Chronic infection with a tissue-invasive helminth attenuates sublethal anaphylaxis and reduces granularity and number of mast cells.

BACKGROUND: Immunoglobulin E (IgE)-mediated anaphylaxis is a potentially fatal condition in which allergy effector cells rapidly discharge pre-formed inflammatory mediators. Treatments that address the immune component of allergic anaphylaxis are inadequate. Helminths have been previously shown to suppress effector cell function; however, their ability to treat pre-existing allergy remains unclear. OBJECTIVE: To evaluate the ability of chronic helminth infection to protect against anaphylaxis in previously sensitized mice. METHODS: A sublethal model of anaphylaxis was used, in which BALB/c mice were sensitized by three intraperitoneal (i.p.) injections of OVA/alum. Temperature drop was then monitored after systemic OVA challenge in uninfected mice and in mice infected chronically with Litomosoides sigmodontis, a tissue-invasive filarial nematode. RESULTS: Litomosoides sigmodontis-infected mice exhibited significantly lower serum levels of mMCP-1 and were less hypothermic at 30-minute post-challenge compared to uninfected OVA-challenged controls. Characterization of anaphylaxis revealed that FcԑR1 and mast cells were required for hypothermia and elevated serum mMCP-1. OVA-IgE and OVA-IgG1 serum levels were not significantly altered by L sigmodontis infection, and experiments with IL-10-/- mice demonstrated that IL-10 was not required for protection against anaphylaxis. However, peritoneal mast cell numbers were significantly lower in infected mice, and those that were present exhibited decreased granularity by flow cytometry and marked depletion of intracytoplasmic granules by light microscopy. Mast cells from infected mice had lower expression of the activation markers CD200R and CD63 and contained significantly lower basal stores of histamine. CONCLUSIONS: Chronic L sigmodontis infection protects against anaphylaxis, likely due to reduction in mast cell numbers and depletion of pre-formed inflammatory mediators in remaining mast cells.

Engineered ovalbumin-expressing regulatory T cells protect against anaphylaxis in ovalbumin-sensitized mice.

Allergy is a major public health concern, the main treatment for which is symptomatic relief with anti-inflammatory drugs. A key clinical challenge is to induce specific tolerance in order to control allergen-specific memory B and T cells, and specifically block effector cell responses. Our lab recently developed antigen-specific regulatory T-cell (Treg) therapies as a treatment for adverse responses. Recently, we created a chimeric antigen receptor (CAR) approach in which we engineered a target protein antigen, ovalbumin (OVA), linked with the transmembrane and signal transduction domains, CD28-CD3ζ to directly target B cells and sensitized mast cells in an allergy model. We named this receptor "BAR" for B-cell Antibody Receptor. Murine or human Tregs, transduced with a BAR containing OVA or control Tregs expressing an unrelated antigen, were successfully expanded in vitro and tested in the murine OVA-alum allergy model with measurable titers of anti-OVA IgE. Because BAR Tregs express the target antigen and could interact with specific IgE on sensitized mast cells, we first demonstrated that intravenously injected OVA-BAR Tregs did not directly lead to a drop in temperature or release of mediators in plasma indicative of anaphylaxis. Forty-eight hours later, mice were challenged intraperitoneally with 200 µg OVA to induce an anaphylactic reaction, and temperature immediately measured for 30 min. We found that OVA-BAR Tregs protected mice from hypothermia, whereas mice given control BARs (expressing an unrelated antigen) or PBS showed substantial temperature drops indicative of anaphylaxis when systemically challenged with OVA. Importantly, this effect was also demonstrated in a passive anaphylaxis model in which mice that received anti-OVA IgE antibody were protected from hypothermia when treated with OVA-BAR Tregs prior to systemic OVA challenge. These results provide proof of principle that engineered allergen-specific T-regulatory cells can provide clinical protection against severe allergic reactions in individuals already IgE-sensitized to an allergen.

Rush desensitization with a single antigen induces subclinical activation of mast cells and protects against bystander challenge in dually sensitized mice.

