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We studied T cell responses to four glutathione S transferase (GST)-fused mite antigens prepared in our laboratory using peripheral blood lymphocytes from mite-sensitive patients with bronchial asthma. Of the four recombinant antigens, purified GST-Mag3 had the strongest ability to cause patients' lymphocytes to proliferate, and its potency was almost comparable to that of crude mite bodies (Dfb) and faeces (Dff) antigens and a purified major antigen, Der f 2. The responder lymphocytes were mainly T cells, because the proliferative response was depleted by the treatment of lymphocytes with anti-CD3 antibody and complement, but not with anti-CD20 antibody and complement. The responsiveness of lymphocytes to GST-Mag3 correlated with that to Der f 2, but GST-Mag3 displayed slightly higher activity to stimulate lymphocytes than Der f 2. Simultaneously, the levels of Dff- and GST-Mag3- specific IgE antibodies correlated with the responsiveness of lymphocytes to GST-Mag3. These results suggest that Mag3 is a new valuable antigen for the response of T cell proliferation in mite-sensitive patients. [ABSTRACT FROM AUTHOR]

House dust mite allergen is thought to be a major cause of asthma. Characterization of these allergen molecules is therefore an important step for the development of effective diagnostic and therapeutic agents against mite-associated allergic disorders. Here we report molecular cloning and expression of the group 6 (chymotrypsin-like) allergen from the house dust mite, Dermatophagoides farinae. Sequencing analysis indicates that cloned cDNA, designated Der f 6, encodes a 279 amino acid polypeptide which conserves a primary structure characteristic for chymotrypsin-like serine proteases found in mammals. Recombinant Der f 6 expressed in Escherichia coli bound IgE in a pool made of 20 sera, and induced histamine release from patients' peripheral blood cells.

Peripheral blood mononuclear cells taken from 38 normal subjects and 50 Japanese cedar pollinosis patients were cultured with or without mitogens or Japanese cedar pollen antigens to study the proliferative response and immunoglobulin production in periphral blood lymphocytes. When stimulated with Con A or anti-CD 40 mAb, the proliferative responses in the patient group were significantly higher than those in the normal group, and when stimulated with anti-CD 40 mAb, the IgG 2 production was markedly dominant among the IgE and IgG subclass productions in both groups. When stimulated with Japanese cedar pollen antigens, the proliferative responses in the patient group were markedly augmented in comparison with the normal group, and showed a correlation with the serum anti-cedar IgE antibody titers in the patient group. In regard to the immunoglobulin production in peripheral blood lymphocytes stimulated with Japanese cedar pollen antigens, the IgE productions in the patient group were significantly higher than those in the normal group, and showed a weak correlation with the serum anti-cedar IgE antibody titers in the patient group. The IgG productions were, however, slightly enhanced by the stimulation with Japanese cedar pollen antigens in both of the normal and patient groups. While it could not be clearly demonstrated which subclass of IgG was responsible for the enhancement of IgG production, there were some cases of relatively higher responses of IgG 2 or IgG 4 production. Though the IgG 4 productions in the patient group were significantly higher than those in normal group, there was no clear correlation with the serum anti-cedar IgE antibody titers in the patient group. On the other hand, it seems to be noteworthy that a weak negative correlation was recognized between the IgG 2 productions and the IgG 4 productions, and between the IgG 4 productions and the IgE productions. Considering these results in the present study, it was concluded to be difficult to identify a IgG 4 high responder in a course of clinical immunotherapy through the in vitro IgG 4 production or other related reactions as attempted in this study.

We examined survival in 53 patients over 60 years old who had chronic obstructive pulmonary disease, and whose FEV1 was less than 60% of the forced vital capacity and was less than 60% of the predicted value. They comprised 34 men and 19 women. The mean age was 75.2 years. The %FEV1 was 39.0% and the body mass index was 19.0. Neither age %FEV1, nor body mass index differed significantly between men and women. However, the number of cigarettes smoked per day, the number of years of cigarette smoking, and the Brinkman index were higher in men than in women. The Brinkman index was 1255.0 in men and 617.8 in women (p = 0.0001). For the group as a whole, the 5-year survival rate was 65% and the 10-year survival rate was 35%. The survival rate of men did not differ from that of women. Survival and %FEV1 did not differ between men and women, despite the significant difference in Brinkman index, which suggests that women were more susceptible to the effects of cigarette smoking than men. Age, one tenth of %FEV1, and body mass index less than 19 were found to be independent predictors of mortality (proportional hazards analysis, p = 0.044, 0.019, and 0.024, respectively). The 5-year survival rate were as follows: 85% in patients less than 75 years of age, 50% in patients more than 75 years of age, 25% in patients with a %EFV1 less than 30%, 80% in patients with a %FEV1 of 30% to 49%, 62% in patients with a %FEV1 of 50% to 60%, 50% in patients with a body mass index of less than 19, and 83% in patients with a body mass index of more than 19.

