also by other mechanisms. Indeed, a real-time quantitative RT-PCR assay disclosed that primary ATL cells had significantly lower MTAP mRNA expression than normal lymphocytes. Since MTAP-negative ATL cell lines also showed much higher sensitivity to L-alanosine than MTAP-positive ATL cell lines, we used these cell lines to investigate whether it is possible to develop selective therapy targeting MTAP deficiency. A substrate of MTAP, methylthioadenosine (MTA) or its substitutes rescued concanavalin A (Con A)-activated normal lymphocyte proliferation from L-alanosine toxicity. All the compounds except 5′-deoxyadenosine, however, also caused the undesirable rescue of MTAP-negative ATL cell lines. 5′-Deoxyadenosine had the desired ability to rescue hematopoietic progenitor cells without rescuing ATL cell lines. These results support the rationale for a chemotherapy regimen of L-alanosine combined with 5′-deoxyadenosine rescue in MTAP-deficient ATL.

Effects of sub-MICs of erythromycin and other macrolide antibiotics on serum sensitivity of Pseudomonas aeruginosa.

We examined the effects of sub-MICs of erythromycin (EM) and other macrolide antibiotics on the serum sensitivity of Pseudomonas aeruginosa. P. aeruginosa S-6 grown for 36 and 48 h on agar with 10 micrograms of EM per ml (1/10th the MIC) showed significantly increased sensitivity to human serum bactericidal activity compared with those of bacteria grown on agar without EM (P < 0.05). No changes in serum sensitivity were observed in bacteria grown for less than 24 h. This increased sensitivity was apparent even at a concentration of 1.5 micrograms of EM per ml (1/67th the MIC) in bacteria grown for 48 h (P < 0.01). Among the other macrolide antibiotics tested, clarithromycin also enhanced sensitivity to serum, but there were no changes in the sensitivities of bacteria grown on agar with kitasamycin, josamycin, rokitamycin, or oleandomycin even at a concentration of 12 micrograms/ml (1/16th, 1/16th, 1/8th, and 1/33rd the MICs, respectively). P. aeruginosa S-6 grown on agar with subinhibitory concentrations of EM showed decreased cell surface hydrophobicity in a dose-dependent manner, whereas oleandomycin and rokitamycin, even at a concentration of 12 micrograms/ml, induced a slight decrease in hydrophobicity which was approximately equivalent to that of 1.5 micrograms of EM per ml. Among six other strains of the nonmucoid phenotype, three strains became more sensitive to serum by exposure to 10 micrograms of EM per ml for 48 h. In contrast, no evident correlation between EM treatment and a change in serum sensitivity was observed in six strains of the mucoid phenotype, as judged by the results of experiments with both 2 and 0.4% serum. These results show that EM at subinhibitory concentrations enhances the serum sensitivity of some P. aeruginosa strains. Since induced serum sensitivity was accompanied by a decrease in bacterial cell surface hydrophobicity, EM may render P. aeruginosa more serum sensitive by changing the cell surface structure(s) of this organism.