BACKGROUND: Rush desensitization can provide short-term tolerance to individuals who are allergic to certain medications in instances where other therapeutic interventions are limited. While rush desensitization (DS) is typically successful in preventing adverse type I hypersensitivity reactions, the mechanism of allergic protection remains unknown. Given the rise in prevalence of individuals displaying multiple allergies, understanding the impact of rush DS on "bystander" allergens, or additional allergens to which an individual is sensitized, could help inform clinical recommendations. OBJECTIVE: To evaluate the effect of rush DS on bystander sensitization. MATERIALS AND METHODS: We used a murine model of rush DS, whereby BALB/c mice were sensitized to ovalbumin (OVA) and desensitized through repeated intraperitoneal injections of OVA. Using a local anaphylaxis assay, we measured ear swelling by Evans blue extravasation following intradermal challenge. In studies to measure the impact on bystander antigens, a modified protocol was used in which mice were dually sensitized to OVA and Keyhole limpet hemocyanin (KLH), and densensitized to either OVA or KLH prior to allergic challenge. RESULTS: The immunological effects of rush DS were independent of changes in Th1 and Th2 cytokine production and circulating OVA-IgE levels. Instead, rush DS resulted in subclinical degranulation of mast cells prior to challenge. In our dual sensitization model, rush DS with a single antigen conferred protection against allergic challenge to a secondary antigen. Bystander protection required prior sensitization, as DS with an irrelevant antigen did not impact allergic responsiveness. CONCLUSIONS AND CLINICAL RELEVANCE: We reveal that a key mechanism of rush DS protection against allergic responsiveness may be the subclinical degranulation of mast cells. Therefore, performing rush DS to a single antigen to which one is IgE-sensitized may be sufficient to desensitize to multiple allergens. Future studies could lead to streamlined protocols of rush DS for patients with multiple allergies.

Identification of the cellular sentinels for native immunogenic heat shock proteins in vivo.

Select members of the heat shock proteins (HSPs) family, such as gp96, elicit immune responses specific to their chaperoned peptides. Although immunologic effects of HSPs on APCs described to date have largely been demonstrated with cell lines or primary cells in culture, their collective responses in vitro have been consistent with priming immune responses. In this study, we examine the physiologically relevant APCs in mice that are targeted after vaccination with native, murine HSPs, and we characterize those cells. Gp96 accesses the subcapsular region of the draining lymph node, and it is internalized predominantly by CD11b(+) cells in this locale. Cells acquiring gp96 can transfer protective antitumor immunity to naive mice by actively cross-presenting gp96-chaperoned peptides and providing costimulation. Our studies illustrate how HSPs act to alert the immune system of cellular damage and will be of paramount importance in immunotherapy of patients with cancer and infectious disease.

Analysis of LMNB1 duplications in autosomal dominant leukodystrophy provides insights into duplication mechanisms and allele-specific expression.

Autosomal dominant leukodystrophy (ADLD) is an adult onset demyelinating disorder that is caused by duplications of the lamin B1 (LMNB1) gene. However, as only a few cases have been analyzed in detail, the mechanisms underlying LMNB1 duplications are unclear. We report the detailed molecular analysis of the largest collection of ADLD families studied, to date. We have identified the minimal duplicated region necessary for the disease, defined all the duplication junctions at the nucleotide level and identified the first inverted LMNB1 duplication. We have demonstrated that the duplications are not recurrent; patients with identical duplications share the same haplotype, likely inherited from a common founder and that the duplications originated from intrachromosomal events. The duplication junction sequences indicated that nonhomologous end joining or replication-based mechanisms such fork stalling and template switching or microhomology-mediated break induced repair are likely to be involved. LMNB1 expression was increased in patients' fibroblasts both at mRNA and protein levels and the three LMNB1 alleles in ADLD patients show equal expression, suggesting that regulatory regions are maintained within the rearranged segment. These results have allowed us to elucidate duplication mechanisms and provide insights into allele-specific LMNB1 expression levels.

Ovalbumin-derived precursor peptides are transferred sequentially from gp96 and calreticulin to MHC class I in the endoplasmic reticulum.

Cellular peptides generated by proteasomal degradation of proteins in the cytosol and destined for presentation by MHC class I (MHC-I) are associated with several chaperones. Heat shock proteins 70, 90, and the TCP-1 ring complex have been implicated as important cytosolic players for chaperoning these peptides. In this study, we report that gp96 and calreticulin are essential for chaperoning peptides in the endoplasmic reticulum. Importantly, we demonstrate that cellular peptides are transferred sequentially from gp96 to calreticulin and then to MHC-I forming a relay line. Disruption of this relay line by removal of gp96 or calreticulin prevents the binding of peptides by MHC-I and hence presentation of the MHC-I-peptide complex on the cell surface. Our results are important for understanding how peptides are processed and trafficked within the endoplasmic reticulum before exiting in association with MHC-I H chains and beta2-microglobulin as a trimolecular complex.

Association Between Use of Acid-Suppressive Medications and Antibiotics During Infancy and Allergic Diseases in Early Childhood.

