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A man in his 70s visited our department for dyspnea with pulmonary infiltrate that was unresolved by antibiotics. He had been taking Sansoninto for five years and doubled its dose a month ago. After discontinuing Sansoninto without any additional medications, his symptoms gradually disappeared, and pulmonary infiltration improved. Drug lymphocyte stimulation tests showed a positive result for Sansoninto. We diagnosed this patient with Sansoninto-induced lung injury. Sansoninto is a combination drug that consists of sansonin, bukuryo, senkyo, chimo, and kanzo. This paper reports the first case of Sansoninto-induced lung injury and discusses the mechanism considering its components.

This paper aims to grasp how the Environmental Impact Assessment (EIA) is implemented for offshore oil and gas developments in five countries advancing in this industry and to help EIA practitioners to develop and review EIA reports for marine resource developments. For this purpose, we reviewed the legislative literature, analyzed EIA reports prepared in the field, and interviewing EIA practitioners. Our results found the similarities and the differences in EIA methodologies, environmental impacts subject to assessment, and management measures by country as well as by project phase. Consistency among the five countries can be an effective reference for any marine resource developments regardless of the country.

33歳，男性。31歳時慢性骨髄性白血病にて非血縁者間同種骨髄移植を施行された。移植40日後，肝酵素の急激な上昇がみられ，急性GVHD(graft-versus-host disease)と診断された。肝·胆道系酵素の上昇，汎血球減少が遷延し，慢性GVHDに移行したと考えられた。プレドニゾロン，シクロスポリン内服にて加療されるも，移植10ヵ月後に全身に紅斑，皮膚硬化が出現した。皮膚症状の改善を認めず，2003年7月当科に入院した。頭部は疎な毛髪，厚い鱗屑と痂皮を認め，顔面に色素脱失と鱗屑や血痂を付着したびらんを認めた。体幹·四肢には広範囲に色素沈着·脱失が存在し，米粒大の小水疱が散在，全指趾で爪甲の消失，指の屈曲制限を認めた。両踵，足底外側縁，母趾球部に最大5×3cmの潰瘍を認めた。組織学的に表皮下水疱，リンパ球の表皮内浸潤，液状変性，コロイド小体，真皮上層に軽度の炎症細胞浸潤，付属器周囲のリンパ球浸潤を認めた。蛍光抗体直接法，抗核抗体は陰性で，貧血，肝·胆道系酵素上昇，CRP高値，低ガンマグロブリン血症を認めた。プレドニゾロンとシクロスポリンの内服を増量し，ジフルプレドナート，アズレン外用にて加療，皮疹は改善した。経過中白血球·血小板減少を認め，シクロスポリンによる副作用と考え，タクロリムス水和物内服に変更した。足底以外の潰瘍，びらんはほぼ上皮化したが，右踵部の潰瘍は，安静や種々の外用剤，被覆剤を使用したが，難治であった。トラフェルミンとトレチノイントコフェリルの併用で踵部の潰瘍は縮小，退院5ヵ月後にはほぼ上皮化した。

Surveillance of Helicobacter pylori antimicrobial susceptibility reflecting the general population in Japan is limited. The antimicrobial susceptibilities of 3,707 H. pylori strains isolated from gastric mucosa samples of previously untreated patients diagnosed with gastroduodenal diseases at 36 medical facilities located throughout Japan between October 2002 and September 2005 were evaluated. Using an agar dilution method for antimicrobial susceptibility testing of H. pylori , the MIC distributions and trends during the study period for clarithromycin, amoxicillin, and metronidazole were studied. While the MIC 50 and MIC 90 for clarithromycin did not change during the 3-year period, the MIC 80 showed a 128-fold increase. Furthermore, the rate of resistance increased yearly from 18.9% (2002 to 2003) to 21.1% (2003 to 2004) and 27.7% (2004 to 2005). With a resistance rate of 19.2% among males compared to 27.0% among females, a significant gender difference was observed ( P < 0.0001). Our study shows that in Japan, there is an evolving trend towards increased resistance to clarithromycin with geographical and gender differences as well as between clinical disease conditions. No significant changes in resistance were observed for amoxicillin and metronidazole during the period. While the benefit of H. pylori antimicrobial susceptibility testing has been debated in Japan, current empirical regimens are not based on susceptibility data representative of the general population. The development of an effective H. pylori eradication regimen in Japan will require continued resistance surveillance as well as a better understanding of the epidemiology of resistance.

SummaryDiffuse panbronchiolitis (DPB) is a distinctive form of small airway disease, which is characterized by chronic inflammation with lymphocyte infiltration around bronchioles. The aim of this study was to evaluate the importance of factors related to apoptosis in peribronchiolar lymphocytes of DPB. We employed immunohistochemical methods for the localization of Bax (a promoter of apoptosis), Bcl-2 (an inhibitor of apoptosis), and caspase-3 (a key executioner molecule of apoptosis) in lung tissues of five patients with DPB. In all patients, immunostaining for Bax was almost completely absent in accumulated lymphocytes around the bronchioles and in lymphocytes of the parafollicular area that correspond to a zone populated by T cells. In contrast to the reaction for Bax, Bcl-2 immunoreactivity was uniformly strong in all of the patients. The pattern of staining for caspase-3 was similar to that for Bax in all of the patients. In normal lung tissue, a few lymphocytes showed negative immunostaining for Bcl-2 and a positive reaction for caspase-3. Our results suggest that Bcl-2 protein may provide T-lymphocyte survival and hypercellularity in the bronchioles, thereby contributing to the progression of DPB.

We examined the protective effect of intratracheal immunization with Pseudomonas aeruginosa pili protein against respiratory infection caused by P. aeruginosa. Mice were immunized intratracheally or subcutaneously with purified pili protein or bovine serum albumin as a control. Intratracheally but not subcutaneously pili protein-immunized mice showed significant improvement of survival after intratracheal challenge with the PAO1 strain. Furthermore, bacterial cell counts in pili protein-immunized murine lungs were significantly decreased compared to controls at 18 h after the challenge. Antipili protein antibody titers in bronchoalveolar lavage fluid of intratracheally pili protein-immunized mice were higher than in bovine serum albumin immunized mice. However, antipili antibody titers were not increased in bronchoalveolar lavage fluid of subcutaneously pili protein-immunized mice, despite the high serum antipili antibody titers. Inoculation of P. aeruginosa induced immediate increases in interleukin-12 and interferon-gamma in bronchoalveolar lavage fluid of pili protein-immunized mice, reflecting an adequate and rapid immune response against P. aeruginosa respiratory tract infection. Our findings suggest that intratracheal pili protein immunization is effective against respiratory tract infection caused by P. aeruginosa in mice.

