Development of noninvasive tonometer using resonance phenomenon.

A tonometer is used to measure ocular pressure either by shooting a short blast of compressed air onto the cornea or by applying pressure directly to the cornea. At present, the tonometer is the primary instrument available for measuring ocular pressure. However, measuring ocular pressure by such means can either frighten or injure the patient. We propose an improved method of measuring ocular pressure in which the tonometer is applied over the patient's closed eyelid for several seconds. The sensor part of the newly developed tonometer contains a bimorph type transducer and weighs only 28.7 (g). When the sensor is placed on the eyelid and the transducer is vibrated by altering the applied voltage, the current flowing through the transducer changes in relation to the ocular pressure. Ocular pressure can thus be determined based on the current flowing through the system. During measurement, patients are generally unaware of the vibrations of the sensor and report no pain.

Functional human IgE specific for Dermatophagoides farinae antigen is produced in SCID mice reconstituted with peripheral mononuclear cells derived from healthy persons and patients with asthma.

BACKGROUND: Whether normal peripheral blood mononuclear cells (PBMCs) transferred to severe combined immunodeficient (SCID) mice produce specific IgE remains unclear. METHODS: Mice received injections of Dermatophagoides farinae antigen (Df)-stimulated PBMCs from healthy persons (IgE RAST score of 0). RESULTS: High titers of Df-specific IgE were detected. The Df-specific IgE activity produced was comparable to or higher than that produced by cells from patients with asthma although the time to maximal production was longer. IgE derived from PMBCs of healthy persons or patients with asthma induced histamine release from cultured human basophils that had been stimulated with Df antigen or an anti-IgE antibody. Treatment of Df-stimulated PBMCs with a high dose, but not a low dose, of interleukin-4 stimulated production of Df-specific IgE by PMBCs from healthy persons or patients with asthma. In contrast, intravenous injection of IFN-gamma into reconstituted SCID mice decreased Df-specific IgE production by PBMCs from patients with asthma. In PMBCs from healthy persons, IgE class-switching may occur later and block the effects of treatment with IFN-gamma. CONCLUSIONS: PBMCs from healthy persons and persons with asthma have clones reactive to allergen and produce functional IgE specific for relevant antigens in mite-sensitive bronchial asthma.

Effect of roxithromycin on T lymphocyte proliferation and cytokine production elicited by mite antigen.

Clinical evidence suggests that roxithromycin (RXM) may be an effective additional therapy for bronchial asthma. However, how it interferes with allergic responses is unclear. To investigate the mechanisms of action of RXM, lymphocyte transformation and interferon (IFN)-gamma, interleukin (IL)-4 and IL-5 synthesis associated with Dermatophagoides farinae (Df), mite antigen in patients with bronchial asthma were evaluated in vitro in the presence of RXM. T cell proliferation in Df antigen-stimulated patients' lymphocytes was suppressed by 50-100 microg/ml of RXM. Production of IL-4 and IL-5 was similarly decreased by 1-10 microg/ml RXM, whereas, IFN-gamma production, which was reduced by Df-stimulation alone, was increased by 50 microg/ml RXM. Our results suggest that skewed cytokine profiles of patients with mite antigen-induced bronchial asthma may be corrected with RXM, which may mimic those of patients in remission, who are tolerant of Df antigen.

Enhancement of mucosal immune response against HIV-1 Gag by DNA immunization.

In order to examine the feasibility of Gag-expression DNA as a potential candidate for HIV vaccine using a mouse model, we injected DNA into mice either intramuscularly or by using a gene gun. Both methods induced a low level of antibody production. However, after booster immunization with p24 protein emulsified with complete Freund's adjuvant via a footpad, we found that only the preceding intramuscular DNA immunization induced an anti-Gag Th1-type (IgG(2a)) antibody response, in addition to the enhancement of a Th2-type (IgG(1)) antibody response. Importantly, when mice were boosted intranasally with p24 and cholera toxin, intramuscular DNA injection was found to enhance both systemic and mucosal Gag-specific immune responses. These results indicate that intramuscular DNA immunization confers the inducibility of memory cells, which circulate around various mucosal tissues. Therefore, intramuscular DNA priming, followed by a mucosal booster immunization, could be considered as a regimen applicable to HIV vaccine.

Fatal buckwheat dependent exercised-induced anaphylaxis.

