Immunological changes during specific immunotherapy of grass pollen allergy: reduced lymphoproliferative responses to allergen and shift from TH2 to TH1 in T-cell clones specific for Phi p 1, a major grass pollen allergen

Background and Objective The mechanisms operative in specific immunotherapy (SIT) of Type I allergy are not completely understood. In the present study we evaluated immunological changes during SIT in pollinosis. Method Eight patients suffering from pollinosis (monosensitized to grass pollen) were treated with conventional SIT. All subjects had IgE specific for Phi p 1. a major allergen of timothy grass. In vitro changes in the immunological reactivity to grass pollen extract and to recombinant Phi p 1 were evaluated. Subjects were examined at three occasions: before, after 3 months and after I year of SIT. Results Serological analysis revealed a marked increase of grass pollen- and Phi p 1-specific IgG, titres of specific IgE did not change significantly. Lymphoproliferative responses to grass pollen extract and rPhl p 1 were reduced already after 3 months of treatment. Accordingly, the cloning efficiency for Ph1 p 1-specific T-cell clones (TCC) dropped markedly in all patients. The majority of allergen-specific TCC raised before SIT revealed a TH₂-like pattern of cytokine production. TCC established after SIT revealed TH₁ characteristics. This shift was due to a decrease in IL-4 rather than an increase in IFN-production by T cells. Investigations of the epitopes recognized by T cells before and after SIT did not reveal the outgrowth of new (ldquo;protecting”) specificities. We could not observe induction of allergen-speeific CD8⁺ lymphocytes (supressor cells). Conclusion Our data indicate that — on the level of TH lymphocytes — SIT induces tolerance to the allergen and a modulation of the cytokine pattern produced in response to allergen stimulation.     

微囊化转mIFN-γ基因CHO细胞皮下移植促进荷瘤小鼠TH1/TH2的漂移

目的：通过观察微囊化转mIFN-γ基因CHO细胞皮下移植对荷瘤小鼠TH1／TH2类细胞因子的影响，进一步了解外源性IFN－γ对TH1／TH2平衡的调节作用，同时验证微囊化基因工程细胞移植治疗恶性肿瘤的可行性。方法：将mIFN-γ基因转染到正常细胞CHO，然后进行微囊化，移植到荷瘤小鼠的皮下，2周后，测定小鼠血清TH1和TH2类细胞因子IFN－γ、IL－2、IL－12、IL－4、IL－10的水平，并与对照组相比较。同时测量肿瘤的平均直径，以观察肿瘤的生长速度，并观察荷瘤小鼠的生存期。结果：经微囊化转mIFN-γ基因CHO细胞皮下移植干预治疗后，小鼠血清TH1类细胞因子IFN－γ、IL－2、IL－12水平明显升高，而TH2类细胞因子IL－4、IL－10变化不明显；小鼠肿瘤的生长速度明显减慢，生存期明显延长。结论：外源性IFN－γ促进荷瘤小鼠TH1细胞的分化，使TH1／TH2向着TH1方向漂移；微囊化基因工程细胞的移植，有望替代基因工程药物，成为治疗恶性肿瘤的又一新平台。     

Effect of allergen immunotherapy on nasal responses in guinea-pigs with allergic rhinitis

Methods We have investigated the effects of allergen immunotherapy on the nasal responses in the guinea-pigs with allergic rhinitis. Thirty-three male Hartley guinea-pigs with allergic rhinitis were divided into three groups; those receiving intradermal injection of saline (Group 1) or 0.1% ovalbumin (Group 2) 6 days after the last intranasal sensitization, and those injected with 0.1% ovalbumin intradermally once daily for 6 consecutive days from the next day after the last intranasal sensitization (Group 3). Results The dye leakage and histamine content into the nasal lavage significantly decreased at 30min after antigen challenge in Group 3, compared with Group 1 or 2. We also observed the change of mast cell numbers in superficial nasal mucosa, lamina propria and injected dorsal skin. The number of mast cells in superficial nasal mucosa significantly decreased in Group 3 compared with Group 1 or 2, but not those in nasal lamina propria or dorsal skin. Conclusions These results suggest that the improvements of nasal responses such as dye leakage and histamine content may be caused by the decrease of mast cell numbers in the superficial mucosal layer after the specific immunotherapy. which may be developing tolerance and one of the mechanisms underlying the beneficial effect of immunotherapy.     

