Assessment of airway distribution of transnasal solutions in mice by PET/CT imaging.

PURPOSE: Transnasal administration is one of the most common routes for allergen challenge in mouse models of airway diseases. Although this technique is widely used, neither the amount of allergen that reaches the lung nor its airway distribution has been well established. We used positron emission tomography (PET) and computed tomography (CT) to examine the anatomical distribution of a solution containing a tracer immediately after transnasal delivery and to determine the possible influence of age and administered volume. PROCEDURES: Forty-six female BALB/c mice were divided into three groups according to instillation volume and age: (A) 15 microl, 8-10 weeks old (N = 10), (B) 30 microl, 8-10 weeks old (N = 20), and (C) 30 microl, 32 weeks old (N = 16). Anesthetized animals underwent a dynamic scan in a dedicated small-animal PET scanner immediately after transnasal administration of a solution containing (18)FDG. Regions of interest were used to obtain quantitative data. Animals were also imaged with a small-animal CT scanner to obtain complementary anatomical information. RESULTS: Mean +/- SD (5.69 +/- 4.51%) of the solution administered reached the lungs in group A, 41.84 +/- 8.03% in group B, and 36.65 +/- 16.15% in group C. A comparable percentage was delivered to the left and right lungs in all the groups. Analysis of variance revealed a significant difference between the groups in the proportion of the solution that reached the lungs depending on the injection volume (P < 0.001), but not depending on animal age. CONCLUSIONS: In this first report on quantitative imaging by PET and CT in small animals, we confirmed the suitability of the transnasal route with an instilled volume of 30 microl delivering fluids into the lower airways, although only about 40% of the dose reaches the lungs.

Pollen-induced airway inflammation, hyper-responsiveness and apoptosis in a murine model of allergy.

BACKGROUND: Previous studies indicate that murine models are useful tools for studying the allergic diseases, including certain aspects of bronchial asthma such as cellular tissue inflammation and pulmonary function. OBJECTIVE: To develop an experimental model of allergic lung inflammation based on a relevant human allergen, olive pollen, and to establish the immunological, cellular and functional airway features of the allergic response in this model. METHODS: Induction of systemic allergic response was achieved by the subcutaneous administration of Olea europaea extract in BALB/c mice. Olea-specific Igs (IgG1, IgG2a and IgE) and cytokines from splenocyte cultures IL-4, IL-5 IL-10, IL-13 and IFN-gamma were measured. Allergic airway response was generated by transnasal instillation of the allergens. Airway responsiveness was monitored by non-invasive methacholine inhalation challenge. Lungs were paraffin embedded and histologically analysed. Apoptosis was studied by the TUNEL technique in the lung tissue and through cell cycle analysis by flow cytometry in splenocytes. RESULTS: Our results demonstrate that Olea-sensitized mice develop a specific allergic antibody (IgG1 and IgE) and cytokine (IL-4, IL-5, IL-10 and IL-13) response. After transnasal Olea instillation, they show inflammatory infiltration of lung tissue, mucus secretion and non-specific hyper-responsiveness in the airway. Concomitantly, differences in the rate of apoptosis are observed in the lung cells as well as a significant reduction of spontaneous apoptosis in the splenocytes of allergic mice. CONCLUSION: We present a novel animal model of olive pollen-allergic disease. This model presents traits associated with human allergic asthma and could be an interesting tool in the study of underlying molecular mechanisms and in exploring the therapeutic approaches to this disease.

Anisakis simplex allergy: a murine model of anaphylaxis induced by parasitic proteins displays a mixed Th1/Th2 pattern.

The study of the singular hypersensitivity reactions to Anisakis simplex (A.s) proteins, may help us to undestand many of the unknown immune interactions between helmiths infections and allergy. We have developed a murine model of allergy to A. simplex, that mimics human A. simplex allergy to study the specific aspects of anaphylaxis induced by parasites. Male C3H/HeJ mice were intraperitoneally sensitized to A. simplex. Mice were then intravenous or orally challenged with A. simplex. Antigen-specific immunoglobulins, polyclonal IgE, anaphylactic symptoms, plasma histamine levels and cytokine profiles were determined. Comparative IgE immunoblot analyses were also performed. Specific IgE, IgG(1) and IgG(2a) were detected in sensitized mice since week 3. Polyclonal IgE raised and peaked with different kinetics. Intravenous A. simplex challenge produced anaphylaxis in mice, accompanied by plasma histamine release. Oral A. simplex challenge in similarly sensitized mice did not caused symptoms nor histamine release. Numerous A. simplex allergens were recognized by sensitized mouse sera, some of them similar to human serum. The A. simplex stimulated splenocytes released IL-10, IFN-gamma, IL-4, IL-13 and IL-5. We describe a new animal model of anaphylaxis. It exhibits characteristics of type I hypersensitivity reactions to Anisakis simplex similar to those observed in allergic humans. Different responses to i.v. or oral A. simplex challenges emerged, which did not reflect a window tolerization period. The cytokine profile developed (mixed Th(1)/Th(2) pattern) differed from the observed in classical models of anaphylaxis or allergy to food antigens. This model may permit to investigate the peculiar allergic reactions to parasitic proteins.