Cases of food-dependent exercise-induced anaphylaxis (FEA) caused by buckwheat have been rare. Clinical, laboratory, and autopsy findings are present on an 8-year old girl with FEA caused by Japanese buckwheat. The patient consumed buckwheat noodles called "zaru soba" and immediately thereafter swam vigorously. Approximately 30 minutes later, she complained of abdominal pain, vomiting, coughing, and chest discomfort. Another ten minutes later her consciousness level deteriorated and she experienced cardiorespiratory arrest. The heart beat was restored and she was admitted to the hospital. She never regained consciousness and expired after another arrest 13 days later. Her IgE level was high (2,840 IU/ml) and the IgE-radioallergosorbent test (RAST) score was 2 for soybeans, 3 for buckwheat, 2 for rice, and 3 for wheat. An exaggerated hematemesis that occurred immediately after hospital admission indicated an inflammatory condition of the digestive tract that was caused by buckwheat. Marked ulceration accompanied with hemorrhage and necrosis was noted at the ileum. Extensive hemorrhage involving the endotracheal pulmonary field and lymphocyte infiltration of the alveolar space likely appeared after the inflammation. The analysis of buckwheat-specific IgE antibody by immunoblotting showed 7 bands that reacted with the IgE of the patient's serum, 4 bands: 16, 20, 24, and 58 kDa, were specific to the patient as compared to subjects not allergic to buckwheat. A first case of fatal FEA by buckwheat is reported with reference to specific IgE.

Studies on the interactions between drugs and estrogen: analytical method for prediction system of gynecomastia induced by drugs on the inhibitory metabolism of estradiol using Escherichia coli coexpressing human CYP3A4 with human NADPH-cytochrome P450 reductase.

To establish a prediction system for drug-induced gynecomastia in clinical fields, a model reaction system was developed to explain numerically this side effect. The principle is based on the assumption that 50% inhibition concentration (IC(50)) of drugs on the in vitro metabolism of estradiol (E2) to its major product 2-hydroxyestradiol (2-OH-E2) can be regarded as the index for achieving this purpose. By using human cytochrome P450s coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli as the enzyme, the reaction was examined. Among the nine enzymes (CYP1A1, 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) tested, CYP3A4 having a V(max)/K(m) (ml/min/nmol P450) value of 0.32 for production of 2-OH-E2 was shown to be the most suitable enzyme as the reagent. The inhibitory effects of ketoconazole, cyclosporin A, and cimetidine toward the 2-hydroxylation of E2 catalyzed by CYP3A4 were obtained, and their IC(50) values were 7 nM, 64 nM, and 290 microM, respectively. The present results suggest that IC(50) values thus obtained can be substituted as the prediction index for gynecomastia induced by drugs, considering the patients' individual information.

Detection and measurement of urinary 2-hydroxyestradiol 17-sulfate, a potential placental antioxidant during pregnancy.

BACKGROUND: Preeclampsia is associated with a quantitative imbalance between lipid peroxide and an antioxidant coproduced in the placenta. To investigate our hypothesis that 2-hydroxyestradiol 17-sulfate (2-OH-ES) is the placental antioxidant during pregnancy, we developed an assay for 2-OH-ES in urine and studied samples from women with and without preeclampsia. METHODS: The detection and measurement of 2-OH-ES in the urine of pregnant women were performed by RIA using highly specific antiserum to 2-OH-ES. To confirm the reliability of the RIA method, the same samples were analyzed by HPLC using an electrochemical detector. RESULTS: Urinary 2-OH-ES values obtained by RIA showed a close relationship to those obtained by HPLC (y = 1.1x - 0.01; r = 0.96). The urinary 2-OH-ES concentrations during the first, second, and third trimesters were 2. 0 +/- 0.6 (mean +/- SE, n = 13), 5.3 +/- 1.3 (n = 21), and 15.3 +/- 2.0 microg/mg creatinine (n = 54), respectively, and <0.15 microg/mg creatinine (n = 10) at 2-24 h after delivery. The concentrations in preeclamptic women during the third trimester were significantly lower, 3.9 +/- 1.9 microg/mg creatinine (mean +/- SE, n = 12). CONCLUSIONS: RIA can be used to measure urinary 2-OH-ES during pregnancy. The increase in urinary 2-OH-ES during gestation, its decrease after delivery, and the lower values in preeclampsia are consistent with a role of 2-OH-ES as a placental antioxidant.

Wild-derived inbred mice have a novel basis of susceptibility to polyomavirus-induced tumors.