DEN-2 NGC株感染BALB/c小鼠TH1/TH2类细胞因子产生的差异

目的通过观察2型登革病毒(DEN2)NGC株初次感染和再次感染BALB/c小鼠后小鼠血浆中TH1和TH2类细胞因子的产生及其动态变化,研究DEN2感染的免疫机制。方法用不同量的DEN2NGC株经皮下多点注射,建立BALB/c小鼠感染模型,间接ELISA法测定感染后不同时间小鼠血浆IL-2、IFN-γ、IL-4、IL-5、IL-6、IL-10的含量。结果(1)在初、再次感染阶段,DEN2NGC株各感染组产生IL-4、IFN-γ、IL-2、IL-5、IL-6、IL-10与正常对照组有显著差异(P<0·05)。①在初次感染阶段,NGC株各感染组血浆IL-2、IL-5、IL-6于第4、6、8天出现;在再次感染阶段,各感染组产生IL-2、IL-5、IL-6均于第1天迅速升高;IL-2水平于第4天达高峰(104448·46±13314·1pg/ml),维持5~7天后迅速下降;IL-5于48小时达到高峰(135·125±46·75pg/ml);各组小鼠IL-6产生的动态相似,在再次感染后第1天,达高峰(555·823±44·639pg/ml)后逐渐下降。②初次感染早期,IL-4水平明显升高,而IFN-γ处于较低水平;再次感染后第1天,IL-4水平达最高峰(4294·668±349·038pg/ml),IFN-γ则于第4天和第11天达较高水平,两者的产生彼消此长,且产生动态与感染病毒量有关。(2)在初、再次感染阶段,用不同量DEN2NGC株感染组产生细胞因子水平有差异。结论DEN2NGC株初次和再次感染BALB/c小鼠后,TH1和TH2应答均可发挥一定作用,二者相互影响,TH细胞及其产生的CK在DEN感染的发病机制中起重要作用。     

Pharmacokinetics of an allergen and a monomeric allergoid for oromucosal immunotherapy in allergic volunteers

BACKGROUND AND OBJECTIVE: Little is known about the pharmacokinetics of allergens for local immunotherapy. Thus, we studied the pharmacokinetics in allergic volunteers of a commercial allergenic vaccine in orosoluble tablets (LAIS(R), Lofarma S.p.A). METHODS: The carbamylated monomeric allergoid derived from Parietaria judaica major allergen (Par j 1), characterized by maintenance of the original molecular size, and the native allergen, were radiolabelled with 123I, then incorporated into the commercial soluble tablets and administered to allergic subjects. Early sequential and late static scintigraphic acquisitions were performed, and plasma radioactivity was measured at different time intervals. RESULTS: No difference in local pharmacokinetics was observed between the allergen and the allergoid: part of the tracer was retained in the mouth for at least 2 h after swallowing. No direct absorption through the oral mucosa could be detected, as plasma radioactivity increased only after swallowing and peaked at 2 h. However, the plasma peak attained with the allergoid in tablets was significantly higher with respect to the native allergen. Finally, some undegraded allergoid, but not the allergen, could be constantly detected in the bloodstream at plasma peak. CONCLUSIONS: The results showed a similar behaviour of the allergoid and the allergen in tablets as far as their local kinetics are concerned, whereas plasma peak was higher with the allergoid than with the allergen. Therefore we conclude that the chemical modification of the allergen may affect its pharmacokinetics, by making it less susceptible to enzymatic degradation.     

TH1/TH2细胞因子与习惯性流产的关系研究

目的通过测定习惯性流产患者（RSA）及正常妊娠妇女外周血血清TH1/TH2细胞因子的含量,探讨TH1/TH2细胞因子与习惯性流产发生的关系。方法采用酶联免疫吸附法检测30例RSA患者,72例正常妊娠妇女血清IL-2、IL-4、IL-10、IFN-γ、TNF-α及TNF-β水平并比较两组之间的差异。结果RSA患者IL-2、IL-4、IL-10、IFN-γ、TNF-α血清水平显著高于正常妊娠妇女（P〈0.05）,TNF-β血清水平显著低于正常妊娠妇女（P〈0.05）。结论血清IL-2、IL-4、IL-10、IFN-γ、TNF-α水平可能在习惯性流产发生中起重要作用。     

