Yao-Pi Hsu

Seasonal asthma in northern California: allergic causes and efficacy of immunotherapy.

Inland areas of northern California have an intense grass pollination in the spring of each year. This is accompanied by a stirking rise in the incidence of asthma. We documented this relationship and designed a trial to test the efficacy of immunotherapy for grass-pollen asthma. Aeroallergen counts were performed on the roof of the allergy clinic of David Grant Medical Center from January 1981 to December 1984 by a gravity collector. These counts were compared to counts done on a Rotorod at a nearby hospital from July 1982 to September 1984. Climatologic factors were also tabulated. Visits for asthma and rhinitis to our emergency room and asthma admissions to our hospital were counted for the 4-year period. A randomized, double-blinded, placebo-controlled trial of immunotherapy with grass-pollen extract was performed from November 1984 to June 1985. Two groups of clinically and immunologically well-matched subjects were started on an accelerated preseasonal trial of immunotherapy. One group received a standardized grass extract, and the other group did not. Both groups received other extracts of aeroallergens to which they were skin test positive that occur locally in the spring and summer. This was done because of our dissatisfaction with a histamine placebo used in a previous pilot study. Symptom medication scores (SMS) and immunologic parameters were followed. For the 4-year period, grass-pollen count (GPC) correlated strongly with asthma emergency room visits (r = 0.90; p less than 0.001) and for rhinitis (r = 0.92; p less than 0.001). Asthma admissions also correlated strongly with GPC (r = 0.72; p less than 0.001). Other aeroallergens either did not correlate significantly or occurred in such small numbers that they could not be seriously considered. Rotorod counts supported these conclusions with the exceptions of some Basidiomycetes. Climatologic factors demonstrated no relationship to the incidence of asthma. Asthma SMS were lower in the grass-treated group, p less than 0.05. Rhinitis SMS were also lower but did not reach significance, p = 0.11. RGGI sIgE did not rise significantly in the grass-treated group but did in the placebo-treated group. RGGI sIgE rose in both groups, although to significantly higher levels in the grass-treated group, p less than 0.001. The asthma SMS were inversely related to increasing RGGI cumulative dose, p less than 0.10. Linear regression analysis of the dose-response scattergram suggests that a cumulative dose of approximately 90 micrograms of RGGI may be desirable.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytokine dysregulation in activated cystic fibrosis (CF) peripheral lymphocytes

Recent studies demonstrate in vivo and in vitro cytokine dysregulation in CF epithelial cells. To see if these abnormalities may be generalized to other cells expressing cystic fibrosis transmembrane conductance regulator (CFTR) but not directly exposed to local inflammation, we studied mRNA transcription, intracellular protein production and extracellular secretion of IL‐2, IL‐4, IL‐5, IL‐10 and interferon‐gamma (IFN‐γ) from freshly isolated blood mononuclear and CD4+ T cells from CF patients and controls. Cells were activated by phorbol myristate acetate (PMA) and anti‐CD3, PMA–ionomycin, or lipopolysaccharide (LPS) and assessed for cytokine mRNA transcription by semiquantitative reverse transcriptase‐polymerase chain reaction, intracellular protein production by flow cytometry, and secretion by supernatant ELISA. Cytokine expression was highly stimulus‐dependent. CF cells showed higher IL‐10 transcription than control cells after maximal activation by LPS (P = 0·01); despite this, cytokine production and secretion were equivalent to controls. CF cells showed lower cellular IL‐10 production after PMA‐anti‐CD3 activation (P = 0·002). CF cells secreted less IFN‐γ than control cells after maximal activation by PMA‐anti‐CD3 (1836 ± 273 pg/ml versus 9635 ± 3437 pg/ml, P = 0·04). IL‐2, IL‐4 and IL‐5 regulation was similar to controls. We conclude that CF mononuclear cells show selective cytokine dysregulation after maximal activation, namely reduced IFN‐γ secretion and increased IL‐10 mRNA without increased production or secretion. These findings extend defects described in respiratory epithelial cells to circulating immunoregulatory cells, suggesting a link between CF genotype and cytokine dysregulation.

