Sublingual grass pollen immunotherapy is associated with increases in sublingual Foxp3‐expressing cells and elevated allergen‐specific immunoglobulin G4, immunoglobulin A and serum inhibitory activity for immunoglobulin E‐facilitated allergen binding to B cells

Background The mechanisms of sublingual immunotherapy (SLIT) are less well understood than those of subcutaneous immunotherapy (SCIT). Objectives To determine the effects of grass‐pollen SLIT on oral mucosal immune cells, local regulatory cytokines, serum allergen‐specific antibody subclasses and B cell IgE‐facilitated allergen binding (IgE‐FAB). Methods Biopsies from the sublingual mucosa of up to 14 SLIT‐treated atopics, nine placebo‐treated atopics and eight normal controls were examined for myeloid dendritic cells (mDCs) (CD1c), plasmacytoid dendritic cells (CD303), mast cells (AA1), T cells (CD3) and Foxp3 using immunofluorescence microscopy. IL‐10 and TGF‐β mRNA expression were identified by in situ hybridization. Allergen‐specific IgG and IgA subclasses and serum inhibitory activity for binding of allergen‐IgE complexes to B cells (IgE‐FAB) were measured before, during and on the completion of SLIT. Results Foxp3⁺ cells were increased in the oral epithelium of SLIT‐ vs. placebo‐treated atopics (P=0.04). Greater numbers of subepithelial mDCs were present in placebo‐treated, but not in SLIT‐treated, atopics compared with normal controls (P=0.05). There were fewer subepithelial mast cells and greater epithelial T cells in SLIT‐ compared with placebo‐treated atopics (P=0.1 for both). IgG₁ and IgG₄ were increased following SLIT (P<0.001). Peak seasonal IgA₁ and IgA₂ were increased during SLIT (P<0.05). There was a time‐dependent increase in serum inhibitory activity for IgE‐FAB in SLIT‐treated atopics. Conclusions SLIT with grass pollen extract is associated with increased Foxp3⁺ cells in the sublingual epithelium and systemic humoral changes as observed previously for SCIT. Cite this as: G. W. Scadding, M. H. Shamji, M. R. Jacobson, D. I. Lee, D. Wilson, M. T. Lima, L. Pitkin, C. Pilette, K. Nouri‐Aria and S. R. Durham, Clinical & Experimental Allergy, 2010 (40) 598–606.     

Extense variant gene family repertoire overlap in Western Amazon Plasmodium falciparum isolates

In order to find a molecular basis for observations of relatively fast developing immunity to malarial infections in the Western Amazon region, the partial var, stevor and rif gene repertoires of nine different Plasmodium falciparum isolates collected in 1985 and 2000–2004 were evaluated. In contrast to previous results from South East Asia, the variant gene repertoire in Brazilian isolates is rather small and redundant. While the individual var repertoire sizes of Brazilian strains did not differ from Southeast Asian/African isolates, we found an over three times higher overlap of var sequence repertoires in Amazonian strains which was also conserved over time, suggesting the ongoing circulation of a similar var gene repertoire. Coincidently, almost 40% of the sequences identified herein showed the highest degree of similarity to var genes from either Brazilian or Venezuelan isolates, indicating a limited var repertoire of P. falciparum in the Amazon Basin as a whole. The intrastrain similarities of var genes were slightly but significantly lower than in Southeast Asian/African samples suggesting a higher selective pressure for diversification in Amazonian isolates. Despite of higher copy numbers per genome, rif genes also showed a significant repertoire overlap. stevor genes, which share the same predominant subtelomeric localization as var and rif genes, showed a still higher repertoire overlap and were highly similar to 3D7 stevor genes, indicating stronger functional conservation than var and rif genes. This is the first study that reveals that P. falciparum variant gene repertoires of certain areas can be limited. This has important implications for the strain-specific immunity against variant antigens occurring in these areas.     

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.     