The effects of peritoneal rest for 24 h during peritoneal dialysis and hemodialysis combination therapy were investigated using cultured human peritoneal mesothelial cell (HPMC) models. Cell activity was investigated by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenylterazolium bromide (MTT) assay after exposing HPMCs to peritoneal dialysis fluids (PDFs) with different pH levels. The following PDFs (50 microl/well) were used for exposure durations of 30 or 240 min: acidic heat-sterilized PDFs (L-H PDF, pH 5.5) and neutral heat-sterilized PDFs (N-H PDF, pH 6.7). Control wells were exposed to M-199 Hanks medium containing 20% fetal bovine serum (FBS) for 30 or 240 min. Supernatants were then aspirated from each well and M-199 culture medium containing 20% FBS (50 microl) was added to each well to rest HPMCs for 24 h before investigation of MTT activity. The activity of HPMCs exposed to L-H PDF for 240 min decreased to approximately 20% and 15% when compared with controls (glucose concentrations of 1.36% and 3.86%, respectively; P < 0.01 versus control, Tukey-Kramer test), and to approximately 60% and 40% after exposure to N-H PDF for 240 min (glucose: 1.36% and 3.86%; P < 0.01). The activity of HPMCs exposed to L-H PDF for 240 min followed by rest was approximately 20% and 4% when compared with controls (glucose: 1.36% and 3.86%; P < 0.01) and was 93% and 96% when compared with controls after exposure to N-H PDF for 240 min followed by rest (glucose: 1.36% and 3.86%). These findings suggest that rest for 24 h after exposure to N-H PDF improves the activity of HPMCs.

Background: Although Helicobacter pylori has been regarded as a pathogen of gastric cancer, the mechanism by which H. pylori is involved in gastric carcinogenesis remains unknown. To clarify the role of H. pylori in carcinogenesis, the expression of tumor suppressor p53 and its regulator multiple double minute 2 (MDM2) in gastric mucosa were examined before and after H. pylori eradication. Methods: Biopsy specimens were obtained from 31 patients with H. pylori-infected gastric mucosa. Endoscopic biopsies were repeated 6 months after successful eradication. In addition, biopsy specimens from 12 patients with non-infected gastric mucosa were obtained. Immunohistochemical analysis was performed on the specimens using primary antibodies specific for p53 and MDM2. Results: Six months after H. pylori eradication, labeling indices for p53 were significantly reduced in the gastric corpus (2.3-fold; P

Long-term administration of macrolide antibiotics reduced the number of lymphocytes in bronchoalveolar lavage fluid in patients with chronic airway inflammatory disease. To evaluate the inflammatory activity of macrolides, their effect on apoptosis of activated lymphocytes isolated from human peripheral blood was compared with that of other antibiotics. Macrolides, including clarithromycin and azithromycin, at a final concentration of 100 μg/ml accelerated apoptosis of activated lymphocytes, while other antibiotics such as fosfomycin sodium, β-lactams — ceftazidime, piperacillin sodium and biapenem, and a quinolone, ofloxacin, did not cause significant induction of apoptosis. Our results suggest that 14- or 15-membered ring macrolides are specifically involved in the augmentation of apoptosis of activated lymphocytes, and this may be of value therapeutically for chronic airway diseases.

Objective In the current studies, we investigated the clinical effects of long-term macrolide antibiotic therapy for patients with chronic small airway disease (CAD) that clinically and radiologically mimics but is pathologically distinct from diffuse panbronchiolitis (DPB).Patients and Methods Twenty-one Japanese patients were selected on the basis of clinical criteria for DPB and were categorized as DPB or CAD following histological evaluation of surgical lung biopsies. All patients received long-term macrolide therapy, and therapeutic results were compared for the DPB and CAD groups.Results Clinical, laboratory, radiological, and bacterial features, as well as neutrophilia in bronchoalveolar lavage fluid were strikingly similar in both groups. Long-term treatment with macrolides improved the clinical symptoms and PaO2 in both groups. There was a significant improvement in forced expiratory volume in one second (FEV1), vital capacity (VC), and %VC in patients with DPB but not in patients with CAD. Neutrophilia in bronchoalveolar lavage fluid was also reduced following therapy in DPB patients but was refractory in CAD patients.Conclusion Based on the different responses to macrolides, CAD might be associated with conditions distinct from those of DPB. Nevertheless, low-dose macrolide therapy may be applied in CAD to achieve clinical improvement, such as in respiratory symptoms and PaO2.

Osteopontin (OPN) produced by alveolar macrophages functions as a fibrogenic cytokine in the development of bleomycin (BLM)-induced murine pulmonary fibrosis, and OPN mRNA is expressed on lung tissues from patients with idiopathic pulmonary fibrosis (IPF). The present study investigates plasma OPN levels in human interstitial pneumonia (IP) and their relationships with disease severity by analyzing the correlation between plasma OPN concentrations and pulmonary functions. The concentrations of OPN in plasma were measured in 17 patients with IP, in 9 with sarcoidosis and in 20 healthy controls using an antigen-capture enzyme-linked immunosorbent assay. The concentrations of OPN in plasma were significantly higher in IP patients than in those with sarcoidosis or in controls. Based on a Receiver Operating Characteristic curve analysis, cut-off points between 300 and 380ng/ml discriminated between IP and control subjects with 100% sensitivity and 100% specificity. In such case, the sensitivity for sarcoidosis decreased (55.5–33.3%) in cut-offs with 100% specificity. Plasma OPN levels inversely and closely correlated with arterial oxygen tension (PaO2) in patients with IP. Immunohistochemically, OPN was localized predominantly in macrophages and airway epithelium. These findings suggest that plasma OPN levels were found to be associated with the presence of IP, and that OPN play an important role in the development of IP.

This study compared the clinical characteristics and the effects of long-term macrolide antibiotic therapy of HLA-B54-positive and -negative cases in patients with diffuse panbronchiolitis (DPB). Thirty-two Japanese patients were enrolled who had the clinical criteria for DPB. All patients received long-term macrolide therapy, and therapeutic results were compared according to the presence or the absence of HLA-B54 antigen. Clinical, laboratory, radiological and bacterial features were strikingly similar in both groups before macrolide therapy. Long-term treatment with macrolides improved clinical symptoms, PaO(2), and forced expiratory volume in 1s (FEV(1)) equally in both groups. This study indicates that genetic susceptibility may not explain the pathogenesis of DPB, and that low-dose macrolide therapy can achieve clinical improvement irrespective of genetic predisposition in DPB.