Polyomavirus induces a broad array of tumors when introduced into newborn mice of certain standard inbred strains, notably those bearing the H-2(k) haplotype. Susceptibility in these mice is conferred by an endogenous mouse mammary tumor virus superantigen (Mtv-7 sag) that acts to delete T cells required for polyomavirus-induced tumor immunosurveillance. In the present study we show that mice of two wild-derived inbred strains, PERA/Ei (PE) and CZECH II/Ei (CZ), are highly susceptible to polyomavirus but carry no detectable Mtv sag-related sequences and show no evidence of Vbeta deletion. C57BR/cdJ (BR) mice, which are H-2(k) but lack the endogenous Mtv-7, are highly resistant based on an effective anti-polyomavirus tumor immune response. When crossed with BR, both PE and CZ mice transmit their susceptibility in a dominant fashion, indicating a mechanism(s) that overrides the immune response of BR. Susceptibility in PE and CZ mice is not based on interference with antigen processing or presentation since cytotoxic T cells from BR can efficiently kill F(1)-derived tumor cells in vitro. The expected precursors of polyomavirus-specific cytotoxic T cells are present in both the wild inbred animals and their F(1) progeny. These findings indicate a novel basis of susceptibility that operates independently of endogenous superantigen and prevents the development of tumor immunity.

Serum 2-hydroxyestradiol 17-sulphate in pregnancy.

2-Hydroxyestradiol 17-sulphate (2-OH E2-17-S) is a catecholized form of sulphated estrogen. In vitro studies showed that its antioxidative effect is almost equal to that of free catecholestrogens, such as 2-OH E2 or 4-OH E2 and alpha-tocoferol, but the existance of 2-OH E2-17-S in human serum has not yet been made clear. 2-OH E2-17-S strongly antagonizes lipid peroxidation, and so it may play an important role in pregnancy, for example as an anti-oxidant in pregnancy-induced hypertension (PIH). The serum level of 2-OH E2-17-S was measured during mid to late pregnancy by a direct radioimmunoassay (RIA) without hydrolysis. The serum levels at 28-31 weeks, 32-35 weeks and 36-40 weeks of gestation were 4.68+/-0.93 (mean+/-SE), 8.38+/-1.21 and 18.31+/-3.41 nmol/l, respectively. The serum level in PIH cases at 36-40 weeks (4.64+/-1.29 nmol/l) was significantly lower than that in normal pregnancy. The 2-OH E2-17-S level in umbilical arteries was significantly higher than that in maternal peripheral vein. These results suggest that the feto-placental unit plays an important role in catecholizing E2-17-S to 2-OH E2-17-S, which may act as an antioxidant in pregnancy.

Synthesis of 6- and 7-hydroxyestradiol 17-sulfates: the potential metabolites of estradiol 17-sulfate by female rat liver microsomes.

The potential ring-B hydroxylated metabolites of estradiol 17-sulfate (1) by female rat liver microsomes were chemically prepared as authentic compounds. They are 6alpha- and 6beta-hydroxyestradiol 17-sulfates (7 and 9), and 7alpha- and 7beta-hydroxyestradiol 17-sulfates (12 and 16), whose synthetic procedures are described.

IL-12 affects Dermatophagoides farinae-induced IL-4 production by T cells from pediatric patients with mite-sensitive asthma.

BACKGROUND: IL-12 is a critical cytokine in the regulation of immune responses produced by phagocytic cells exposed to microorganism infection. OBJECTIVE: We sought to study the effect of low doses and high doses of IL-12 on TH1 versus TH2 cytokine expression to elucidate the etiology of mite antigen-sensitive bronchial asthma in infants. METHODS: We studied the effect of IL-12 on Dermatophagoides farinae (Df) antigen-induced IL-4 production and subsequent production of IgE by PBMCs from pediatric patients with asthma. RESULTS: Simultaneous addition of 1 to 10 ng/mL IL-12 to cultures enhanced Df-induced IL-4 production, although low doses (0.05 to 0.1 ng/mL) of IL-12 downregulated IL-4 production. Endogenous IL-12 is required for such production. These phenomena were not observed in Df-stimulated control PBMCs. In contrast, on stimulation with the same dose of Df, IFN-gamma production by patient PBMCs was enhanced in a dose-dependent fashion by addition of IL-12. Quantification analysis of RT-PCR-amplified DNA fragments by laser-induced fluorescence showed that a high dose of IL-12 augments mRNA expression for IL-4 protein synthesis, whereas a low dose of IL-12 inhibits IL-4 mRNA expression, and that the signal of mRNA for IFN-gamma protein synthesis was increased on Df stimulation in a dose-dependent fashion. Df-induced in vitro production of IgE and Df-specific IgE in serum from severe combined immunodeficient mice reconstituted with PBMCs were increased by treatment with high doses of IL-12, whereas low doses of IL-12 inhibited that production. The combined results indicate that at a low dose of IL-12, IL-4 and IFN-gamma production was regulated reciprocally; however, at high doses of IL-12, cells produced IL-4 and IFN-gamma simultaneously, and neither cytokine was regulated. CONCLUSION: Low-dose and high-dose IL-12 induce TH1 responses, and high-dose IL-12 induces both TH1 responses and TH2 or TH0 responses. Consequently, the IL-4 production may overcome TH1-type cell activation of IgE production in patients with mite-sensitive bronchial asthma.

