Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity

Peanut allergy is one of the most life-threatening food allergies and one of the serious challenges facing the peanut and food industries. Current proposed solutions focus primarily on ways to alter the immune system of patients allergic to peanut. However, with the advent of genetic engineering novel strategies can be proposed to solve the problem of peanut allergy from the source. The objectives of this study were to eliminate the immunodominant Ara h 2 protein from transgenic peanut using RNA interference (RNAi), and to evaluate the allergenicity of resulting transgenic peanut seeds. A 265-bp-long PCR product was generated from the coding region of Ara h 2 genomic DNA, and cloned as inverted repeats in pHANNIBAL, an RNAi-inducing plant transformation vector. The Ara h 2-specific RNAi transformation cassette was subcloned into a binary pART27 vector to construct plasmid pDK28. Transgenic peanuts were produced by infecting peanut hypocotyl explants with Agrobacterium tumefaciens EHA 105 harbouring the pDK28 construct. A total of 59 kanamycin-resistant peanut plants were regenerated with phenotype and growth rates comparable to wild type. PCR and Southern analyses revealed that 44% of plants stably integrated the transgene. Sandwich ELISA performed using Ara h 2-mAbs revealed a significant ( P <  0.05) reduction in Ara h 2 content in several transgenic seeds. Western immunobloting performed with Ara h 2-mAb corroborated the results obtained with ELISA and showed absence of the Ara h 2 protein from crude extracts of several transgenic seeds of the T₀ plants. The allergenicity of transgenic peanut seeds expressed as IgE binding capacity was evaluated by ELISA using sera of patients allergic to peanut. The data showed a significant decrease in the IgE binding capacity of selected transgenic seeds compared to wild type, hence, demonstrating the feasibility of alleviating peanut allergy using the RNAi technology.     

应用流式细胞术分析林麝外周血 CD4+、CD8+ T淋巴细胞

通过对圈养林麝(Moschusberezovskii)外周血淋巴细胞CD4~+、CD8~+亚群的检测,探讨林麝细胞免疫功能状态,并探索应用流式细胞仪分析其淋巴细胞亚群的方法,为研究林麝重大疾病的病理机制及诊断方法提供科学依据。本研究选取健康林麝和患呼吸道疾病林麝各5头,以双色流式细胞术检测其外周血淋巴细胞CD4~+、CD8~+亚群的含量,并进行比较。结果显示,羊源CD4、CD8的流式荧光抗体能够标记林麝细胞并有效检测;患病林麝与健康林麝相比,外周血CD4~+细胞含量无差异(P 0.05),CD8~+细胞含量则显著降低(P 0.01),CD4~+/CD8~+比值显著增高(P 0.01)。结果表明,患呼吸系统炎性疾病的林麝其外周血淋巴细胞CD8~+亚群变化显著,检测淋巴细胞亚群对林麝疾病的诊断有重要意义。     

A comparative study of pollen trends in Genoa and Sanremo (Italy) from 1981 to 1989

Summary Trends of the atmospheric dispersion of the pollens of Urticaceae, Poaceae, Asteraceae,Plantago, Chenopodiaceae-Amaranthaceae, Polygonaceae, Oleaceae, Betulaceae-Corylaceae,Quercus, Castanea, and Cupressaceae-Taxaceae were studied from 1981 to 1989 in two localities (Genoa and Sanremo) about 120 km apart on the coast of Liguria in Italy. Statistical analysis revealed, in particular, and overall increase in pollen rain in Genoa during the period considered. The following observations were carried out on the pollens of greatest allergological interest: Urticaceae, a slightly increasing trend in Genoa; Poaceae, a moderately increasing trend in Sanremo; Oleaceae, almost parallel, uniform trendsin both centres. The values of this study lies in its ability to point out any increase which may occur over the years in the dispersal of certain allergenic pollens.     

Acute exacerbations of chronic obstructive pulmonary disease are associated with decreased CD4+ & CD8+ T cells and increased growth & differentiation factor-15 (GDF-15) in peripheral blood

Background

Although T cells, especially CD8+, have been implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, their role during acute exacerbations (AE-COPD) is uncertain.

