IgE and IgG antibody responses to recombinant Alt a 1 as a marker of sensitization to Alternaria in asthma and atopic dermatitis.

BACKGROUND: Sensitization to Alternaria alternata is a risk factor for the development of wheezing and asthma. Alt a 1 is the major Alternaria allergen causing sensitization in asthmatics. Some atopic dermatitis (AD) patients have very high immunoglobulin (Ig)E antibody (ab) to Alternaria as analysed by Pharmacia CAP, however, it is not clear whether these are specific responses or whether Alt a 1 is involved in disease symptoms. OBJECTIVE: The aim of this study was to analyse specific IgE and IgG ab responses to recombinant Alt a 1 in asthmatic and AD patients and to compare these results to IgE ab against Alternaria measured by CAP. METHODS: Sera from individuals who were IgE positive to Alternaria by CAP were obtained from 58 patients with asthma/rhinitis, 19 patients with AD, and 20 patients with cystic fibrosis (CF) who were included as specificity controls. IgE and IgG ab to recombinant Alt a 1 were measured by radioimmunoassay (RIA). RESULTS: Of 43 asthma/rhinitis patients having an Alternaria CAP score > 2, a high percentage (93%) had both IgE and IgG ab to Alt a 1, emphasizing its importance as a major allergen. Only, 47% of AD patients with CAP score greater than 2 had ab to Alt a 1, and their levels were low when compared to the asthmatics. For CF controls, 75% of these patients had no IgE ab to Alt a 1, and those which were positive to Alt a 1 by RIA were also positive by CAP. Overall, patients with a low CAP (1-2) had a low prevalence (20-30%) of IgE or IgG ab to Alt a 1. CONCLUSION: IgE and IgG ab to Alt a 1 in asthmatics are good markers for sensitization to Alternaria. Although AD patients gave high Alternaria CAP scores, they had low or undetectable levels of IgE to Alt a 1, suggesting that other Alternaria allergens may be important in AD or that the CAP results are non-specific. Recombinant allergens may provide more specific measures of sensitization to fungi.

Use of specific IgE in assessing the relevance of fungal and dust mite allergens to atopic dermatitis: a comparison with asthmatic and nonasthmatic control subjects.

BACKGROUND: Although allergens have been implicated as aggravating factors in atopic dermatitis (AD), there is little epidemiologic data on the significance of specific IgE. OBJECTIVE: We sought to compare sensitization to dust mite and fungi between patients with AD and asthmatic and nonasthmatic control subjects. METHODS: Total IgE and specific IgE to Dermatophagoides pteronyssinus, Alternaria alternata, Aspergillus fumigatus, Candida albicans, Malassezia furfur, and Trichophyton rubrum were measured in 73 patients with moderate to severe AD. Total IgE and IgE specific for D pteronyssinus, A alternata, and M furfur were also measured in sera from 156 asthmatic and 212 nonasthmatic control subjects. RESULTS: Positive correlations were found between total IgE and IgE antibodies specific for each of the antigens. IgE specific for M furfur was observed more frequently in adults compared with children with AD (P <.01). AD sera had higher levels of total IgE and a higher prevalence of positive sera to D pteronyssinus (95% vs 42% and 17% for subjects with AD, asthmatic subjects, and nonasthmatic subjects, respectively), M furfur (53% vs 1% and 0.5%), and A alternata (49% vs 29% and 18%). Among the sera from subjects allergic to mites, the contribution of IgE specific for D pteronyssinus to the total IgE levels was similar regardless of the clinical status. CONCLUSIONS: Our results demonstrate that moderate-to-severe AD is strongly associated with sensitization to dust mite andM furfur (odds ratios, 45.6 and 132 vs pooled control sera). These results suggest that both environmental allergens and colonizing fungi contribute to the severity of disease, which is consistent with the view that mite avoidance and antifungal treatment can be beneficial in the treatment of these patients.

Acetylcholine receptor alpha subunit mRNA expression in human thymus: augmented expression in myasthenia gravis and upregulation by interferon-gamma.

