Serological, genomic and structural analyses of the major mite allergen Der p 23.

BACKGROUND: Der p 23 was recently identified in a European population as a major allergen and potentially a chitin binding protein. OBJECTIVE: This study sought to assess the importance of Der p 23 among other Dermatophagoides allergens in a North American population and to determine the structure for functional characterization. METHODS: IgE binding to Der p 23, Der p 1, Der p 2, Der p 5, Der p 7 and Der p 8 was measured by ELISA. RNA-seq data from D. pteronyssinus were compared as estimates of allergen expression levels. The structure was analysed by X-ray crystallography and NMR. RESULTS: Despite a high prevalence of Der p 23, (75% vs. 87% and 79% for Der p 1 and Der p 2, respectively), the anti-Der p 23 IgE levels were relatively low. The patient response to the 6 allergens tested was variable (n = 47), but on average anti-Der p 1 and anti-Der p 2 together accounted for 85% of the specific IgE. In terms of abundance, the RNA expression level of Der p 23 is the lowest of the major allergens, thirty fold less than Der p 1 and sevenfold less than Der p 2. The structure of Der p 23 is a small, globular protein stabilized by two disulphide bonds, which is structurally related to allergens such as Blo t 12 that contain carbohydrate binding domains that bind chitin. Functional assays failed to confirm chitin binding by Der p 23. CONCLUSIONS AND CLINICAL RELEVANCE: Der p 23 accounts for a small percentage of the IgE response to mite allergens, which is dominated by Der p 1 and Der p 2. The prevalence and amount of specific IgE to Der p 23 and Der p 2 are disproportionately high compared to the expression of other Dermatophagoides allergens.

Identification of Maillard reaction products on peanut allergens that influence binding to the receptor for advanced glycation end products.

BACKGROUND: Recent immunological data demonstrated that dendritic cells preferentially recognize advanced glycation end product (AGE)-modified proteins, upregulate expression of the receptor for AGE (RAGE), and consequently bias the immune response toward allergy. METHODS: Peanut extract was characterized by mass spectrometry (MS) to elucidate the specific residues and specific AGE modifications found in raw and roasted peanuts and on rAra h 1 that was artificially glycated by incubation with glucose or xylose. The binding of the RAGE-V1C1 domain to peanut allergens was assessed by PAGE and Western analysis with anti-Ara h 1, 2, and 3 antibodies. IgE binding to rAra h 1 was also assessed using the same methods. RESULTS: AGE modifications were found on Ara h 1 and Ara h 3 in both raw and roasted peanut extract. No AGE modifications were found on Ara h 2. Mass spectrometry and Western blot analysis demonstrated that RAGE binds selectively to Ara h 1 and Ara h 3 derived from peanut extract, whereas the analysis failed to demonstrate Ara h 2 binding to RAGE. rAra h 1 with no AGE modifications did not bind RAGE; however, after AGE modification with xylose, rAra h 1 bound to RAGE. CONCLUSIONS: AGE modifications to Ara h 1 and Ara h 3 can be found in both raw and roasted peanuts. Receptor for AGE was demonstrated to selectively interact with AGE-modified rAra h 1. If sensitization to peanut allergens occurs in dendritic cells via RAGE interactions, these cells are likely interacting with modified Ara h 1 and Ara h 3, but not Ara h 2.

Improvement of renal graft function after conversion from a calcineurin inhibitor including immunosuppression to a mycophenolate sodium including regimen: a 4-year follow-up.

BACKGROUND: The most common immunosuppressive regimens after renal transplantation include calcineurin inhibitors (CNI). However, due to renal toxicity long-term graft survival does not seem to be positively affected by CNIs. METHODS: In the present study, we investigated 17 patients, in which the CNI immunosuppression was converted to a CNI-free, mycophenolate sodium (MPS) regimen. Conversion was performed due to progressive impairment of the graft function from suspected CNI toxicity. We retrospectively analyzed graft function as well as toxicity and surrogate markers for 4 years before and 4 years after conversion using a repeated-measures mixed model data analysis and/or a paired sample t-test. RESULTS: The mean time point of therapy conversion was 11.2 ± 4.6 years after transplantation. Within 1 month of CNI discontinuation, allograft function improved significantly, remaining at a significant level for 2 years. The estimated glomerular filtration rate increased from 43.4 ± 14.8 to a maximum of 55.7 ± 21.7 mL/min at 1 year after conversion (P = .0027). After 4 years, the end of the observation period, renal function was similar to the baseline. There were no significant side effects. CONCLUSION: These data suggested that, when chronic CNI-toxicity is suspected, renal allograft recipients may benefit from CNI withdrawal in favor of a MPS-including immunosuppressive regimen.