This study compares the characteristics of fatal asthma patients and those of near-fatal asthma patients who had experienced severe life threatening attacks. Data from 67 fatal asthma patients and 80 near-fatal asthma patients were analyzed. The mean age of fatal asthma cases were older than near-fatal asthma cases, 51.9 yrs and 44.3 yrs, respectively. Similarities of the two cases were seen in sex, type of asthma and severity of asthma. The experiences of previous life-threatening asthma did not differ in both groups (43.3% of fatal vs 40.0% of near-fatal). From the viewpoint of type of development of severe exacerbation, rapid exacerbation was more frequent in fatal cases than near-fatal cases, but acute exacerbation after unstable asthma was more in near-fatal asthma. Seventy-six percent of all asthma deaths occurred at home or on the way to the emergency department. The rate of delay of receiving medical care was significantly higher in fatal cases (72%) than near-fatal cases (26%). There was no significant difference in the frequency of medical management before fatal or near-fatal episode such as oxygen therapy, mechanical ventilation. Use of ambulance, visiting emergency department, previous hospitalization between the two groups. The profile of fatal cases was almost same to that of near-fatal cases except delay of receiving medical care. In conclusion, the analysis of near-fatal cases was very important to make clear the causes of asthma death.

At present, we are performing desensitization immunotherapy on patients with mite-positive bronchial asthma using purified mite feces antigen fractions. We obtained the following results in 13 patients 4 to 12 months after the start of treatment. 1) The fraction showing the maximum reaction in the skin test was HM1-2 (molecular weight: 150-155 kD) in two patients, HM2 (30-40 kD) in nine patients and HM3 (10-20 kD) in two patients, and immunotherapy was performed using these fractions. None of the patients showed the maximum skin reactions or was treated with HM4 (less than 10 kD). 2) In nine patients with perennial asthma, the effects of treatment were excellent in four and moderate in three, while two cases remained unchanged. In four patients with seasonal asthma, the treatment was effective in all cases. 3) In patients in whom immunotherapy was effective, specific IgG antibody increased after the treatment, but it did not increase in the unchanged cases. Among the subclasses of specific IgG antibody increased by the treatment, no clear changes were seen in IgG4 antibody and no conclusion has been reached at present. 4) None of the patients had definite adverse reactions such as the occurrence of asthma attacks or anaphylaxis. These results suggested that purified mite feces antigens are safe and effective. We plan to continue this study with more patients in the future.

A series of mite allergens with high carbohydrate content was fractionated from mite extract containing mite feces by ultrafiltration, and successive chromatography on Ultrogel AcA 54, Sepharose CL-6B, and DEAE Toyopearl 650S. The final fractions isolated were termed HM1-2-Eff, -0.1A, -0.1B, -0.3A, -0.3B, and -1M according to the eluted salt concentration. These six fractions were stained weakly with Coomassie Brilliant Blue but strongly with periodate-Schiff's reagent on SDS-PAGE. Their molecular weight were estimated to be 150-155 KD on Sepharose CL-6B. All of these six fractions provoked strong erythema in skin of mite-sensitive and asthmatic patients. HM1-2-Eff, -0.1A, -0.1B and -0.3B reacted with 73% of mite-sensitive patients' IgE by the nitrocellulose dot blotting. One ng/ml of 4 fractions (HM1-2-0.1A, 0.3A, 0.3B and -1M) released histamine from mite allergic patient's blood cells. HM1-2-0.3A induced proliferation of T cell from immunized mouse by mite extract containing mite feces (Dff).

A low-molecular-size antigen, LM2, which possesses a significant activity to elicit histamine release, was isolated from an ultrafilterable (Mr-cutoff < 10k) fraction of a mite extract containing feces by consecutive chromatography on Ultrogel AcA 54 and Sephadex G-25. The isolated antigen was still heterogeneous glycoproteins, giving a smeary band around pI 3-5 on an Ampholine PAG plate, with molecular weights of 6-8k and 4k on SDS-PAGE and Sephadex G-50 gel filtration, respectively. Chemical treatments of the antigen suggested that the epitope responsible for the elicitation of histamine release resided in the protein moiety. The antigen had activities to provoke conjunctival congestion and histamine release in mite-allergic patients, but not immunogenicity by itself. The antigen competitively inhibited the reactions of HM1, HM2, and HM3 fractions to corresponding antisera, but did not cross-react with anti-Der f I or anti-Der f II sera.