To elucidate the mechanisms underlining alpha3(V) collagen chain expression, we performed an initial analysis of the structure and function of the core promoter of the human COL5A3 gene. The core promoter, which lacks a typical TATA motif and has a high GC content, was defined within the -129 bp immediately upstream from the major transcription start site by transient transfection experiments. In this region, we identified four DNA-protein complexes, named A, B, C, and D, by a combination of DNase I footprinting and electrophoretic mobility shift assays. Electrophoretic mobility shift assays using mutant oligonucleotide revealed that the complexes A, B, C, and D bind to -122 to -117, the -101 to -96, the -83 to -78, and the -68 to -57 bp, respectively. The competition assays using consensus oligonucleotides and supershift assays with specific antibodies showed that complex A consists of CBF/NF-Y. In a chromatin immunoprecipitation assay, CBF/NF-Y protein directly bound to this region, in vivo. Functional analysis showed that CBF/NF-Y activated the gene, whereas the proteins of complexes B and C repressed its activity. Furthermore, overexpression of a mutant form of the CBF-B/NF-YA subunit, which forms CBF/NF-Y with CBF-A/NF-YB and CBF-C/NF-YC subunits, inhibited promoter activity.

Of the nonphysiological compounds in glucose-rich peritoneal dialysate, we investigated the cytotoxicity and synergistic cytotoxicity of acidity and glucose degradation products (GDPs) using human peritoneal mesothelial cells (HPMC). The effect of pH on cell viability was examined by adding 1N HCl to a phosphate-buffered solution (pHor = 5.5). We also examined the cytotoxic effects of various GDPs [glyoxal (GO), methylglyoxal (MGO), and 3-deoxyglucosone (3DG), alone or in combination] and pH (5.5 or 6.7). The cells were exposed to these solutions for 2 or 4 h. Cell viability was determined by 3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay. Although the MTT viability of HPMC was not decreased by GDP or acidity alone, the combination of acidity and GDP markedly decreased MTT viability, strongly suggesting the synergistic cytotoxicity of GDP and acidity.

To clarify the association of Chlamydia pneumoniae DNA in peripheral blood mononuclear cells (PBMCs) with acute coronary syndrome (ACS) and stable coronary artery disease (CAD) in Japanese adults, touchdown-nested polymerase chain reaction was used to detect the presence of C. pneumoniae DNA. The prevalence of C. pneumoniae DNA in PBMCs was similar in a comparison of 88 patients (52.3%) with ACS, 164 patients (50.0%) with stable CAD, and 88 control subjects (50.0%). Temporal changes in the prevalence of C. pneumoniae DNA in PBMCs were also assessed every 3 months during a 1-year period (n = 59). The prevalence was significantly higher in the first 3-month period (January through March) than in any of the other 3-month periods. In conclusion, the prevalence of C. pneumoniae DNA in PBMCs in patients with ACS or stable CAD was comparable to that in control populations. Furthermore, the presence of circulating C. pneumoniae was strongly associated with seasonal variability.

MICs of clarithromycin and amoxycillin for 253 isolates of Helicobacter pylori were measured by an air-dried microplate method and compared with the results obtained by the agar plate dilution method. The air-dried microplate method is performed by coating each well of a 96-well microplate with the test antibiotic and air-drying it. There were no marked differences between the air-dried microplate method and agar plate dilution methods in the MIC50 and MIC90 values or MIC ranges of clarithromycin obtained for the 253 isolates of H. pylori. More specifically, the MICs of clarithromycin for 114 (45.1 %) of the 253 isolates were the same by the air-dried microplate method as the agar plate dilution method, and the differences in the MICs of clarithromycin for a further 114 isolates (45.1 %) varied within one twofold dilution. The MICs of amoxycillin by the former method were in close agreement with the MICs obtained by the latter method: MICs of amoxycillin for 199 (78.7 %) of the 253 isolates were the same by both methods, and the differences in the MICs of amoxycillin for 42 isolates (16.6 %) varied within one twofold dilution. These results indicate that the air-dried microplate method is a useful method for determination of MICs, because the results obtained were in close agreement with those obtained by the standard agar plate dilution method. The air-dried microplate method is, therefore, a convenient and reliable method for determining the MICs of clarithromycin and amoxycillin for H. pylori isolates.

A 70-year-old man presented with pancytopenia in August 2000, and the results of a bone marrow examination performed in January 2001 confirmed the diagnosis of refractory anemia. He was treated with cyclosporine (CsA) at 3.3 mg/kg per day, and the pancytopenia improved. The patient complained of epigastralgia 21 months later, and a gastric endoscopic examination showed an ulcer with a cleaved bank. A biopsy revealed diffuse large B-cell lymphoma. In situ hybridization analysis detected no Epstein-Barr virus (EBV) in the lymphoma. CsA treatment was discontinued, and a gastrectomy was performed 31 days later. A detailed histologic examination revealed no infiltration of abnormal B-cells in the resected stomach. Although EBV-positive lymphoma is a known complication of immunosuppressive therapy, no causal association between immunosup-pressants and EBV-negative lymphoma has been established. The spontaneous remission observed after the withdrawal of CsA treatment suggests that immunosuppressive therapy can be a pathogenic factor in a subset of EBV-negative lymphomas.

Object It was reported that the liver fibrosis is reduced in patients with chronic liver disease type C after eradication of HCV. Degree of fibrosis was expressed by the classical fibrosis scoring system (F0-F4) at present. However a change of subtle reduction of fibrosis cannot be expressed in its fibrosis score and thus we measured an area of fibrosis and compared detailed change of the fibrosis area in paired liver biopsy. Methods For quantitative assessment of liver fibrosis, we measured an area of fibrosis using computed digital image analysis and pursued the rate of a fibrosis area for whole biopsied liver tissue of 25 patients with HCV infection (20 complete response patients to IFN therapy who showed viral eradication: CR and 5 non-treated ones with IFN but had conservative therapy: CT). Results In CR group, fibrosis rate was 7.2±1.5% before therapy, 2.7±0.5% after the therapy and they were significantly regressed. In CT group, fibrosis rate was 8.4±4.3% at the first biopsy, 15.9±7.7% at the second biopsy. Conclusions Regression of liver fibrosis could be confirmed in patients with virologic response to IFN even in patients with liver cirrhosis. Our quantitative evaluation provided information superior to that obtained by the classical fibrosis staging score system.

SUMMARY The balance between Th1 and Th2 response determines the outcome of Helicobacter pylori infection. Interferon (IFN)-γ plays an inductive role in gastric inflammation, whereas interleukin (IL)-4 counterbalances Th1 response and suppresses the development of gastritis. Th cell response is regulated by co-stimulatory factors. A co-stimulatory molecule, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), plays an inhibitory role in IL-2-dependent cell growth and mediates an optimal inhibitory signal to Th1 and Th2 cells. We administered anti-CTLA-4 monoclonal antibody (MoAb), which blocks CTLA-4 signalling, to examine the relative role for this signalling during maturation of Th1 and Th2 cells in H. pylori infection in mice. Mice treated by anti-CTLA-4 MoAb within the first week of infection showed an inhibition of gastric inflammation, accompanied by an increasing ratio of H. pylori-specific IgG1/IgG2a in serum following infection. Furthermore, the treatment resulted in the higher ratio of IL-4/IFN-γ by splenocytes in response to H. pylori antigen at 6 weeks after infection, compared with untreated mice. These results suggest that the predominance of Th2 response by CTLA-4 blockade leads to an inhibition of the development of gastric inflammation. CTLA-4 signalling could contribute to the regulation of Th subsets and the development of gastric inflammation in H. pylori infection.