Balanced TH1 and TH2 immunopotentiating effects of silicates partly containing nanoparticles present in calcined serpentine

Calcined Serpentine (CS) is used in various formulations of alternative systems of medicine as a tonic to vital organs and as an anti-inflammatory agent. The process of calcination or incineration is believed to render non-toxic, gently absorbable, adaptable and digestible properties to the mineral compounds. The present study characterized CS and also evaluated its immunostimulatory potential. CS was characterized by using transmission electron microscopy (TEM), X-ray powder diffraction, atomic absorption spectroscopy and CHNS analysis. The characterized CS was further evaluated for its immunomodulatory potential in Swiss mice. X-Ray diffraction analysis revealed that the CS contained silicates of magnesium, calcium and iron as major minerals. Elemental composition and heavy metal analyses showed a presence of various inorganic elements/heavy metals, albeit at levels well below daily permissive intake values. TEM analysis of the test CS revealed a presence of nano particles with an average size of 10–20?nm (≈ 26% of total material). Oral administration of CS to mice at 50, 75, 100 or 200?μg/kg body weight for 10 days led to enhanced levels of total IgG, IgG₁, IgG_2a and IgG_2b in ovalbumin-immunized mice as well as ex vivo lymphocyte proliferation and levels of T_H1 (IL-2, IFNγ) and T_H2 (IL-4, IL-10) cytokines produced by their cultured splenocytes. Similarly, CS treatment resulted in enhanced delayed-type hypersensitivity responses in GRBC-primed hosts. CS also activated host peritoneal macrophages, as indicated by increases in phagocytic activity and in TLR-2, CD80 and CD86 expression. The CS did not affect liver, kidney and spleen histology. Taken together, the results indicated that absorbed CS was stimulatory of host cell-mediated immune responses. It is hypothesized for now that the immunomodulatory effect of CS may have been due, in part, to a presence of nanoparticles on the CS; further study is required to validate this viewpoint.     

强直性脊柱炎TH亚群激活及T细胞活化状态研究

目的 :研究强直性脊柱炎 (AS)患者TH1 TH2细胞激活状态及T细胞活化状况 ,探讨其发病机理。方法 :运用流式细胞仪 (CBA)法检测 35例AS患者TH1(INF γ、TNF α、IL 2 )、TH2 (IL 10、IL 5、IL 4 )细胞因子水平以及外周血淋巴细胞CD3 、CD4 、CD8 T细胞、B细胞 (CD19 )、NK细胞 (CD16 5 6 )和CD3 HLA DR 、CD4 HLA DR 、CD8 HLA DR T细胞百分率 ,并与健康对照组比较。结果 :AS患者血浆TNF α水平、IL 2水平均显著低于健康对照组 (P <0 0 1,P <0 0 5 ) ,IL 10水平显著高于健康对照组 (P <0 0 5 )。CD3 、CD3 CD8 T细胞百分率显著低于健康对照。CD8 HLA DR T细胞百分率均显著低于健康对照 (P <0 0 5 ) ,CD4 HLA DR T显著高于健康对照 (P <0 0 5 )。结论 :AS患者血浆低水平的TNF α、IL 2和高水平的IL 10提示其体内存在着TH1 TH2平衡的偏移 ;TH1激活程度低下 ,而TH2激活程度增强 ,细胞因子水平的改变尤以TH1细胞因子TNF α改变特别明显。AS患者在多个层面存在细胞免疫功能紊乱     

Liposomes as vehicle for allergen presentation in the immunotherapy of allergic diseases

Liposomes are non-toxic, biodegradable and weakly immunogenic lipid vesicles which can be used as immunomodulating agents. In the present study, multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) were used to incorporate an allergenic protein from Artemisia scoparia pollen. MLV incorporated more allergenic protein than SUV. To assess the immunomodulating effect of allergen entrapped in liposomes, Swiss strain mice (made IgE responders) were injected with either free allergen or liposome-entrapped allergen (LEA) and their immune response was measured in terms of specific IgG and specific IgE levels. Results indicated that specific IgE response was significantly lower in mice injected LEA (P less than 0.02) than in mice injected free allergenic protein. Although specific IgG response was higher in mice injected LEA, there was no statistically significant difference between the two groups. Potential use of liposomes as non-immunogenic biocompatible vehicle for antigen presentation in immunotherapy will be discussed.     

