Interferon-gamma (IFN-gamma) production by human T lymphocytes upon Legionella pneumophila stimulation in vitro.

In vitro immune responses to Legionella pneumophila were investigated. When human peripheral blood lymphocytes (PBL) from healthy volunteers were stimulated with formalin-killed L. pneumophila for 7 days in vitro, strong proliferative responses were observed. The responding cells were shown to be a CD4 T cell subset. It was also found that the CD4 T cells secreted significant amounts of IFN-gamma into the PBL culture supernatant. The production of IFN-gamma and IL-4 by PBL was measured semiquantitatively by reverse transcriptase-assisted polymerase chain reaction (RT-PCR) methods. Formalin-killed or live L. pneumophila-stimulated PBL expressed the mRNA for IFN-gamma but not the mRNA for IL-4. The results suggest that the whole bacterium, as opposed to the supernatant, predominantly stimulates Th1 type helper T cells. The cloned T cells specific for L. pneumophila expressed the mRNA for IFN-gamma but not for IL-4. In contrast to formalin-killed or live L. pneumophila stimulation, when PBL were stimulated with the bacterial culture supernatant, the proliferating T cells produced the mRNA for IL-4 as well as for IFN-gamma. A significant correlation between the proliferative response to formalin-killed L. pneumophila and IFN-gamma release in culture was observed (r = 0.6932, P < 0.001) in PBL from 30 healthy volunteers. From these in vitro studies, it is suggested that the whole L. pneumophila bacterium and their soluble antigens stimulate T cells in a manner which results in a different pattern of cytokine production.

[Serodiagnosis of Legionella pneumonia--data of our laboratory in the recent 3 years].

We had examined antibody titers against Legionella spp. of patients' sera which were mainly sent from other hospitals, performed with the indirect fluorescent antibody (IFA) method. The clinical status of the cases diagnosed as Legionella pneumonia serologically, were also studied. Out of 105 cases with clinically suspected Legionella pneumonia, 15 cases (14.3%) were seropositive. In 9 out of the 15 cases (60.0%) were caused by Legionella pneumophila serogroup 1. Clinical outline of these 15 cases did not contradict those reported in the literature, and erythromycin was effective in many cases. Significant rises (more than four times) of the titer were observed 3 to 4 weeks after onset in most of cases. We would like to emphasize that this should be performed serodiagnosis of Legionella pneumonia.

[Usefulness of polymerase chain reaction method in the early diagnosis of Pneumocystis carinii pneumonia].

We evaluated the sensitivity and specificity of polymerase chain reaction (PCR) method in the detection of Pneumocystis carinii, and compared its clinical usefulness with that of a previous method, toluidine blue-O (TBO) staining in diagnosis of P. carinii pneumonia (PCP) using clinical samples such as sputum and bronchoalveolar lavage fluid (BALF). Using P. carinii obtained from BALF of the infected rats, we showed that PCR showed 100 fold more sensitivity than TBO staining as indicated by the comparison of their detection limits (78 cysts/ml vs 7800 cysts/ml). In addition, the detection using PCR in this study was specific for P. carinii because all other pathogens examined were not positive. In the clinical study we analyzed 44 patients including 10 with definite PCP, 8 with portable PCP and 26 with other diagnosis. In the sputum, the sensitivity and specificity were 100% and 95%, respectively, in PCR, while 13% and 100% in TBO staining. These results revealed that PCR was much superior to TBO staining in sensitivity especially when the sputum was used as samples, and suggested that examination of the sputum by PCR was clinically very useful in early diagnosis of P. carinii pneumonia.

[An animal model of Pneumocystis carinii pneumonia and therapeutic efficacy of interferon-gamma in this model].

To evaluate the efficacy of drugs for treatment, we tried to make an animal model of Pneumocystis carinii (P. carinii) pneumonia. Specific pathogen free (SPF) rats immunosuppressed with corticosteroid were intratracheally inoculated with P. carinii. Six weeks after the inoculation, the lung sections of infected rat lung showed increased numbers of P. carinii in the alveoli and thickening of alveolar septa with mononuclear cell infiltration. From 7 to 9 weeks after inoculation, the intensity of infection became more severe, and some rats died at this period. Then, to evaluate drug efficacy in this model, we finished the drug therapy by the 6th week and used the number of P. carinii in the lung and the inflammation score as an indicator of drug efficacy. In this P. carinii pneumonia animal model, both sulfamethoxazole-trimethoprim clinically established anti P. carinii drug and interferon-gamma which is one of the lymphokines mainly produced by T lymphocytes indicated therapeutic and synergistic efficacy against P. carinii pneumonia.

Influence of methylprednisolone on the intracellular antimicrobial activity of erythromycin and clindamycin against Legionella pneumophila.