We analyzed the efficiency of the quantitative real-time PCR assay for cytomegalovirus (CMV) reactivation in adult T-cell leukemia-lymphoma (ATL) patients and compared the results with those obtained with qualitative nested PCR and antigenemia assays. The viral load obtained by the real-time PCR assay closely paralleled the number of antigen-positive cells obtained with the antigenemia assay. Real-time PCR revealed that a large number of DNA copies could be present even in samples assessed as negative or low in antigen-positive cells (0 to 10 antigen-positive cells/50,000 cells) by antigenemia assay. CMV copy numbers did not differ between the negative and low-antigen-positive groups. When the input concentration for real-time PCR assay was 2,500 to 5,000 copies/ml, the positivity rate for the nested PCR assay was 47.3%, while the positivity rate was more than 90% at an input concentration of ≥50,000 copies/ml. Real-time PCR is more sensitive than the antigenemia and nested PCR assays. Moreover, real-time PCR was able to detect CMV reactivation earlier than the antigenemia and nested PCR assays through the use of longitudinal analysis in four ATL patients with CMV pneumonia. In longitudinal assessments, analysis of the results suggested that a cutoff level of 5,000 copies/ml might be used to initiate treatment. Real-time PCR is more suitable for monitoring CMV reactivation in ATL patients than the antigenemia and nested PCR assays. CMV viral loads of 5,000 copies/ml are proposed as the cutoff for initiating antiviral therapy in ATL patients.

The aim of the present study was to test the hypothesis that inadequate improvement in heart rate variability (HRV) in the healing stage of acute myocardial infarction (AMI) is associated with left ventricular (LV) remodeling. The study group comprised 20 patients (14 men, 6 women; mean age, 61+/-12 years) with a reperfused first anterior AMI (/=24 h). The standard deviation of normal RR intervals (SDNN) was calculated from the 24-h ambulatory electrocardiogram recorded on day 3 of admission and at predischarge from the hospital. Left ventriculography was performed immediately after primary angioplasty and at predischarge. The change in SDNN (deltaSDNN) was compared with the change in the LV end-systolic volume index (deltaLVESVI), a parameter of LV remodeling. SDNN increased from 73+/-19 ms on day 3 to 109+/-35 ms at predischarge (p=0.0003). SDNN at predicharge and deltaSDNN correlated negatively with deltaLVESVI (r=-0.52, p=0.02, and r=-0.61, p=0.004, respectively), whereas SDNN on day 3 did not correlate with deltaLVESVI. Multiple regression analysis selected deltaLVESVI (p=0.02) as an independent factor of deltaSDNN. This study indicates that deltaSDNN and SDNN at predicharge are associated with deltaLVESVI in patients with a reperfused first anterior wall AMI, indicating that persistently reduced HRV is associated with postinfarction LV remodeling.

Deep-seated trichosporonosis is a lethal opportunistic infection in immunocompromised patients. Trichosporon asahii and T. mucoides are the most common strains of fungi that cause disseminated trichosporonosis. Thirteen patients were diagnosed with disseminated trichosporonosis over a 20 year period in Oita Medical University Hospital. The majority of them had underlying hematologic malignancies, for which they received cytotoxic chemotherapy resulting in neutropenia. For the rapid diagnosis of this condition, we developed a novel nested-PCR assay that detected DNA specific for Trichosporon asahii and Trichosporon mucoides in the serum of patients with the condition. In a retrospective study using these serum samples, the specific DNA fragment was detected a few days to a few weeks earlier than blood culture. To treat this infection, we studied the efficacy of granulocyte colony-stimulating factor (GCS-F) alone and in combination with antifungal agents against disseminated trichosporonosis in neutropenic mice. The results suggested that GCS-F might be a useful immunomodulator against Trichosporon infections in neutropenic mice and the therapeutic outcome improved when used in combination with fluconazole. Furthermore our experimental animal model demonstrated that disseminated trichosporonosis is induced by immunosuppresion in hosts with latent trichosporonemia which was detectable by the nested PCR but not by blood culture. We found that there is a critical period for the progression of disseminated trichosporonosis after entry of fungi into the bloodstream.

Summary Background : The widespread use of eradication therapy for Helicobacter pylori in Japan has led to an increase in antibiotic-resistant strains and the problem of re-treatment in cases of eradication failure. Aim : To perform drug sensitivity testing for metronidazole in 92 H. pylori-positive patients who had failed eradication treatment with first-line triple therapy, consisting of a proton pump inhibitor, amoxicillin and clarithromycin, and were administered metronidazole-containing second-line therapy. Methods : Second-line eradication therapy, consisting of rabeprazole (20 mg b.d.), amoxicillin (750 mg b.d.) and metronidazole (250 mg b.d.), was administered for 1 week and the eradication rates and influence of metronidazole resistance were determined. Results : The eradication rates for rabeprazole–amoxicillin–metronidazole were 88% (81/92) using intention-to-treat analysis and 91% (81/89) using per protocol analysis. The eradication rates were 97% (61/63) for metronidazole-sensitive strains and 82% (18/22) for metronidazole-resistant strains. Conclusions : As second-line H. pylori eradication treatment in Japan, rabeprazole–amoxicillin–metronidazole triple therapy is effective, even with metronidazole-resistant strains.

Summary Background : The resistance of Helicobacter pylori to clarithromycin has become one of the primary reasons for eradication failure. Aim : To compare the eradication rates of triple therapy using amoxicillin (A), clarithromycin (C) and rabeprazole (R) or lansoprazole (L) against clarithromycin-sensitive and clarithromycin-resistant strains. Methods : Two hundred and ninety-five patients were randomly divided into four groups and treated for 1 week: 147 cases were treated with RAC, i.e. 49 cases with R20C400 (10 mg R + 750 mg A + 200 mg C, twice daily), 48 cases with R40C400 (20 mg R + 750 mg A + 200 mg C, twice daily) and 50 cases with R40C800 (20 mg R + 750 mg A + 400 mg C, twice daily); 148 cases with treated with LAC (30 mg L + 750 mg A + 200 mg C, twice daily). Results : According to intention-to-treat and per protocol analyses, the eradication rates were 88% and 91% with RAC and 78% and 81% with LAC; the eradication rates with R20C400, R40C400 and R40C800 were 94%, 81% and 86%, respectively, in the intention-to-treat analysis. In addition, the eradication rates for clarithromycin-sensitive strains with RAC and LAC were 98% and 89%, respectively, and for clarithromycin-resistant strains with RAC and LAC were 8.1% and 0%, respectively. Conclusions : The eradication rate was significantly higher with RAC than LAC. The eradication rate for clarithromycin-resistant strains was low in both groups, and an improved eradication rate could not be achieved by changing the dose of clarithromycin or proton pump inhibitor.