Methods

We recruited subjects with COPD and a history of previous AE-COPD and studied them quarterly to collect blood and spontaneously expectorated sputum while stable. During exacerbations (defined by a change in symptoms plus physician diagnosis and altered medications), we collected blood and sputum before administering antibiotics or steroids. We used flow cytometry to identify leukocytes in peripheral blood, plus Luminex® analysis or ELISA to determine levels of inflammatory biomarkers in serum and sputum supernatants.

Results

Of 33 enrolled subjects, 13 participated in multiple stable visits and had ≥1 AE-COPD visit, yielding 18 events with paired data. Flow cytometric analyses of peripheral blood demonstrated decreased CD4+ and CD8+ T cells during AE-COPD (both absolute and as a percentage of all leukocytes) and significantly increased granulocytes, all of which correlated significantly with serum C-reactive protein (CRP) concentrations. No change was observed in other leukocyte populations during AE-COPD, although the percentage of BDCA-1+ dendritic cells expressing the activation markers CD40 and CD86 increased. During AE-COPD, sICAM-1, sVCAM-1, IL-10, IL-15 and GDF-15 increased in serum, while in sputum supernatants, CRP and TIMP-2 increased and TIMP-1 decreased.

Conclusions

The decrease in CD4+ and CD8+ T cells (but not other lymphocyte subsets) in peripheral blood during AE-COPD may indicate T cell extravasation into inflammatory sites or organized lymphoid tissues. GDF-15, a sensitive marker of cardiopulmonary stress that in other settings independently predicts reduced long-term survival, is acutely increased in AE-COPD. These results extend the concept that AE-COPD are systemic inflammatory events to which adaptive immune mechanisms contribute.

Trial registration

{"type":"clinical-trial","attrs":{"text":"NCT00281216","term_id":"NCT00281216"}}NCT00281216, ClinicalTrials.gov.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0251-1) contains supplementary material, which is available to authorized users.     

Rapid turnover of the CD8(+)CD28(-) T-cell subset of effector cells in the circulation of patients with head and neck cancer

CD8⁺ T cells in the circulation of patients with head and neck cancer (HNC) were previously shown to be significantly more sensitive to, and preferentially targeted for, apoptosis than CD4⁺ T cells (Hoffmann et al., Clin Cancer Res, 8:2553–2562, 2002). To distinguish global from CD8⁺ subset-specific apoptosis, we studied Annexin-binding to naïve, memory, and effector subsets of CD8⁺ cells by multicolor flow cytometry. Age-related changes in naïve and effector CD8⁺ cell subsets were observed in patients and normal controls (NC). The frequencies of naïve (CD28⁺CD45RO^-) CD8⁺ T cells were lower and those of memory (CD28⁺CD45RO⁺) and effector (CD28^-) CD8⁺ T cells significantly higher in the circulation of HNC patients relative to age-matched NC. Among CD8⁺ T cells, the CD28^- effector cell subset contained the highest proportion of Annexin-binding cells, while the naïve CD28⁺CD45RO^- subset contained the lowest. This suggested a high turnover rate of the CD8⁺CD28^- effector cell subset in patients with HNC, which was being compensated by a rapid transition of naïve CD8⁺ T cells to the effector cell pool. Following tumor resection, the frequency of CD8⁺CD28^- T cells normalized in the patients, an indication that the presence of tumor had an influence on the size of CD8⁺CD28^- T-cell pool. Ex vivo, in mixed lymphocyte-tumor cultures (MLTC) with semiallogeneic T cells as responders, CD8⁺CD28^- T cells could be generated from CD8⁺CD28⁺ cells by repeated stimulations with tumor cells. These CD8⁺CD28^- effector cells lysed the tumor, produced IFN- in response to the tumor, and strongly expressed granzyme B. Thus, the high rate of their apoptosis in the circulation of patients with HNC might be expected to contribute to tumor progression. However, the ex vivo generation of this cell subset was suppressed by strong CD28/B7 ligation or by overexpresson of MHC molecules on tumor cells, suggesting that adequate costimulation is necessary for protection from apoptosis. It appears that interactions of immune and tumor cells might determine the fate of this terminally differentiated effector cell subset.Supported in part by NIH grants: PO-1 DE 12321 and RO-1 CA 82016 to Theresa L. Whiteside.     