We have investigated the effect of methylprednisolone on the intracellular activity of erythromycin and clindamycin in vitro. An assay system was developed for the determination of intracellular activity of antibiotics against Legionella pneumophila using guinea pig resident alveolar macrophages. Erythromycin at a concentration of 0.625 mg/L (5 x MIC) and clindamycin at a concentration of 8 mg/L (MIC) inhibited the growth of a single strain of L. pneumophila in macrophages, whilst ceftizoxime at a concentration of 0.625 mg/L (5 x MIC) did not. Methylprednisolone at therapeutic concentrations did not affect the intracellular antibacterial activity of either erythromycin or clindamycin against L. pneumophila. We found no direct effect of methylprednisolone on the intracellular antibacterial activity of either erythromycin or clindamycin.

[Human lymphocyte proliferative response and gamma-interferon production to Pneumocystis carinii antigen in vitro].

In vitro proliferative response and gamma-interferon (IFN-gamma) production to Pneumocystis carinii antigen were investigated in peripheral blood mononuclear cells (PBMC) from healthy volunteers and cord blood mononuclear cells (CBMC) from umbilical cords after delivery. Twenty three of 25 samples of PBMC had strong proliferative response, and 15 of 25 samples of PBMC produced IFN-gamma. In the samples showing strong proliferative response, CD4 lymphocytes expressed interleukin 2 receptors. None of the 3 CBMC samples had proliferative response nor production of IFN-gamma. When CD4 lymphocytes were depleted in PBMC, the proliferative response was markedly decreased and IFN-gamma production was abolished. In conclusion, the lymphocytes of healthy adults are activated and produce IFN-gamma by stimulation with P. carinii antigen, and these two response are both induced by CD4 lymphocytes.

[Detection of Legionella pneumophila using a nested polymerase chain reaction].

We evaluated the usefulness of a Nested PCR method for detecting Legionella pneumophila. This method resulted in L. pneumophila specific detection as far as we evaluated. The first and second step PCR achieved the sensitivity as small as 10 pg and 10 fg of the target DNA, respectively. In the detection from Legionella seeded sputa, the method could detect 0.1 cfu/ml of the bacteria, and it took about 12 hours to detect the target DNA. We demonstrated that the Nested PCR method was superior in sensitivity and rapidity for isolation of the bacteria to the conventional using low pH treatment and selective media for Legionella.

[Correlation of tuberculin skin reaction with lymphocyte proliferation, interferon-gamma production and tumor necrosis factor-alpha production after in vitro stimulation with PPD and killed Mycobacterium tuberculosis using peripheral blood of healthy donors].

The quantitative relationships among the in vitro lymphocyte proliferation in peripheral blood in 19 healthy donors to purified protein derivative (PPD) and the killed Mycobacterium tuberculosis, interferon-gamma (IFN gamma) and tumor necrosis factor-alpha (TNF alpha) production in these culture supernatants, and the in vivo skin reaction to PPD which were simultaneously measured were studied. Statistical analysis was performed with t-test and multiple regression analysis: The results obtained were as follows; 1) The magnitude of the in vitro lymphocyte proliferation by PPD and the killed M. tuberculosis failed to correlate with the erythema and the induration of the in vivo skin reaction to PPD. 2) The erythema of skin test correlates with TNF alpha production in the culture supernatants that the lymphocytes in peripheral blood were cocultured with these antigens for 7 days. (R = 0.566062, 0.01 less than p less than 0.02) 3) There is a correlation between the erythema and the induration of skin test. (R = 0.526662, 0.02 less than p less than 0.05). 4) Though the magnitude of the lymphocyte proliferation to PPD correlates IFN gamma production in the culture supernatants (R = 0.525915, 0.02 less than p less than 0.05), these response to the killed M. tuberculosis correlates both IFN gamma production (R = 0.55049, 0.01 less than p less than 0.02) and TNF alpha production (R = 0.51283, 0.02 less than p less than 0.05) in the culture supernatants.(ABSTRACT TRUNCATED AT 250 WORDS)

[Mechanism of acquired resistance against Legionella--blastogenic responses of murine spleen lymphoid cells following the stimulation with Legionella antigens].

We have tried to characterize the blastogenic responses of murine spleen lymphoid cells from BALB/c mice immunized with Legionella pneumophila serogroup (SG) 1 (Philadelphia 1 strain) and non-treated mice. Lymphoid cells from immunized mice showed stronger blastogenic responses following stimulation with concanavalin A or formalin-treated L. pneumophila SG1 whole cell antigen than those showed by lymphoid cells from non-treated mice. These cells from immunized mice also responded strongly when stimulated in vitro with other SGs of L. pneumophila, while these responded weakly when stimulated with other species of Legionella. Serum antibody titers of immunized mice against each SG of L. pneumophila were examined and the cross reactions were also recognized. However, the relatedness of serum antibody titers and the blastogenic responses against each serogroup of L. pneumophila was small. The epitopes recognized by the cellular immunity might be different in part from those recognized by serum antibodies, and investigations should be made on what the cellular immunity recognizes and how it works.