Ara h 2: crystal structure and IgE binding distinguish two subpopulations of peanut allergic patients by epitope diversity.

BACKGROUND: Peanut allergy affects 1% of the population and causes the most fatal food-related anaphylactic reactions. The protein Ara h 2 is the most potent peanut allergen recognized by 80-90% of peanut allergic patients. METHODS: The crystal structure of the major peanut allergen Ara h 2 was determined for the first time at 2.7 Å resolution using a customized maltose-binding protein (MBP)-fusion system. IgE antibody binding to the MBP fusion construct vs the natural allergen was compared by ELISA using sera from peanut allergic patients. RESULTS: The structure of Ara h 2 is a five-helix bundle held together by four disulfide bonds and related to the prolamin protein superfamily. The fold is most similar to other amylase and trypsin inhibitors. The MBP--Ara h 2 fusion construct was positively recognized by IgE from 76% of allergic patients (25/33). Two populations of patients could be identified. Subpopulation 1 (n = 14) showed an excellent correlation of IgE antibody binding to natural vs recombinant Ara h 2. Subpopulation 2 (n = 15) showed significantly reduced IgE binding to the MBP fusion protein. Interestingly, about 20% of the IgE binding in subpopulation 2 could be recovered by increasing the distance between MBP and Ara h 2 in a second construct. DISCUSSION: The reduced IgE binding to the MBP--Ara h 2 of subpopulation 2 indicates that the MBP molecule protects an immunodominant epitope region near the first helix of Ara h 2. Residues involved in the epitope(s) are suggested by the crystal structure. The MBP--Ara h 2 fusion constructs will be useful to further elucidate the relevance of certain epitopes to peanut allergy.

NorthernLights in slide-based cytometry and microscopy.

In recent years, slide-based cytometry has become a key technology for polychromatic cytometric investigations, and many efforts have been made to increase the number of measurable fluorochromes for multiparametric analysis. Sequential photobleaching of fluorochromes next to very photostable dyes is one approach for this technology. As the ALEXA dyes are known to be photostable as compared to the conventional fluorochromes FITC, PE (Riggs et al., Am J Pathol 1958;34:1081-1097), and APC, a differentiation within a fluorochrome pair is possible. Here, we have analyzed the newly available NorthernLights secondary antibodies for use in slide-based cytometry and microscopy. Currently, these fluorochrome-conjugates are now available with three distinct excitation- and emission maxima (NL493, NL557, NL637). Their spectral properties are similar to the frequently used fluorochromes FITC, PE, and APC and can, therefore, be used with most common excitation sources of cytometers or microscopes. As the NorthernLights are bright, resistant to photobleaching, stable in alcohols and xylene and of affordable price, these dyes are promising candidates for use with most laser- and HBO/XBA-based fluorescence microscopy-like techniques.

Decrease in serum tacrolimus level and rise in serum creatinine under late addition of cinacalcet in a renal transplant recipient with hyperparathyroidism: a case report.

Cinacalcet is a calcimimetic drug that has been approved for treatment of secondary and tertiary hyperparathyroidism in patients with renal failure requiring renal replacement therapy. A few cases of successful treatment in renal transplant patients immunosuppressed with cyclosporine have been reported. Herein we have reported the case of a 48-year-old renal transplant recipient presenting with secondary hypercalcemic hyperparathyroidism (parathyroid hormone [PTH] 896 pg/mL; total calcium, up to 3.3 mmol/L) under immunosuppressive therapy with tacrolimus. Owing to substantial comorbidity and a high operative risk, we decided to initiate a therapeutic trial with cinacalcet. Using a daily dose of 30 mg of Cinacalcet, normal calcium levels and a mild fall in PTH levels (decline of 62 pg/mL) were achieved within the first week of treatment. At this point, we also observed a marked decrease in tacrolimus levels (from 6.3 to 2.6 mg/dL) without any change in concomitant medications. Thus, we adapted the tacrolimus dosage. Concurrent with cinacalcet therapy, there was a rise in serum creatinine levels (from 3.9 to 4.9 mg/dL before discontinuation of cinacalcet), which was not reversible after termination of 3 weeks of treatment with cinacalcet, but continued. Cinacalcet and tacrolimus are both metabolized via cytochrome P 450. The documented decrease in tacrolimus serum levels, suggested a drug-drug interaction between tacrolimus and cinacalcet. The irreversible deterioration in renal function may be attributed to nephrotoxic properties of cinacalcet, but may also indicate an acceleration of the natural course of chronic allograft nephropathy.