Successful prophylactic administration of Helicobacter pylori whole cell sonicate (WCS) plus complete Freund's adjuvant (CFA) or aluminum hydroxide (ALM) against subsequent H. pylori infection was reported recently. Here we tested the effect of WCS plus TiterMax Gold (TMX) or ALM in mice with chronic H. pylori infection. Mice with chronic (18 weeks) H. pylori infection were injected intraperitoneally with H. pylori (Sydney strain) WCS plus ALM or TMX once weekly for three times. The number of colonizing H. pylori in the stomach, IgG1 and IgG2a levels, and local inflammatory status were determined after therapeutic immunization. H. pylori specific IgG1, but not IgG2a, was significantly induced in mice immunized with H. pylori WCS plus TMX or ALM. Immunization did not result in reduction of bacterial count or recruiting inflammatory cells to the stomach. Adjuvant H. pylori WCS resulted in induction of CD4+ Th2 cell-mediated immunity although it did not reduce bacterial density in mice with chronic H. pylori infection. Our results implied that CD4+ Th1 cell-mediated immunity, rather than Th2 cell dominant immunity, might play a role in reducing the number of bacteria in chronic H. pylori infection.

Background.Helicobacter pylori is accepted as a definite human gastric carcinogen from an epidemiological point of view despite insufficient experimental data. Although we previously showed that the number of p53 immunopositive cells in the atrophic gastric mucosa of H. pylori-infected Japanese monkeys gradually increased over time, data on p53 gene mutations were not obtained in that study. To obtain direct evidence of carcinogenesis associated with H. pylori infection, we investigated whether p53 gene mutations are present in the gastric mucosa of a nonhuman primate model susceptible to H. pylori. Materials and Methods. Using the DNA from gastric tissues obtained from six H. pylori-uninfected monkeys of different ages, nucleotide sequence of the wild-type p53 gene was determined by amplification of exons (Ex) 5, 6, 7 and 8 and sequencing. Gastric specimens obtained from eight Japanese monkeys that had been infected with H. pylori for different lengths of time (1.5–7.5 years), were analyzed for mutations in exons 5–8 of p53. Results. In the six H. pylori-uninfected monkeys, nucleotide sequences of p53 Ex 5–8 were completely common and no mutations were noted. However, among the monkeys that were infected with H. pylori over various periods of time, there was an accumulation of p53 nucleotide (amino acid) substitutions as the gastric atrophy score increased. Conclusions. We conclude that the appearance of p53 gene mutation may be closely associated with the degree of gastric mucosal atrophy, which depends on the duration of H. pylori infection. Searching for p53 gene mutations may be useful for studying the progression of gastric carcinogenesis associated with H. pylori.

Pseudomonas aeruginosa is a common causative agent of septicemia in compromised host and the entry site of organism is most commonly the respiratory and genitourinary tract. P. aeruginosa septicemia is often associated with vesicular or pustular skin lesions, subcutaneous nodules, deep abscess, cellulites and bullae. We report a case of P. aeruginosa pneumonia with multiple pustular skin lesions on the chest and leg. A 77-year-old male was admitted to our hospital complaining of fever, productive cough and eruptions. Laboratory findings revealed a leucocytosis (14,830/microliter) and an elevated CRP (21.72 mg/dl). The chest radiograph and computed tomography revealed a fluid level in preexisting bullae and a consolidation shadow with multiple cavities in the right upper lobe and nodular shadow with cavity in the left lower lobe. P. aeruginosa strain was isolated from the bronchial lavage and pustule. Blood cultures were negative. Skin biopsy specimens showed histologically a dense infiltrate of neutrophils in the horny cell layer. He was diagnosed as Pseudomonas aeruginosa pneumonia complicated with multiple pustular skin lesions. He was treated with antimicrobial agents for 24 days and his clinical condition improved.

Background. p53 gene mutations are believed to play a critical role in the development of gastric carcinoma. We examined the relation betweenHelicobacter pylori infection andp53 gene mutations of the gastric mucosa in human and animal models.Methods. To detect the originalp53 DNA sequences of the Japanese monkey and Mongolian gerbil, thep53 genes of these animals were amplified using the nested polymerase chain reaction method with primers for the humanp53 gene. Direct DNA sequencing of exons 5, 6, 7, and 8 of thep53 genes was performed by the dyedeoxy terminator method for gastric mucosa of humans, the Japanese monkey, and the Mongolian gerbil. The expression ofp53 was examined immunohistochemically in a Japanese monkey model.Results. Mutations of thep53 gene were identified in 52.4% of humanH. pylori-positive mucosa and in 100% of monkeyH. pylori-positive mucosa. However, no mutations of thep53 gene were found in theH. pylori-positive gastric mucosa of Mongolian gerbils. There were no mutations inH. pylori-negative gastritis mucosa of humans, monkeys, or Mongolian gerbils. Nuclear staining of p53 was seen in the glandular cells of theH. pylori-infected mucosa of Japanese monkeys, especially in the neck region of the glands.Conclusions. These findings demonstrate that theH. pylori infection can inducep53 point mutations in humans and the Japanese monkey and appear to be involved in the pathway leading to dysplasia or carcinoma. However, our direct DNA sequencing method showed nop53 mutations in the Mongolian gerbil model at present. Further studies with this model are needed.

We conducted a study for the growth of and selectivity for the desired microorganisms using a newly developed selective culture medium for Helicobacter pylori, Columbia horse blood agar HP (CHBHP), at three different Japanese clinical laboratories, Hokkaido, Kanto and Kyusyu. When standard strains and clinical isolates of H. pylori were examined, the recovery of the organism on the CHBHP media was comparable to that of conventional selective and nonselective media. However, colonies were obviously larger on the CHBHP media. These media yielded the highest H. pylori positive rate for clinical specimens at all the three laboratories. The detection rate of the CHBHP media in H. pylori-positive specimens was higher than that of media commonly used at the three laboratories (98.1% to 100% vs. 88.0% to 96.2%). The CHBHP media also achieved a higher detection rate for specimens from H. pylori-infected animals. CHBHP media have an excellent growth supporting ability and selectivity originating from Columbia agar base and do not require the combined use of non-selective media for the growth and isolation of the organism, resulting in lower cost. Thus, they are useful media for the selective culture and isolation of H. pylori from clinical and animal specimens.

