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Candida albicans-induced histamine release from basophils was studied in 54 patients with bronchial asthma in comparison with the release caused by house dust and anti-IgE. The release of histamine induced by C. albicans and that induced by house dust were closely related to the serum levels of specific IgE antibodies as expressed by RAST scores. A correlation of C. albicans-induced histamine release with the release caused by anti-IgE was not generally observed. On the other hand, a close correlation was found between house dust- and anti-IgE-induced histamine release. It was suggested from these results that the differences between C. albicans- and house dust-induced histamine release might be due to the different antigenicity of the two allergens.

The inhibitory effect of nicardipine, a calcium antagonist, on the antigen- and anti-IgE-induced histamine release from basophilic leucocytes of patients with bronchial asthma was examined. The agent significantly inhibited both antigen-stimulated and anti-IgE-induced histamine release from basophils (the maximum percent inhibition was 57.8 +/- 7.2% and 56.0 +/- 8.8%, respectively). Pre-incubation of basophils with nicardipine for periods of up to 120 min did not alter the inhibitory effect. These results suggest that nicardipine modifies the histamine release from basophils which closely participate in an attack of bronchial asthma.

Helicobacter pylori (H. pylori) infection in the stomach is etiologically closely associated with chronic active gastritis, peptic ulcer, gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. In this study, we examined the antibody responses and cytokine profiles of three strains of mice (BALB/c, C3H/He, and C57BL/6) infected with H. pylori. Following this, correlations between host-immune reactions and intensity of inflammation were analyzed. H. pylori (ATCC43504) was intragastrically administered once a week to the mice from 4 weeks of age, and they were sacrificed at the ages of 4 and 7 months. In these mice, we examined the histology of the stomach, antibody titers against H. pylori, and serum levels of cytokines (IL-4, IL-10, TNF-alpha, IL-2 and Interferon-gamma). In BALB/c mice, inflammation of the stomach was minimal. Inflammation was observed in 63.6% of C57BL/6 mice and 33.3% of C3h/He mice. In C57BL/6 and C3H/He mice, all the cytokines tended to increase. In contrast, BALB/c mice were inactive in cytokine production except for IL-2. Two C3H/He mice developed severe inflammation with lymph follicles; one showed a response largely typical of Th-1, and the other showed a response largely typical of Th-2. Although a definite correlation was not shown between Th-1/Th-2 response evaluated by cytokine production and intensity of inflammation, it appears that in H. pylori-induced inflammation both cell-mediated (Th-1) and humoral (Th-2) immunity play a role in pathogenesis.

A sensitive method of HLA-DRB1 typing was devised using a semi-nested polymerase chain reaction (PCR) followed by a restriction fragment length polymorphism (RFLP) analysis (semi-nested PCR-RFLP method). The first-round amplification (30 cycles) of the semi-nested PCR was performed using DRB generic primer pairs and the second round of PCRs (20 cycles) were performed using DRB1 group-specific primers. The products of the second round PCRs were digested with restriction endonucleases for the typing of HLA-DRB1 alleles. By this method, HLA-DRB1 typing was possible from 10 pg of genomic DNA extracted from lymphocytes and from 0.5 microliter of 1,000 times diluted blood without DNA extraction. HLA-DRB1 alleles could be typed from a 2-mm long bloodstained cotton thread prepared from 10 times diluted blood and from a 2-mm thread of whole blood bloodstains stored at room temperature for 2 years. From the mixture of blood of two individuals with different genotypes, DRB1 alleles of the minor component were detected down to 1/1,000 of the major component. This semi-nested PCR-RFLP method is useful for HLA-DRB1 typing from extremely small amounts of DNA and from mixed samples.