In vivo imaging in experimental preclinical tumor research--a review.

The multiparametric molecular cell and tissue analysis in vitro and in vivo is characterized by rapid progress in the field of image generation technologies, sensor biotechnology, and computational modeling. Fascinating new potentials in unraveling the detailed functions of single cells, organs, and whole organisms are presently emerging and permit the close monitoring i.e. tumor development or basic cell development processes with an unprecedented multiplicity of promising investigative possibilities. To answer basic questions of in vivo tumor development and progression fluorescence based imaging techniques provide new insights into molecular pathways and targets. Genetic reporter systems (eGFP, DsRED) are available and high sensitive detection systems are on hand. These techniques could be used for in vitro assays and quantified e.g. by microscopy and CCD based readouts. The introduction of novel fluorescent dyes emitting in the near infrared range (NIR) combined with the development of sensitive detector systems and monochromatic powerful NIR-lasers for the first time permits the quantification and imaging of fluorescence and/or bioluminescence in deeper tissues. Laser based techniques particularly in the NIR-range (like two-photon microscopy) offer superb signal to noise ratios, and thus the potential to detect molecular targets in vivo. In combination with flat panel volumetric computed tomography (fpVCT), questions dealing e.g. with tumor size, tumor growth, and angiogenesis/vascularization could be answered noninvasively using the same animal. The resolution of down to 150 microm/each direction can be achieved using fpVCT. It is demonstrated by many groups that submillimeter resolutions can be achieved in small animal imaging at high sensitivity and molecular specificity. Since the resolution in preclinical small animal imaging is down to approximately 10 microm by the use of microCT and to subcellular resolutions using ( approximately 1 microm) microscope based systems, the advances of different techniques can now be combined to "multimodal" preclinical imaging and the possibilities for in vivo intravital cytometry now become within one's reach.

Effective treatment of hepatitis C-associated immune-complex nephritis with cryoprecipitate apheresis and antiviral therapy.

A 38-year-old pregnant woman (19th week of pregnancy) complained of fatigue, cold inducible paresthesias, generalized edema and mild arterial hypertension. Her past medical history was notable for frequent episodes of polyarthralgia and positivity for rheumatoid factor. On admission, acanthocyturia and unselective glomerular-tubular proteinuria with 19 g/d were detected with a slight decrease in creatinine clearance. Rheumatoid factor was robustly elevated and a cryocrit of 1.5 vol%, caused by a so far unknown replicative hepatitis C, was detected. Renal biopsy yielded membrano-proliferative glomerulonephritis. During pregnancy, high-dose corticosteroid therapy was administered. Edema disappeared and blood pressure normalized under albumin substitution and low-dose furosemide application. However, Cesarian section became necessary due to placental insufficiency at 27 weeks of gestation. Thereafter, neither virus load, cryocrit nor proteinuria decreased significantly under a combined therapy with pegylated interferon-a and ribavirin. Thus, cryoprecipitate apheresis was initiated resulting in robust decreases of clinical complaints, viral load, cryocrit and proteinuria. Cryoglobulinemia with renal involvement caused by hepatitis C is difficult to treat due to limitations of immunosuppressive and anti-viral therapy. In our patient, cryoprecipitate apheresis was a safe and effective therapeutic addition to standard therapy.

Communication using eye roll reflective signalling.