Inflammatory cytokines determine the susceptibility of human CD8 T cells to Fas-mediated activation-induced cell death through modulation of FasL and c-FLIP(S) expression

The nature of inflammatory signals determines the outcome of T cell responses. However, little is known about how inflammatory cytokines provided to human CD8 T cells during activation affects their susceptibility to post-activation cell death. We have examined and compared the effects of the inflammatory cytokine IL-12, as well as the combination of IL-1, IL-6, and IL-23 (IL-1/6/23) on the susceptibility of primary human CD8 T cells to post-activation cell death. Human CD8 T cells activated in the presence of IL-1/6/23 underwent significantly less cell death after activation as compared with those activated in IL-12. This was due to reduced susceptibility to Fas-mediated activation-induced cell death (AICD). Mechanistically, the reduced level of cell death in CD8 T cells activated in IL-1/6/23 was a result of a low level of FasL expression and high level of c-FLIP(S) expression. When the effect of IL-1, IL-6, and IL-23 individually was examined, IL-1 or IL-6 alone was sufficient to inhibit CD8 T cell death that occurs after activation in IL-12. IL-1, but not IL-6, inhibited expression of FasL, whereas IL-6, but not IL-1, increased c-FLIP(S) expression. Our findings show that the presence of IL-1 and/or IL-6 during activation of human CD8 T cells attenuates Fas-mediated AICD, whereas IL-12 increases the susceptibility of activated CD8 T cells to this form of cell death.     

Extracellular 5-Hydroxytryptamine in Median Raphe Nucleus of the Conscious Rat Is Decreased by Nanomolar Concentrations of 8-Hydroxy-2-(Di-n-Propylamino)tetralin and Is Sensitive to Tetrodotoxin

Abstract: Extracellular 5-hydroxytryptamine (5-HT) in the median raphe and dorsal hippocampus was measured using in vivo microdialysis. Administration of 60 m M K⁺ through the probe into the median raphe region significantly increased 5-HT output from the median raphe and the right dorsal hippocampus. Local infusion of 10 µ M tetrodotoxin into the median raphe region substantially decreased 5-HT in the median raphe and left and right dorsal hippocampus. Systemic administration (0.3 mg/kg s.c.) of 8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT) decreased the 5-HT levels in the dialysates from both the median raphe region and dorsal hippocampus. Administration of 30 n M 8-OH-DPAT through the dialysis probe into the median raphe region decreased 5-HT output from the median raphe and dorsal hippocampus significantly, whereas at concentrations from 60 n M to 10 µ M , no significant effects were found in either region. With 100 µ M 8-OH-DPAT, a significant increase was seen in the median raphe region, but not in dorsal hippocampus. Similar findings were obtained following microinjections of different doses of the compound into the median raphe region. The results of this study indicate that the somatodendritic release of 5-HT is impulse flow-dependent. Moreover, the decrease of 5-HT in the median raphe region by low nanomolar concentrations of 8-OH-DPAT supports the notion that somatodendritic 5-HT release is subject to a local negative feedback mechanism through 5-HT_1A autoreceptors.     

Occurrence of pre-MBT synthesis of caspase-8 mRNA and activation of caspase-8 prior to execution of SAMDC (S-adenosylmethionine decarboxylase)-induced, but not p53-induced, apoptosis in Xenopus late blastulae

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus fertilized eggs activates caspase-9 and executes maternal program of apoptosis shortly after midblastula transition (MBT). We find that overexpression of caspase-8 and p53, like that of SAMDC, induces apoptosis in Xenopus late blastulae. The apoptosis induced by p53 was abolished by injection of mRNA for xdm-2, a negative regulator of p53, and by injection of a peptide inhibitor or a dominant-negative type mutant of caspase-9, but not caspase-8. The apoptosis induced by SAMDC was not abolished by injection of xdm-2 mRNA, but was abolished by injection of a peptide inhibitor or a dominant-negative type mutant mRNA of both caspase-9 and caspase-8. Unlike caspase-9 mRNA, caspase-8 mRNA did not occur as a maternal mRNA rather induced to be expressed during cleavage stage (pre-MBT stage) by overexpression of SAMDC but not p53. Furthermore, while activities to process procaspase-8 and procaspase-9 appeared in SAMDC-overexpressed apoptotic embryos, the activity to process procaspase-8 did not appear in p53-overexpressed apoptotic embryos. We conclude there are at least two pathways in the execution of the maternal program of apoptosis in Xenopus embryos; one being through do novo expression of caspase-8 gene during cleavage stage, and the other without involvement of caspase-8.