Importance: Allergic diseases are prevalent in childhood. Early exposure to medications that can alter the microbiome, including acid-suppressive medications and antibiotics, may influence the likelihood of allergy. Objective: To determine whether there is an association between the use of acid-suppressive medications or antibiotics in the first 6 months of infancy and development of allergic diseases in early childhood. Design, Setting, and Participants: A retrospective cohort study was conducted in 792 130 children who were Department of Defense TRICARE beneficiaries with a birth medical record in the Military Health System database between October 1, 2001, and September 30, 2013, with continued enrollment from within 35 days of birth until at least age 1 year. Children who had an initial birth stay of greater than 7 days or were diagnosed with any of the outcome allergic conditions within the first 6 months of life were excluded from the study. Data analysis was performed from April 15, 2015, to January 4, 2018. Exposures: Exposures were defined as having any dispensed prescription for a histamine-2 receptor antagonist (H2RA), proton pump inhibitor (PPI), or antibiotic. Main Outcomes and Measures: The main outcome was allergic disease, defined as the presence of food allergy, anaphylaxis, asthma, atopic dermatitis, allergic rhinitis, allergic conjunctivitis, urticaria, contact dermatitis, medication allergy, or other allergy. Results: Of 792 130 children (395 215 [49.9%] girls) included for analysis, 60 209 (7.6%) were prescribed an H2RA, 13 687 (1.7%) were prescribed a PPI, and 131 708 (16.6%) were prescribed an antibiotic during the first 6 months of life. Data for each child were available for a median of 4.6 years. Adjusted hazard ratios (aHRs) in children prescribed H2RAs and PPIs, respectively, were 2.18 (95% CI, 2.04-2.33) and 2.59 (95% CI, 2.25-3.00) for food allergy, 1.70 (95% CI, 1.60-1.80) and 1.84 (95% CI, 1.56-2.17) for medication allergy, 1.51 (95% CI, 1.38-1.66) and 1.45 (95% CI, 1.22-1.73) for anaphylaxis, 1.50 (95% CI, 1.46-1.54) and 1.44 (95% CI, 1.36-1.52) for allergic rhinitis, and 1.25 (95% CI, 1.21-1.29) and 1.41 (95% CI, 1.31-1.52) for asthma. The aHRs after antibiotic prescription in the first 6 months of life were 2.09 (95% CI, 2.05-2.13) for asthma, 1.75 (95% CI, 1.72-1.78) for allergic rhinitis, 1.51 (95% CI, 1.38-1.66) for anaphylaxis, and 1.42 (95% CI, 1.34-1.50) for allergic conjunctivitis. Conclusions and Relevance: This study found associations between the use of acid-suppressive medications and antibiotics during the first 6 months of infancy and subsequent development of allergic disease. Acid-suppressive medications and antibiotics should be used during infancy only in situations of clear clinical benefit.

A Proteomic Analysis of the Body Wall, Digestive Tract, and Reproductive Tract of Brugia malayi.

Filarial worms are parasitic nematodes that cause devastating diseases such as lymphatic filariasis (LF) and onchocerciasis. Filariae are nematodes with complex anatomy including fully developed digestive tracts and reproductive organs. To better understand the basic biology of filarial parasites and to provide insights into drug targets and vaccine design, we conducted a proteomic analysis of different anatomic fractions of Brugia malayi, a causative agent of LF. Approximately 500 adult female B. malayi worms were dissected, and three anatomical fractions (body wall, digestive tract, and reproductive tract) were obtained. Proteins from each anatomical fraction were extracted, desalted, trypsinized, and analyzed by microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry. In total, we identified 4,785 B. malayi proteins. While 1,894 were identified in all three anatomic fractions, 396 were positively identified only within the digestive tract, 114 only within the body wall, and 1,011 only within the reproductive tract. Gene set enrichment analysis revealed a bias for transporters to be present within the digestive tract, suggesting that the intestine of adult filariae is functional and important for nutrient uptake or waste removal. As expected, the body wall exhibited increased frequencies of cytoskeletal proteins, and the reproductive tract had increased frequencies of proteins involved in nuclear regulation and transcription. In assessing for possible vaccine candidates, we focused on proteins sequestered within the digestive tract, as these could possibly represent "hidden antigens" with low risk of prior allergic sensitization. We identified 106 proteins that are enriched in the digestive tract and are predicted to localize to the surface of cells in the the digestive tract. It is possible that some of these proteins are on the luminal surface and may be accessible by antibodies ingested by the worm. A subset of 27 of these proteins appear especially promising vaccine candidates as they contain significant non-cytoplasmic domains, only 1-2 transmembrane domains, and a high degree of homology to W. bancrofti and/or O. volvulus.