Cytotoxicity of mebendazole against established cell lines from the human, rat, and mouse liver.

The direct cytotoxicity of mebendazole (MBZ) was investigated by using cell lines derived from human, mouse and rat liver. It was demonstrated that Chang liver cells (derived from human liver) were more sensitive to the cytotoxic effects of MBZ than the other two cell lines. Longer incubation of the cells with MBZ resulted in stronger toxicity, and the cytotoxicity was dependent on the MBZ concentration above a certain threshold value (0.25-0.50 mg/l in a 42-h culture). Inhibition of the proliferation of Chang liver cells by MBZ was detected at a concentration of 0.008 mg/l, a lower concentration than that having a cytotoxic effect. The other two cell lines were less sensitive to the inhibitory effect of MBZ. Proliferation of human mononuclear cells following stimulation by phytohemagglutinin (PHA) was inhibited by MBZ, and this inhibition was more extensive than that of cells stimulated with whole formalin-treated Pseudomonas aeruginosa. It is suggested that dividing cells may be more sensitive to MBZ cytotoxicity. This anti-proliferative effect may be related to its clinically known side effects, such as hepatotoxicity and bone marrow suppression.

[Adoptive immunotherapy for pulmonary tuberculosis caused by multi-resistant bacteria using autologous peripheral blood leucocytes sensitized with killed Mycobacterium tuberculosis bacteria].

A patient with pulmonary tuberculosis caused by bacteria resistant to various anti-microbial agents was treated with adoptively transferred autologous peripheral blood leucocytes (PBL) sensitized with killed Mycobacterium tuberculosis organisms in vitro. The 32-year-old man was admitted to our hospital from National Sanitarium Okinawa Hospital with weight loss, high fever, and rapid aggravation on chest X-ray. Patient's PBL obtained by leukapheresis and separated with Ficoll-Hypaque solution were cultured with killed Mycobacterium tuberculosis bacteria of 0.4 microgram per ml at 1 x 10(6) cells per ml for 7 days in media containing 0.5 U recombinant 1L-2 per ml. After incubation, PBL were layered and centrifuged on Ficoll-Hypaque solution and washed three times with saline. PBL (1-3 x 10(8)) were combined and concentrated for infusion in 20 to 30 ml saline. After injection, patient displayed fever and transitory drop of PaO2. Although the patient did not have an improved on chest X-ray, his fever was alleviated, weight was increased, accelerated ESR was slightly improved, and the number of organisms in sputum (Number of Gaffky) temporarily decreased. Adoptive immunotherapy using the autologous PBL which were sensitized with killed bacteria may be an effective anti-tuberculous immunotherapy.

[New trial with thiabendazole for treatment of human strongyloidiasis].

Okinawa prefecture is well known as an endemic area of Strongyloides stercoralis infection, and its recent infection rate was reported 6.2%, which was investigated by a new technique to detect S. stercoralis, agar plate method. Traditional treatment with thiabendazole was temporarily effective for S. stercoralis, but the recurrence rate was extremely high. We tried the new treatment for the purpose of complete eradication of the parasite. The patients were divided into two groups, who were given 500 mg of thiabendazole three times daily for 5 days and not medicated for the following 9 days. The medication was repeated 3 times in group 1 which consisted of 92 patients and 4 times in group 2 which consisted of 70 patients. Obtained results were as follows: 1) Six months after treatment, the cure rate was 89.5% in the only one course treatment, and 100% in more than 2 course treatments. 2) Side effects such as nausea, vomiting, anorexia or general fatigue were noted in 67.5% of all the patients after initial treatment, and 45.1% of the patients were dropped out of this trial. The dose of the drug was reduced in 32.1% of the patients, and only 22.8% were treated with full course of the regimen. 3) The elevation of S-GPT was observed in 33.8% of all patients. After initial treatment the rate was only 8.1%, but after 3 or 4 repeated course of treatments the rate was elevated to 39.0% and 45.4%, respectively. The liver injury was closely related to the total dose of thiabendazole and the period of the medication.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of Legionella pneumophila in guinea pig peritoneal macrophages by new quinolone, macrolide and other antimicrobial agents.