A significant activity to elicit histamine release was found in an ultrafilterable (Mr-cutoff < 10 kD) fraction of a mite feces extract from a spent mite medium. The activity was divided into two fractions (LM1 and LM2) on an Ultrogel AcA 54 column when monitored by histamine release assay using washed peripheral blood cells from mite-allergic patients. The larger-molecular-size antigen, LM1 was further separated into three allergenic fractions by consecutive chromatography on Sephadex G-50, GM-DEAE, and TSKgel ODS-120T. All the purified allergens, LM1s (LM1a, LM1b, and LM1c), which produced positive reactions in skin tests on allergic patients, were glycoproteins (molecular weight: 8 kD on SDS-PAGE; 12.5 kD on Gel filtration) with different carbohydrate contents and pI values ranging from 4 to 5 on an IEF plate. LM1s were cross-reactive with anti-Der f II but not with anti-Der f I. The reaction of LM1c to anti-Der f II serum was completely inhibited with the other antigens, while the reactions of LM1a and LM1b to the serum were partially inhibited with LM1c or the other antigens, respectively.

An immunoreactive clone, which shares no homology with the major allergens, Der f I and Der f II, was isolated by screening of a Dermatophagoides farinae cDNA library with rabbit antiserum raised against an extract of the house dust mite and sera from patients with mite allergy. The deduced amino acid sequence of the cDNA of the clone is significantly homologous, up to 65.5%, to the carboxyl-terminal region of the heat shock cognate protein (hsc) 71 in the heat shock protein (hsp) 70 family. A pool of the recombinant protein-specific IgE purified from mite-allergic sera recognized a 67 kDa protein on a blot of ATP-binding components partially purified from the mite body extract, while the antibody did not bind to the major allergens. We conclude that the recombinant protein, one of several important allergens, may be a protein in the heat shock protein 70 family from the house dust mite, D. farinae.

A cDNA clone encoding a new allergen from the Dermatophagoides farinae cDNA lambda gt11 library was isolated and sequenced. There was no amino acid sequence homology with other known allergens. The gene product, beta-galactosidase fusion protein, of the truncated cDNA on blot reacted with IgE in 13 of 43 sera from patients allergic to mites. The affinity-purified fusion protein had a potent ability to release histamine from washed blood cells of the mite-allergic patients. Human specific IgE eluted from the fusion protein band on blots recognized a 39-kD component on blots of a mite body extract.

Two IgE epitope sequences comprising Ser56-Pro-Val-Thr-Lys-Arg-Ala-Ser-Leu-Lys-Ile-Asp-Ser-Lys-Lys70 and Asp104-Val-Glu-Leu-Ser-Leu-Arg-Ser-Ser-Asp-Ile-Ala115 were identified by deletion analysis of the cDNA encoding a new 39-kD protein of mite allergen. A synthetic dodecapeptide corresponding to the latter epitope sequence functioned as a monovalent and mite-specific hapten. Replacement of each of the 12 amino acid residues with Gly, using site-directed mutagenesis, indicated that Arg110 may play a central role in IgE binding. However, the 8 allergic sera tested exhibited a wide variation in their amino acid residues required for reactivity to IgE in allergic sera.

A HPLC method was developed for the determination of histamine released from washed blood cells of allergic patients after exposure to allergens in the presence or absence of plasma. The blood cells used were preserved in a vital state at 4 degrees C during at least 6 hours after blood-collecting. The responses of blood cells to an appropriately diluted series of a mite feces extract (Dff) solution were evaluated in three patient's groups (68 cases of hyposensitized, 59 cases of newly visited and 44 cases of the others) with bronchial asthma. Washed blood cells from the hyposensitized groups had a tendency to release histamine easily even with lower concentration of the Dff solution. Both the sensitivities of washed blood cells to Dff or mite body extract (Dfb) and the responses of the cells to diverse antigens in neither extract differed largely in individual cases. The titer of blocking antibody could be expressed as the amount proportional to the concentration of Dff or Dfb.