We performed haptoglobin (Hp) genotyping by polymerase chain reaction using allele-specific primer-pairs. The major six genotypes of Hp were identified using this method. Among Japanese individuals living in Ehime and Okayama Prefectures, the allele frequencies were estimated to be Hp2 = 0.723 and Hp1s = 0.277. Genotyping of Hp was possible with 0.3 ng of DNA and with 0.125 microliter of blood. It was also possible with whole blood left at room temperature for a month and also with the bloodstains left at room temperature for three years. In the heated blood samples, both alleles, Hp2 and Hp1s, were detected in those heated at 100 degrees C for 2 h. In bloodstains, Hp2 and Hp1s were detected in samples heated at 100 degrees C for 2 h and 120 degrees C for 30 min. In addition, the genotype could be detected in samples other than blood such as saliva, hair roots, tissue sections and dental pulps. The present method for Hp genotyping is expected to become a useful method in forensic analysis.

A carboxyfluorescein (CF)-enveloping soybean phosphatidylcholine liposome was used as a model of physicochemical damage of biomembranes. The liposomes were exposed to a metal-chelate complex [2 mM of ferric nitrilotriacetate (FeNTA) or cupric nitrilotriacetate (CuNTA)] plus a reductant (2 mM of ascorbate or various concentrations of reduced glutathione), and CF release from damaged liposomal membranes and the generation of thiobarbituric acid-reactive substances (TBARS) were measured. In the presence of a reducing agent, both FeNTA and CuNTA stimulated markedly CF release and an increase in the TBARS level, while in the absence of a reducing agent both of the chelate complexes showed little CF release and TBARS. The effects of H2O2 addition to the reaction system containing liposome with FeNTA or CuNTA plus ascorbate were also examined. The CF release was slightly increased by the addition of a smaller dose (0.5 mM) of H2O2 and it was inhibited by 8 mM of H2O2. A similar result was obtained in the TBARS test. These results suggest that FeNTA- or CuNTA-mediated lipid peroxidation can damage liposomal membranes physicochemically, and the redox reaction of the chelated metal itself is more important than a Fenton-type reaction in the process.

Previously, we reported that interleukin-2 (IL-2)-stimulated helper T cells produced an unknown soluble factor which induced dendritic cell-like differentiation in primary cultures of monocytic leukemia cells and we referred to this factor as dendritic cell differentiation factor (DCDF). In this study, we attempted to purify and characterize DCDF and investigated its biological effect on normal human monocytes. Gel filtration chromatography indicated that the molecular weight of DCDF is approximately 30-35 kDa. Chromatofocusing indicated that the isoelectric point of DCDF is approximately 5.0. DCDF, partially purified by subsequent gel filtration, chromatofocusing, and hydrophobic chromatography, significantly enhanced the HLA-DR expression of normal human monocytes and a human monocytic leukemia cell line, THP-1. This biological activity was not neutralized by any known antibodies to human cytokines. DCDF significantly amplified the T-cell stimulatory activity of monocytes in the allogeneic mixed leukocyte reaction (MLR). Moreover, DCDF significantly enhanced IL-1 beta and IL-6 production by monocytes in a dose-dependent manner. These results suggest that DCDF is a novel human cytokine which stimulates the accessory cell function of monocytes.

We have already developed the liposome immune lysis assay (LILA) for the determination of C-reactive protein (CRP) by employing an inhibition method and a sandwich method. We herein report a new LILA system involving the use of monoclonal antibodies-bearing liposomes. We established five monoclonal antibodies to CRP antigen, AC-1, -2, -3, -4, -5 which had the capacity to activate complement and form antigen-antibody complex. Each of these antibodies was covalently coupled to carboxyfluorescein-entrapped multilamellar liposomes. When the liposomes were incubated with CRP antigen in the presence of guinea pig complement, CRP antigen-dependent liposome lysis was observed but the sensitivity was not great enough for practical use. On the other hand, when liposomes coupling two monoclonal antibodies (AC-1, AC-2) which recognized distinct CRP antigenic determinants were employed in the assay, the sensitivity increased compared with that using only one monoclonal antibody, and the detectable concentration range was 5-300 ng/ml. These results indicated that the combination of two or more monoclonal antibodies which recognize distinct CRP antigenic determinants is effective for increasing the sensitivity of the assay.