Body reflections in the ultraviolet (UV) are a common occurrence in nature. Despite the abundance of such signals and the presence of UV cones in the retinas of many vertebrates, the function of UV cones in the majority of taxa remains unclear. Here, we report on an unusual communication system in the razorback sucker, Xyrauchen texanus, that involves flash signals produced by quick eye rolls. Behavioural experiments and field observations indicate that this form of communication is used to signal territorial presence between males. The flash signal shows highest contrast in the UV region of the visual spectrum (lambdamax approximately 380 nm), corresponding to the maximum wavelength of absorption of the UV cone mechanism in suckers. Furthermore, these cones are restricted to the dorsal retina of the animal and the upwelling light background is such that their relative sensitivity would be enhanced by chromatic adaptation of the other cone mechanisms. Thus, the UV cones in the sucker have optimal characteristics (both in terms of absorbance and retinal topography) to constitute the main detectors of the flash signal. Our findings provide the first ecological evidence for restricted distribution of UV cones in the retina of a vertebrate.

Bioimpedance analysis and intradialytic hypotension in intermittent hemodialysis.

BACKGROUND: Intradialytic hypotension (IDH) is one of the most severe complications during hemodialysis. Its appearance is caused in part by rapid fluid removal with concomitant failure in blood pressure regulation but also by other dialytic-dependent and independent factors. PATIENTS AND METHODS: We investigated total (TBW), extracellular (ECW) and intracellular water (ICW) in chronic intermittent hemodialysis dialysis hypotension-prone (CRF-HP, n = 11) and nonhypotension-prone (CRF-NHP, n = 10) patients with end-stage renal disease before, every 30 minutes during, as well as after dialysis and within onset of intradialytic hypotension by multifrequent bioimpedance analysis (BIA). Additionally, intradialytic time course of BIA in patients with acute renal failure (ARF) and septic shock (n = 10) was observed. RESULTS: IDH occurred in 72.1% of CRF-HP and in 80% of ARF patients. In CRF-HP and CRF-NHP, ECW significantly decreased by -12.44 +/- 4.22% in CRF-HP and -9.0 +/- 6.2% in CRF-NHP comparing pre- and post-dialysis values (each p < 0.01). Conversely, ICW increased by +11.5 +/- 11.3% in CRF-HP and +18.4 +/- 25.2% in CRF-NHP (each p < 0.05). In patients with ARF no significant changes could be detected. Calculated ECW/ICW and ECW/TBW ratio significantly decreased in CRF patients with a higher rate in CRF-HP patients (p < 0.05). Neither ECW/ICW nor ECW/TBW ratio correlated with mean arterial pressure. The onset of intradialytic hypotension (n = 35) did not differ intraindividually compared to normotensive periods (n = 411). Fluid removal in CRF patients seems to be mainly from the extracellular space. The reduced decreases in ECW/ICW and ECW/TBW ratios in CRF-HP compared to CRF-NHP may indicate an insufficient refilling from intra- to extracellular compartment in CRF-HP. CONCLUSION: In conclusion, multifrequent BIA is not capable to predict hypotension in the individual patient during a particular dialysis session.

The crystal structure of a major dust mite allergen Der p 2, and its biological implications.

The crystal structure of the common house mite (Dermatophagoides sp.) Der p 2 allergen was solved at 2.15 A resolution using the MAD phasing technique, and refined to an R-factor of 0.209. The refined atomic model, which reveals an immunoglobulin-like tertiary fold, differs in important ways from the previously described NMR structure, because the two beta-sheets are significantly further apart and create an internal cavity, which is occupied by a hydrophobic ligand. This interaction is structurally reminiscent of the binding of a prenyl group by a regulatory protein, the Rho guanine nucleotide exchange inhibitor. The crystal structure suggests that binding of non-polar molecules may be essential to the physiological function of the Der p 2 protein.

Direct structure refinement of high molecular weight proteins against residual dipolar couplings and carbonyl chemical shift changes upon alignment: an application to maltose binding protein.

The global fold of maltose binding protein in complex with beta-cyclodextrin has been determined using a CNS-based torsion angle molecular dynamics protocol involving direct refinement against dipolar couplings and carbonyl chemical shift changes that occur upon alignment. The shift changes have been included as structural restraints using a new module, CANI, that has been incorporated into CNS. Force constants and timesteps have been determined that are particularly effective in structure refinement applications involving high molecular weight proteins with small to moderate numbers of NOE restraints. Solution structures of the N- and C-domains of MBP calculated with this new protocol are within approximately 2 A of the X-ray conformation.

Comparison of a neural net-based QSAR algorithm (PCANN) with Hologram- and multiple linear regression-based QSAR approaches: application to 1,4-dihydropyridine-based calcium channel antagonists.

