Immune Tolerance Network

BACKGROUND: Cyclophosphamide and glucocorticoids have been the cornerstone of remission-induction therapy for severe antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis for 40 years. Uncontrolled studies suggest that rituximab is effective and may be safer than a cyclophosphamide-based regimen. METHODS: We conducted a multicenter, randomized, double-blind, double-dummy, noninferiority trial of rituximab (375 mg per square meter of body-surface area per week for 4 weeks) as compared with cyclophosphamide (2 mg per kilogram of body weight per day) for remission induction. Glucocorticoids were tapered off; the primary end point was remission of disease without the use of prednisone at 6 months. RESULTS: Nine centers enrolled 197 ANCA-positive patients with either Wegener's granulomatosis or microscopic polyangiitis. Baseline disease activity, organ involvement, and the proportion of patients with relapsing disease were similar in the two treatment groups. Sixty-three patients in the rituximab group (64%) reached the primary end point, as compared with 52 patients in the control group (53%), a result that met the criterion for noninferiority (P<0.001). The rituximab-based regimen was more efficacious than the cyclophosphamide-based regimen for inducing remission of relapsing disease; 34 of 51 patients in the rituximab group (67%) as compared with 21 of 50 patients in the control group (42%) reached the primary end point (P=0.01). Rituximab was also as effective as cyclophosphamide in the treatment of patients with major renal disease or alveolar hemorrhage. There were no significant differences between the treatment groups with respect to rates of adverse events. CONCLUSIONS: Rituximab therapy was not inferior to daily cyclophosphamide treatment for induction of remission in severe ANCA-associated vasculitis and may be superior in relapsing disease. (Funded by the National Institutes of Allergy and Infectious Diseases, Genentech, and Biogen; ClinicalTrials.gov number, NCT00104299.)

BACKGROUND: The prevalence of peanut allergy among children in Western countries has doubled in the past 10 years, and peanut allergy is becoming apparent in Africa and Asia. We evaluated strategies of peanut consumption and avoidance to determine which strategy is most effective in preventing the development of peanut allergy in infants at high risk for the allergy. METHODS: We randomly assigned 640 infants with severe eczema, egg allergy, or both to consume or avoid peanuts until 60 months of age. Participants, who were at least 4 months but younger than 11 months of age at randomization, were assigned to separate study cohorts on the basis of preexisting sensitivity to peanut extract, which was determined with the use of a skin-prick test--one consisting of participants with no measurable wheal after testing and the other consisting of those with a wheal measuring 1 to 4 mm in diameter. The primary outcome, which was assessed independently in each cohort, was the proportion of participants with peanut allergy at 60 months of age. RESULTS: Among the 530 infants in the intention-to-treat population who initially had negative results on the skin-prick test, the prevalence of peanut allergy at 60 months of age was 13.7% in the avoidance group and 1.9% in the consumption group (P<0.001). Among the 98 participants in the intention-to-treat population who initially had positive test results, the prevalence of peanut allergy was 35.3% in the avoidance group and 10.6% in the consumption group (P=0.004). There was no significant between-group difference in the incidence of serious adverse events. Increases in levels of peanut-specific IgG4 antibody occurred predominantly in the consumption group; a greater percentage of participants in the avoidance group had elevated titers of peanut-specific IgE antibody. A larger wheal on the skin-prick test and a lower ratio of peanut-specific IgG4:IgE were associated with peanut allergy. CONCLUSIONS: The early introduction of peanuts significantly decreased the frequency of the development of peanut allergy among children at high risk for this allergy and modulated immune responses to peanuts. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00329784.).

BACKGROUND: Islet transplantation offers the potential to improve glycemic control in a subgroup of patients with type 1 diabetes mellitus who are disabled by refractory hypoglycemia. We conducted an international, multicenter trial to explore the feasibility and reproducibility of islet transplantation with the use of a single common protocol (the Edmonton protocol). METHODS: We enrolled 36 subjects with type 1 diabetes mellitus, who underwent islet transplantation at nine international sites. Islets were prepared from pancreases of deceased donors and were transplanted within 2 hours after purification, without culture. The primary end point was defined as insulin independence with adequate glycemic control 1 year after the final transplantation. RESULTS: Of the 36 subjects, 16 (44%) met the primary end point, 10 (28%) had partial function, and 10 (28%) had complete graft loss 1 year after the final transplantation. A total of 21 subjects (58%) attained insulin independence with good glycemic control at any point throughout the trial. Of these subjects, 16 (76%) required insulin again at 2 years; 5 of the 16 subjects who reached the primary end point (31%) remained insulin-independent at 2 years. CONCLUSIONS: Islet transplantation with the use of the Edmonton protocol can successfully restore long-term endogenous insulin production and glycemic stability in subjects with type 1 diabetes mellitus and unstable control, but insulin independence is usually not sustainable. Persistent islet function even without insulin independence provides both protection from severe hypoglycemia and improved levels of glycated hemoglobin. (ClinicalTrials.gov number, NCT00014911 [ClinicalTrials.gov].).