The effect of 14 antimicrobial agents, including new quinolones and a new macrolide, on the intracellular multiplication of Legionella pneumophila in cultured guinea pig peritoneal macrophages was examined. Gentamicin and beta-lactam antibiotics did not inhibit the intracellular growth of L. pneumophila. Minocycline, erythromycin and DR-3355 inhibited multiplication at concentrations of 1, 0.5 and 0.1 mg/l respectively. Rifampicin, the new macrolide roxithromycin, and the new quinolones ofloxacin, ciprofloxacin and AT-4140 all inhibited the intracellular growth of L. pneumophila at concentrations of less than 0.03 mg/l. The minimal extracellular concentration inhibiting intracellular multiplication (MIEC), compared with conventional MIC measurements, provides a better indication of antimicrobial efficacy against bacteria, such as L. pneumophila, which can multiply in phagocytes.

[A case of Pneumocystis carinii pneumonia with hyperinfection of Strongyloides stercoralis complicated with smoldering adult T-cell leukemia].

A 43-year-old woman visited a clinic for an attack of bronchial asthma which she had been suffering since her childhood. She was treated with prednisolone which was used for the first time. Two weeks later, she had a fever and her chest X-ray showed diffuse reticulonodular shadows on both middle to lower lung fields. In spite of the use of antibacterial drugs, her symptoms such as cough, dyspnea, malaise and fever increased. It was revealed that she had Stronglyoides sterocoralis in the stool. She was referred to our department for treatment and further examination. Transbronchial lung biopsy (TBLB) was performed, and cyst of Pneumocystis carinii were histologically detected in the lung specimen. Anti-human T-lymphotropic virus type 1 (HTLV-1) antibody in the serum was 1:4,096 less than. Typical adult T-cell leukemia (ATL) cells were also observed in the peripheral blood smear at the rate of 10-15% of leukocytes. The parasite was observed in the sputum too. We diagnosed her as having Pneumocystis carinii pneumonia with hyperinfection of Strongyloides stercoralis complicated with smoldering ATL, and the pneumonia might have been induced by steroid therapy (total doses of 500 mg, for 25 days). After sulfamethoxazole-trimethoprim (ST compound) was used for the Pneumocystis carinii pneumonia, her symptoms markedly subsided, and the chest X-ray findings turned to normal by 45 days after the treatment. Thiabendazole was initially administered for the Strongyloidiasis and the parasite temporarily disappeared from both sputum and stool. Then pyrvinium pamoate and mebendazole were used, but the parasite could not be completely eradicated in the stool. We did not treat the smoldering ATL because there were no symptoms. We have been looking after her as an outpatient now, and she has neither symptoms nor signs.

[A retrospective study of hospitalized patients with fever of unknown origin (FUO) past six years].

We analysed retrospectively 48 hospitalized patients with fever of unknown origin (FUO) from 1982 through 1988. The criteria of FUO were (1) temperature of more than 38.3 degrees C documented on several occasions (2) overall duration of illness more than three weeks, (3) uncertain diagnosis till one week after hospitalization. Of this group of FUO, 25 patients (52.1%) were found to have infections, 8 patients (16.7%) had collagen disorders, 7 patients (14.6%) had neoplastic disorders, 3 patients (6.3%) were crohn disease and 5 patients (10.4%) were undiagnosed. Among infectious diseases, chronic tonsillitis was the most frequent (5 patients: 20%) and they were diagnosed by the provocative examination. Non bacterial meningitis and cervical lymphadenitis were diagnosed in all 3 patients (12% in all), Adult Still's disease was found in 3 patients (37.5%) and systemic lupus erythematosus (SLE) in 2 patients (25%) in collagen disease. Immunoblastic lymphadenopathy was diagnosed in 3 patients (42.9%) of malignant diseases. Three cases of Crohn disease were revealed in all the patients of the miscellaneous group. Duration of fever was relatively short in infection diseases compared to malignant and Crohn diseases. The most common laboratory abnormality is an elevated erythrocyte sedimentation rate (89.6%). As the final diagnosis of FUO are changing with the development of diagnostic techniques, a new criteria of FUO is necessary.

[A case of bronchiolitis obliterans organizing pneumonia in a patient with rheumatoid arthritis].

A 54-year-old man with rheumatoid arthritis visited his general practitioner because of fever and cough. Chest X-ray showed an infiltrative shadow in the right lower field. Antibiotic treatment was not effective, and the specimens obtained by transbronchial lung biopsy was not diagnostic. The patient was transferred to our hospital for further examination and treatment. Previously he had been treated with prednisolone in the knee joint for rheumatoid arthritis. Open lung biopsy was performed. The specimen obtained showed bronchiolitis obliterans organizing pneumonia (BOOP) histologically. The patient recuperated and the chest X-ray shadow decreased with no therapy except the previous treatment with prednisolone.