The sensitivity and specificity of single cell polymerase chain reaction (PCR) were studied. Its high sensitivity enabled detection of a single-copy gene, such as human T-lymphotropic virus type I genome in paraffin sections. The rate of obtaining positive signals with this method was affected by the number of copies of the gene in the target cell. Specificity was satisfactory if the procedure was properly and carefully followed. Since the single cell PCR is a time-consuming method which requires skill and experience to pick up the target cells accurately, the applicability of this method is limited. It works best when it is used to analyze a single or a few copy genes in histologically identified cells.

We report the first case in Japan, i.e., the first case among oriental subject of Farber's disease. This is a rare disorder of lipid metabolism in infancy subsequent to a genetically-determined defect in ceramide degradation. Main features are characterized clinically by hoarseness, joint swelling, subcutaneous nodules and retarded psychomotor development. Lipid analysis and pathological investigation on the material obtained from a subcutaneous nodule confirmed clearly the presence of ceramide and intracytoplasmic inclusion bodies characteristic for Farber's disease. In this case, we experienced also corneal opacity and striking abnormalities in electroencephalogram, which have apparently not been noticed in the 17 cases hitherto reported.

The arylsulfatase activity and histamine concentration of bronchoalveolar lavage fluid (BALF) were examined in patients with bronchial asthma in relation to the eosinophil count and asthma type (atopic and non-atopic). The BALF arylsulfatase activity and histamine concentration were significantly higher in atopic asthmatics than in non-atopic asthmatics. In atopic asthmatics, the activity of arylsulfatase was significantly increased in patients with a higher eosinophil count (10% or more). However, the BALF histamine concentration did not correlate with the eosinophil count. In non-atopic asthmatics, there was no significant correlation between arylsulfatase activity and the eosinophil count. The results show that arylsulfatase participates in IgE-mediated allergic reactions.

We investigated the effects of pentoxifylline (PTX) on endotoxin-induced diaphragmatic dysfunction in vitro. Seventy-two rats were divided into 3 groups: a group in which endotoxin (20 mg/kg) was injected intraperitoneally (endotoxin-group), a group in which PTX (100 mg/kg) was injected intraperitoneally 30 min before injection of endotoxin (endotoxin-PTX group), and a group in which only saline was given (sham group). Left hemidiaphragms were removed 4 h after injection of endotoxin. We evaluated the diaphragmatic contractility by twitch characteristics and force-frequency curves in vitro. We measured serum TNF-alpha concentrations, diaphragm malondialdehyde (MDA) levels (an index of oxygen-derived free radical-mediated lipid peroxidation), and diaphragm cAMP concentrations. Diaphragmatic force generation capacity was signifi cantly reduced after injection of endotoxin. Serum TNF-alpha concentrations and diaphragmatic MDA levels were significantly elevated after injection of endotoxin. PTX administration significantly improved diaphragmatic contractility and prevented the elevation in TNF-alpha concentrations and MDA levels after injection of endotoxin. There were no significant changes in the diaphragm cAMP concentrations among the 3 groups. These results demonstrated that PTX administration prevented endotoxin-induced diaphragmatic dysfunction without changing diaphragm muscle cAMP concentrations. The protective effects of PTX against endotoxininduced diaphragmatic contractile deterioration might be caused by attenuating TNF-alpha-mediated oxygen-derived free radical production.