As remarkable improvements have been made to the working environment and working methods of oyster shucking workers during the past 30 years, a study was made on the effects of these improvements on hoya (sea-squirt) asthma and the following results were obtained. 1) The prevalence of hoya (sea-squirt) asthma among oyster shucking workers was 36.0%, 30.1%, 21.7%, 22.0%, 18.0%, and 26.6% in 1963, 1968, 1976, 1984, 1988, and 1992, respectively. Accompanying the improvements made to the working environment and working methods, asthmatic symptoms failed to develop in some of the patients through engaged in oyster shucking work. Thus, those with symptoms actually accounted for 36.0%, 18.7%, 15/8%, 7.4%, 8.4%, and 8.0% of the workers, respectively. 2) The proportion of serious cases among the patients rapidly decreased from 29.2% in 1963 to 0% after 1984. On the other hand the proportion of slight cases was 35.4% in 1963, but after 1988 all the cases were slight cases. 3) During the period from 1984 to 1992, the number of those newly engaged in oyster shucking work was 74. The number of those who developed hoya (sea-squirt) asthma during this period was 8 or 10.1%. All the cases were asthma of the rhinitis type. 4) The number of workers who did not develop asthmatic symptoms though engaged in oyster shucking work was 53 in 1984, 40 in 1988, and 49 in 1992. Of this number, 31% are now under hyposensitization therapy, 57.0% have received this therapy in the past, and 12.0% have not received any therapy.

Hoya (sea-squirt) asthma is a typical type I occupational asthma. A total of 22 females with this disease whose age ranged from 22 to 69 years were treated for two years with high concentration purified sea-squirt antigen named Ei-M having a molecular weight of 22,800. One case who developed ordinary bronchial asthma abandoned the immunotherapy during the early stage of the therapy, but in the remaining 21 cases remarkable effects were rapidly observed from the early stage of treatment. These cases did not develop asthmatic attacks even through they engaged in oyster shucking work and no symptomatic therapy was indicated. Serum anti-Ei-M IgG antibody was significantly elevated after the therapy. None of the treated cases developed any side effects.

Among three glycoproteins, DIIIa, Ei-M, and Gi-rep, isolated from sea squirt as antigens capable of eliciting skin reaction specific to sea squirt allergy, only DIIIa induces asthmatic attack and conjunctival reaction, whereas Ei-M and Gi-rep do not. Periodate oxidation eliminated not only the conjunctival allergenicity but also the skin allergenicity from DIIIa, suggesting that the conjunctival and skin allergenicity could both be ascribed to the epitope residing in the carbohydrate chains of DIIIa. On the other hand, proteolysis with Pronase E and chemical modifications of the carboxyl or amino groups by various methods eliminated the conjunctival allergenicity from DIIIa, whereas the skin allergenicity was still retained. Therefore, the expression of conjunctival reaction was expected to be supported by a relatively small structural factor residing in the protein moiety of DIIIa, which we have tentatively designated as conjunctival allergenicity-supporting factor (CASF). CASF might contribute to the permeability of the antigen into mucosa, enabling the carbohydrate epitope to interact with the allergy-specific IgE distributed in conjunctiva and bronchi . In specificity analysis of a rabbit anti-DIIIa serum, a periodate oxidation- and acid-stable but heat- and alkali-unstable epitope (type gamma) in the protein moiety of DIIIa was also characterized as a useful marker of the antigen. However, the type gamma epitope was apparently independent of CASF that was substantially stable to alkali and heat.

Two antisera were prepared in rabbits against substantially purified preparations of sea-squirt antigens, Gi-rep and Ei-M, which were apparently discriminated from each other in antigenic behavior in radioimmunoassay experiments. Radioimmunometric analysis of the interaction between the two anti-sea-squirt sera and the two antigens revealed that Gi-rep induced in rabbit only one type of antibody (type A) which reacted with Gi-rep and Ei-M in common, while Ei-M induced not only type A antibody having the common reactivity but also another antibody (type B) which was strictly specific to Ei-M. This observation supported the preceding suggestion that Gi-rep and Ei-M carried a common antigenic determinant (type alpha) but that Ei-M had an additional determinant (type beta) specific to Ei-M. In this connection, in view of the similarity of type A antibody in specificity to the blocking antibody induced in asthmatic patients with sea-squirt allergy on hyposensitization therapy with these antigen preparations, it was suggested that the presumed allergenic substance in sea-squirt also carries the type alpha determinant.

The accuracy of radioimmunoassay (RIA) has been much improved for sea-squirt antigen Gi-x, which is considered to be of higher molecular weight than antigen Ei-M, by using radiolabeled Gi-x as a tracer. Gel chromatography monitored by the improved RIA revealed a wide distribution of molecular weight of Gi-x type antigens, in contrast to Ei-M. However, since a considerable portion of the anti-genic activity gave a single peak in gel chromatography with Sepharose 6B, the substance in the peak fractions was isolated as a fairly homogeneous preparation and referred to as Gi-rep. Gi-rep showed distinct characteristics of Gi-x type antigens and was clearly discriminated from Ei-M by radioimmunometry in vitro. The in vitro observation also suggested that Gi-rep and Ei-M carried a common antigenic determinant (type alpha), but that Ei-M also carried a specific determinant (type beta). The weight-average molecular weights were 1.1 x 10(5) for Gi-rep and 2.3 x 10(4) for Ei-M. Both preparations consisted of acidic glycoproteins with considerable amounts of sulfate and phosphate.