We purified a 29-kDa Helicobacter pylori outer membrane protein (Omp29 protein) and cloned the gene encoding the protein from H. pylori strain ATCC 43504. The Omp29 gene corresponded to the reported JHP73 and the HP78–79 genes of H. pylori strains. A corresponding nucleotide fragment was detected in all 150 tested H. pylori clinical isolates by PCR or Southern blotting. The amplified Omp29-corresponding fragments were categorized into a ca. 770-bp-long group and a larger-fragment group. Sequence analysis indicated that the larger fragments were likely synthesized from the 770-bp fragments by insertion of an irrelevant fragment via 17-bp-long repeat sequences. Immunoblot analysis implies that the ca. 770-bp fragment is responsible for the protein homologous to Omp29, whereas the larger fragments are not responsible for those proteins or encoding antigenically distinct proteins. We postulate that the H. pylori outer membrane protein Omp29 can alter its antigenicity through gene modifications mediated by nucleotide transfer.

Suppression of endotoxin release and subsequent production of inflammatory cytokines is crucial in the treatment of septic shock. We investigated the effect of clindamycin (CLI) on endotoxic shock induced in mice byEscherichia colilipopolysaccharide (LPS). Mice were treated with CLI (160 to 600 mg/kg) or saline and then injected withE. coliLPS andd-(+)-galactosamine intraperitoneally 0.5 h after CLI administration. Pretreatment with CLI significantly improved survival in a dose-dependent manner (CLI, at 160, 300, and 440 mg/kg) and significantly lowered the peak concentrations of tumor necrosis factor alpha and interleukin-1β(IL-1β) in serum. However, the peak concentrations of IL-6 in the sera of CLI-treated mice were higher than in control mice. Our findings suggest that CLI alters LPS-induced inflammatory cytokine production and suppresses endotoxin-induced mortality in this murine model.

The blood coagulation cascade is activated following vascular-wall injury. The serine protease thrombin is the final protease in this cascade that causes the formation of fibrin from fibrinogen. Thrombin also causes the activation of platelets, which are trapped in a fibrin net followed by hemostasis. Platelets gathered into fibrin clots release several growth factors such as platelet-derived growth factor and transforming growth factor β. In the present study, we demonstrated that the vascular endothelial growth factor (VEGF) could be bound to fibrin clots in the plasma, and that incubation of the endothelial cells with these VEGF-bound fibrin clots induced proliferation of endothelial cells. Thus, it suggests that clot-bound VEGF may play a role in wound healing through the proliferation of endothelial cells and vascular smooth-muscle cells. On the other hand, a noticeable migration of monocytes was observed when they were cultured on dishes in the presence of VEGF-bound fibrin clots. Moreover, peripheral blood monocytes incubated in the presence of VEGF-bound fibrin clots strikingly increased the production of IL-6 and IL-8, demonstrating that VEGF trapped in fibrin clots not only induces proliferation of human umbilical vein endothelial cells and migration of monocytes but also enhances secretion of IL-6 and IL-8. Thus, our data suggest that fibrin clots that contain several growth factors act as a bioactive reservoir and may play an important role in hemostasis as well as wound healing.

We detected the metallo-beta-lactamase gene blaIMP positive strains of the gram-negative rods (GNR) isolated in Oita Medical University Hospital between 1993 and 1999 and studied the clinical characteristics of patients infected or colonized with blaIMP positive GNR. 25 strains (20 Pseudomonas aeruginosa and 5 Serratia marcescens) were detected and most of them were isolated from urinary samples after 1997. In the studies of antimicrobial susceptibility, some strains had sensitivity to aztreonum or imipenem although most of the strains showed multidrug resistance. When blaIMP positive GNR were isolated from patients, these strains were thought to have caused infection in 88% of the patients. About half of the patients were over 65 years old and had malignant diseases. Most of the patients had inserted urinary tract catheters, intratracheal tube or intravernous catheters. It was suggested that the insertion of the catheters were related to infection of blaIMP positive GNRs. Two patients were not treated with any antibiotics before the isolation of blaIMP positive GNRs although more than half of the patients were administered carbapenems and cephems. Most of strains were isolated in the same department and showed the same genotype by pulsed field gel electrophoresis.

In order to clarify the time course of left ventricular (LV) wall motion in patients with anterior acute myocardial infarction (AMI) showing terminal QRS distortion on the admission electrocardiogram (ECG), the present study examined 106 patients with their first anterior AMI (≤6 h) who underwent emergency coronary arteriography and cardiac cathetherization at 1 and 6 months after the infarction. The patients were classified into 2 groups according to the presence (group A, n=23) or absence (group B, n=83) of terminal QRS distortion (emergence of the J point at ≥50% of the R-wave amplitude in leads with QR configuration and/or absence of S waves in leads with RS configuration) on the admission ECG. Group A had a lower LV ejection fraction and more reduced regional wall motion (RWM) in the infarct region at both 1 and 6 months after AMI than group B. The degree of improvement in RWM between 1 and 6 months after AMI was less in group A than in group B (−0.1 ±0.5 vs 0.4±0.6 SD/chord, p

Objective The carbonyl group of glucose reacts nonenzymatically with the amino group of protein to form advanced glycosylation end-products (AGEs). AGEs are found in the peritoneum of continuous ambulatory peritoneal dialysis patients, and this AGE formation is suspected to be one of the causes of impaired peritoneal function. In order to control AGE formation in the peritoneum, AGE formation and ultrafiltration in rats were examined with peritoneal dialysates using as osmotic agents saccharides that lack a carbonyl group, the saccharic acid lactobionate [molecular weight (MW) 358.30], the sugar alcohol maltitol (MW 344.32), and the nonreducing sugar nistose (MW 666.58). Design Bovine serum albumin (BSA) (25 mg/mL) was incubated with 18, 36, and 72 mg/mL maltitol, lactobionate, nistose, and glucose at 37°C. After 3 or 6 weeks, amounts of furosine and N-(carboxymethyl) lysine were measured. A 20-mL intraperitoneal injection of a lactate-based dialysate (osmotic pressure 388 mOsm/kg) containing 4.34% maltitol, 4.52% lactobionate, or 8.4% nistose was given to Sprague–Dawley rats and, after 2, 4, or 6 hours, the quantity of effluent and levels of urea nitrogen and creatinine in the effluent and in serum were measured. Results No increases in furosine or N-(carboxymethyl) lysine were seen with maltitol, lactobionate, or nistose after 3 and 6 weeks of incubation with BSA; AGE formation was not observed. In the study in rats, the quantity of abdominal fluid after a 6-hour dwell was nistose > lactobionate > maltitol > glucose. No differences in dialysate-to-plasma ratios for urea nitrogen or creatinine were seen in any group. Conclusions AGE formation in peritoneal tissue might be controlled by using saccharides with a modified carbonyl group as osmotic agents for peritoneal dialysates. Nistose is considered to yield the most efficient dialysis.

