350 Human dendritic cells in the SCID mouse model: Their potentiating role in the allergic reaction

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Abstracts

Human Airway Epithelial Cells Are Lysed by Peripheral Blood Mononuclear Cells after Incubation with a Bacterial Lectin Terry Chin*, No@ Alonazi§ *Loma Linda University School of Medicine, Loma Linda, CA tArmed Force Hospital Riyadh. Kingdom of Sauda Arabia Plant lectins such as phytohemagglutinin (PHA) are well known to induce lymphocytes to become cytotoxic cells or lectin-dependent cellular cytotoxicity (LDCC). The precise mechanisms of lung destruction in cystic fibrosis (CF) are unknown. Since airway inflammation has been speculated to play a major role in the pathogenesis of lung injury in CF, we examined whether an internal bacterial antigen of Pseudomonas aeroginosa (PSA)-PA-I, can induce peripheral blood mononuclear cells (PBMC) to be cytotoxic toward 3 human airway epithelial cell lines (A549. NHBE and BEAS-2B). PBMC were isolated from 20 normal donors by density gradient centrifugation and incubated in the presence of PA-I (in RPMI-1640 with 10% FCS) for 3-5 days at 37°C. Cell concentration was then adjusted and stimulated and nonstimulated PBMC were added to epithelial target cells at different effector:to:target (E:T) cell ratios. Cellular cytotoxicity was assayed by LDH release measured by ELISA kit (Promega) and specific lysis calculated. Optimal cytotoxicity was observed after 3 day incubation for A549 targets and 5 days for BEAS-2B targets. There was an increase in cytotoxicity toward A549 (p=O.O12) and BEAS-2B (p=O.O09) at an E:T ratio of 4: I (see Figure). These results demonstrate that bacterial lectins are capable of eliciting lectin-dependent cellular cytotoxicity (LDCC) toward airway epithelial cells. Whether CF patients will show a difference in LDCC activity requires further study. In addition, other lectins such as those from Aspergillus may play a role in allergic bronchopulmonary aspergillosis (ABPA).

J ALLERGY CLIN IMMUNOL JANUARY 2000

A statistical analisys of the results for specific IgE were performed for each allergen. We concluded from the statistical study that there is an association between values obtained by the two systems for each allergen. We concluded from this study that both methods can be used in clinical practice showing good correlation with skin prick tests and clinical history. 349

The Immune Responses in Asthmatic Mice with DNA Vaccine Containing Allergen and IL-12 cDNA Sequences ML Kuo. LC Chen, CC Huang. JL Huang Medical Research Center, Chang Gung Memorial Hospital. Bronchial asthma is a chronic inflammatory disorder of the airways. It causes a great public health concern and potential preventive method is not available currently. Many cells contribute to the onset of the disease. in particular. T lymphocytes, mast cells, and eosinophils. It has been well established that cytokines produced by Th2 type cells are responsible for clinical symptoms, IL-4 and IL-13 can enhance the IgE production by B cells and IL-5 promotes the differentiation, maturation, survival and recruitment of eosinophils to the airways. Among those possible approaches in the treatment or prevention of asthma, DNA vaccine containing allergen cDNA sequence has been tested in animal models related to airway hypersensitivity. In addition, IL-12 has been approved to be an important factor to lead the precursor T helper cells to differentiate into Thl population. In this study, we examined the preventive effect of airway hypersensitiveness with the injection of plasmid DNA containing chicken ovalbumin (OVA) cDNA and/or IL- 12 sequences. We used the DNA vector that expresses IL-12 fusion protein to ensure the equal expression of both ~35 and p40 peptides. Purified plasmid DNA was injected intramuscularly 3 weeks before OVA sensitization. The percentage of eosinophils in bronchoalveolar lavage fluid (BALF) and serum OVAspecific IgE. IgGl and IgG2a were examined in each group. The results indicate that percentage of eosinophils reduced from 23.6% of the control group to 14.9% of the group injected with the combination of OVA and IL-12 DNA. More significant reduction (3.7%) of eosinophils was seen with the injection of IL- I2 sequence plus control plasmid. The injection of OVA sequence plus control plasmid DNA did not affect eosinophil cell counts in BALE Similar results were obtained with OVA-specific IgGl level. The group with the ILI2 sequences plus control plasmid gave the most reduction of OVAspecific IgGl level. This indicates that IL-12 could inhibit Th2-type activation. However, this group also showed the least level of OVAspecific IgGZa. On the other hand, the combination of OVA and IL- I2 stimulated the most OVA-specific IgG2a production, which is an indicator of Thl-type response. These preliminary results indicate that plasmid DNA containing IL-I 2 sequence is more potent to affect the ThZ-type immune responses. The OVA sequence alone is not sufficient to change Thl or Th2 polarity in OVA-sensitized mice in this study. The injection of plasmid DNA containing allergen and IL- 12 sequences showed immunoregulatory effect to asthmatic mice. The results of this study suggest that cytokine gene injection is effective modulating allergic responses and may provide high risk asthmatic children an alternative choice to prevent the onset of asthma.