A randomized,double‐blind,placebo‐controlled,dose‐finding trial with Lolium perenne peptide immunotherapy

Background

A novel subcutaneous allergen immunotherapy formulation (gpASIT+?) containing Lolium perenne peptides (LPP) and having a short up‐dosing phase has been developed to treat grass pollen–induced seasonal allergic rhinoconjunctivitis. We investigated peptide immunotherapy containing the hydrolysate from perennial ryegrass allergens for the optimum dose in terms of clinical efficacy, immunogenicity and safety.

Methods

This prospective, double‐blind, placebo‐controlled, phase IIb, parallel, four‐arm, dose‐finding study randomized 198 grass pollen–allergic adults to receive placebo or cumulative doses of 70, 170 or 370 μg LPP. All patients received weekly subcutaneous injections, with the active treatment groups reaching assigned doses within 2, 3 and 4 weeks, respectively. Efficacy was assessed by comparing conjunctival provocation test (CPT) reactions at baseline, after 4 weeks and after completion. Grass pollen–specific immunoglobulins were analysed before and after treatment.

Results

Conjunctival provocation test (CPT) response thresholds improved from baseline to V7 by at least one concentration step in 51.2% (170 μg; P = .023), 46.3% (370 μg), and 38.6% (70 μg) of patients receiving LPP vs 25.6% of patients receiving placebo (modified per‐protocol set). Also, 39% of patients in the 170‐μg group became nonreactive to CPT vs 18% in the placebo group. Facilitated allergen‐binding assays revealed a highly significant (P < .001) dose‐dependent reduction in IgE allergen binding across all treatment groups (70 μg: 17.1%; 170 μg: 18.8%; 370 μg: 26.4%). Specific IgG₄ levels increased to 1.6‐fold (70 μg), 3.1‐fold (170 μg) and 3.9‐fold (370 μg) (mPP).

Conclusion

Three‐week immunotherapy with 170 μg LPP reduced CPT reactivity significantly and increased protective specific antibodies.

     

T follicular helper (Tfh) cells in normal immune responses and in allergic disorders

Follicular helper T cells (T_fh) are located within germinal centers of lymph nodes. Cognate interaction between T_fh, B cells, and IL‐21 drives B cells to proliferate and differentiate into plasma cells thereby leading to antibody production. T_fh cells and IL‐21 are involved in infectious and autoimmune diseases, immunodeficiencies, vaccination, and cancer. Human peripheral blood CXCR5⁺ CD4⁺ T cells comprise different subsets of T_fh‐like cells. Despite the importance of the IgE response in the pathogenesis of allergic disorders, little is known about the role of follicular and blood T_fh cells and IL‐21 in human and experimental allergic disease. Here, we review recent advances regarding the phenotypic and functional characteristics of both follicular and blood T_fh cells and of the IL‐21/IL‐21R system in the context of allergic disorders.     

ASRI2005-88 Comparable levels of CD4+CD25+ CTLA4+ Treg cells in peripheral blood of pre-eclampsia and normal pregnant patients

The acceptance of the semiallogeneic fetus within the maternal environment requires tolerance mechanisms not fully characterized yet. Normal pregnancy is known to be associated with a Th2 profile. Furthermore, T-regulatory cells were proposed to regulate the Th2/Th1 balance at early stages of pregnancy. Treg may avoid the shift to a Th1 profile preventing miscarriage. Accordingly, spontaneous abortion is characterized by a Th1 dominance and diminished levels of Tregulatory cells (Treg). The major aim of the present work was to investigate if pre-eclampsia, a late immunological complication of pregnancy, is characterized by similar hallmarks. Therefore, we measured the surface antigens CD4, CD25, CD8, CTLA4 (as well as the secretion of IL-10) in peripheral blood from patients suffering from pre-eclampsia (n = 8) and age-matched patients undergoing normal pregnancies (n = 9) by 4-colour flow-cytometry. We were not able to find any significant differences in the levels of CD4⁺, CD25⁺, CD8⁺, CTLA4, CD4⁺/CD25⁺, CD4⁺/CD25^bright, CD4⁺/CTLA4, CD25⁺/CTLA4, CD4⁺/CD25⁺/CTLA4, CD8⁺/CD25⁺, CD8⁺/CTLA4 or CD8⁺/CD25⁺/CTLA4 cell subsets. Our data suggest that Treg may not participate in the onset of pre-eclampsia and suggest other regulatory mechanisms during late pregnancy.     