The diagnosis of tuberculous peritonitis is quite difficult because the symptoms are not specific for the disease and the incidence of occurrence are relatively rare. We report a case of tuberculous peritonitis diagnosed by ultrasonography-guided peritoneal biopsy. A 64-year-old male was admitted to our hospital because of fever, dyspnea and abdominal pain. Laboratory findings revealed an elevated ESR (53 mm/1 hr.) and positive CRP. The tuberculin skin test was negative. The chest radiograph revealed bilateral pleural effusion. Abdominal ultrasonographic examination and computed tomography showed ascitic fluid, thickening of the mesentery and peritoneum, and inflammatory pseudotumor of the omentum. Ascitic fluid was exudate with a high lymphocyte count and elevated ADA (184 IU/l). Microbiological studies with the fluid were negative. Peritoneal biopsy guided by ultrasonography was performed, and the specimens showed central caseous necrosis surrounded by epitheloid cells and acid-fast bacilli were demonstrated. The size of the pseudotumor, pleural effusion and ascites decreased after antituberculous chemotherapy with corticosteroid was given. Diagnosis of tuberculous peritonitis has often been made by laparotomy or laparoscopy. In a case of this kind, percutaneous peritoneal biopsy guided by ultrasonography is safe and useful.

The present study examined the relation of plasma oxidized low-density lipoprotein (LDL) levels to plasma LDL cholesterol levels and the impairment of endothelium-dependent coronary vasorelaxation in patients with coronary artery disease (CAD). In the first study, the relationship between plasma levels of oxidized LDL and LDL cholesterol were investigated in 88 patients with CAD. In the second study, the changes in the diameter of the left anterior descending (LAD) and the left circumflex (LCX) coronary arteries were measured after intracoronary administration of acetylcholine (15 microg) and isosorbide dinitrate (2.5 mg) in 15 patients with CAD. Plasma oxidized LDL levels were determined with a sandwich enzyme-linked immunosorbent assay. Plasma oxidized LDL levels did not correlate with plasma LDL cholesterol levels (r=-0.03, p=NS). The % diameter changes (mean+/-SEM) in the LAD and LCX after intracoronary acetylcholine were -8.3+/-3.5% and -10+/-4.2%, respectively. The % diameter changes in the LAD and LCX after intracoronary isosorbide dinitrate were 23+/-4.8% and 23+/-5.1%, respectively. The % diameter changes in the LAD and LCX inversely correlated with plasma oxidized LDL levels after intracoronary acetylcholine (LAD: r=-0.55, p=0.03; LCX: r=-0.59, p=0.02), but were not after intracoronary isosorbide dinitrate. Plasma LDL cholesterol, triglyceride, and high-density lipoprotein cholesterol levels did not correlate with the coronary vasoreaction to acetylcholine. In conclusion, plasma oxidized LDL levels do not correlate with plasma LDL-cholesterol levels and are related to impairment of endothelium-dependent coronary vasodilation in patients with CAD.

Deep-seated trichosporonosis is a lethal opportunistic infection in immunocompromised patients. For the rapid diagnosis of this condition, we developed a novel nested-PCR assay that detects DNA specific for clinically important strains of Trichosporon in serum of patients with disseminated trichosporonosis. In this assay, two sets of oligonucleotide primers were derived from the sequence of 26 S ribosomal RNA genes of T. asahii. The specific fragment was amplified from T. asahii and T. mucoides but not from other microorganisms. In a retrospective study using serum samples of patients with disseminated trichosporonosis, the specific fragment was detected in 64% (7 of 11). To treat this infection, we studied the efficacy of rhG-CSF alone and in combination with antifungal agents against disseminated trichosporonosis in neutropenic mice. The results suggested that rhG-CSF might be a useful immunomodulator against Trichosporon infections and the therapeutic outcome might be better when used in combination with antifungal agents.

The effect of Helicobacter pylori infection on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric cancer was studied using a Mongolian gerbil model. Five-week-old male Mongolian gerbils were divided into four groups of 25-30 animals each and challenged for 20 weeks with H.pylori, MNNG, a combination of H.pylori and MNNG, or neither of them. Four to 20 animals from each group were killed at 16, 24 and 52 weeks after H.pylori inoculation, and histopathological changes in their stomachs were examined. A well-differentiated adenocarcinoma was first observed 24 weeks after inoculation in the combination group. At 52 weeks, only six of 15 animals were colonized with H.pylori persistently, and four of them showed well-differentiated adenocarcinomas; on the other hand, neither of the animals with disappearance of H.pylori from the combination group showed adenocarcinoma. At the same observation time, three of 17 animals from MNNG group showed poorly differentiated adenocarcinomas. The incidence of gastric carcinoma in the combination group was significantly higher than that in the MNNG group (P < 0.05). However, no tumors were seen in the control and H.pylori groups. The present findings demonstrate that H.pylori infection enhances the carcinogenic action of MNNG.

C57BL/6 mice were orally immunized with five weekly doses of 2 mg, 200 μg, or 2 μg of Helicobacter pylori (Sydney strain) whole-cell sonicate combined with cholera toxin. One week after the last vaccination, mice were challenged with 5 × 10 7 CFU of live H. pylori three times at 2-day intervals. At 6 or 18 weeks after the challenge, mice were sacrificed and bacterial cultures and histological studies of the stomach were performed. Vaccination with 2 mg/session or 200 μg/session inhibited H. pylori colonization by 90 and 100%, respectively. These mice were considered protected. Lower levels of H. pylori -specific immunoglobulin A (IgA) were detected in fecal and saliva samples before challenge. However, a significant increase in IgA secretion in mucosal tissue and a higher labeling index for IgA-positive lumina of pyloric glands were noted in these mice in response to challenge and in a vaccine dose-dependent manner. In protected mice, however, severe gastritis characterized by marked infiltration of inflammation mononuclear cells was noted at 6 weeks after challenge, compared with the gastritis seen in unprotected mice or nonvaccinated, ordinarily infected mice. Marked expression of gamma interferon mRNA was detected in the stomach of all protected mice, and 50% of these mice expressed interleukin 4 (IL-4) or IL-5 mRNA. Our findings suggest that local secretory IgA antibody and severe postimmunization gastritis correlate well with protection of mice against H. pylori infection.