BACKGROUND: The 18-month efficacy of a single course of rituximab as compared with conventional immunosuppression with cyclophosphamide followed by azathioprine in patients with severe (organ-threatening) antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis is unknown. METHODS: In a multicenter, randomized, double-blind, double-dummy, noninferiority trial, we compared rituximab (375 mg per square meter of body-surface area administered once a week for 4 weeks) followed by placebo with cyclophosphamide administered for 3 to 6 months followed by azathioprine for 12 to 15 months. The primary outcome measure was complete remission of disease by 6 months, with the remission maintained through 18 months. RESULTS: A total of 197 patients were enrolled. As reported previously, 64% of the patients in the rituximab group, as compared with 53% of the patients in the cyclophosphamide-azathioprine group, had a complete remission by 6 months. At 12 and 18 months, 48% and 39%, respectively, of the patients in the rituximab group had maintained the complete remissions, as compared with 39% and 33%, respectively, in the comparison group. Rituximab met the prespecified criteria for noninferiority (P<0.001, with a noninferiority margin of 20%). There was no significant difference between the groups in any efficacy measure, including the duration of complete remission and the frequency or severity of relapses. Among the 101 patients who had relapsing disease at baseline, rituximab was superior to conventional immunosuppression at 6 months (P=0.01) and at 12 months (P=0.009) but not at 18 months (P=0.06), at which time most patients in the rituximab group had reconstituted B cells. There was no significant between-group difference in adverse events. CONCLUSIONS: In patients with severe ANCA-associated vasculitis, a single course of rituximab was as effective as continuous conventional immunosuppressive therapy for the induction and maintenance of remissions over the course of 18 months. (Funded by the National Institute of Allergy and Infectious Diseases and others; RAVE ClinicalTrials.gov number, NCT00104299.)