Previous studies by us and others have demonstrated the expression of acetylcholine receptors on epithelial cells in the thymus of myasthenia gravis (MG) and control subjects. In the present experiments, we used a reverse transcription-polymerase chain reaction (RT-PCR) to analyze the profile of the two major isoforms of the alpha chain of these receptors (AChRalpha), P3A- and P3A+, in thymus tissue obtained from MG and control subjects and a human thymic epithelial cell line (TEC9). In addition, using a semiquantitative RT-PCR, we compared the amounts of P3A- and P3A+ mRNA expressed in thymic tissue obtained from these two sources and determined if their expression in TEC9 is modulated by cytokines. We found that mRNAs encoding P3A- and P3A+ are expressed at approximately a 5:1 ratio in both MG and control thymus tissue. This contrasts with skeletal muscle where mRNAs encoding these isoforms are expressed equally. A pattern of preferential P3A- vs P3A+ mRNA expression was also observed in TEC9. We observed 2.8-fold greater expression of both isoforms in MG than in control thymus. Expression of both isoforms in TEC9 was enhanced significantly by treatment with interferon-gamma whereas IL-1alpha, IL-4, and IL-6 had no effect. Thus, there is differential regulation of AChRalpha variants in thymus and TEC relative to muscle and interferon-gamma represents a novel regulator of AChRalpha mRNA expression. MG thymus is distinguished by increased expression of both isoforms of this autoantigen, a finding that may reflect enhancement of transcription by local microenvironmental factors.

Trichophyton antigens associated with IgE antibodies and delayed type hypersensitivity. Sequence homology to two families of serine proteinases.

The dermatophyte fungus Trichophyton exhibits unique immunologic properties by its ability to cause both immediate and delayed type hypersensitivity. An 83-kDa Trichophyton tonsurans allergen (Tri t 4) was previously shown to elicit distinct T lymphocyte cytokine profiles in vitro. The homologous protein, Tri r 4, was cloned from a Trichophyton rubrum cDNA library, and the recombinant protein was expressed in Pichia pastoris. This 726-amino acid protein contained an arrangement of catalytic triad residues characteristic of the prolyl oligopeptidase family of serine proteinases (Ser-Asp-His). In addition, a novel Trichophyton allergen, encoding 412 amino acids, was identified by its human IgE antibody-binding activity. Sequence similarity searches showed that this allergen, designated Tri r 2, contained all of the conserved residues characteristic of the class D subtilase subfamily (41-58% overall sequence identity). Forty-two percent of subjects with immediate hypersensitivity skin test reactions to a Trichophyton extract exhibited IgE antibody binding to a recombinant glutathione S-transferase fusion protein containing the carboxyl-terminal 289 amino acids of Tri r 2. Furthermore, this antigen was capable of inducing delayed type hypersensitivity skin test reactions. Our results define two distinct antigens derived from the dermatophyte Trichophyton that serve as targets for diverse immune responses in humans.

Future directions for allergen immunotherapy.

Over the last 30 years several approaches to modify immunotherapy have been tested, including allergoids, alum precipitation, and most recently peptides. However, none of these have replaced the traditional regimens. Over the same period our scientific understanding of allergic disease has been transformed. Today it is possible to identify and monitor changes occurring during treatment and to target many different aspects of the immune system. Recombinant technology provides a powerful technique both for sequencing proteins and producing allergens in commercial quantities. The recombinant proteins can be modified by site-directed mutagenesis so as to decrease their reactivity with IgE antibodies while maintaining reactivity with T cells. Knowledge of the tertiary structure of allergens will make it simpler to identify and change surface epitopes. A completely different approach is to use plasmids to introduce the genes for an allergen. The strength of this technique is that the plasmid can be designed to control expression and also to influence the cytokine profile of the response or the isotype of antibodies produced. Finally, different adjuvants can be used with proteins to alter the response. These include IL-12, immunostimulatory sequences of DNA, and bacterial proteins such as those used in HibVax. It is now possible to identify the cells that control the immune response to allergens and to design treatments that will either downregulate or change the response of T cells. The challenge is to transform this information into an effective treatment for allergic disease.