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Human Dendritic Cells in the SCID Mouse Model: Their Potentiating Role in the Allergic Reaction Hamida Hammad& C Duez’, 0 Fahy$. A-B Tonne/S;, JOY Pestef$ *INSERM U416 tNational Jewish Center tDenver SINSERM U416. Lille, France ‘National Jewish Center, Denver, CO Dendritic cells (DC) are present in the lungs and in the airways from normal and allergic subjects where they are exposed to inhaled antigens. The DC function is mainly dependent on their migratory properties. To better understand the role of DC in the allergic reaction, DC were generated in vitro from purified monocytes from healthy donors and allergic patients after 14 days of culture in the presence of GM-

348 Specific

IgE Determination by Two Different Methods AlaSTAT, DPC and UniCAP, Pharmacia AP Pires, R Murta. F India Immunoallergology Department, Hospital S. Bernardo, Settibal, Portugal IgE antibodies have been considered as a marker of atopy. UniCAP System from Pharmacia has been considered a reference system for total and specific IgE in atopy. The appearance of new methodologies for specific and total IgE determination can be of great economical and research interest for laboratories involved in allergy, bringing another way to quantify IgE. In this study we compared two different methods to quantify total and specific IgE in serum: the UniCAP System (Pharmacia, Upsalla, Sweden) and AlaSTAT method (DPC, LA, California, USA). We studied 248 serum samples from allergic patients quantifying the specific IgE to Der p, Dac g, Phl p and Bet v as well as total IgE in 43 samples. We observed for Der p allergens a good correlation between methods to classes 0,1,2 and 3. To the classes 4.5 6 the AlaSTAT method showed higher values than UniCAP. To the Dac g and Phl p allergens we observed a good correlation between methods in classes 4.5 and 6. In UniCAP we observed higher absolute values of specific IgE than AlaSTAT. Concerning the Bet v allergens we observed higher values inside each class and in all classes (except class 0) with UniCAP System comparing to the AlaSTAT method. With the total IgE we could observe a linear regression with a r2 value of 0.91.

Abstracts

J ALLERGY CLIN IMMUNOL VOLUME 105, NUMBER 1, PART 2

CSF and IL-4. Their migratory behaviour was analyzed in the SCID mouse model. At day 0, SCID mice were injected intraperitoneally with I x I06 monocyte-derived DC and at day 4, were exposed or not to the aeroallergen Dermatophagoides pteronyssinus (Dpt). At day 7, mice were sacrificed and the presence of the markers CD I a, CD 14. CD68 was investigated by immunohistochemistry on frozen or paraffin sections of thymus, lungs and spleen. Only CDla-positive DC were detectable in the thymus, the lungs and the spleen of reconstituted SCID mice. After exposure to Dpt, the number of CDla+ DC increased in the lymphoid organs and decreased in the lungs, suggesting a selective DC traffic. Indeed, Der p I. the major allergen of Dpt, was only detected in the spleen of mice exposed to Dpt. This migration capacity could be dependent on two factors: the adhesion molecule CD 1Ic and TNFa, both highly expressed by DC in vitro exposed to Der p I. Moreover, when SCID mice reconstituted with PBMC from allergic patients were previously injected with DC from the same donor, the IgE production was highly increased compared to the IgG production. In contrast, with cells from healthy donors, an opposite effect was observed. In addition, DC were also detected in SCID mice only reconstituted with PBMC from allergic patients and producing human IgE and developping an inflammatory pulmonary infiltrate. At least one month after the Dpt exposure, DC were located in the alveolar spaces and walls of the lungs and preferentially expressed the phenotype of mature DC. Taken together, all these results suggest that in SCID mice, human DC may play a role in the development of the allergic reaction in response to Dpt. aaaaaaa 351 Residential Exposures Associated with Asthma in U.S. Children Bruce Lunphear, Michael Weirzmon