Grass pollen immunotherapy: A single year double-blind, placebo-controlled study in patients with grass pollen-induced asthma and rhinitis

Fifteen grass pollen--sensitive asthmatic patients were selected from 200 patients with grass pollenosis on the basis of positive SPTs and RASTs that were restricted to grass pollens (except Bermuda grass), no previous IT, and residence and occupation in an area monitored by serial pollen counts. They underwent a double-blind trial of specific IT with a mixture of three grass pollen--aqueous extracts (velvet, sweet vernal, and timothy) or placebo. After 10 mo, the mean maintenance dose of pollen extract (assayed by RAST inhibition) in eight actively treated patients was 6000 RAST units (range 3000 to 8000) and the mean total dose was 18,700 RAST units (range 10,200 to 30,000). Results were assessment done by the following clinical and immunological data: (1) during the pollen season, daily symptom scores; (2) PD 20% FEV1, IgE antibody to timothy by RAST in serum and in nasal secretions, serum IgG antibody to purified timothy allergen D by solid-phase radioimmunoassay, and the four IgG subclass antibodies by enzyme immunoassay were all measured before treatment and before and after the pollen season. Symptom scores of both treated patients and controls correlated with pollen counts (R = 0.88, p less than 0.05 and R = 0.71, p less than 0.05, respectively). There was a significant difference between the mean symptom score values of treated patients versus controls (Kruskal-Wallis test, p less than 0.001). No significant differences or changes either in the PD 20% FEV1 or IgE antibody to timothy in serum and nasal secretions were found in the two groups before or after IT.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced IL‐10 secretion by CD4+ T lymphocytes expressing mutant cystic fibrosis transmembrane conductance regulator (CFTR)

Expression of the CFTR protein is thought to be physiologically important only in exocrine epithelial cells. However, chronic respiratory inflammation and infection remain unexplained phenomena in disease pathogenesis. Non‐transformed, antigen‐responsive CD4+ T cells cloned from healthy controls and CF patients homozygous or heterozygous for the δF508 mutation transcribed CFTR mRNA and expressed immunoreactive cytoplasmic CFTR protein. T cell clones (TCC) from controls and CF patients displayed equivalent Ca2+‐mediated Cl− current; however, TCC from patients with CF but not controls displayed defective cAMP‐mediated Cl− current. Although CF‐derived TCC preserved mitogen and antigen proliferative responses and specificity to tetanus toxoid epitopes, they selectively secreted ≈ 45% less IL‐10 compared with control TCC after activation with concanavalin A (Con A) (624 ± 101 versus 1564 ± 401 pg/ml per 106 cells, respectively; P = 0.04) or anti‐CD3/phorbol ester (5148 ± 1634 versus 11 788 ± 2390 pg/ml; P = 0.05). This difference was independent of atopy. Secretion of interferon‐gamma, IL‐2, and IL‐4 was comparable in CF and control TCC after both forms of activation, while IL‐5 was reduced in CF TCC following anti‐CD3/phorbol myristate acetate (PMA) but not after Con A. We conclude that expression of mutant CFTR in human TCC is accompanied by ion channel dysfunction characteristic of the CF phenotype, and is accompanied by a reduction in IL‐10 secretion after polyclonal activation. It is possible that disruption of IL‐10‐mediated anti‐inflammatory homeostasis may contribute to early onset sustained inflammation in CF airways.

Allergy to semisynthetic penicillins in cystic fibrosis.

Allergic reactions to anti-Pseudomonal penicillin derivatives are an increasing problem in therapy of cystic fibrosis lung disease. We evaluated 15 patients, ages 12 to 37 years, with documented allergic reactions to carbenicillin, ticarcillin, or piperacillin. Intradermal skin test reactions were positive for benzylpenicillin in seven patients, penicilloyl-polylysine in one, and ticarcillin or piperacillin in eight, for a total of 11 of 11 tested. Results of radioallergosorbent testing to penicilloyl conjugates were positive in eight of 14 patients and equivocal in four others. Overall, skin tests or RAST results were positive in 13 of 15 patients. All patients were desensitized with a semisynthetic penicillin by continuous serial intravenous infusion of 10-fold dose increments, beginning with 10(-6) of the therapeutic dose. Desensitization was successful in 25 of 26 instances. After intravenously administered therapy, maintenance of desensitization with dicloxacillin orally was unsuccessful in four of six patients. We conclude that (1) allergy to semisynthetic penicillins in cystic fibrosis usually is IgE mediated; (2) such allergy can be evaluated by skin testing; (3) it can be safely and in most cases successfully treated by intravenous desensitization; and (4) allergic patients should be desensitized on each subsequent admission for intravenously administered therapy.