Tracheal intubation in ambulatory surgery patients: using remifentanil and propofol without muscle relaxants.

UNLABELLED: Using alfentanil followed by an anesthetic induction dose of propofol provides adequate conditions for tracheal intubation without neuromuscular relaxants. Remifentanil, which has a clinical onset similar to that of alfentanil, has not been investigated for this indication. Accordingly, 80 ASA physical status I and II premedicated outpatients were randomly assigned to one of four groups (n = 20/group). Remifentanil 1, 2, 3, or 4 micrograms/kg (Groups I-IV, respectively) was infused intravenously over 90 s. Sixty seconds after beginning the remifentanil infusion, propofol 2 mg/kg was infused over 5 s. Ninety seconds after the administration of propofol, laryngoscopy and tracheal intubation were attempted and graded. Clinically acceptable intubating conditions (i.e., jaw relaxed, vocal cords open, and fewer than two coughs in response to intubation) were observed in 35%, 75%, 100%, and 95% of patients in Groups I-IV, respectively. Clinically acceptable intubating conditions were significantly (P < 0.05) less likely to occur in Group I compared with all other groups. Excellent intubating conditions (i.e., vocal cords open, no movement in response to intubation) were observed in 30%, 50%, 80%, 80% of patients in Groups I-IV, respectively. Overall conditions at intubation were significantly (P < 0.05) better in Groups III and IV compared with Groups I and II. The mean time to resumption of spontaneous ventilation after induction was < 5 min in all groups. No patient manifested clinically significant muscle rigidity. The mean arterial pressure decreased 16%, 20%, 28%, 26% immediately before tracheal intubation in Groups I-IV, respectively. No patient was treated for hypotension or bradycardia. In conclusion, healthy, premedicated patients with favorable airway anatomy can be reliably intubated with good or excellent conditions 90 s after the administration of remifentanil 3-4 micrograms/kg and propofol 2 mg/kg. IMPLICATIONS: Remifentanil 3 micrograms/kg and propofol 2 mg/kg co-administered intravenously may reliably provide adequate conditions for tracheal intubation in healthy patients without neuromuscular relaxants. This combination of drugs may allow the rapid return of spontaneous ventilation.

Indoor versus outdoor allergens in allergic respiratory disease.

Immediate hypersensitivity to indoor or outdoor allergens is strongly associated with asthma or hay fever, respectively. Recent progress has defined the sequences, tertiary structures and enzymatic functions of many of the proteins involved; furthermore, the immune responses to these proteins have been examined; however, the mechanisms responsible for the dichotomous response remain elusive. The resolution of such mechanisms may explain the large increase in the prevalence of eosinophil-rich inflammation of the lower respiratory tract.

Chronic sinusitis: risk factors for extensive disease.

BACKGROUND: Chronic sinus disease is one of the most common diseases in the United States, and little is understood about its pathogenesis. OBJECTIVE: To characterize prospectively the immunologic parameters that accompany extensive chronic sinusitis. METHODS: Eighty adult patients with chronic sinus symptoms had a complete blood count with differential, coronal computed tomographic (CT) scan of the sinuses, and serum assays for total IgE, specific IgE, IgA, IgG, IgG subclasses, and pneumococcal titers. RESULTS: Thirty-seven (46%) patients had extensive sinusitis, defined as a CT score > or = 12. A highly significant correlation was noted between the extent of disease and the peripheral eosinophil count (r = 0.53, p < 0.0001). In keeping with this finding, an eosinophil count > or = 200/microl was strongly associated with extensive disease (odds ratio [OR] = 19.2, 95% confidence interval [CI] = 5.4 to 72.7). Asthma (OR = 6.8, 95% CI = 2.2 to 22.0), atopy (OR = 4.3, 95% CI, 1.5 to 12.8), and age > or = 50 years (OR = 6.5, 95% CI = 2.0 to 22.2) were also associated with extensive disease. However, the association of eosinophils with extent of disease was independent of asthma, atopy, or age. Levels of IgG1, IgG2, and IgG3 subclasses did not correlate with extent of disease seen on CT scan. Although total IgE and IgG4 levels did correlate with disease, on multiple stepwise regression they did not add to the predictive value of the eosinophil count in identifying patients with extensive disease. CONCLUSIONS: The association of asthma, atopy, eosinophilia, and elevated levels of IgE and IgG4 with extensive disease on CT scan is compatible with the hypothesis that chronic sinusitis may be a disease of immune activation of the T(H2) type.