In modern emergency and critical care, physicians tend to choose the mode of mechanical ventilation based on spontaneous breathing for the purpose of promoting discharge of pulmonary secretion and preventing atelectasis in patients with acute respiratory insufficiency. However, we often observe "differences in recovery" among patients treated using the same PSV settings beyond "differences in individual characteristics." We evaluated the Pressure Support Ventilation (PSV) mode aiming to certify the difference among 7 representative mechanical ventilators using the Active Servo Lung 5000 (ASL5000) respiratory simulation system. The following parameters were measured: The time delay that resulted in the lowest inspiratory pressure from the point at which the ventilator recognized spontaneous breathing (TD), the lowest inspiratory airway pressure (cmH2O) generated prior to the initiation of PSV (DeltaPaw), the work of breathing while triggering required to achieve the lowest inspiratory negative pressure from the beginning of inspiratory support (WOBtrig), and the inspiratory work of breathing (WOBi). The mean TD of the Puritan-Bennett type 840 (PB840) was signifi cantly shorter than those of other ventilators (p0.01). The WOBtrig of the PB840 was significantly lower than those of others (p0.01). However, the WOBi values of the Servo-I and T-Bird were greater than the others, with the Evita series showing the smallest WOBi of the 7 ventilators tested. According to this simulation study using ASL 5000, we concluded that PB840 was the most rapid response ventilator, but the Evita series was the gentlest mechanical ventilator among 7 ventilators from the standpoint of the total work of breathing during the inspiration phase in the setting of PSV.

Percutaneous cardiopulmonary support (PCPS) has been applied for cardiopulmonary arrest (CPA). We have developed a novel method of cardiopulmonary resuscitation using PCPS combined with liposome-encapsulated hemoglobin (TRM645) to improve oxygen delivery to vital organs. Ventricular fibrillation was electrically induced to an adult goat for 10 min. Next, PCPS (30 ml/kg/min, V/Q: 1) was performed for 20 min. Then, external defibrillation was attempted and observed for 120 min. The TRM group (n5) was filled with 300 mL of TRM645 for the PCPS circuit. The control group (n5) was filled with the same volume of saline. The delivery of oxygen (DO2) and oxygen consumption (VO2) decreased markedly by PCPS after CPA, compared to the preoperative values. DO2 was kept at a constant level during PCPS in both groups, but VO2 slowly decreased at 5, 10, and 15 min of PCPS in the control groups, demonstrating that systemic oxygen metabolism decreased with time. In contrast, the decreases in VO2 were small in the TRM group at 5, 10, and 15 min of PCPS, demonstrating that TRM645 continuously maintained systemic oxygen consumption even at a low flow rate. AST and LDH in the TRM group were lower than the control. There were significant differences at 120 min after the restoration of spontaneous circulation (p<0.05).

The mechanism of action of the drug was investigated from various points of view. The findings may be summarized as follows: 1. In the experiments of the degranulation of mesenteric mast cells of rats, menaquinone proved to significantly inhibit the degranulation either in active or passive sensitization with the reagin-like antibody. 2. Menaquinone did not inhibit the formation of the reagin-like antibody. 3. In the experiements of the degranulation of basophilic granulocytes from patients of bronchial asthma, the rate of appearance of A form basophilic cells upon addition of the antihuman IgE goat serum was not markedly but significantly inhibited in the patients treated with menaquinone for long periods, as compared with that in the control, whereas the in vitro addition of menaquinone did not exert a significant inhibitory action.

A case of ovarian leiomyoma is reported, together with histologic, immunohistologic and electron microscopic findings. A solid firm tumor, measuring 6.5 X 5 X 5 cm, was found in the right ovary of a 65-year-old woman. The tumor had an obvious whorled pattern on the cut-surface. Well-differentiated, long spindle-shaped neoplastic cells revealed positive immunoreactivity for anti-desmin. Ultrastructural observations included numerous microfilaments with dense patches in the cytoplasm, micropinocytotic vesicles beneath plasma membranes and continuous basal laminae around neoplastic cells. These findings were compatible with leiomyoma. The possible histogenesis of ovarian leiomyoma was discussed.