Functional interleukin-5 activity in peripheral blood mononuclear cells from adolescents with mite antigen asthma in remission.

BACKGROUND: Asthma improves in most children during adolescence such that a small minority of patients exhibit clinically significant symptoms by the age of 20 years. OBJECTIVE AND METHODS: To investigate late allergic reactions, including eosinophil inflammation, associated with outgrowing mite antigen-induced bronchial asthma during adolescence, the relationship between clinical status and functional activity of interleukin (IL)-5 produced by Dermatophagoides farinae (Df)-stimulated peripheral mononuclear cells (PBMCs) in culture was assessed in mouse IL-3-dependent cells transfected with the human IL-5 receptor gene. RESULTS: Activity of IL-5 spontaneously produced by PBMCs from either patients with mite-sensitive bronchial asthma or nonatopic control subjects was low. The activity of IL-5 produced by PBMCs stimulated with concanavalin A was significantly higher. Upon challenge with specific allergens, such as Df antigen, but not with irrelevant antigens, including ovalbumin, the in vitro activity was increased in patients with active disease and decreased in patients in remission. CONCLUSION: Results suggest that the antigen-specific up-regulation of functional IL-5 activity in late allergic reactions is reduced in patients in remission and likely to result in an improvement in clinical status. The Df antigen may suppress Df-induced responses in patients with asthma in remission.

A comparative investigation of the restorative effects of roxithromycin on neutrophil activities.

The effects of roxithromycin, a macrolide antibiotic, on neutrophil activities were investigated in six seriously handicapped patients with severe mental retardation. Neutrophil activities were evaluated by flow cytometry using a heparinized blood analysis method. All six patients showed decreased levels of neutrophil phagocytosis, intracellular killing, and CD11b expression. Treatment with roxithromycin in vitro selectively restored the decreased phagocytic and bactericidal activities of neutrophils in these patients. There was no significant restorative effect with cefaclor, ofloxacin, or aztreonam. These results suggest the need to consider therapeutic effects of antibiotics on neutrophil functions in patients at increased risk for bacterial infections due to decreased neutrophil activities.

Identification of estrogen-modified nucleosides from calf thymus DNA reacted with 6-hydroxyestrogen 6-sulfates.

Two estrogen sulfates, pyridinium 3-methoxyestra-1,3, 5(10)-trien-6alpha-yl sulfate (3MeE-6alpha-S) and its 6beta-isomer (3MeE-6beta-S), synthesized as model compounds to demonstrate the carcinogenesis of estrogen, were found to react with calf thymus DNA to produce steroid-modified DNA adducts. Digestion of the DNA by nuclease P1 and phosphodiesterase I followed by alkaline phosphatase gave a deoxyribonucleoside fraction, of which N2-[3-methoxyestra-1,3, 5(10)-trien-6alpha-yl]deoxyguanosine, N2-[3-methoxyestra-1,3, 5(10)-trien-6beta-yl]deoxyguanosine, N6-[3-methoxyestra-1,3, 5(10)-trien-6beta-yl]deoxyadenosine, and N6-[3-methoxyestra-1,3, 5(10)-trien-6alpha-yl]deoxyadenosine (identified as a base adduct) were identified using HPLC by comparing them with authentic specimens prepared by reacting dG and dA with both sulfates. No steroid-dC adduct was detected in the digestion products of the DNA adduct, although dC reacted with the sulfates to form N4-[3-methoxyestra-1,3,5(10)-trien-6beta-yl]deoxycytidine. These results mean that estrogen 6-sulfate has an ability to modify DNA via the amino group of a guanine or adenine residue in DNA. The present studies imply that a sequential metabolism (hydroxylation and sulfation) at the C6-position of the estrogen molecule causes damage to DNA.

Correlation between antigen-specific IL-2 response test and provocation test for egg allergy in atopic dermatitis.