Semiautomatic quantitation of macrophages in human renal biopsy specimens in proteinuric states.

AIMS: To develop and validate a rapid and economical semiautomated approach to the measurement of immunostainable tissue components which is applicable to routine diagnostic practice. To apply this approach to the measurement of macrophages in renal biopsy specimens in nephrotic states, as protein in the renal tubules may induce macrophage infiltration, and the morphology of macrophages in tissue sections does not lend itself to cell counting. METHODS: Macrophages were identified by immunostaining with a pan-macrophage marker, followed by digital image capture and analysis using a macro procedure written for the freeware image analysis program NIH-Image. RESULTS: The method was rapid, robust and accurate to within the limits imposed by sampling error inherent in the use of small needle biopsy specimens. Very few macrophages are found in normal kidney (mean volume fraction (+/- 95% confidence limits) 0.04% (0.02%)) but infiltration of macrophages was detected in minimal change nephropathy (0.29% (0.12%)) and in membranous glomerulonephritis (0.42% (0.11%)). A statistically significant correlation was found between macrophage volume fraction and weight of proteinuria in minimal change nephropathy but not in membranous glomerulonephritis. Correlations were found in both diseases between macrophage volume fraction and serum creatinine at time of biopsy. CONCLUSIONS: The equipment is inexpensive and measurement takes less than one minute per biopsy specimen. The results indicate that macrophage infiltration is part of the pathological process in minimal change nephropathy and membranous glomerulonephritis. The correlation with creatinine at time of biopsy suggests that renal impairment in minimal change nephropathy may result from infiltration by immunologically active cells and not merely from haemodynamic changes in nephrons. However, the correlation is not close, indicating that the relation between macrophage infiltration and disease severity is not a simple one.

Iron-based second-sphere contrast agents for magnetic resonance imaging: development of a model system and evaluation of iron (III) tris (tironate) complex in rats.

RATIONALE AND OBJECTIVES: Iron(III) complexes have been developed as magnetic resonance (MR) contrast agents based on the use of ligands capable of providing coordinative saturation at iron and allowing the second-sphere hydrogen bonding of water molecules. METHODS: Studies of solution-phase binding of a probe ion to a diamagnetic metal catecholate complex and molecular orbital calculations were performed. R1 values were determined. Toxicity studies of iron(III) tris(tironate) were conducted with rats. Excretion studies were performed by analysis of urine samples. MR measurements were made. RESULTS: Second-sphere coordination of a probe ion to a metal catecholate complex occurred in solution. Molecular orbital calculations suggested flexibility in design. Iron(III) tris (catecholate) complexes were shown to have high R1 values. Images of the kidneys and liver showed dose-dependent enhancement in T1-weighted images. The onset of toxicity was at approximately 0.30 mmol/kg. Urine analysis indicated essentially complete clearance (0.1-0.2 mmol/kg) within 24-48 hours. CONCLUSION: Interactions between water molecules and basic sites within a paramagnetic metal-ligand complex allow for application of suitable complexes as T1 agents.

The role of allergy and atopy in asthma.

Asthma has become a very common disease and despite new treatments continues to cause very large numbers of hospital admissions, school absences and so on. Recent evidence from many parts of the world has established that the single most important risk factor for perennial asthma is sensitization to one of the major indoor allergens: dust mite, cat, dog, or cockroach. However, there is also striking evidence about the role of rhinoviruses and ozone as enhancers of the immune response. Over the past 2 years, the ways in which these enhancers interact with the primary immune response both to cause asthma and to influence the severity of the disease have started to become clear.

