Single-cell profiling of peanut-responsive T cells in patients with peanut allergy reveals heterogeneous effector TH2 subsets

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Mutation of POC1B in a Severe Syndromic Retinal Ciliopathy

We describe a consanguineous Iraqi family with Leber congenital amaurosis (LCA), Joubert syndrome (JBTS), and polycystic kidney disease (PKD). Targeted next‐generation sequencing for excluding mutations in known LCA and JBTS genes, homozygosity mapping, and whole‐exome sequencing identified a homozygous missense variant, c.317G>C (p.Arg106Pro), in POC1B, a gene essential for ciliogenesis, basal body, and centrosome integrity. In silico modeling suggested a requirement of p.Arg106 for the formation of the third WD40 repeat and a protein interaction interface. In human and mouse retina, POC1B localized to the basal body and centriole adjacent to the connecting cilium of photoreceptors and in synapses of the outer plexiform layer. Knockdown of Poc1b in zebrafish caused cystic kidneys and retinal degeneration with shortened and reduced photoreceptor connecting cilia, compatible with the human syndromic ciliopathy. A recent study describes homozygosity for p.Arg106Pro_POC1B in a family with nonsyndromic cone‐rod dystrophy. The phenotype associated with homozygous p.Arg106Pro_POC1B may thus be highly variable, analogous to homozygous p.Leu710Ser in WDR19 causing either isolated retinitis pigmentosa or Jeune syndrome. Our study indicates that POC1B is required for retinal integrity, and we propose POC1B mutations as a probable cause for JBTS with severe PKD.     

Randomized, open-label study of the impact of two doses of subcutaneous recombinant interleukin-2 on viral burden in patients with HIV-1 infection and CD4+ cell counts of > or = 300/mm3: CPCRA 059

The effect of intermittent courses of recombinant interleukin-2 (rIL-2) on HIV-1 load in patients receiving combination antiretroviral therapy remains uncertain. CPCRA 059 was an open-label, randomized, multicenter trial in which 511 patients with HIV-1 infection and CD4+ cell counts of > or = 300/mm3 who were receiving antiretroviral therapy were assigned to receive no rIL-2 (255 patients [controls]) or subcutaneous rIL-2 in dosages of 4.5 MIU (130) or 7.5 MIU (126) twice daily for 5-day courses every 8 weeks to maintain CD4+ cell counts that were twice the baseline value or > or = 1,000/mm3. The primary objective of this study was to compare the effects of the two doses of rIL-2 and no rIL-2 on viral load and CD4+ cell counts over 12 months. There was no difference in the following viral load measurements between the rIL-2 treatment groups and the control treatment group: percentage of patients with viral loads of <50 copies/mL at 12 months (p =.55), time to viral load of > or = 50 copies/mL for patients who had baseline viral loads of <50 copies/mL (p =.35), and change in viral load from baseline for patients who had viral loads of > or = 50 copies/mL at baseline (p =.63). At each follow-up visit, the change in CD4+ cell count from baseline was significantly greater in the rIL-2 treatment groups than in the control treatment group, with a mean difference of 251/mm3 at month 12 (95% confidence interval, 207-295; p <.0001). No unanticipated adverse experiences were seen in this trial, to our knowledge the largest randomized evaluation of rIL-2 treatment conducted to date.     

Microevolution of the direct repeat region of Mycobacterium tuberculosis: implications for interpretation of spoligotyping data

The direct repeat (DR) region has been determined to be an important chromosomal domain for studying the evolution of Mycobacterium tuberculosis. Despite this, very little is known about microevolutionary events associated with clonal expansion and how such events influence the interpretation of both restriction fragment length polymorphism (RFLP) and spoligotype data. This study examined the structure of the DR region in three independently evolving lineages of M. tuberculosis with a combination of DR-RFLP, spoligotyping, and partial DNA sequencing. The results show that the duplication of direct variable repeat (DVR) sequences and single-nucleotide polymorphisms is rare; conversely, the deletion of DVR sequences and IS6110-mediated mutation is observed frequently. Deletion of either single or contiguous DVR sequences was observed. The deletion of adjacent DVR sequences occurred in a dependent manner rather than as an accumulation of independent events. Insertion of IS6110 into either the direct repeat or spacer sequences influenced the spoligotype pattern, resulting in apparent deletion of DVR sequences. Homologous recombination between adjacent IS6110 elements led to extensive deletion in the DR region, again demonstrating a dependent evolutionary mechanism. Different isolates from the same strain family and isolates from different strain families were observed to converge to the same spoligotype pattern. In conclusion, the binary data of the spoligotype are unable to provide sufficient information to accurately establish genotypic relationships between certain clinical isolates of M. tuberculosis. This has important implications for molecular epidemiologic strain tracking and for the application of spoligotype data to phylogenetic analysis of M. tuberculosis isolates.     

Identification of sequential IgE-binding epitopes on bovine alpha(s2)-casein in cow's milk allergic patients

BACKGROUND: Caseins are the major allergens responsible for cow's milk allergy (CMA). We have previously identified the IgE-binding epitopes of the major cow's milk (CM) proteins except for alpha(s2)-casein. METHODS: Overlapping decapeptides representing the entire length of alpha(s2)-casein were synthesized on a cellulose-derivatized membrane. Sera from 13 CM-allergic children, 4-15 years of age, with a median level of CM-specific IgE >100 kU/l (range 33.7 to > 100 kU/l) were used to identify IgE-binding epitopes. RESULTS: Four major and six minor sequential IgE-binding regions were identified on alpha(s2)-casein. The first major region is located in the middle of the protein at amino acids (AA) 83-100, and the other three major regions are located in the carboxy terminal portion of the protein at AA 143-158, 157-172 and 165-188. The minor IgE-binding regions were identified at AA 31-44, 43-56, 93-106, 105-114, 117-128, and 191-200. CONCLUSION: We identified 10 sequential IgE-binding regions on alpha(s2)-casein and performed the first crucial step in the development of immunotherapeutic interventions for CMA.     