The novel coronavirus SARS-CoV-2 (COVID-19) was recognized in December 2019 as a cause of severe pneumonia and has now led to a global pandemic.1Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (14613) Google Scholar Respiratory illnesses caused by COVID-19 cover a range of severity. The identification of risk and protective factors for disease severity from COVID-19 is critical to direct development of new treatments and infection prevention strategies. Early large case series have identified a number of risk factors for severe disease, including older age, hypertension, diabetes, cardiovascular disease, tobacco exposure, and chronic obstructive pulmonary disease.2Wu Z, McGoogan JM. Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72314 cases from the Chinese Center for Disease Control and Prevention [e-pub ahead of print]. JAMA https://doi.org/10.1001/jama.2020.2648. Accessed March 25, 2020.Google Scholar The US Centers for Disease Control and Prevention lists asthma as a risk factor for severe COVID-19 illness, which is logical given that many respiratory viruses have been well established to cause more serious illnesses in those with chronic airway diseases such as asthma. However, asthma and respiratory allergy have not been identified as significant risk factors for severe COVID-19 illness in case series from China.2Wu Z, McGoogan JM. Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72314 cases from the Chinese Center for Disease Control and Prevention [e-pub ahead of print]. JAMA https://doi.org/10.1001/jama.2020.2648. Accessed March 25, 2020.Google Scholar These preliminary reports led us to question whether we could identify features of allergy and/or asthma that could be associated with potential for reduced severity of COVID-19 illness. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as its cellular receptor, as do SARS-CoV and the coronavirus NL63.1Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (14613) Google Scholar Higher ACE2 expression increases in vitro susceptibility to SARS-CoV,3Jia H.P. Look D.C. Shi L. Hickey M. Pewe L. Netland J. et al.ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia.J Virol. 2005; 79: 14614-14621Crossref PubMed Scopus (663) Google Scholar and studies examining factors that affect ACE2 gene expression have revealed that its upregulation is associated with smoking, diabetes, and hypertension, all of which are associated with increased severity of COVID-19 illness.4Brake S.J. Barnsley K. Lu W. McAlinden K.D. Eapen M.S. Sohal S.S. Smoking upregulates angiotensin-converting enzyme-2 receptor: a potential adhesion site for novel coronavirus SARS-CoV-2 (Covid-19).J Clin Med. 2020; 9: 841Crossref PubMed Scopus (368) Google Scholar We hypothesized that 1 potential explanation for the unexpected observation that asthma and other allergic diseases may not be a risk factor for severe COVID-19 disease is a reduced ACE2 gene expression in airway cells and thus decreased susceptibility to infection. To test this hypothesis, we examined whether asthma and respiratory allergy are associated with reduced ACE2 expression in airway cells from 3 different cohorts of children and adults. In all 3 studies, total RNA was extracted from nasal or lower airway epithelial brush samples, with RNA sequencing performed independently for each study as previously described and provided in detail in the Supplementary Information (available in this article’s Online Repository at www.jacionline.org).5Larson D, Patel P, Salapatek AM, Couroux P, Whitehouse D, Pina A, et al. Nasal allergen challenge and environmental exposure chamber challenge: a randomized trial comparing clinical and biological responses to cat allergen. J Allergy Clin Immunol https://doi.org/10.1016/j.jaci.2020.02.024. Accessed March 25, 2020.Google Scholar Differential expression of ACE2 was assessed by using a weighted linear mixed effects model (limma) appropriate for RNA sequencing data and an empiric Bayes method. Children at high risk for asthma based on parental histories and living in urban neighborhoods were enrolled prenatally and followed prospectively in the Urban Environment and Childhood Asthma (URECA) cohort; 318 of them had nasal epithelial brushes obtained at 11 years of age. Prevalence of asthma was assessed at 10 years of age, and atopic status was defined by allergic sensitization trajectories (no or minimal, low, medium, and high) as previously described.6Bacharier L.B. Beigelman A. Calatroni A. Jackson D.J. Gergen P.J. O'Connor G.T. et al.Longitudinal phenotypes of respiratory health in a high-risk urban birth cohort.Am J Respir Crit Care Med. 2019; 199: 71-82Crossref PubMed Scopus (67) Google Scholar Additional type 2 biomarkers, including fractional exhaled nitric oxide, peripheral blood eosinophil level, and total IgE level, were evaluated by using standard methods. In URECA, allergic sensitization was inversely related to ACE2 expression in the nasal epithelium regardless of asthma status (Fig 1, A). In children with asthma, moderate allergic sensitization (fold change [FC] = 0.70; P = 4.2E–3) and high allergic sensitization (FC = 0.54; P = 6.4E–5) were associated with progressively greater reductions in ACE2 expression compared with in children with asthma but no/minimal allergic sensitization (Fig 1, B). ACE2 expression was also significantly inversely associated with type 2 biomarkers (see Table E1 in this article’s Online Repository at www.jacionline.org), including the number of positive allergen-specific IgE test results (β coefficient –0.089; P = 3.1E–5), total IgE level (β coefficient –0.31; P = 5.1E–6), fractional exhaled nitric oxide (β coefficient –0.45; P = 3.4E–3), and nasal epithelial expression of IL13 (β coefficient –0.123; P = 8.6E–5). ACE2 expression was not significantly correlated with peripheral blood eosinophil level (β coefficient –0.13; P = .07). Although male sex has been associated with increased severity of COVID-19 illness,2Wu Z, McGoogan JM. Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72314 cases from the Chinese Center for Disease Control and Prevention [e-pub ahead of print]. JAMA https://doi.org/10.1001/jama.2020.2648. Accessed March 25, 2020.Google Scholar no sex-based differences in ACE2 expression were found in URECA. Of note, 10 participants reported nasal corticosteroid use at the time of nasal sampling, and it was not associated with alterations in ACE2 expression. We also evaluated 24 adult participants with allergic rhinitis to cat who had no asthma symptoms in the prior year, were enrolled in a study in which they underwent nasal cat allergen challenge (NAC), and had been exposed to cat allergen through an environmental exposure chamber (EEC), as previously described.5Larson D, Patel P, Salapatek AM, Couroux P, Whitehouse D, Pina A, et al. Nasal allergen challenge and environmental exposure chamber challenge: a randomized trial comparing clinical and biological responses to cat allergen. J Allergy Clin Immunol https://doi.org/10.1016/j.jaci.2020.02.024. Accessed March 25, 2020.Google Scholar Pre–allergen challenge and post–allergen challenge nasal brush samples were obtained. Allergen exposure by both NAC and EEC led to significant reductions in ACE2 expression (Fig 2, A) (with NAC, FC = 0.81 and P = 2.4E–3; with exposure through an EEC, FC = 0.79 and P = 1.6E–3). An additional cohort of 23 adult participants with mild asthma that was not treated with asthma controller therapy underwent segmental allergen bronchoprovocation to dust mite, ragweed, or cat, as previously described.7Kelly E.A. Esnault S. Liu L.Y. Evans M.D. Johansson M.W. Mathur S. et al.Mepolizumab attenuates airway eosinophil numbers, but not their functional phenotype, in asthma.Am J Respir Crit Care Med. 2017; 196: 1385-1395Crossref PubMed Scopus (99) Google Scholar Pre–allergen challenge and post–allergen challenge bronchial brushings were obtained and demonstrated significantly reduced ACE2 expression in lower airway epithelium in the post–allergen challenge samples (Fig 2, B) (FC 0.64; P = .01). From in vitro models obtained from the Gene Expression Omnibus, we assessed the effects of IL-13, a type 2 cytokine strongly related to allergic asthma, on ACE2 expression in differentiated airway epithelial cells. IL-13 significantly reduced ACE2 expression (see Fig E1 in this article’s Online Repository at www.jacionline.org) in both nasal (FC = 0.44; P = 5.8E–4) and bronchial (FC = 0.80; P = 5.1E–3) epithelium. Viral respiratory infections are the most common trigger of severe asthma exacerbations in children and adults. Unexpectedly, large epidemiologic studies of the COVID-19 pandemic in China did not identify asthma as a risk factor of severe COVID-19–related illnesses.2Wu Z, McGoogan JM. Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72314 cases from the Chinese Center for Disease Control and Prevention [e-pub ahead of print]. JAMA https://doi.org/10.1001/jama.2020.2648. Accessed March 25, 2020.Google Scholar Here, we report that respiratory allergy and controlled allergen exposures are each associated with significant reductions in ACE2 expression. ACE2 expression was lowest in those with both high levels of allergic sensitization and asthma. Importantly, nonatopic asthma was not associated with reduced ACE2 expression. Given that ACE2 serves as the receptor for SARS-CoV-2, our findings suggest a potential mechanism of reduced COVID-19 severity in patients with respiratory allergies. However, it is likely that additional factors beyond ACE2 expression modulate the response to COVID-19 in individuals with allergy, and elucidation of these factors may also provide important insights into COVID-19 disease pathogenesis. The strengths of our study include carefully phenotyped cohorts of children and adults. Further, the allergen challenge studies included both upper and lower airway samples, with each demonstrating a consistent impact on ACE2 expression. The limitations include lack of clinical information to directly link ACE2 expression to SARS-CoV-2 infection and illness severity in our study populations. In addition, we do not have data on the ACE2 protein levels to confirm the gene expression data, although previous work suggests a direct association between ACE2 mRNA levels and ACE2 protein levels in the lung.8Li Y. Zeng Z. Cao Y. Liu Y. Ping F. Liang M. et al.Angiotensin-converting enzyme 2 prevents lipopolysaccharide-induced rat acute lung injury via suppressing the ERK1/2 and NF-kappaB signaling pathways.Sci Rep. 2016; 6: 27911Crossref PubMed Scopus (134) Google Scholar It is important to note that early data in the United States suggest a higher rate of asthma in patients hospitalized for severe COVID-19 illness, but the data do not specify whether the asthma was allergic, which is an important differentiation that relates to our findings. Nor do the data mention the potential presence of other comorbidities, such as obesity, that have been identified as risk factors for COVID-19 illness.9Centers for Disease Control and PreventionHospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019 - COVID-NET, 14 States, March 1-30, 2020.MMWR. 2020; 69Google Scholar Future studies focused on respiratory allergy, asthma, and perhaps other allergic disorders are needed to provide greater understanding of the impact of underlying allergy on COVID-19 susceptibility and disease severity. The modulation of ACE2 expression by type 2 inflammatory processes suggests the need to comprehensively evaluate the role of type 2 immune regulation in COVID-19 pathogenesis. Further elucidation of these relationships could identify novel therapeutic strategies to more effectively control this pandemic. In all 3 studies, total RNA was extracted from epithelial brush samples preserved in RLT buffer (Qiagen, Germantown, Md). Samples were thawed, vortexed, and quick-spun, after which the supernatant was transferred to fresh tubes. The samples were then spun through a Qiashredder column (Qiagen) and extracted by using RNeasy mini kits (Qiagen) with 25-μL elution volumes following the manufacturer’s protocol. In the cat allergy upper airway challenge study, sequencing libraries were constructed from total RNA by using TruSeq RNA Sample Preparation Kits v2 (Illumina). In the URECA and adult asthma studies, sequencing libraries were constructed from total RNA by using SMART-Seq v4 Ultra Low Input RNA Kit (Takara). For each study, libraries were clustered onto a flow cell by using a cBOT amplification system with a HiSeq SR v4 Cluster Kit (Illumina). Single-read sequencing was carried out on a HiSeq2500 sequencer (Illumina), using a HiSeq SBS v4 Kit to generate 58-bp reads, with a target of approximately 10 million reads per sample. Samples for each study were processed and sequenced independently. Reads were processed by using workflows managed on the Galaxy platform. Reads were trimmed by 1 base at the 3' end and then trimmed from both ends until base calls had a minimum quality score of at least 30 (Galaxy FASTQ Trimmer tool [version 1.0.0]). FastqMcf (version 1.1.2) was used to remove any remaining adapter sequence. To align the trimmed reads, we used the STAR aligner with the GRCh38 reference genome and gene annotations from ensembl, release 91. Gene counts were generated by using HTSeq-count (version 0.4.1). For quality control, the samples kept were those that had counts greater than 1 million, more than 80% of reads aligned, and a median coefficient of variation (CV) coverage less than 1. Genes were filtered to include those that had a trimmed mean of M-value (TMM) normalization count of at least 1 in at least 10% of libraries and were classified as protein coding by using BioMart.E1Smedley D. Haider S. Durinck S. Pandini L. Provero P. Allen J. et al.The BioMart community portal: an innovative alternative to large, centralized data repositories.Nucleic Acids Res. 2015; 43: W589-W598Crossref PubMed Scopus (531) Google Scholar Counts were transformed to log2 counts per million along with observation-level weights by using voomWithQualityWeights from the limma R packageE2Liu R. Holik A.Z. Su S. Jansz N. Chen K. Leong H.S. et al.Why weight? Modelling sample and observational level variability improves power in RNA-seq analyses.Nucleic Acids Res. 2015; 43: e97Crossref PubMed Scopus (304) Google Scholar to create a weighted gene expression matrix suitable for downstream analyses. Differential expression of ACE2 was assessed independently in each data set by using a weighted linear mixed effects model (limma) appropriate for RNA sequencing data and an empiric Bayes method.E2Liu R. Holik A.Z. Su S. Jansz N. Chen K. Leong H.S. et al.Why weight? Modelling sample and observational level variability improves power in RNA-seq analyses.Nucleic Acids Res. 2015; 43: e97Crossref PubMed Scopus (304) Google Scholar, E3Ritchie M.E. Phipson B. Wu D. Hu Y. Law C.W. Shi W. et al.Limma powers differential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res. 2015; 43: e47Crossref PubMed Scopus (20650) Google Scholar Mixed effects linear regression models were used; they included relevant clinical or technical variables (for URECA, cytologically determined cell percentages in the brush and the clinical site were used; for the upper airway challenge study, processing batch was used; and for the adult asthma study, no fixed effects were included) and a random effect of participant in both of the airway challenge studies. P values less than .05 were considered statistically significant. We searched the National Center for Biotechnology Information Gene Expression Omnibus for the terms IL13 and epithelial subset to the organism Homo sapiens.E4Barrett T. Wilhite S.E. Ledoux P. Evangelista C. Kim I.F. Tomashevsky M. et al.NCBI GEO: archive for functional genomics data sets--update.Nucleic Acids Res. 2013; 41: D991-D995Crossref PubMed Scopus (6441) Google Scholar From this we identified 2 studies investigating the effects of IL-13 stimulation on human airway epithelial cells grown at an air-liquid interface that had repeated measures in the IL-13 stimulation and unstimulated groups. In the GSE110799 study, human nasal epithelial cells isolated from nasal turbinates were cultured in an air-liquid interface until the full differentiation was complete, after which the differentiated cells at ALI-D47 were incubated with 100 ng/mL of IL-13 for 3 days. In GSE37693, RNA was isolated from primary culture airway epithelial cells grown at an air-liquid interface treated with or without IL-13 for 21 days.E5Alevy Y.G. Patel A.C. Romero A.G. Patel D.A. Tucker J. Roswit W.T. et al.IL-13-induced airway mucus production is attenuated by MAPK13 inhibition.J Clin Invest. 2012; 122: 4555-4568Crossref PubMed Scopus (152) Google Scholar Differential expression analysis was performed by using GEO2R, which performs voom and limmaE2Liu R. Holik A.Z. Su S. Jansz N. Chen K. Leong H.S. et al.Why weight? Modelling sample and observational level variability improves power in RNA-seq analyses.Nucleic Acids Res. 2015; 43: e97Crossref PubMed Scopus (304) Google Scholar, E3Ritchie M.E. Phipson B. Wu D. Hu Y. Law C.W. Shi W. et al.Limma powers differential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res. 2015; 43: e47Crossref PubMed Scopus (20650) Google Scholar in the National Center for Biotechnology Information's Gene Expression Omnibus browser.Table E1Association of T2 biomarkers and nasal brush ACE2 expression in the URECA cohortBiomarkerAssociation with ACE2 expression (β coefficient)P valuePositive allergen-specific IgE–0.0893.1E–5Total IgE level–0.315.1E–6Fractional exhaled nitric oxide–0.453.4E–3Blood eosinophils–0.13.07Nasal epithelial IL-13 expression–0.1238.6E–5 Open table in a new tab COVID-19: Start with the noseJournal of Allergy and Clinical ImmunologyVol. 146Issue 5PreviewWe read with interest the data from Jackson et al1 that revealed lower angiotensin-converting enzyme 2 (ACE2) expression in nasal brushings from children with allergic sensitization, along with a progressive decline in ACE2 expression in relation to increasing IgE sensitization in those with asthma. Moreover, in nasal brushings from adults with allergic rhinitis, ACE2 expression was lower after exposure to cat allergen. Full-Text PDF