We studied the effects of a new antiarrhythmic and antianginal agent, bepridil, on the intracellular calcium transient and contraction of cultured neonatal rat ventricular cells. and compared the effects with those caused by an authentic Ca 2+ -entry blocker, D600 (methoxyverapamil). The Ca 2+ transient was measured by using dual-wavelength microfluorometry of fura-2. The contraction was measured as a shortening of cell aggregates with the use of a video image-analyzing system. Both bepridil (1-30 μM) and D600 (1-30 μM) decreased the peak systolic amplitude of the Ca 2+ transient in a concentration- and frequency-dependent manner. Bepridil, but not D600, significantly shortened the half-decay time of the Ca 2+ transient and prolonged the time course of the contraction. D600 decreased the contraction in parallel with the decrease in the peak Ca 2+ transient, whereas bepridil exerted no significant effect on the contraction. Bepridil (10 μM) induced a leftward shift (to lower amplitude of peak systolic Ca 2+ transient) of the relation between the magnitude of contraction and the peak systolic Ca 2+ transient, which was obtained by changing external Ca 2+ concentration. In contrast. D600 (10 μM) did not affect the relation. The results suggest that the negative inotropic effect of bepridil (caused by its Ca 2+ channel-blocking effect) is offset by its simultaneous increase in the sensitivity of contractile protein(s) to intracellular Ca 2+ , which may be a unique characteristic of this antiarrhythmic agent in a clinical setting.

Background: No previous report has shown the relationship between Helicobacter pylori infection and a direct sequence analysis of p53 gene mutation in a biopsy sample of human gastric mucosa. Methods: A total of 60 endoscopic biopsies samples (21 patients with H. pylori-positive gastritis and 9 patients with H. pylori-negative gastritis), including antral mucosa and corpus mucosa, were used in this study. Direct DNA sequencing of exons 5, 6, 7, and 8 of the p53 gene was performed by the dyedeoxy terminator method. Results: Mutations in the p53 gene were identified in non-hot spot codons in exon 7 and 8 in 11 of 21 samples (52.4%) from H. pylori-positive gastritis patients. There was no mutation in H. pylori-negative gastritis patients. Conclusions: This finding shows that H. pylori infection can induce p53 point mutations and appears to be involved in the pathway leading to dysplasia or carcinoma.

Pulmonary aspergillosis is classified into invasive, saprophytic, and allergic forms. In this study, we evaluated the usefulness of PCR for differentiating between different forms of aspergillosis or in monitoring disease activity during treatment by detecting DNA specific for Aspergillus species in the serum. Nested PCR was used to detect Aspergillus DNA in the sera of 30 patients with various forms of pulmonary aspergillosis. The results were compared with those of latex agglutination tests for detecting galactomannan antigen. We also examined the serial changes in the results of nested PCR during and after treatment of a subgroup of patients with invasive pulmonary aspergillosis with amphotericin B. The highest proportion of positive nested PCR results were in patients with invasive aspergillosis (10 of 12; 83%), while patients with pulmonary aspergilloma had the lowest frequency of positive tests (1 of 9; 11%). These results suggested that the sensitivity of the nested PCR depends on the extent of invasion by Aspergillus species. Serial assays showed that the results of nested PCR became negative shortly after commencement of antifungal treatment and that such changes did not correlate with clinical responsiveness to treatment. Our results indicate the potential usefulness of nested PCR with serum samples for the diagnosis of invasive aspergillosis and the detection of a shift in the status of infection from a noninvasive type to invasive aspergillosis. However, the results of the nested PCR did not correlate with the response to antifungal treatment.

To clarify the mechanism of eosinophilia in adult T-cell leukemia (ATL), we studied three ATL patients having marked eosinophilia. Eosinophil-predominant colony-stimulating activity was detected in the serum of one patient and in the conditioned media (CM) from cultured ATL cells from two patients. Soluble interleukin 5 (IL-5), but no interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), was detected in sera from all patients. On the other hand, GM-CSF was produced in vitro by ATL cells from all cases, whereas detectable IL-3 and IL-5 was produced by cells from only one, suggesting that in the other two cases, the serum IL-5 was produced by the normal reacting lymphocytes. The fact that no patient showed marked neutrophilia supports the possibility that IL-5 may have a leading role in the development of eosinophilia, with GM-CSF produced by ATL cells playing a complementary role.

New methods for the possible presence DNA specific for Aspergillus or Trichosporon species were developed. In aspergillus PCR, Aspergillus l8S rRNA encoding gene was amplified from five strains of Aspergillus species by the nested PCR but not from other microorganisms. Results of preliminary investigation of this method demonstrated efficient detections of Aspergillus species in serum samples of rats model of aspergillosis and 29 patients with invasive aspergillosis. In trichosporon PCR, Trichosporon 26S rRNA encoding gene was amplified from two strains of Trichosporon species, and detected by the nested PCR in 64 % of serum samples of patients with trichosporonosis, while glucuronoxylomannan antigen was detected in 55 % of samples. The high sensitivity and specificity of the nested PCR indicate that the assay can provide early diagnosis with sufficient accuracy to be clinically useful for patients with opportunistic fungal infection.

We compared PCR, galactomannan detection assay using a latex agglutination test and (1→3)‐β‐d‐glucan detection assay in detecting infection in rats experimentally infected with Aspergillus fumigatus. On day 2 after inoculation, (1→3)‐β‐d‐glucan and nested PCR were positive for 80%, while galactomannan detection assay was positive for 60%. In addition, the positive result of nested PCR (87.5%) was higher than those of galactomannan detection assay (75%) and (1→3)‐β‐d‐glucan (71.4%) on day 3 after inoculation. The sensitivity of nested PCR was superior to those of galactomannan detection assay and (1→3)‐β‐d‐glucan detection assay. The three diagnostic tests were compared with histopathological findings, and the sensitivity of three diagnostic tests was correlated with histopathological changes. In addition, the elevated levels of (1→3)‐β‐d‐glucan paralleled the development and progression of pulmonary aspergillosis. Our results indicate that a combination of two or three of these tests seems to provide a rapid diagnosis of invasive aspergillosis and assist in the evaluation of the development and severity of invasive aspergillosis. J. Clin. Lab. Anal. 12:257–262, 1998. © 1998 Wiley‐Liss, Inc.

The in vitro effect of macrolides at concentrations below the minimum inhibitory concentration (sub-MIC) on the interaction between Pseudomonas aeruginosa biofilm and human polymorphonuclear leukocytes (PMNs) was investigated by using a chemiluminescence assay. The PMN response to either mucoid or nonmucoid P. aeruginosa biofilm was strongly reduced compared with the response to planktonic bacteria (p < 0.01, p < 0.001, respectively). When biofilms were treated with erythromycin, clarithromycin, roxithromycin and azithromycin prior to incubation with PMNs, the chemiluminescence response was markedly enhanced as compared to untreated controls, and a dose-dependent effect was noted over the range of sub-MIC concentrations studied. In general, macrolides appeared to be slightly more active against mucoid biofilm. Azithromycin was shown to be the most active macrolide against P. aeruginosa biofilms. However, the treatment with sub-MICs of rokitamycin did not have any effect. On the other hand, treatment of planktonic bacteria with macrolides before being exposed to the PMNs did not affect the chemiluminescence response as compared to untreated controls. These findings suggest that macrolides inhibiting the biofilm formation of P. aeruginosa may facilitate the phagocytosis of bacteria by PMNs.