In the human lymphoreticular system, the alpha and beta subunits of S-100 protein are found in ordinary monocyte-macrophages and non-phagocytic histiocytes such as Langerhans cells and interdigitating reticulum cells, respectively. The beta subunit is also present in some CD8+ T cells. In the present study, we investigated the ontogeny of these histiocytes and lymphocytes in humans. Yolk sacs and 4 to 21-week fetuses were examined immunohistochemically for the presence of S-100 protein subunits using antisera monospecific to each subunit. S-100 alpha + macrophages were present in the yolk sacs and the hepatic sinusoids of the 4th week embryos prior to bone marrow hematopoiesis. These macrophages later appeared in other lymphoid organs when anlagen of these organs were formed. No S-100 beta + cells were found in the yolk sacs. S-100 beta+ histiocytes were first detected in the hepatic sinusoids of the 5th week embryo, and after the 8th week of gestation, they were distributed in other lymphoid organs. S-100 beta+ lymphocytes were not found in the liver. They were first detected in the thymus at the 12th week of gestation, and were subsequently distributed in other lymphoid organs. These results suggest that S-100 beta+ lymphocytes and histiocytes may belong to different cell lineages, and the former may not be the precursor of the latter.

We compared the transfection efficiency of four types of positively charged liposomes composed of (i) N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dilauroylphosphatidylcholine (DLPC), and dioleoylphosphatidylethanolamine (DOPE) (1:2:2 molar ratio); (ii) 3β [N-(N′, N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and DOPE (3:2 molar ratio); (iii) dimethyldioctadecylammonium bromide (DDAB) and DOPE (1:2.2 molar ratio); (iv) N-[1-(2,3-dioleyloxy) propyl] -N,N,N-trimethylammonium chloride (DOTMA) and DOPE (1:1, w/w; lipofectin). Luciferase gene was used as a reporter gene. Among the cationic liposomes used, the liposomes composed of TMAG, DOPE and DLPC showed a much higher efficiency of plasmid DNA entrapment than the other cationic liposomes tested. In the absence of serum, the cationic multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) composed of TMAG, DOPE and DLPC gave highly efficient transfection. On the other hand, MLV, dehydration-rehydration vesicles (DRV), and SUV liposomes prepared with the mixtures of DC-Chol and DOPE showed similar levels of transfection efficiency. However, the cationic liposomes composed of DDAB and DOPE showed inferior efficiency, whether in the form of DRV, SUV or MLV. The transfection efficiency of lipofectin was also low. In the presence of serum, on the other hand, a considerable (about 30-50%) amount of transfection activity was still observed at 10% fetal calf serum in the cationic MLV and SUV composed of TMAG, DOPE and DLPC. Cationic MLV, composed of TMAG, DOPE and DLPC, can transfect plasmid DNA, not only in the adherent cell lines but also in the suspension cell lines. These findings indicate that the transfection efficiency of cationic liposomes is affected by the lipid composition, the type of liposome, or the presence or absence of serum. They also indicate that the cationic liposomes containing TMAG, DOPE and DLPC are efficient vectors for gene transfer into cells.

A simian cell line, Si-IIA, harboring Epstein-Barr-virus (EBV) -related herpesvirus (Si-IIA-EBV), produces malignant lymphoma in rabbits when administered by intravenous inoculation. In this study, we analyzed the Si-IIA-EBV genome and compared it with human EBV and herpesvirus macaca fascicularis 1 (HVMF 1 ), which is associated with B-cell lymphoma developing in SIV-infected immunosuppressed monkeys. DNA from Si-IIA-EBV was amplified by the polymerase chain reaction using three different primer pairs complementary to human EBV (B95-8) DNA; two of the primer pairs covered part of the long internal repeat 1 region (IR 1) and the third covered part of the BRRF 1 region. Direct sequencing of the three PCR products revealed that Si-IIA-EBV DNA had about 82% nucleotide homology to the human EBV DNA in all three regions and 92.4% homology to HVMF1 in the IR1 region. The blotting pattern by Southern blot analysis was different between Si-IIA-EBV and human EBV.

Sixty-four patients with confirmed bronchial asthma were treated with HC 20-511 (Ketotifen). HC20-511 was evaluated to be very effective in 6.3%, effective in 50.0% and slightly effective in 10.9% of these patients. The appearance of reactive basophils was inhibited by HC 20-511 in 5 out of 6 cases of reaction to house dust, in all three cases with buckwheat allergy to their allergen and in 7 out of 11 cases to anti-IgE. These results confirm that HC 20-511 inhibits type I allergic reactions induced by specific allergen and IgE.