BACKGROUND: Conjugating immunostimulatory sequences of DNA to specific allergens offers a new approach to allergen immunotherapy that reduces acute allergic responses. METHODS: We conducted a randomized, double-blind, placebo-controlled phase 2 trial of a vaccine consisting of Amb a 1, a ragweed-pollen antigen, conjugated to a phosphorothioate oligodeoxyribonucleotide immunostimulatory sequence of DNA (AIC) in 25 adults who were allergic to ragweed. Patients received six weekly injections of the AIC or placebo vaccine before the first ragweed season and were monitored during the next two ragweed seasons. RESULTS: There was no pattern of vaccine-associated systemic reactions or clinically significant laboratory abnormalities. AIC did not alter the primary end point, the vascular permeability response (measured by the albumin level in nasal-lavage fluid) to nasal provocation. During the first ragweed season, the AIC group had better peak-season rhinitis scores on the visual-analogue scale (P=0.006), peak-season daily nasal symptom diary scores (P=0.02), and midseason overall quality-of-life scores (P=0.05) than the placebo group. AIC induced a transient increase in Amb a 1-specific IgG antibody but suppressed the seasonal increase in Amb a 1-specific IgE antibody. A reduction in the number of interleukin-4-positive basophils in AIC-treated patients correlated with lower rhinitis visual-analogue scores (r=0.49, P=0.03). Clinical benefits of AIC were again observed in the subsequent ragweed season, with improvements over placebo in peak-season rhinitis visual-analogue scores (P=0.02) and peak-season daily nasal symptom diary scores (P=0.02). The seasonal specific IgE antibody response was again suppressed, with no significant change in IgE antibody titer during the ragweed season (P=0.19). CONCLUSIONS: In this pilot study, a 6-week regimen of the AIC vaccine appeared to offer long-term clinical efficacy in the treatment of ragweed allergic rhinitis. (ClinicalTrials.gov number, NCT00346086 [ClinicalTrials.gov] .).

Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to alpha-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.

A fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users, and publishers by providing a specification of what is required to unambiguously interpret experimental findings. Here, we present the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, stating the minimum information required to report flow cytometry (FCM) experiments. We brought together a cross-disciplinary international collaborative group of bioinformaticians, computational statisticians, software developers, instrument manufacturers, and clinical and basic research scientists to develop the standard. The standard was subsequently vetted by the International Society for Advancement of Cytometry (ISAC) Data Standards Task Force, Standards Committee, membership, and Council. The MIFlowCyt standard includes recommendations about descriptions of the specimens and reagents included in the FCM experiment, the configuration of the instrument used to perform the assays, and the data processing approaches used to interpret the primary output data. MIFlowCyt has been adopted as a standard by ISAC, representing the FCM scientific community including scientists as well as software and hardware manufacturers. Adoptionof MIFlowCyt by the scientific and publishing communities will facilitate third-party understanding and reuse of FCM data.

BACKGROUND: Growing knowledge about cellular interactions in the immune system, including the central role of cytokine networks, has lead to new treatments using monoclonal antibodies that block specific components of the immune system. Systemic cytokine concentrations can serve as surrogate outcome parameters of these interventions to study inflammatory pathways operative in patients in vivo. This is now possible due to novel technologies such as multiplex immunoassays (MIA) that allows detection of multiple cytokines in a single sample. However, apparently trivial underappreciated processes, (sample handling and storage, interference of endogenous plasma proteins) can greatly impact the reliability and reproducibility of cytokine detection.Therefore we set out to investigate several processes that might impact cytokine profiles such as blood collecting tubes, duration of storage, and number of freeze thawing cycles. RESULTS: Since under physiological conditions cytokine concentrations normally are low or undetectable we spiked cytokines in the various plasma and serum samples. Overall recoveries ranged between 80-120%. Long time storage showed cytokines are stable for a period up to 2 years of storage at -80 degrees C. After 4 years several cytokines (IL-1alpha, IL-1beta, IL-10, IL-15 and CXCL8) degraded up to 75% or less of baseline values. Furthermore we show that only 2 out of 15 cytokines remained stable after several freeze-thawing cycles. We also demonstrate implementation of an internal control for multiplex cytokine immunoassays. CONCLUSION: All together we show parameters which are essential for measurement of cytokines in the context of clinical trials.