Activation of CFTR chloride current by nitric oxide in human T lymphocytes.

Nitric oxide, which is produced by cytokine‐activated mononuclear cells, is thought to play an important role in inflammation and immunity. While the function of nitric oxide as a direct cytotoxic effector molecule is well established, its function as a transducer molecule in immune cells is not. By use of whole‐cell patch clamp recordings, we show that nitric oxide activates cystic fibrosis transmembrane conductance regulator CI‐ currents in normal human cloned T cells by a cGMP‐dependent mechanism. This pathway is defective in cystic fibrosis‐derived human cloned T cells. These findings not only delineate a novel transduction mechanism for nitric oxide but also support the hypothesis that an intrinsic immune defect may exist in cystic fibrosis.

Isotypic and antigenic restriction of the blocking antibody response to ryegrass pollen: Correlation of rye group I antigen-specific IgG1 with clinical response

To investigate the role of blocking antibodies in allergen immunotherapy (IT), we analyzed IgE, IgG, and IgG subclass 1 to 4 antibody responses to ryegrass group I antigen (RGGI) in a prospective double-blind, heterologous allergen, allergen-controlled trial of grass-pollen IT in 18 adults with seasonal rhinitis and asthma. Serum was assayed preseasonally before starting IT and again in midseason at time of documented highest natural exposure. Antibodies were measured by ELISA, and immunogenic specificities of ryegrass extract were examined by Western immunoblots. Nine subjects receiving grass-pollen IT and nine control subjects had similar clinical and immunologic status before IT. RGGI-specific IgE antibodies (sIgE) did not change from pretreatment levels in actively treated subjects but increased in control subjects (p less than 0.002). RGGI sIgG increased approximately thirteen-fold with active IT versus threefold during natural seasonal exposure (p less than 0.0005). The IgG-blocking response to RGGI was restricted to IgG1 and IgG4. Ten nonatopic subjects had similar RGGI sIgG1 but lower or undetectable sIgE and sIgG4 than the 18 atopic study subjects. Active IT dramatically increased RGGI sIgG4 (p less than 0.001) and to a lesser extent RGGI sIgG1 (p less than 0.01). Immunoblots demonstrated eight IgE-binding ryegrass-polypeptide allergens, with RGGI ubiquitous, and 11 IgG-binding polypeptides, including all eight allergens. A negative correlation between seasonal rhinitis symptom-medication scores and RGGI sIgG1 levels was found (r = -0.62, p less than 0.01), but no other immunologic parameters assayed were related to clinical improvement. Although RGGI sIgG4 predominates in the blocking response and is a useful marker of effective IT, early beneficial biologic effects may involve IgG1 antibodies.

Altered Antibody Isotype in Cystic Fibrosis. Possible Role in Opsonic Deficiency

ABSTRACT. Patients with cystic fibrosis (CF) whose respiratory tracts are colonized with Pseudomonas aeruginosa (PA) may develop a specific opsonic deficiency for alveolar macrophage phagocytosis of PA. We examined the possible role of altered antibody (Ab) isotype in this phenomenom by measuring serum levels and distribution of IgG and IgG subclass Ab (IgGl, IgG2, IgG3, and IgG4) to the major opsonic immunodeterminant, serotype-specific lipopolysaccharide (LPS), by means of enzyme-linked immunosorbent assays employing monoclonal secondary antibodies, and comparing these results to the serum opsonic capacity in an in vitro murine alveolar macrophage phagocytic assay. Twenty-one patients with CF who were colonized with PA had approximately a 30-fold elevation of PA LPS IgG Ab levels and higher IgG subclass 1-4 Ab compared to 10 uncolonized patients with CF and 11 healthy controls (p<0.05-0.0005 depending on the isotype). Colonized patients with CF had a shift in PA LPS Ab distribution toward IgG3 compared to uncolonized patients with CF (p<0.02). A surprising finding was that uncolonized patients with CF had lower levels (p<0.05) and proportion (p<0.002) of PA LPS IgG2 Ab than controls, with an apparent shift to higher levels and proportion of PA LPS IgG4 (p<0.01). Serum from colonized patients with CF showed diminished opsonic capacity for phagocytosis of PA compared to uncolonized patients and controls (p<0.005), with 42% showing inhibitory activity. Functional Ab was also found to be inhibitory at high (> 500 ng/ml) concentrations. Serum opsonic capacity appeared to include a noncomplement cofactor for optimal activity. Levels of PA LPS IgG4 but not IgGl-3 subclass Ab correlated inversely with opsonic capacity. We conclude that high levels of PA LPS IgG4 Ab may be inhibitory to normal pulmonary clearance of PA in colonized patients with CF, and that high levels of functional antibodies may also contribute to this specific acquired deficiency. The role of deficient IgG2 Ab responses to PA LPS and possibly other polysaccharide antigens in CF requires further study.