Susceptibility to severe ulcerative colitis is associated with polymorphism in the central MHC gene IKBL.

BACKGROUND & AIMS: IKBL gene lies telomeric of the tumor necrosis factor cluster in the central major histocompatibility complex and carries a structural polymorphism at position +738. In the Spanish white population, we found the IKBL+738(C) allele in haplotypes carrying either HLA-DRB1(*)1501 or -DRB1(*)0103. Because these HLA class II alleles may confer susceptibility to ulcerative colitis, we investigated an association between IKBL+738(C) and this disease. METHODS: DNA-based techniques were used to type individual alleles of HLA-DRB1 and IKBL+738. The frequencies of these alleles were compared among ethnically matched populations comprising 155 patients and 298 controls. RESULTS: IKBL+738(C) allele was exclusively increased in patients with extensive and/or intractable disease. HLA-DRB1(*)0103 was the only HLA-DRB1 allele to be significantly increased in frequency in patients with UC compared with controls. It was found in patients with extensive and distal disease. In the HLA-DRB1(*)0103-negative population, patients with extensive disease still had a significant association with IKBL+738(C). The difference between the 2 groups of patients was statistically significant (13.7% vs. 1.7% in patients with distal disease; odds ratio, 9.25; P = 0.01). CONCLUSIONS: HLA-DRB1(*)0103 is associated with susceptibility to ulcerative colitis, and IKBL+738(C) marks a propensity to extensive and more severe disease.

Presence of a protective allele for achalasia on the central region of the major histocompatibility complex.

Idiopathic achalasia is a motility disorder of the esophagus whose etiology is unknown. An association between HLA genes and susceptibility to achalasia which suggests a possible immunogenetic mechanism has been reported recently. This study was designed to examine the HLA class II association in a large group of achalasia patients further and to investigate the distribution of TNFa and TNFb microsatellites in these patients. The study population, all Spanish, white and unrelated, consisted of 115 consecutive patients and 339 healthy controls. All of the patients had been diagnosed with primary achalasia of the esophagus with manometric, radiographic and endoscopic studies. All studies were performed on DNA samples after locus-specific amplification with the polymerase chain reaction: HLA-DRB1, DQA1 and DQB1 were typed by dot-blot hybridization and the size of the TNFa and TNFb microsatellites was measured using a semiautomatic method. The broad allele HLA-DQ1 was seen to be weakly associated with achalasia. The TNFa11 allele and the DRB1*1501-DQA1*0102-DQB1*0602 haplotype were reduced in achalasia patients but the stratified analyses showed that this was true only when both were present in the same individual. These results confirm the association between achalasia and HLA-DQ1 allele and suggest that TNFa11 is a marker for a protective allele for the disease, present on the B7-DRB1*1501 (7.1) ancestral haplotype in our population.

Primary association of tumor necrosis factor-region genetic markers with susceptibility to rheumatoid arthritis.

OBJECTIVE: To determine whether tumor necrosis factor (TNF) polymorphisms are associated with susceptibility to rheumatoid arthritis (RA) independently of the HLA-DR shared epitope. METHODS: Fifty-two Spanish families with one or more affected members were typed for HLA-DRB1, TNF promoter polymorphisms, and TNFa and TNFb microsatellites. We performed an association analysis comparing transmitted versus not transmitted haplotypes, with or without shared epitope, to determine whether there is an independent effect of TNF genetic markers on RA susceptibility. RESULTS: TNFa6,b5 was significantly associated with susceptibility to RA. The haplotypes containing these markers were preferentially transmitted to the affected offspring, even if these haplotypes lacked the HLA-DR shared epitope. TNF promoter polymorphisms were not associated with susceptibility to RA. CONCLUSION: The data suggest that TNFa/b is an independent marker of RA susceptibility, pointing to a genetic role of the TNF region in the pathogenesis of RA.

Primary association of a TNF gene polymorphism with susceptibility to multiple sclerosis.