BACKGROUND: This guideline from the European Academy of Allergy and Clinical Immunology (EAACI) recommends approaches to prevent the development of immediate-onset / IgE-mediated food allergy in infants and young children. It is an update of a 2014 EAACI guideline. METHODS: The guideline was developed using the AGREE II framework and the GRADE approach. An international Task Force with representatives from 11 countries and different disciplinary and clinical backgrounds systematically reviewed research and considered expert opinion. Recommendations were created by weighing up benefits and harms, considering the certainty of evidence and examining values, preferences and resource implications. The guideline was peer-reviewed by external experts, and feedback was incorporated from public consultation. RESULTS: All of the recommendations about preventing food allergy relate to infants (up to 1 year) and young children (up to 5 years), regardless of risk of allergy. There was insufficient evidence about preventing food allergy in other age groups. The EAACI Task Force suggests avoiding the use of regular cow's milk formula as supplementary feed for breastfed infants in the first week of life. The EAACI Task Force suggests introducing well-cooked, but not raw egg or uncooked pasteurized, egg into the infant diet as part of complementary feeding. In populations where there is a high prevalence of peanut allergy, the EAACI Task Force suggests introducing peanuts in an age-appropriate form as part of complementary feeding. According to the studies, it appears that the most effective age to introduce egg and peanut is from four to 6 months of life. The EAACI Task Force suggests against the following for preventing food allergy: (i) avoiding dietary food allergens during pregnancy or breastfeeding; and (ii) using soy protein formula in the first 6 months of life as a means of preventing food allergy. There is no recommendation for or against the following: use of vitamin supplements, fish oil, prebiotics, probiotics or synbiotics in pregnancy, when breastfeeding or in infancy; altering the duration of exclusive breastfeeding; and hydrolysed infant formulas, regular cow's milk-based infant formula after a week of age or use of emollients. CONCLUSIONS: Key changes from the 2014 guideline include suggesting (i) the introduction of peanut and well-cooked egg as part of complementary feeding (moderate certainty of evidence) and (ii) avoiding supplementation with regular cow's milk formula in the first week of life (low certainty of evidence). There remains uncertainty in how to prevent food allergy, and further well-powered, multinational research using robust diagnostic criteria is needed.

BACKGROUND: In a randomized trial, the early introduction of peanuts in infants at high risk for allergy was shown to prevent peanut allergy. In this follow-up study, we investigated whether the rate of peanut allergy remained low after 12 months of peanut avoidance among participants who had consumed peanuts during the primary trial (peanut-consumption group), as compared with those who had avoided peanuts (peanut-avoidance group). METHODS: At the end of the primary trial, we instructed all the participants to avoid peanuts for 12 months. The primary outcome was the percentage of participants with peanut allergy at the end of the 12-month period, when the participants were 72 months of age. RESULTS: We enrolled 556 of 628 eligible participants (88.5%) from the primary trial; 550 participants (98.9%) had complete primary-outcome data. The rate of adherence to avoidance in the follow-up study was high (90.4% in the peanut-avoidance group and 69.3% in the peanut-consumption group). Peanut allergy at 72 months was significantly more prevalent among participants in the peanut-avoidance group than among those in the peanut-consumption group (18.6% [52 of 280 participants] vs. 4.8% [13 of 270], P<0.001). Three new cases of allergy developed in each group, but after 12 months of avoidance there was no significant increase in the prevalence of allergy among participants in the consumption group (3.6% [10 of 274 participants] at 60 months and 4.8% [13 of 270] at 72 months, P=0.25). Fewer participants in the peanut-consumption group than in the peanut-avoidance group had high levels of Ara h2 (a component of peanut protein)-specific IgE and peanut-specific IgE; in addition, participants in the peanut-consumption group continued to have a higher level of peanut-specific IgG4 and a higher peanut-specific IgG4:IgE ratio. CONCLUSIONS: Among children at high risk for allergy in whom peanuts had been introduced in the first year of life and continued until 5 years of age, a 12-month period of peanut avoidance was not associated with an increase in the prevalence of peanut allergy. Longer-term effects are not known. (Funded by the National Institute of Allergy and Infectious Diseases and others; LEAP-On ClinicalTrials.gov number, NCT01366846.).

Trials of immune therapies in new-onset type 1 diabetes (T1D) have shown success, but not all subjects respond, and the duration of response is limited. Our aim was to determine whether two courses of teplizumab, an Fc receptor-nonbinding anti-CD3 monoclonal antibody, reduces the decline in C-peptide levels in patients with T1D 2 years after disease onset. We also set out to identify characteristics of responders. We treated 52 subjects with new-onset T1D with teplizumab for 2 weeks at diagnosis and after 1 year in an open-label, randomized, controlled trial. In the intent to treat analysis of the primary end point, patients treated with teplizumab had a reduced decline in C-peptide at 2 years (mean -0.28 nmol/L [95% CI -0.36 to -0.20]) versus control (mean -0.46 nmol/L [95% CI -0.57 to -0.35]; P = 0.002), a 75% improvement. The most common adverse events were rash, transient upper respiratory infections, headache, and nausea. In a post hoc analysis we characterized clinical responders and found that metabolic (HbA1c and insulin use) and immunologic features distinguished this group from those who did not respond to teplizumab. We conclude that teplizumab treatment preserves insulin production and reduces the use of exogenous insulin in some patients with new-onset T1D. Metabolic and immunologic features at baseline can identify a subgroup with robust responses to immune therapy.