Andrew

Al&e,

Peggy

Auingec

Robert

Byrd,

CONTEXT. Residential exposures such as indoor allergens and environmental tobacco smoke are recognized risk factors for asthma, but the contribution of such exposures to asthma in U.S. children is unknown. OBJECTIVE. To estimate the population attributable risk of residential exposures to doctor-diagnosed asthma for U.S. children younger than 6 years of age. DESIGN. Cross-sectional survey conducted from 1988 to 1994. Setting and Participants. A total of 8,257 children less than 6 years of age who participated in the Third National Health and Nutrition Examination Survey, a survey of the health and nutritional status of children and adults in the United States. MAIN OUTCOME MEASURE. Doctor-diagnosed asthma, as reported by the parent. RESULTS. Six percent of children had doctor-diagnosed asthma. The prevalence of asthma was higher among boys (6.7%) than girls (5. I %), and higher among Black children (8.9%) than White children (5.2%). Independent predictors of doctor-diagnosed asthma included a family history of atopy (OR=2.2, 95%CI=1.5, 3.1). child’s history of allergy to a pet (08=24.2,95%CI=8.4,69.5), exposure to environmental tobacco smoke (OR=l.8. CI=1.2-2.6). use of a gas stove or oven for heat OR=1.8 (CI=l.O2-3.2) and presence of a dog in the household (OR= 1.6, CI=I. I, 2.3). The population attributable fraction of z I or more residential exposure for doctor-diagnosed asthma in U.S. children c 6 years of age was 39%. or an estimated 532,972 excesscases.In contrast, having a family history of atopy accounted for 300,CKKlexcesscases. CONCLUSIONS. The elimination of identified residential exposures - if causally associated with asthma - would result in a 40% decline in doctor-diagnosed asthma among U.S. children younger than 6 years of age. 352 Hypersensitivity Pneumonitis From Pezizia Domiciliana: A Case of El Nifio Lung 24 Dyer; RS Wright, MI Liebhaber Sansum-Santa Barbara Medical Foundation Clinic, Santa Barbara, CA

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We present an unusual case of extrinsic allergic alveolitis in the home setting. An unprecedented high rainfall during the 1998 El Nigo season and the flooding of a domestic residence provided the conditions for an unusual caseof hypersensitivity pneumonitis in the Santa Ynez Mountains near Santa Barbara, California. A previously healthy 54 year-old woman developed severe dyspnea over a three month period and was found to have restrictive lung disease on pulmonary function testing with evidence of alveolitis. Her diffusing capacity was markedly reduced at 14% predicted. A high-resolution chest tomogram showed ground-glass haziness and small ce.ntriIobular nodules. An open lung biopsy revealed extrinsic allergic alveolitis with moderate lymphoplasmacytic infiltrate and non-necrotizing granulomas (hypersensitivity pneumonitis). The etiology was not initially apparent. A home inspection showed an unusual mushroom identified as Pezizia donziciliana (a Discomycete) growing in the basement. Volumetric air sampling and serum precipitins, obtained specific for fungal samples in the home, confirmed that Pezizia domiciliana was the likely cause of hypersensitivity pneumonitis. The patient subsequently improved with avoidance measures and a course of steroid therapy. This is the first described case.of hypersensitivity pneumonitis due to Pezizia domiciliana. We speculate that the unprecedented rainfall and flooding in her basement due to the El Niilo rains produced ideal factors for the growth of this fungus. The home visit led to mitigating the exposure and prevention of disease progression.

353 Effect of Cigarette

Smoke (CS) on Intracelhlar Reduced-Glutathione Levels of Primary Cultures of Human Bronchial Epithelial Cells of Never-Smokers, Smokers and Patients with COPD C Rusznak*t, PR Mills*. JL Devalia*, RJ Sapsford*. RJ Davies*, S Lozewicz* *Academic Department of Respiratory Medicine, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, London, UK tA1lergy Center, Vanderbilt University Medical Center, Nashville, TN, USA Although cigarette smoking is of paramount importance in the development of chronic obstructive pulmonary disease.(COPD), only a small proportion of smokers develop the disease. We have recently demonstrated, that primary cultures of epithelial cells from patients with COPD have a greater susceptibility to the effects of CS causing increased permeability and release of pro-inflammatory mediators such asIL- IO; and sICAM- I. To investigate the putative underlying mechanisms responsible for the observed differences, we established primary explant cultures of human bronchial epithelial cells (HBEC) from biopsy material obtained from never-smokers, who had normal pulmonary function, smokers with normal pulmonary function and smokers with COPD. The HBEC cultures were exposed for 20 minutes to CS or air, and intracellular reduced glutathione (GSH) levels measured after 24 hour incubation. Levels of intracellular GSH following exposure to air were significantly higher in cultures of HBEC derived from both smokers with normal pulmonary function and COPD patients, than in cultures from healthy never-smokers. Exposure to CS significantly decreased the concentration of intracellular GSH in all cultures when compared to exposure to air. The fall in intracellular GSH was significantly greater in cells from smokers with COPD (mean 72.9% decrease) than in cells from smokers with normal pulmonary function (mean 43.9% decrease, p=O.O2) or cells from never-smokers (mean 50.6% decrease,p=O.O48).These results suggest that whereas smokers with and without COPD demonstrate increased levels of GSH within bronchial epithelial cell cultures, those with COPD have a greater susceptibility to the effect of CS in reducing intracellular GSH levels. Whether in patients with COPD the greater susceptibility of bronchial epithelial cells to CS exposure contributes to the develop ment ofCOPD, or is a secondary characteristic of this condition, need to be determined.