Effects of Glucocorticoids on Humoral and Cellular Immunity and on Airway Inflammation in Patients with Steroid-Dependent Intractable Asthma

The effects of glucocorticoids on the proportion of lymphocytes in bronchoalveolar lavage (BAL) fluid in relation to humoral and cellular immunity were studied in 56 patients with steroid-dependent intractable asthma. To analyze the mechanism responsible for reduced numbers of BAL lymphocytes, we divided the subjects into 4 groups according to their BAL lymphocyte proportions: 0—4.9%, 5.0-9.9%, 10.0-14.9%, and 15.0-20.0%. Serum IgG levels and the peripheral lymphocyte count were significantly reduced in patients with a low proportion of BAL lymphocytes (less than 9.9%) than in those with more than 10% BAL lymphocytes. Delayed cutaneous reactivity to purified protein derivative was suppressed in patients with a low proportion of BAL lymphocytes (less than 4.9%). The mean proportion of BAL neutrophils tended to increase as the proportion of BAL lymphocytes decreased. These results show that the reduction in BAL lymphocytes produced by glucocorticoids is associated with suppressed humoral and cellular immunity, and that under such conditions the proportion of BAL neutrophils increases.     

Effects of Long-Term Glucocorticoid Therapy on Bronchoalveolar Cells in Adult Patients with Bronchial Asthma

The effects of long-term glucocorticoid therapy on airway inflammation were examined in 84 asthma patients. The proportion of lymphocytes in bronchoalveolar lavage (BAL) fluid was significantly decreased in patients with steroid-dependent intractable asthma (SDIA) compared to results in non-SDIA patients, while BAL neutrophils were significantly increased in SDIA patients compared to results in non-SDIA patients. Regarding age, in patients under the age of 69 (except those between 30 and 39), BAL lymphocyte number was significantly decreased in SDIA compared with non-SDIA subjects, and in patients between 50 and 69, BAL neutrophils were significantly increased in SDIA compared with non-SDIA subjects. The number of BAL lymphocytes was significantly lower in patients with serum cortisol levels of less than 5.0 μg/dl than in those with levels of more than 5.1 μg/dl. BAL lymphocyte number was also significantly lower in patients who had received glucocorticoid therapy for more than 6 years than in those who had received such therapy for 2 years. These results show that long-term glucocorticoid therapy decreases the number of lymphocytes and increases neutrophil numbers in the airways.     

Local secretory immunoglobulin A and postimmunization gastritis correlate with protection against Helicobacter pylori infection after oral vaccination of mice

C57BL/6 mice were orally immunized with five weekly doses of 2 mg, 200 microgram, or 2 microgram of Helicobacter pylori (Sydney strain) whole-cell sonicate combined with cholera toxin. One week after the last vaccination, mice were challenged with 5 x 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 microgram/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.     

Endogenous peroxidase activity in primary culture of human thyroid cells. Localization and measurement

Intracellular localization of and an assay method for endogenous peroxidase (PO) activity were studied using primary culture of thyroid cells obtained from patients with hyperthyroidism. PO activity was visualized by cytochemical reaction and was located mainly in perinuclear cisternae and rough endoplasmic reticulum. With increased culture time, the number of cells showing positive PO activity and amount of the enzyme reaction product in individual cells showed a parallel decrease. For measurement of PO activity, cultured thyroid cells were frozen and thawed and then incubated with citric acid buffer solution containing o-phenylenediamine (opd) and hydrogen peroxide. After incubation, the optical density (OD) of the solution colorized by endogenous peroxidase was measured at 405 nm using a microplate reader. About 1 X 10(4) cells were sufficient for assay of PO activity. Using the above method to assay PO activity and sandwich enzyme immunoassay for thyroglobulin (TG), chronological changes in the PO activity and TG concentration in the culture medium were examined. Although the cells showed no decrease in number, PO activity and TG concentration decreased chronologically. When the ratio of PO activity to TG concentration was calculated, in 3 cases the ratio was almost constant, and in the remaining two, it decreased chronologically. The present biochemical method thus seems useful for determining peroxidase activity of cultured thyroid en masse.     

Comparative sequence analysis of the M gene among rabies virus strains and its expression by recombinant vaccinia virus

Nucleotide sequences and the deduced amino acid sequences of the gene encoding the matrix (M) protein of the Nishigahara and the CVS strains of rabies virus have been determined. The M gene is 609 nucleotides long and is capable of coding for a peptide composed of 202 amino acids. Sequence comparison of these M genes with those of other stains [Pasteur (PV), ERA, Avol] revealed that there is 89.7–91.5% homology at the nucleotide level, and 90.1–92.1% homology at amino acid level, between almost all combinations of these strains. However, in the combinations of the PV and ERA strains, and the virulent CVS and the avirulent CVS-derived Avol strains, much higher homology was observed both at the nucleotide and amino acid levels. The predicted secondary structure and hydropathy profiles also exhibited similar features. Recombinant vaccinia virus containing the M gene was constructed. Sodium dodecyl sulfate (NaDodSO₄)-polyacrylamide gel electrophoresis of the precipitates obtained by immune reaction of the recombinant virus-infected cell lysate with a monoclonal antibody against the M protein revealed that electrophoretic mobility of the expressed protein is indistinguishable from that of the authentic M protein from rabies virions.The nucleotide sequence data of the M genes of the CVS and Nishigahara (RCEH) strains reported in this paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under the accession numbers D90450 and D90451.     