Free fatty acids sensitize dendritic cells to amplify TH1/TH17‐immune responses

Obesity is associated with body fat gain and impaired glucose metabolism. Here, we identified both body fat gain in obesity and impaired glucose metabolism as two independent risk factors for increased serum levels of free fatty acids (FFAs). Since obesity is associated with increased and/or delayed resolution of inflammation observed in various chronic inflammatory diseases such as psoriasis, we investigated the impact of FFAs on human monocyte‐derived and mouse bone marrow‐derived dendritic cell (DCs) functions relevant for the pathogenesis of chronic inflammation. FFAs such as palmitic acid (PA) and oleic acid (OA) did not affect the pro‐inflammatory immune response of DCs. In contrast, PA and OA sensitize DCs resulting in augmented secretion of TH1/TH17‐instructive cytokines upon pro‐inflammatory stimulation. Interestingly, obesity in mice worsened a TH1/TH17‐driven psoriasis‐like skin inflammation. Strong correlation of the amount of total FFA, PA, and OA in serum with the severity of skin inflammation points to a critical role of FFA in obesity‐mediated exacerbation of skin inflammation. Our data suggest that increased levels of FFAs might be a predisposing factor promoting a TH1/TH17‐mediated inflammation such as psoriasis in response to an inflammatory danger signal.     

Modified T-cell activation pattern during specific immunotherapy (SIT) in cat-allergic patients

OBJECTIVE: The aim of the study was to analyse early effects of specific immunotherapy (SIT) on immune functions in cat-allergic patients. METHODS: Immunological responses of peripheral blood mononuclear cells from eight cat-allergic patients were analysed before and after SIT in comparison with 11 nonallergic controls. Cells were stimulated in vitro with either bacterial superantigen, mitogen, or cat allergen. Production of IL-12 and TH1/TH2 cytokines was analysed by ELISA and lymphocyte subset distribution was assessed by flow-cytometry. RESULTS: We found a significantly reduced secretion of IL-12 (P < 0.05) from cells of allergic individuals compared with the controls. This finding was associated with significantly lower IFN-gamma production after stimulation with allergen (P < 0.05) that did not increase during SIT. However, no significant differences were seen after stimulation with mitogen indicating an allergen specific IFN-gamma secretion response in allergic individuals. Prior to SIT IL-5 production was significantly higher in cells of allergic donors stimulated with allergen < 0.005 or mitogen (< 0.05). After reaching the maintenance dose for SIT, allergen-induced IL-5 production returned to normal levels, whereas it remained elevated after stimulation with mitogen. These changes were associated with a reduced frequency of CD45 RO T cells following SIT. CONCLUSION: These results suggest that SIT exerts early effects on allergen-specific T-cell responses with selective inhibition of the up-regulated TH2 immune response.     

ORIGINAL ARTICLE: TH1 – TH2 Response and the Atopy Risk in Patients with Reproduction Failure

Problem Enhanced TH2 activity is characteristic for atopic diseases and is observed also in physiological pregnancy. The immune causes of repeated pregnancy losses and/or repeated in vitro fertilization failure may be associated with TH2 hypoactivity. The association with frequency of atopic diseases is unclear. Method of Study Intracellular production of IL‐4 and IFN‐γ by peripheral CD4+ T lymphocytes was studied, as well as serum levels of total and allergen specific IgE. Simultaneously skin prick tests with inhalant allergens were performed, and clinical features of atopy were registered by means of a questionnaire. Results Lower intracellular production of IL‐4 by peripheral CD4+ T cells and lower frequency of elevated total and allergen specific IgE were found in women with reproduction failure compared to controls, as well as lower frequency of some symptoms possibly associated with atopy. Conclusion Our study showed the presence of TH2 hypoactivity in women with reproduction failure, which may be associated with lower occurrence of atopic diseases.     