Under monitoring the antigenic activity by radioimmunoassay (RIA), one of the major sea-squirt antigens was isolated by gel chromatography after treatment of the extract from internal organs of sea-squirt with QAE-Sephadex. This antigen was fairly homogenous as judged from its elution profile in chromatography on a column of Ultrogel AcA 44 with respect not only of the antigenic activity but also to UV absorption. Furthermore, the antigen inhibited the binding of the 125I-labeled tracer antigen to rabbit anti-sea-squirt antibody in a similar mode to the case of Ei-2, one of the sea-squirt antigens previously prepared, but its specific activity was as high as 2.33 times that of Ei-2. Accordingly this isolated antigen was corresponded to the major antigenic substance in Ei-2 and referred to as Ei-M. In addition, the other antigen of Gi-x was also isolated by chromatography on a column of Sepharose 6B as the major antigenic substance in Gi-2, the other one of the previous antigens. Gi-x was apparently different from E-M in the mode of inhibition of the binding of the tracer antigen to the antibody, although its antigenic activity against the asthmatic patients in vivo was as high as that of Ei-M. Thus, it was considered that sea-squirt contained at least two types of antigens of Ei-M and GI-X.

The clinical effects of cefoxitin (CFX) were studied in 31 cases of respiratory tract infections. The results were as follows: As for the clinical effects, CFX was excellent in 5 cases, good in 13, fair in 8 and poor in 5 out of 31 patients; the efficacy rate was 58.1%. The efficacy rate was 57.1% in bronchopneumonia, 61.1% in pneumonia and 50.0% in acute exacerbation of chronic respiratory tract infections. The efficacy rate was 70.6% in the group of 4 g/day or less and 42.9% in the group of 6 g/day or more. The efficacy rate was 50.0% in 6 cases that had not been responded to other antibiotics previously. As for side effects, skin eruption was observed in only 1 patient. No abnormality was observed in laboratory tests due to CFX. In conclusion, CFX is a useful drug in the treatment of respiratory tract infections.

Norfloxacin (NFLX), a new quinolone antibacterial agent, was investigated for its antibacterial activity and clinical efficacy on respiratory tract infections. The results obtained are summarized as follows: 1. Antibacterial activities were evaluated against 127 strains of various bacteria isolated from clinical sources. MIC80's of this drug were: against Staphylococcus aureus and Streptococcus pyogenes 1.56 micrograms/ml; Haemophilus influenzae 0.05 microgram/ml or less; and Klebsiella sp. and Enterobacter sp. 0.10 microgram/ml. These antibacterial activities were superior to these of ampicillin and cephalexin, except against S. pyogenes. 2. Clinical responses to NFLX in a total of 32 cases with respiratory tract infections were excellent in 9 cases, good in 12, fair in 9, poor in 2, with an efficacy rate of 65.6%. Neither adverse reactions nor abnormalities of laboratory test results were observed.

An antigen capable of inducing asthmatic attack in patients with sea-squirt allergy has been isolated from body fluid of sea-squirt, Styela plicata, and designated as DIIIa. The chemical composition and the behavior in anion-exchange chromatography showed that the antigen was a weakly acidic glycoprotein. The weight-average molecular weight of DIIIa was estimated to be 9,880 by the sedimentation equilibrium method. This antigen was apparently discriminated from the previously isolated sea-squirt antigens, Gi-rep and Ei-M, by its activity to induce asthmatic attack and conjunctival congestion, though it gave a strongly positive reaction in skin tests against allergic patients, similarly to the previous antigens. From the cross-reaction of DIIIa to rabbit anti-Gi-rep and anti-Ei-M sera in vitro, it was confirmed that the antigen carried essentially the same antigenic determinant as Gi-rep and Ei-M, and this was termed type alpha determinant. Furthermore, the allergenic activities detectable in vivo and the reactivity to the rabbit antisera in vitro were simultaneously absorbed by immobilized immunoglobulin from anti-Gi-rep serum. Thus, it was suggested that the antigenic determinant responsible for the allergic reactions in vivo corresponded to that specified as type alpha in vitro on the basis of the reactivity against the rabbit anti-Gi-rep serum.