CONTEXT: Although life-saving, liver transplantation burdens children with lifelong immunosuppression and substantial potential for morbidity and mortality. OBJECTIVE: To establish the feasibility of immunosuppression withdrawal in pediatric living donor liver transplant recipients. DESIGN, SETTING, AND PATIENTS: Prospective, multicenter, open-label, single-group pilot trial conducted in 20 stable pediatric recipients (11 male; 55%) of parental living donor liver transplants for diseases other than viral hepatitis or an autoimmune disease who underwent immunosuppression withdrawal. Their median age was 6.9 months (interquartile range [IQR], 5.5-9.1 months) at transplant and 8 years 6 months (IQR, 6 years 5 months to 10 years 9 months) at study enrollment. Additional entry requirements included stable allograft function while taking a single immunosuppressive drug and no evidence of acute or chronic rejection or significant fibrosis on liver biopsy. Gradual immunosuppression withdrawal over a minimum of 36 weeks was instituted at 1 of 3 transplant centers between June 5, 2006, and November 18, 2009. Recipients were followed up for a median of 32.9 months (IQR, 1.0-49.9 months). MAIN OUTCOME MEASURES: The primary end point was the proportion of operationally tolerant patients, defined as patients who remained off immunosuppression therapy for at least 1 year with normal graft function. Secondary clinical end points included the durability of operational tolerance, and the incidence, timing, severity, and reversibility of rejection. RESULTS: Of 20 pediatric patients, 12 (60%; 95% CI, 36.1%-80.9%) met the primary end point, maintaining normal allograft function for a median of 35.7 months (IQR, 28.1-39.7 months) after discontinuing immunosuppression therapy. Follow-up biopsies obtained more than 2 years after completing withdrawal showed no significant change compared with baseline biopsies. Eight patients did not meet the primary end point secondary to an exclusion criteria violation (n = 1), acute rejection (n = 2), or indeterminate rejection (n = 5). Seven patients were treated with increased or reinitiation of immunosuppression therapy; all returned to baseline allograft function. Patients with operational tolerance compared with patients without operational tolerance initiated immunosuppression withdrawal later after transplantation (median of 100.6 months [IQR, 71.8-123.5] vs 73.0 months [IQR, 57.6-74.9], respectively; P = .03), had less portal inflammation (91.7% [95% CI, 61.5%-99.8%] vs 42.9% [95% CI, 9.9%-81.6%] with no inflammation; P = .04), and had lower total C4d scores on the screening liver biopsy (median of 6.1 [IQR, 5.1-9.3] vs 12.5 [IQR, 9.3-16.8]; P = .03). CONCLUSION: In this pilot study, 60% of pediatric recipients of parental living donor liver transplants remained off immunosuppression therapy for at least 1 year with normal graft function and stable allograft histology.

T cell subsets indicated that partially exhausted effector cells were associated with clinical response. Thus, this trial showed improvement in metabolic responses and delay of diabetes with immune therapy.

Allergen immunotherapy is a cornerstone in the treatment of allergic children. The clinical efficiency relies on a well-defined immunologic mechanism promoting regulatory T cells and downplaying the immune response induced by allergens. Clinical indications have been well documented for respiratory allergy in the presence of rhinitis and/or allergic asthma, to pollens and dust mites. Patients who have had an anaphylactic reaction to hymenoptera venom are also good candidates for allergen immunotherapy. Administration of allergen is currently mostly either by subcutaneous injections or by sublingual administration. Both methods have been extensively studied and have pros and cons. Specifically in children, the choice of the method of administration according to the patient's profile is important. Although allergen immunotherapy is widely used, there is a need for improvement. More particularly, biomarkers for prediction of the success of the treatments are needed. The strength and efficiency of the immune response may also be boosted by the use of better adjuvants. Finally, novel formulations might be more efficient and might improve the patient's adherence to the treatment. This user's guide reviews current knowledge and aims to provide clinical guidance to healthcare professionals taking care of children undergoing allergen immunotherapy.

OBJECTIVE: There is a need to determine which response measures in lupus nephritis trials are most predictive of good long-term renal function. We used data from the Euro-Lupus Nephritis Trial to evaluate the performance of proteinuria, serum creatinine (Cr), and urinary red blood cells (RBCs) as predictors of good long-term renal outcome. METHODS: Patients from the Euro-Lupus Nephritis Trial with proteinuria, serum Cr, and urinary RBC measurements at 3, 6, or 12 months and with a minimum of 7 years of followup were included (n = 76). We assessed the ability of these clinical biomarkers at 3, 6, and 12 months after randomization to predict good long-term renal outcome (defined as a serum Cr value ≤1.0 mg/dl) at 7 years. Receiver operating characteristic curves were generated to assess parameter performance at these time points and to select the best cutoff for individual parameters. Sensitivity and specificity were calculated for the parameters alone and in combination. RESULTS: A proteinuria value of <0.8 gm/day at 12 months after randomization was the single best predictor of good long-term renal function (sensitivity 81% and specificity 78%). The addition of serum Cr to proteinuria as a composite predictor did not improve the performance of the outcome measure; addition of urinary RBCs as a predictor significantly decreased the sensitivity to 47%. CONCLUSION: This study demonstrates that the level of proteinuria at 12 months is the individual best predictor of long-term renal outcome in patients with lupus nephritis. Inclusion of urinary RBCs as part of a composite outcome measure actually undermined the predictive value of the trial data. We therefore suggest that urinary RBCs should not be included as a component of clinical trial response criteria in lupus nephritis.