The associations of three promoter polymorphisms in the tumor necrosis factor (TNFA) gene have been studied in 238 patients and 324 control subjects. A significant correlation was found between MS susceptibility and the TNFA-376 polymorphism. This association was independent of the human leukocyte antigen (HLA) class II association and the combined inheritance of HLA-DRB1*1501 and the TNFA-376A allele more than additively increased susceptibility to MS.

Pancolitis and genetic markers in the Spanish population.

BACKGROUND: although the etiology of ulcerative colitis disease remains an enigma, the importance of the major histocompatibility complex genes has been described, as in many other autoimmune diseases. AIM: we investigated the contribution of HLA-DRB1, DQA1 and DQB1 genes (HLA region) in patients with pancolitis. METHODS: we studied a total of 89 patients diagnosed as having ulcerative colitis (34 pancolitis and 55 left colitis) and 275 healthy control subjects. Complete information on sex, age, family history, age of onset, localization, complications, surgery and treatment was obtained from all patients. DNA was extracted from peripheral blood leukocytes and all individuals were HLA-DRB1 genotyped. RESULTS AND CONCLUSIONS: there was an association between pancolitis and the presence of DR4-Val86 (p = 0.009; OR = 3.3) and DRB1*0103 (p = 0.02; OR = 5.1) alleles. In patients with left colitis an association with DRB1*1501 (p = 0.03; OR = 1.9) and DRB1*0103 alleles (p = 0.03; OR = 3.8) was observed. We conclude that a strong association between DR4-Val86 and pancolitis exists.

HLA class II homozygosity confers susceptibility to common variable immunodeficiency (CVID).

Most cases of CVID occur sporadically, but familial cases do also occur and 15% of the patients with the disease have first degree relatives with IgA deficiency (IgAD). Our purpose was to study CVID association with HLA class II alleles and to ascertain whether this disease shares a common genetic background with IgAD in our population. Patients with CVID (n = 42), were typed using gene amplification and sequence-specific oligonucleotide probing for HLA-DRB1, DRB3, DQA1 and DQB1 loci and their typing compared with that of 96 IgAD and 334 healthy controls. We observed a positive association between non-Asp residues at position 57 of the HLA-DQbeta chain and CVID, although much weaker than in IgAD. Further, we found an association between CVID and homozygosity for genes encoding HLA class II molecules, especially HLA-DQ, not seen in IgAD. The data support the hypothesis that a restricted diversity of HLA class II molecules may contribute to susceptibility to CVID.

Susceptibility to multiple sclerosis mediated by HLA-DRB1 is influenced by a second gene telomeric of the TNF cluster.

Susceptibility to multiple sclerosis (MS) is clearly associated with human leukocyte antigen (HLA)-DRB1*1501, but some studies show associations with HLA-B7 and -B18. These are often co-expressed with DRB1*1501 in the ancestral haplotypes (AH) denoted 7.1 (HLA-A3, B7, tumor necrosis factor [TNF]a11b4, DRB1*1501) and 18.1 (HLA-A25, B18, TNFa10b4, DRB 1*1501). Here we present a systematic study of 218 patients and 274 controls typed at all standard class II and TNF microsatellite loci, and a novel non-synonymous polymorphism in the central major histocompatibility complex gene, inhibitor of kappa B-like protein (IKBL). The C allele at IKBL+738 is only found on the 7.1 haplotype. HLA-DRB1*1501 was associated with disease, as expected. When subjects expressing DRB 1*501 were analyzed separately, TNFa11b4 and IKBL+738C were less common in the patients and, hence, mark an allele that mediates resistance which lies telomeric of IKBL. TNFa10b4 and TNFa1b5 were more common in DRB1*1501 patients than in controls. These alleles have been associated with the 18.1 and 18.2 AH, respectively. Since no component of these haplotypes was an independent risk factor in this study, it appears likely that a gene linked to TNFa10b4 and TNFa1b5 modifies the effect of the susceptibility locus marked by HLA-DRB1*1501. Potential candidate genes telomeric of the TNF cluster are discussed.

Contribution of HLA class II genes to susceptibility in achalasia.

Achalasia is a motor disorder of the esophagus resulting in functional obstruction. The cause of the lesion is unknown although genetic and immunologic factors have been suggested. An association with serological HLA epitopes has been previously reported. In this study, we have further examined this HLA class II association with susceptibility to achalasia by DNA based methods. Achalasia patients (n=40) and healthy controls (n=275), all Caucasians and unrelated, were included in the analysis. The strongest associations were with HLA-DQA1*0101 and two HLA-DQ alphabeta heterodimers having their alpha chain encoded by this allele. Moreover, relative risk was significantly higher in DQA1*0101 homozygotes as compared to heterozygotes and results suggested that DQB1*02 may have a protective role.