Immunotherapy of tumors with α2-macroglobulin-antigen complexes pre-formed in vivo.

The cell surface receptor CD91/LRP-1 binds to immunogenic heat shock proteins (HSP) and α(2)M ligands to elicit T cell immune responses. In order to generate specific immune responses, the peptides chaperoned by HSPs or α(2)M are cross-presented on MHC molecules to T cells. While the immunogenic HSPs naturally chaperone peptides within cells and can be purified as an intact HSP-peptide complex, the peptides have had to be complexed artificially to α(2)M in previous studies. Here, we show that immunogenic α(2)M-peptide complexes can be isolated from the blood of tumor-bearing mice without further experimental manipulation in vitro demonstrating the natural association of tumor antigens with α(2)M. The naturally formed immunogenic α(2)M-peptide complexes are effective in prophylaxis and therapy of cancer in mouse models. We investigate the mechanisms of cross-presentation of associated peptides and co-stimulation by APCs that interact with α(2)M. These data have implications for vaccine design in immunotherapy of cancer and infectious disease.

Characterization, distribution, and expression of novel genes among eight clinical isolates of Streptococcus pneumoniae.

Eight low-passage-number Streptococcus pneumoniae clinical isolates, each of a different serotype and a different multilocus sequence type, were obtained from pediatric participants in a pneumococcal vaccine trial. Comparative genomic analyses were performed with these strains and two S. pneumoniae reference strains. Individual genomic libraries were constructed for each of the eight clinical isolates, with an average insert size of approximately 1 kb. A total of 73,728 clones were picked for arraying, providing more than four times genomic coverage per strain. A subset of 4,793 clones were sequenced, for which homology searches revealed that 750 (15.6%) of the sequences were unique with respect to the TIGR4 reference genome and 263 (5.5%) clones were unrelated to any available streptococcal sequence. Hypothetical translations of the open reading frames identified within these novel sequences showed homologies to a variety of proteins, including bacterial virulence factors not previously identified in S. pneumoniae. The distribution and expression patterns of 58 of these novel sequences among the eight clinical isolates were analyzed by PCR- and reverse transcriptase PCR-based analyses, respectively. These unique sequences were nonuniformly distributed among the eight isolates, and transcription of these genes in planktonic cultures was detected in 81% (172/212) of their genic occurrences. All 58 novel sequences were transcribed in one or more of the clinical strains, suggesting that they all correspond to functional genes. Sixty-five percent (38/58) of these sequences were found in 50% or less of the clinical strains, indicating a significant degree of genomic plasticity among natural isolates.

Extensive genomic plasticity in Pseudomonas aeruginosa revealed by identification and distribution studies of novel genes among clinical isolates.

The distributed genome hypothesis (DGH) states that each strain within a bacterial species receives a unique distribution of genes from a population-based supragenome that is many times larger than the genome of any given strain. The observations that natural infecting populations are often polyclonal and that most chronic bacterial pathogens have highly developed mechanisms for horizontal gene transfer suggested the DGH and provided the means and the mechanisms to explain how chronic infections persist in the face of a mammalian host's adaptive defense mechanisms. Having previously established the validity of the DGH for obligate pathogens, we wished to evaluate its applicability to an opportunistic bacterial pathogen. This was accomplished by construction and analysis of a highly redundant pooled genomic library containing approximately 216,000 functional clones that was constructed from 12 low-passage clinical isolates of Pseudomonas aeruginosa, 6 otorrheic isolates and 6 from other body sites. Sequence analysis of 3,214 randomly picked clones (mean insert size, approximately 1.4 kb) from this library demonstrated that 348 (10.8%) of the clones were unique with respect to all genomic sequences of the P. aeruginosa prototype strain, PAO1. Hypothetical translations of the open reading frames within these unique sequences demonstrated protein homologies to a number of bacterial virulence factors and other proteins not previously identified in P. aeruginosa. PCR and reverse transcription-PCR-based assays were performed to analyze the distribution and expression patterns of a 70-open reading frame subset of these sequences among 11 of the clinical strains. These sequences were unevenly distributed among the clinical isolates, with nearly half (34/70) of the novel sequences being present in only one or two of the individual strains. Expression profiling revealed that a vast majority of these sequences are expressed, strongly suggesting they encode functional proteins.