BACKGROUND: The antigen-specific interleukin-2 response (AIR) test using lymphocytes is effective in searching for the antigen which causes allergic diseases and understanding their disease activity. OBJECTIVE AND METHODS: The correlation between the raw egg oral provocation test and egg white antigen-specific interleukin-2 (IL-2) response test was investigated in 123 children with infantile atopic dermatitis and 13 children with bronchial asthma. RESULTS: Among the 83 who showed positive reactions to provocation, 75 also reacted positively to the AIR test (sensitivity, 90.4%), while among the 53 children who showed negative responses to antigen provocation, 45 produced negative responses to the AIR test (specificity, 84.9%). The specificity of egg white IgE RAST score and skin-prick test are 88.7 and 81.3% which are comparable to that of the AIR test. However, their sensitivity was low (38.6 and 66.7%). In the patterns of symptom developed in the provocation AIR displayed late and delayed type allergic responses in addition to the immediate type which RAST reflected. The RAST-negative group composed of 98 patients included 51 (52.0%) who exhibited positive reactions to the provocation test. Among these 44 responded positively to the AIR test (86.3%). CONCLUSION: The AIR test is effective for screening egg white antigen as part of the tests for antigens responsible for allergic diseases and as a test to ascertain the relevant antigens, and that the conditions that could not be diagnosed by RAST can be detected by the AIR test.

Differential recognition of MBP epitopes in BALB/c mice determines the site of inflammatory disease induction.

Although myelin basic protein (MBP)-recognizing T cells are not readily obtained after immunization of BALB/c mice with MBP (reflecting the BALB/c resistance to actively induced experimental autoimmune encephalomyelitis (EAE)), they can be expanded and cloned after several rounds of in vitro culture. The majority of BALB/c-derived clones recognize an epitope defined by MBP peptide 59-76. When transferred to naive BALB/c recipients, these clones cause classical EAE, with characteristic inflammation and demyelination of the central nervous system (CNS). We previously showed that two related clones recognizing a minor epitope, defined by MBP peptide 151-168, cause inflammation and demyelination preferentially of the peripheral nervous system (PNS). Because MBP has alternatively spliced isoforms, residues 151-168 are not present contiguously in all MBP isoforms. In order to determine whether induction of PNS disease is idiosyncratic to these sister clones, or related to their properties of epitope recognition, an independent T-cell line with similar recognition properties was studied. Clone 116F, derived from a BALB/c shiverer mouse, expresses a different T-cell receptor (TCR), with distinct TCR contact residues, but like the previously described T cells, this clone requires residues from both exons 6 and 7 for optimal stimulation. When adoptively transferred to BALB/c recipients, this clone preferentially induces disease of the PNS. A control BALB/c shiverer-derived MBP 59-76-recognizing clone, in contrast, induces CNS disease. These data strongly suggest that the site of disease initiation may correlate with epitope recognition, particularly when alternative isoforms are involved.

Reduced IL-1 production in adolescents with mite antigen asthma in remission.

To determine the immunological mechanisms associated with outgrowing mite antigen-induced bronchial asthma during adolescence, we studied the relationship between clinical status and Dermatophagoides farinae (Df) antigen-induced peripheral cell activation by measuring IL-1alpha and IL-1beta production in patients with bronchial asthma. After antigen-driven restimulation in vitro, there was increased IL-1alpha, IL-1beta production by peripheral blood mononuclear cells (PBMC) from patients with active bronchial asthma, while cellular IL-1alpha, IL-1beta production was reduced in patients with asthma in remission. IL-1alpha and IL-1beta production by PBMC (possibly reflecting airway inflammation) after exposure to Df antigen might be down-regulated in patients outgrowing mite antigen-induced asthma, because lipopolysaccharide-induced IL-1alpha, IL-1beta production (seen in both normal individuals and patients with active asthma) was also reduced when patients were in remission.

T-cell responses to myelin basic protein in normal and MBP-deficient mice.

BALB/c mice are resistant to the development of experimental autoimmune encephalomyelitis (EAE) after immunization with myelin basic protein (MBP). Previous studies of BALB/c mice suggest that MBP-specific T-cells can eventually be cloned from these mice, although they are either initially present in very low frequencies or are functionally anergic. To determine what role endogenous MBP expression plays in shaping the BALB/c T-cell repertoire, MBP-deficient BALB/c mice were constructed by breeding the shiverer (shi/shi) mutation onto the BALB/c background. These mice lack all conventional isoforms of MBP due to a deletion of MBP exons 3-7. Studies of the MBP-directed response of these mice suggest that endogenous MBP expression is directly responsible for EAE resistance in BALB/c mice, by quantitatively affecting expression of the T-cell repertoire. In contrast to wild-type BALB/c T-cells, uncloned T-cells from BALB/c shi/shi mice immunized with MBP proliferate in vitro to MBP and MBP peptides 59-76 and 89-101 and are able to induce severe EAE upon transfer to BALB/c recipients expressing MBP.