A QSAR algorithm (PCANN) has been developed and applied to a set of calcium channel blockers which are of special interest because of their role in cardiac disease and also because many of them interact with P-glycoprotein, a membrane protein associated with multidrug resistance to anticancer agents. A database of 46 1,4-dihydropyridines with known Ca2+ channel binding affinities was employed for the present analysis. The QSAR algorithm can be summarized as follows: (1) a set of 90 graph theoretic and information theoretic descriptors representing various structural and topological characteristics was calculated for each of the 1,4-dihydropyridines and (2) principal component analysis (PCA) was used to compress these 90 into the eight best orthogonal composite descriptors for the database. These eight sufficed to explain 96% of the variance in the original descriptor set. (3) Two important empirical descriptors, the Leo-Hansch lipophilic constant and the Hammet electronic parameter, were added to the list of eight. (4) The 10 resulting descriptors were used as inputs to a back-propagation neural network whose output was the predicted binding affinity. (5) The predictive ability of the network was assessed by cross-validation. A comparison of the present approach with two other QSAR approaches (multiple linear regression using the same variables and a Hologram QSAR model) is made and shows that the PCANN approach can yield better predictions, once the right network configuration is identified. The present approach (PCANN) may prove useful for rapid assessment of the potential for biological activity when dealing with large chemical libraries.

Hydrogen exchange nuclear magnetic resonance spectroscopy mapping of antibody epitopes on the house dust mite allergen Der p 2.

New strategies for allergen-specific immunotherapy have focused on reducing IgE reactivity of purified recombinant allergens while maintaining T-cell epitopes. Previously, we showed that disrupting the disulfide bonds of the major house dust mite allergen Der p 2 resulted in 10-100-fold less skin test reactivity in mite-allergic subjects but did not change in vitro T-cell proliferative responses. To provide a more complete picture of the antigenic surface of Der p 2, we report here the identification of three epitopes using hydrogen protection nuclear magnetic resonance spectroscopy. The epitopes are defined by monoclonal antibodies that are able to inhibit IgE antibody binding to the allergen. Each monoclonal antibody affected the amide exchange rate of 2-3 continuous residues in different regions of Der p 2. Based on these data, a number of other residues were predicted to belong to each epitope, and this prediction was tested for monoclonal antibody 7A1 by generating alanine point mutants. The results indicate that only a small number of residues within the predicted epitope are functionally important for antibody binding. The molecular definition of these three epitopes will enable us to target limited positions for mutagenesis and to expand our studies of hypoallergenic variants for immunotherapy.

Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein.

The global fold of maltose-binding protein in complex with the substrate beta-cyclodextrin was determined by solution NMR methods. The two-domain protein is comprised of a single polypeptide chain of 370 residues, with a molecular mass of 42 kDa. Distance information in the form of H(N)-H(N), H(N)-CH(3) and CH(3)-CH(3) NOEs was recorded on (15)N, (2)H and (15)N, (13)C, (2)H-labeled proteins with methyl protonation in Val, Leu, and Ile (C(delta1) only) residues. Distances to methyl protons, critical for the structure determination, comprised 77 % of the long-range restraints. Initial structures were calculated on the basis of 1943 NOEs, 48 hydrogen bond and 555 dihedral angle restraints. A global pair-wise backbone rmsd of 5.5 A was obtained for these initial structures with rmsd values for the N and C domains of 2.4 and 3.8 A, respectively. Direct refinement against one-bond (1)H(N)-(15)N, (13)C(alpha)-(13)CO, (15)N-(13)CO, two-bond (1)H(N)-(13)CO and three-bond (1)H(N)-(13)C(alpha) dipolar couplings resulted in structures with large numbers of dipolar restraint violations. As an alternative to direct refinement against measured dipolar couplings we have developed an approach where discrete orientations are calculated for each peptide plane on the basis of the dipolar couplings described above. The orientation which best matches that in initial NMR structures calculated from NOE and dihedral angle restraints exclusively is used to refine further the structures using a new module written for CNS. Modeling studies from four different proteins with diverse structural motifs establishes the utility of the methodology. When applied to experimental data recorded on MBP the precision of the family of structures generated improves from 5.5 to 2.2 A, while the rmsd with respect to the X-ray structure (1dmb) is reduced from 5.1 to 3.3 A.

Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: differences in solution and crystal forms of maltodextrin binding protein loaded with beta-cyclodextrin.

Protein function is often regulated by conformational changes that occur in response to ligand binding or covalent modification such as phosphorylation. In many multidomain proteins these conformational changes involve reorientation of domains within the protein. Although X-ray crystallography can be used to determine the relative orientation of domains, the crystal-state conformation can reflect the effect of crystal packing forces and therefore may differ from the physiologically relevant form existing in solution. Here we demonstrate that the solution-state conformation of a multidomain protein can be obtained from its X-ray structure using an extensive set of dipolar couplings measured by triple-resonance multidimensional NMR spectroscopy in weakly aligning solvent. The solution-state conformation of the 370-residue maltodextrin-binding protein (MBP) loaded with beta-cyclodextrin has been determined on the basis of one-bond (15)N-H(N), (15)N-(13)C', (13)C(alpha)-(13)C', two-bond (13)C'-H(N), and three-bond (13)C(alpha)-H(N) dipolar couplings measured for 280, 262, 276, 262, and 276 residues, respectively. This conformation was generated by applying hinge rotations to various X-ray structures of MBP seeking to minimize the difference between the experimentally measured and calculated dipolar couplings. Consistent structures have been derived in this manner starting from four different crystal forms of MBP. The analysis has revealed substantial differences between the resulting solution-state conformation and its crystal-state counterpart (Protein Data Bank accession code 1DMB) with the solution structure characterized by an 11(+/-1) degrees domain closure. We have demonstrated that the precision achieved in these analyses is most likely limited by small uncertainties in the intradomain structure of the protein (ca 5 degrees uncertainty in orientation of internuclear vectors within domains). In addition, potential effects of interdomain motion have been considered using a number of different models and it was found that the structures derived on the basis of dipolar couplings accurately represent the effective average conformation of the protein.

A method for incorporating dipolar couplings into structure calculations in cases of (near) axial symmetry of alignment.

A method for incorporating dipolar coupling restraints into structure calculations is described which follows closely on methodology that has been recently presented for orienting peptide planes using dipolar couplings [Mueller et al. (2000) J. Mol. Biol., 300, 197-212] and is specifically developed for use in cases of an axially symmetric alignment tensor. Modeling studies on an all alpha-helical protein, farnesyl diphosphate synthase, establish the utility of the approach. A global fold of the 370-residue maltose binding protein in complex with beta-cyclodextrin is obtained from experimentally derived restraints. The average pairwise rmsd values between the N- and C-terminal domains in this NMR structure and the corresponding regions in the X-ray structure of the protein are 2.8 and 3.1 A, respectively.

A robust and cost-effective method for the production of Val, Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-labeled proteins.

A selective protonation strategy is described that uses [3-2H] 13C alpha-ketoisovalerate to introduce (1H-delta methyl)-leucine and (1H-gamma methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of approximately 100 mg/L alpha-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] alpha-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-delta1 methyl)-isoleucine (> 90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.

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.

Tertiary structure of the major house dust mite allergen Der p 2: sequential and structural homologies.

Sensitization to indoor allergens, especially those of the house dust mite, is strongly correlated with the development of asthma. We report the tertiary structure of the major house dust mite allergen, Der p 2, determined by NMR methods. The structure of Der p 2 is a beta-barrel and is composed of two three-stranded antiparallel beta-pleated sheets. This arrangement of beta-strands is similar to the immunoglobulin fold with respect to the orientation of the two sheets and the interactions of the strands. However, the three-dimensional structure of Der p 2 aligns equivalently with a number of proteins from different families within the immunoglobulin superfamily. The structural homology with the highest significance score from analysis by DALI is to Der f 2. Although Der p 2 and Der f 2 are 87% identical in amino acid sequence, they align in three dimensions rather poorly (4.85 A RMSD; Z-score, 8.58). This unexpected finding is likely due to the different solution conditions used during structure determination by NMR for both proteins. While the structural comparisons did not elucidate a clear homologue for the function of Der p 2 in mites, we report that Der p 2 is sequentially homologous to esr16. This is a protein from moths that is expressed coincident with molting. Thus, this homology has important ramifications for the study of mite allergy. The structure of Der p 2 provides a useful tool in the design of recombinant immunotherapeutics for the group 2 allergens.