TH2 activated cells prevent experimental autoimmune uveoretinitis,a TH1-dependent autoimmune disease

Mercuric chloride (HgCl₂) injections protect (Lewis x Brown-Norway) F1 (F1) rats against experimental autoimmune uveoretinitis (EAU) induced by immunization with the retinal S antigen (S-Ag); in contrast HgCl₂-injected F1 rats develop EAU following transfer of lymph node (LN) cells from rats immunized with S-Ag alone. In the present study we demonstrate that the ability of LN cells from rats protected against EAU to transfer the disease into naive F1 rats was considerably reduced. These LN cells neither produced interleukin (IL)-2 nor (interferon (IFN)-γ but exhibited mRNA for IL-4. In contrast, LN cells from diseased rats easily transferred EAU into naive F1 rats, produced significant IL-2 and IFN-γ levels but barely exhibited mRNA for IL-4. Furthermore protected rats predominantly produced IgG2b anti-S-Ag antibodies, while diseased rats produced IgG2b anti-S-Ag antibodies and the increase in expression of MHC class II molecules on B cells was higher in protected rats than in diseased rats. These data suggest that (1) to exert a protective effect, HgCl₂ must act at an early stage of differentiation of precursors of S-Ag specific T cells, and (2) this effect is related to the preferential activation of TH2 cells to the detriment of uveitogenic TH1 cells. Finally, these results indicate that activation of TH2 cells protect from a TH1-dependent autoimmune disease.     

Methods for the in vitro determination of an individual disposition towards TH1- or TH2-reactivity by the application of appropriate stimulatory antigens

In this study we performed several methods for the determination of cytokines (RT-PCR for the demonstration of cytokine mRNA and flow cytometry for the analysis of intracellular cytokines) and compared them with a recently established test system stimulating peripheral blood mononuclear cells (PBMC) with TH1- and TH2-relevant recall antigens and analysing type 1 and type 2 cytokines by ELISA. Aim of the study was therefore to evaluate the reliability of TH1/TH2 cytokine profiles in two individuals with different types of an allergic/atopic disposition: one of them showed a strong TH1/type 1-mediated tuberculin-reaction (subject A), the other (subject B) revealed elevated IgE-levels and eosinophil counts (TH2/type 2-mediated). PBMC were incubated with the type 1-antigen purified protein derivative (PPD) and the type 2-antigen tetanus-toxoid (TT) for seven days. From the comparison of ELISA with RT-PCR and flow cytometry-analysis it became evident that all three methods allowed the definition of subject A as a 'type 1-responder'. Subject B showed a pure type 2-response in the ELISA method; PCR and flow cytometry analysis revealed the simultaneous production of type 1- and type 2-cytokines resulting in a mixed type 1/type 2-profile. Active immunization of subject A with TT at the end of the observation period of 12 months resulted in a transient shift from type 1- to a mixed type 1/type 2-profile (simultaneous PPD-induced IFN-gamma- and TT-induced IL-5 production). From this pilot study based on clear cut clinical criteria concerning either a humoral or cellular immunological reactivity towards allergens/antigens it is suggested that the determination of type 1/type 2-cytokines by ELISA in supernatants of PBMC stimulated with type 1/type 2-relevant antigens is a useful approach for a better classification of 'type1-' or 'type 2-responder'.     

Novel approaches and perspectives in allergen immunotherapy

In this review, we report on relevant current topics in allergen immunotherapy (AIT) which were broadly discussed during the first Aarhus Immunotherapy Symposium (Aarhus, Denmark) in December 2015 by leading clinicians, scientists and industry representatives in the field. The aim of this symposium was to highlight AIT‐related aspects of public health, clinical efficacy evaluation, mechanisms, development of new biomarkers and an overview of novel therapeutic approaches. Allergy is a public health issue of high socioeconomic relevance, and development of evidence‐based action plans to address allergy as a public health issue ought to be on national and regional agendas. The underlying mechanisms are in the focus of current research that lays the ground for innovative therapies. Standardization and harmonization of clinical endpoints in AIT trials as well as current knowledge about potential biomarkers have substantiated proof of effectiveness of this disease‐modifying therapeutic option. Novel treatments such as peptide immunotherapy, intralymphatic immunotherapy and use of recombinant allergens herald a new age in which AIT may address treatment of allergy as a public health issue by reaching a large fraction of patients.