B-cell superantigens: definition and potential impact on the immune response.

Superantigens have been extremely helpful tools in exploring fundamental questions in immunobiology including mechanisms of cell activation, tolerance, and autoimmunity. Until recently, attention has been focused exclusive on T-cell superantigens. However, new data suggest that there are superantigens that directly activate B cells. By definition, these agents (1) stimulate a high frequency of B cells, (2) target B cells that have restricted usage of VH or VL family genes, and (3) bind to immunoglobulins outside the sites that bind conventional antigens. A candidate B-cell superantigen that has received considerable attention in this laboratory is staphylococcal protein A. This agent is best known to the immunologist because of its ability to bind to the Fc fragment of IgG. This binding has been localized to two alpha-helical structures on each of four or five homologous regions that comprise the extracellular domain of protein A. However, it is now clear that protein A contains a second site that binds to determinants on the Fab regions of certain immunoglobulins independently of their heavy-chain isotype. In man this so-called alternative site appears to bind only to immunoglobulins that utilize heavy-chain genes of the VH3 subfamily. In the mouse this type of binding is restricted to immunoglobulins using heavy chains belonging to the S107 and J606 VH families. In this review, we examine the growing list of microbial products that dominate B-cell superantigenic properties. Using staphylococcal protein A as a model for a B-cell superantigen, we consider the potential impact of this novel class of antigens on the immune response. We focus on the ability of B-cell superantigens to influence the expression of the B-cell repertoire. In addition, we consider the hypothesis that the interaction of a B-cell superantigen with its reactive serum immunoglobulins activates the classical complement cascade and thus represents a powerful stimulant of tissue inflammation.

Staphylococcus aureus Cowan I-induced human immunoglobulin responses: preferential IgM rheumatoid factor production and VH3 mRNA expression by protein A-binding B cells.

Protein A (PA), a cell wall component of SAC, activates human B cells by cross-linking the Fabs of membrane immunoglobulins. Recent data indicate that PA binds only to Fabs that use VH3 heavy chains, and thus it has been designated as a B-cell superantigen. We previously reported that Staphylococcus aureus Cowan I (SAC)-induced IgM rheumatoid factor (RF) by human PBMC was mediated by PA. Therefore, we sought to determine if SAC-induced IgMRF production was confined to PA-binding B cells and if these B cells were enriched for the expression of VH3 heavy chains. We observed that the elicitation of IgMRF in response to SAC was limited to a subset of B cells that bind PA and that this subset was enriched for VH3 mRNA expression. Taken together, these results suggest that IgMRFs produced in response to SAC are enriched for usage of VH3 heavy chains. Thus, this SAC-induced autoantibody response appears to represent a new B-cell superantigenic property of PA.

Molecular evidence for the expression of nicotinic acetylcholine receptor alpha-chain in mouse thymus.

The presence and structure of nicotinic acetylcholine receptor (nAChR) in the thymus has been a subject of interest for many years because of its possible role in the pathogenesis of the autoimmune disease myasthenia gravis. Using the polymerase chain reaction with primers specific for the alpha-chain of nAChR (nAChR-alpha), an 880-bp homologous band was found after amplification of cDNA prepared from mouse thymus, thymic medullary and cortical epithelial cell lines, but not from thymocytes or kidney. Sequencing of the polymerase chain reaction product from the thymus and thymic medullary and cortical epithelial lines showed identity with skeletal muscle nAChR-alpha over the region examined. This region includes the domains of the molecule on which B cell and T cell autoantigenic targets have been described. No evidence was found in mouse tissue for the exon 3A, which has been described in human muscle and the human rhabdomyosarcoma cell line TE671. Our results provide evidence at the RNA level for the expression of the nAChR-alpha on stromal cells but not on thymocytes in normal murine thymus and are consistent with a role for intrathymic autoantigen expression in the pathogenesis of myasthenia gravis.