Novel approaches for the treatment of food allergy

At present there is no effective therapy for IgE-mediated food allergy, and patients must rely upon food-allergen avoidance. Unfortunately, the accidental ingestion of allergen-containing foods leading to potentially severe reactions is common. Definitive therapies for food allergy are desperately needed. Although rush immunotherapy has been reported to induce tolerance in some patients, the rate of maintenance of tolerance was low, and there was an undesirably high incidence of adverse reactions. New knowledge of the immunological mechanisms underlying allergic disease has expanded the potential therapeutic options for food allergy. The establishment of animal models of food hypersensitivity, which include sensitization by the oral route and anaphylaxis upon oral challenge, have facilitated the investigation of therapies for food allergy. Novel approaches under investigation include the reduction of IgE by the infusion of anti-IgE antibodies, vaccination with plasmid DNA, the use of anti-allergy immunostimulatory sequences, cytokines and bacterial agents, immunotherapy with mutated proteins and peptides, and complementary medicine such as Chinese herbs.     

Comparison of rapid,automated ribotyping and DNA macrorestriction analysis of Burkholderia pseudomallei

An automated ribotyping device (RiboPrinter) was used to determine the ribotypes of a collection of Burkholderia pseudomallei isolates. In a preliminary evaluation with the restriction enzymes BamHI and EcoRI, the protocol with EcoRI was more discriminating. The reproducibilities of the ribotypes obtained with EcoRI (EcoRI ribotypes) were determined by testing three levels of bacterial loads. The performance of the manufacturer's software was assessed by comparing the machine-optimized ribotypes with the type determined from the original gel image analyzed with Bionumerics software. The library of B. pseudomallei EcoRI ribotypes was then compared with the ribotypes obtained by DNA macrorestriction analysis of XbaI digests by pulsed-field gel electrophoresis. The typeability of B. pseudomallei by EcoRI ribotyping was 100%, and the discrimination index was 0.94. The slightly greater discrimination provided by DNA macrorestriction analysis (0.96) was achieved at the expense of a significantly longer processing time of 6 days, although the method was only half the cost of automated ribotyping. Typeability by macrorestriction analysis was lower (97%) unless a thiourea step was added to neutralize the action of Tris-dependent endonucleases. The digital record of B. pseudomallei isolates analyzed thus far provides a useful resource for future epidemiological studies and will help shorten the response time in the event of a further melioidosis outbreak or the deliberate release of B. pseudomallei as a biohazard.     

Modification of peanut allergen Ara h 3: effects on IgE binding and T cell stimulation

BACKGROUND: Peanut allergy is a major health concern due to the increased prevalence, potential severity, and chronicity of the reaction. The cDNA encoding a third peanut allergen, Ara h 3, has been previously cloned and characterized. Mutational analysis of the Ara h 3 IgE-binding epitopes with synthetic peptides revealed that single amino acid changes at critical residues could diminish IgE binding. METHODS: Specific oligonucleotides were used in polymerase chain reactions to modify the cDNA encoding Ara h 3 at critical IgE binding sites. Four point mutations were introduced into the Ara h 3 cDNA at codons encoding critical amino acids in epitopes 1, 2, 3 and 4. Recombinant modified proteins were used in SDS-PAGE/Western IgE immunoblot, SDS-PAGE/Western IgE immunoblot inhibition and T cell proliferation assays to determine the effects of these changes on in vitro clinical indicators of peanut hypersensitivity. RESULTS: Higher amounts of modified Ara h 3 were required to compete with the wild-type allergen for peanut-specific serum IgE. Immunoblot analysis with individual serum IgE from Ara-h-3-allergic patients showed that IgE binding to the modified protein decreased approximately 35-85% in comparison to IgE binding to wild-type Ara h 3. Also, the modified Ara h 3 retained the ability to stimulate T cell activation in PBMCs donated by Ara-h-3-allergic patients. CONCLUSIONS: The engineered hypoallergenic Ara h 3 variant displays two characteristics essential for recombinant allergen immunotherapy; it has a reduced binding capacity for serum IgE from peanut-hypersensitive patients and it can stimulate T-cell proliferation and activation.     

Differential PsaA-, PspA-, PspC-, and PdB-specific immune responses in a mouse model of pneumococcal carriage

Larger numbers of pneumococci were detected in the nasal tract compared to the lung, cervical lymph nodes, and spleen 1, 2, 4, 7, 14, and 21 days after nasal challenge with Streptococcus pneumoniae strain EF3030. In this mouse model of pneumococcal carriage, peripheral S. pneumoniae pneumococcal surface adhesin A (PsaA)-specific humoral responses (immunoglobulin G2a [IgG2a] > IgG1 = IgG2b > IgG3) were significantly higher than pneumococcal surface protein A (PspA)-specific, genetic toxoid derivative of pneumolysin (PdB)-specific, or pneumococcal surface protein C (PspC)-specific serum antibody levels. However, PspA-specific mucosal IgA antibody levels were significantly higher than those against PsaA, PdB, and PspC. In general, both PsaA- and PspA-specific lung-, cervical lymph node-, nasal tract-, and spleen-derived CD4(+) T-cell cytokine (interleukin-4, interleukin-6, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha) and proliferative responses were higher than those for either PspC or PdB. Taken together, these findings suggest that PsaA- and PspA-specific mucosal responses as well as systemic humoral and T helper cell cytokine responses are predominantly yet differentially induced during pneumococcal carriage.