Effect of exogenous interferon on rubella virus production in carrier cultures of cells defective in interferon production

An established cell line (Vero) defective in interferon production was used to evaluate the role of interferon in chronic rubella virus infections of cell cultures. Inoculation of Vero cells with a low multiplicity of virus resulted in the development of carrier cultures which had the characteristics of a regulated infection. Although added interferon did not alter rubella virus production in carrier cultures of cells capable of producing interferon, such added interferon caused a dramatic reduction of virus production in the carrier cultures of Vero cells. There was a reduction of the fraction of cells producing virus in Vero carrier cultures, but not in carrier cultures of other cells upon incubation in the continual presence of rubella virus antibodies. In addition, the fraction of infected cells fluctuated in carrier cultures in Vero cells. The data indicate that interferon is not necessary for maintaining a chronic rubella virus infection in vitro and suggest an instability of the virus genome in Vero cells.     

Significance of flow cytometric DNA analysis from freshly aspirated samples

The procedure of aspiration biopsy cytology by fine needle aspiration (ABC) is as option in establishing definitive diagnoses for breast cancers. In this series, a needle size of 21G was considered most suitable for ABC as well as flow cytometric DNA analysis. Histograms from fresh samples aspirated by fine needle clearly delineated a sharp peak in G&sup0;G(1) phases and also a better CV was obtained than with paraffin-embedded preparations. In addition, fresh samples gave more reliable DI and suggested the value of measuring nuclear DNA contents. It is believed that the prognoses of breast cancers are closely associated with DNA ploidy patterns. In this sense, flow cytometric DNA analysis of fresh samples of ABC is regarded as important in clinical use.     

Usefulness of magnetic resonance imaging of the wrist for the early diagnosis of dialysis-related amyloidosis

Dialysis-related amyloidosis (DRA) is a major complication of long-term hemodialysis patients. The onset of arthropathy is frequently preceded by carpal tunnel syndrome, but the early non-invasive diagnosis of DRA remains unclear. beta 2-microglobulin amyloid deposits in joint synovia and soft tissue precede radiological abnormalities. Magnetic resonance imaging (MRI) may play a more important role in the early diagnosis of DRA, because it allows direct visualization of synovitis and deposition of abnormal soft tissue. The purpose of this study was to evaluate the usefulness of MRI of the wrist for the early diagnosis of DRA. The study included 72 patients (male 37, female 35) undergoing hemodialysis from initiation to 20 years. The patients were examined by MR images of synovitis, deposition of abnormal soft tissue and cystic bone lesions at the wrists. Normal MR images of synovia and soft tissue were defined in 6 control subjects (2 normal 4 non-dialysis patients). Synovitis of the carpal bones was found in 23% of the patients at the start of hemodialysis. Deposition of abnormal soft tissue in the carpal canal and cystic bone lesions were detected after 1 and 2 years of hemodialysis, respectively. All findings were increased significantly with an increasing duration of dialysis. Synovitis was present in 90% of the patients with deposition of abnormal soft tissue, and in 80% of the patients with cystic bone lesions. beta 2-microglobulin value was significantly higher in patients with synovitis, deposition of abnormal soft tissue and cystic bone lesions than in patients without these findings. Our experience suggest that synovitis examined by MRI of the wrists is useful for the early diagnosis of DRA. Thereby, intensive follow-up and management of DRA are required in patients with synovitis at the start of hemodialysis.     

Lysophosphatidic acid transactivates both c-Met and epidermal growth factor receptor, and induces cyclooxygenase-2 expression in human colon cancer LoVo cells

AIM: To examine whether lysophosphatidic acid (LPA) induces phosphorylation of c-Met and epidermal growth factor receptor (EGFR), both of which have been proposed as prognostic markers of colorectal cancer, and whether LPA induces cyclooxygenase-2 (COX-2) expression in human colon cancer cells. METHODS: Using a human colon cancer cell line, LoVo cells, we performed immunoprecipitation analysis, followed by Western blot analysis. We also examined whether LPA induced COX-2 expression, by Western blot analysis. RESULTS: Immunoprecipitation analysis revealed that 10 umol/L LPA induced tyrosine phosphorylation of c-Met and EGFR in LoVo cells within a few minutes. We found that c-Met tyrosine phosphorylation induced by LPA was not attenuated by pertussis toxin or a matrix metalloproteinase inhibitor, in marked contrast to the results for EGFR. In addition, 0.2-40 umol/L LPA induced COX-2 expression in a dose-dependent manner. CONCLUSION: Our results suggest that LPA acts upstream of various receptor tyrosine kinases (RTKs) and COX-2, and thus may act as a potent stimulator of colorectal cancer.     

OP-1206, a prostaglandin E1 derivative. Effects of oral administration to patients with chronic lung disease

In order to assess the clinical effectiveness of an oral prostaglandin E1 derivative, OP-1206, five patients with chronic obstructive pulmonary disease and two with pulmonary fibrosis were studied from the standpoint of hemodynamics and nine others with chronic lung disease from the standpoint of respiratory function. Oral intake of OP-1206 resulted in a significant decrease in the pulmonary arterial pressure (p less than 0.01), total pulmonary vascular resistance (p less than 0.01), pulmonary arteriole resistance (p less than 0.01), and total systemic vascular resistance (p less than 0.05), and an increase in the cardiac index (p less than 0.05) and oxygen delivery (p less than 0.01) with insignificant changes of PaCO2, PaO2 and pH. There was no clinical improvement of lung function after OP-1206 intake. OP-1206 is a potent vasodilator, improving cardiac performance in patients with chronic lung disease and possibly preventing the progress of cor pulmonale in this kind of patient.