Nonopsonic antibodies in cystic fibrosis. Pseudomonas aeruginosa lipopolysaccharide-specific immunoglobulin G antibodies from infected patient sera inhibit neutrophil oxidative responses.

Antibody opsonins from cystic fibrosis (CF) patients were investigated using nonmucoid and mucoid lipopolysaccharide (LPS) immunotype 1 Pseudomonas aeruginosa as bacterial ligands and PMN phagocytes. CF sera were compared to normal sera, polyvalent PA LPS hyperimmune globulin, and isotype switch variant monoclonal antibodies (MAbs) specific for type 1 PA LPS. Sera from PA-infected CF patients (CF PA+) had elevated levels of PA LPS and alginate IgG antibodies and promoted significantly greater antibody-dependent PMN chemiluminescence responses than sera from uninfected CF patients (CF PA-) or normal human sera (NHS). After adjustment for autologous IgG PA LPS antibody content, however, CF PA+ sera had less antibody-dependent opsonic activity than sera from CF PA- patients (P less than 0.025) or NHS (P less than 0.0025), suggesting qualitative opsonic defects of IgG PA LPS antibodies in CF PA+ sera. Antigen-specific immunoprecipitation of PA LPS antibodies enhanced opsonization by 40% of CF PA+ sera while uniformly reducing that from CF PA- sera (P less than 0.01), indicating LPS-specific nonopsonic antibodies in some CF PA+ sera. Alginate antibodies were not critical opsonins in most uninfected CF patient sera. PA LPS IgG antibodies isolated by immunoaffinity chromatography from NHS, hyperimmune globulin, and CF PA- sources were opsonic and had greater activity at equal antigen-binding concentration than identical antibodies isolated from infected CF patients (P less than 0.01-0.05); the majority of isolates from CF PA+ sera did not promote PMN oxidative responses above nonopsonic baseline. A potential isotypic basis for these findings was supported by differences in PMN responses to PA opsonized with MAbs of identical specificity but differing isotypes. PA LPS-specific IgG antibodies inhibiting PMN oxidative responses in infected patient sera demonstrate antigen-specific immunomodulation of host responses by chronic bacterial parasitism in CF, which may play a role in the pathophysiology of lung disease.

Altered Antibody Isotype in Cystic Fibrosis: Impaired Natural Antibody Response to Polysaccharide Antigens

ABSTRACT. Patients with cystic fibrosis (CF) have impaired natural (preinfection) IgG2 antibody responses to Pseudomonas aeruginosa lipopolysaccharide. To investigate the basis for this defect, we measured natural IgG and IgGl-4 antibody levels to Haemophilus influenzae type b polyribophosphate (PRP) and tetanus toxoid by enzymelinked immunosorbent assay in 24 adult CF patients and 20 normal controls. Immunoglobulin heavy-and light-chain allotypes were determined on 146 Caucasian CF patients and 96 controls. The tetanus toxoid-specific IgG response was predominantly IgGl. CF and control subjects had similar IgG and IgGl antibody levels. The PRP-specific IgG response was predominantly IgG2. In contrast to tetanus toxoid results, CF patients had lower geometric mean level of PRP-specific IgG compared to normal controls (p = 0.0036). ELISA results were confirmed by liquid-phase 3H-PRP-binding assay: CF patients had a geometric mean serum antibody level of 395 versus 922 ng/ml in controls (p = 0.0044). PRP-specific IgG2 levels were also depressed in CF patients (p = 0.03). CF patients had a lower prevalence of the A2m(2) allotype than the local racially matched control sample (p < 0.025). Other allotype prevalences including G2m(n) and Km(l) were similar. Impaired IgG2 antibody responses to microbial polysaccharide surface antigens in CF patients might predispose them to persistent endobronchial infection and lead to production of nonopsonizing isotype responses. The potential role of A2m(2), coded for in the II chain locus on chromosome 14, is unknown, but could be related to mucosal IgA2 antibody responses.