Autosomal glycogenosis of liver and muscle due to phosphorylase kinase deficiency is caused by mutations in the phosphorylase kinase beta subunit (PHKB)

Glycogen storage disease due to phosphorylase kinase deficiency occurs in several variants that differ in mode of inheritance and tissue- specificity. This heterogeneity is suspected to be largely due to mutations affecting different subunits and isoforms of phosphorylase kinase. The gene of the ubiquitously expressed beta subunit, PHKB, was a candidate for involvement in autosomally transmitted phosphorylase kinase deficiency of liver and muscle. To identify such mutations, the complete PHKB coding sequence was amplified by RT-PCR of RNA isolated from blood samples of patients and analyzed by direct sequencing of PCR products. The characterization of mutations was complemented by PCR of genomic DNA. In one female and four male patients, we identified five independent nonsense mutations (Y418ter; R428ter; Y974H+E975ter; Q656ter in two cases), one single-base insertion in codon N421, one splice-site mutation affecting exon 31, and a large deletion involving the loss of exon 8. Although these severe translation-disrupting mutations occur in constitutively expressed sequences of the only known beta subunit gene of phosphorylase kinase, PHKB, they are associated with a surprisingly mild clinical phenotype, affecting virtually only the liver, and relatively high residual enzyme activity of approximately 10%.      

Mechanisms of immunotherapy: IgG revisited

PURPOSE OF REVIEW: This paper will review historical and recent evidence for the induction of 'blocking' IgG antibodies during successful specific immunotherapy. RECENT FINDINGS: Specific immunotherapy is frequently associated with a rise in allergen-specific IgG4 antibodies and a modest reduction in specific IgE titres, although this does not always correlate with clinical efficacy. There is accumulating evidence that specific immunotherapy also influences the blocking activity on IgE-mediated responses by IgG4, and cellular assays are commonly used to investigate these changes. Recently, a novel assay, which detects allergen-IgE binding using flow cytometry, has been used to detect 'functional' specific immunotherapy-induced changes in IgG antibody activity. Results suggest that successful specific immunotherapy is associated with an increase in IgG blocking activity that is not solely dependent on the quantity of IgG antibodies. SUMMARY: Successful immunotherapy is associated with quantitative and qualitative changes in the allergen-specific IgG antibody response. The induction of IgG antibodies with blocking activity may have a protective role not only through the inhibition of allergen-induced, IgE-mediated release of inflammatory mediators from mast cells and basophils, but also through the inhibition of IgE-facilitated antigen presentation to T cells. Qualitative changes in the allergen-specific IgG antibody response may possibly be an important mechanism underlying the clinical efficacy of specific immunotherapy. Monitoring changes in blocking activity using cellular assays may give an early indication of the potential success of treatment.     

Adjuvants for allergen immunotherapy: experimental results and clinical perspectives

PURPOSE OF REVIEW: Inclusion of adjuvants in immunotherapy vaccines are important to enhance immune responses to allergens. This article will cover the recent advances in adjuvant formulations described in published articles primarily over the past 2 years. RECENT FINDINGS: Traditionally, allergen immunotherapy preparations utilize aluminium hydroxide as an adjuvant. These have generally proved efficacious and have a good safety profile. However, recent advances in the understanding of immunological mechanisms underlying immunotherapy and in the design of new adjuvants may allow a more rational approach to adjuvant use. One approach is to use adjuvants such as immunostimulatory sequences or monophosphoryl lipid A, which can deviate allergy-associated Th2 immune responses towards a Th1 phenotype. Both of these adjuvants have been used in pilot controlled clinical trials which have demonstrated clinical efficacy and the induction of protective IgG antibodies. Other approaches to improve immunotherapy vaccines include microencapsulation of allergen to allow delivery of the allergen directly to the gut in order to induce immunological tolerance and vaccination with heat-killed mycobacteria. SUMMARY: There is great interest in newly designed adjuvants to improve the efficacy and safety of allergen immunotherapy. A better understanding of immunological mechanisms and further clinical trials utilizing new adjuvants are needed.