Rapamycin/interleukin-2 (IL-2) combination treatment of NOD mice effectively treats autoimmune diabetes. We performed a phase 1 clinical trial to test the safety and immunologic effects of rapamycin/IL-2 combination therapy in type 1 diabetic (T1D) patients. Nine T1D subjects were treated with 2-4 mg/day rapamycin orally for 3 months and 4.5 × 10(6) IU IL-2 s.c. three times per week for 1 month. β-Cell function was monitored by measuring C-peptide. Immunologic changes were monitored using flow cytometry and serum analyses. Regulatory T cells (Tregs) increased within the first month of therapy, yet clinical and metabolic data demonstrated a transient worsening in all subjects. The increase in Tregs was transient, paralleling IL-2 treatment, whereas the response of Tregs to IL-2, as measured by STAT5 phosphorylation, increased and persisted after treatment. No differences were observed in effector T-cell subset frequencies, but an increase in natural killer cells and eosinophils occurred with IL-2 therapy. Rapamycin/IL-2 therapy, as given in this phase 1 study, resulted in transient β-cell dysfunction despite an increase in Tregs. Such results highlight the difficulties in translating therapies to the clinic and emphasize the importance of broadly interrogating the immune system to evaluate the effects of therapy.

We report here the long-term results of HLA-mismatched kidney transplantation without maintenance immunosuppression (IS) in 10 subjects following combined kidney and bone marrow transplantation. All subjects were treated with nonmyeloablative conditioning and an 8- to 14-month course of calcineurin inhibitor with or without rituximab. All 10 subjects developed transient chimerism, and in seven of these, IS was successfully discontinued for 4 or more years. Currently, four subjects remain IS free for periods of 4.5–11.4 years, while three required reinstitution of IS after 5–8 years due to recurrence of original disease or chronic antibody-mediated rejection. Of the 10 renal allografts, three failed due to thrombotic microangiopathy or rejection. When compared with 21 immunologically similar living donor kidney recipients treated with conventional IS, the long-term IS-free survivors developed significantly fewer posttransplant complications. Although most recipients treated with none or two doses of rituximab developed donor-specific antibody (DSA), no DSA was detected in recipients treated with four doses of rituximab. Although further revisions of the current conditioning regimen are planned in order to improve consistency of the results, this study shows that long-term stable kidney allograft survival without maintenance IS can be achieved following transient mixed chimerism induction. We report here the long-term results of HLA-mismatched kidney transplantation without maintenance immunosuppression (IS) in 10 subjects following combined kidney and bone marrow transplantation. All subjects were treated with nonmyeloablative conditioning and an 8- to 14-month course of calcineurin inhibitor with or without rituximab. All 10 subjects developed transient chimerism, and in seven of these, IS was successfully discontinued for 4 or more years. Currently, four subjects remain IS free for periods of 4.5–11.4 years, while three required reinstitution of IS after 5–8 years due to recurrence of original disease or chronic antibody-mediated rejection. Of the 10 renal allografts, three failed due to thrombotic microangiopathy or rejection. When compared with 21 immunologically similar living donor kidney recipients treated with conventional IS, the long-term IS-free survivors developed significantly fewer posttransplant complications. Although most recipients treated with none or two doses of rituximab developed donor-specific antibody (DSA), no DSA was detected in recipients treated with four doses of rituximab. Although further revisions of the current conditioning regimen are planned in order to improve consistency of the results, this study shows that long-term stable kidney allograft survival without maintenance IS can be achieved following transient mixed chimerism induction.

Autologous hematopoietic stem cell transplantation (HSCT) is commonly employed for hematologic and non-hematologic malignancies. In clinical trials, HSCT has been evaluated for severe autoimmunity as a method to "reset" the immune system and produce a new, non-autoimmune repertoire. While the feasibility of eliminating the vast majority of mature T cells is well established, accurate and quantitative determination of the relationship of regenerated T cells to the baseline repertoire has been difficult to assess. Here, in a phase II study of HSCT for poor-prognosis multiple sclerosis, we used high-throughput deep TCRβ chain sequencing to assess millions of individual TCRs per patient sample. We found that HSCT has distinctive effects on CD4+ and CD8+ T cell repertoires. In CD4+ T cells, dominant TCR clones present before treatment were undetectable following reconstitution, and patients largely developed a new repertoire. In contrast, dominant CD8+ clones were not effectively removed, and the reconstituted CD8+ T cell repertoire was created by clonal expansion of cells present before treatment. Importantly, patients who failed to respond to treatment had less diversity in their T cell repertoire early during the reconstitution process. These results demonstrate that TCR characterization during immunomodulatory treatment is both feasible and informative, and may enable monitoring of pathogenic or protective T cell clones following HSCT and cellular therapies.

There is increasing evidence regarding the importance of allergic sensitization through the skin. In this review, we provide an overview of the atopic march and immune mechanism underlying the sensitization and effector phase of food allergy. We present experimental models and human data that support the concept of epicutaneous sensitization and how this forms one half of the dual-allergen exposure hypothesis. We discuss specific important elements in the skin (FLG and other skin barrier gene mutations, Langerhans cells, type 2 innate lymphoid cells, IL-33, TSLP) that have important roles in the development of allergic responses as well as the body of evidence on environmental allergen exposure and how this can sensitize an individual. Given the link between skin barrier impairment, atopic dermatitis, food allergy, allergic asthma, and allergic rhinitis, it is logical that restoring the skin barrier and prevention or treating atopic dermatitis would have beneficial effects on prevention of related allergic diseases, particularly food allergy. We present the experimental and human studies that have evaluated this approach and discuss various factors which may influence the success of these approaches, such as the type of emollient chosen for the intervention, the role of managing skin inflammation, and differences between primary and secondary prevention of atopic dermatitis to achieve the desired outcome.