Sublingual allergen immunotherapy prevents house dust mite inhalant type 2 immunity through dendritic cell-mediated induction of Foxp3+ regulatory T cells.

Sublingual allergen immunotherapy (SLIT) is an emerging treatment option for allergic asthma and a potential disease-modifying strategy for asthma prevention. The key cellular events leading to such long-term tolerance remain to be fully elucidated. We administered prophylactic SLIT in a mouse model of house dust mite (HDM)-driven allergic asthma. HDM extract was sublingually administered over 3 weeks followed by intratracheal sensitization and intranasal challenges with HDM. Prophylactic SLIT prevented allergic airway inflammation and hyperreactivity with a low lab-to-lab variation. The HDM-specific T helper (Th)2 (cluster of differentiation 4 Th) response was shifted by SLIT toward a regulatory and Th17 response in the lung and mediastinal lymph node. By using Derp1-specific cluster of differentiation 4+ T cells (1-DER), we found that SLIT blocked 1-DER T cell recruitment to the mediastinal lymph node and dampened IL-4 secretion following intratracheal HDM sensitization. Sublingually administered Derp1 protein activated 1-DER T cells in the cervical lymph node via chemokine receptor7+ migratory dendritic cells (DC). DCs migrating from the oral submucosa to the cervical lymph node after SLIT-induced Foxp3+ regulatory T cells. When mice were sensitized with HDM, prior prophylactic SLIT increased Derp1 specific regulatory T cells (Tregs) and lowered Th2 recruitment in the lung. By using Foxp3-diphtheria toxin receptor mice, Tregs were found to contribute to the immunoregulatory prophylactic effect of SLIT on type 2 immunity. These findings in a mouse model suggest that DC-mediated functional Treg induction in oral mucosa draining lymph nodes is one of the driving mechanisms behind the disease-modifying effect of prophylactic SLIT.

Type 2-polarized memory B cells hold allergen-specific IgE memory.

Allergen-specific immunoglobulin E (IgE) antibodies mediate pathology in diseases such as allergic rhinitis and food allergy. Memory B cells (MBCs) contribute to circulating IgE by regenerating IgE-producing plasma cells upon allergen encounter. Here, we report a population of type 2-polarized MBCs defined as CD23hi, IL-4Rαhi, and CD32low at both the transcriptional and surface protein levels. These MBC2s are enriched in IgG1- and IgG4-expressing cells while constitutively expressing germline transcripts for IgE. Allergen-specific B cells from patients with allergic rhinitis and food allergy were enriched in MBC2s. Furthermore, MBC2s generated allergen-specific IgE during sublingual immunotherapy, thereby identifying these cells as a major reservoir for IgE. The identification of MBC2s provides insights into the maintenance of IgE memory, which is detrimental in allergic diseases but could be beneficial in protection against venoms and helminths.

Factors Influencing Patient Satisfaction With Breast Augmentation: A BREAST-Q Effect of Magnitude Analysis.

BACKGROUND: Breast augmentation is one of the most performed cosmetic surgeries. Despite this, patient satisfaction following breast augmentation is poorly understood. OBJECTIVES: The aim of this study was to investigate what patient and surgical factors influence patient satisfaction following primary breast augmentation. METHODS: The BREAST-Q Augmentation module was sent to all females undergoing primary breast augmentation at a single private clinic (Amalieklinikken, Copenhagen, Denmark) between 2012 and 2019. Patient and surgical characteristics at the time of surgery were obtained from the patients' medical records, and data on factors that occurred after the surgery (eg, breastfeeding) were obtained by patient contact. Multivariate linear regression modeled the impact of these factors on BREAST-Q outcomes. RESULTS: A total of 554 females with a mean follow-up time of 5 years after primary breast augmentation were included in this study. Implant type and volume did not affect patient satisfaction. However, higher patient age was associated with significantly higher postoperative patient satisfaction, psychosocial well-being, and sexual well-being (P < .05). Conversely, higher patient BMI, postoperative weight gain, and breastfeeding were associated with significantly lower satisfaction (P < .05). Additionally, subglandular implant placement was associated with significantly lower satisfaction than submuscular implant placement (P < .05). CONCLUSIONS: Implant type and volume did not affect patient satisfaction with breast augmentation. However, young age, higher BMI, subglandular implant placement, and postoperative weight gain were associated with lower patient satisfaction. These factors should be considered when aligning outcome expectations with breast augmentation.

Hematoma and deep surgical site infection following primary breast augmentation: A retrospective review of 1128 patients.

BACKGROUND: Breast augmentation is one of the most frequently performed cosmetic surgery worldwide. Some of the most severe short-term complications after breast augmentation are hematoma and deep surgical site infection. However, these complications are relatively rare; therefore, large patient populations are required to perform statistical analyses. In this study, we provide a detailed analysis of the complications after primary breast augmentation with an emphasis on deep surgical site infection and hematoma. METHOD: We retrospectively reviewed the medical records of women who underwent primary breast augmentation without the use of pocket irrigation between 2012 and 2019 in a single private clinic. A cumulative hazard function and a multivariate analysis on the risk of hematoma were performed. RESULTS: We included 1128 patients in the study. Thirty patients (2.7%) developed postoperative hematoma after a median time of 14 h (IQR 5 h-9 days). Six patients (0.5%) contracted a deep surgical site infection after a median time of 14 days (range 4-41 days). Age, BMI, implant volume, or implant placement was not significantly associated with hematoma. CONCLUSION: Our findings support that the risk of hematoma after primary breast augmentation is highest within the first 24 h after the surgery. This time period should be considered when planning postoperative care for these patients. We did not find an increased rate of deep surgical site infection compared with studies of breast augmentations with pocket irrigation. Further studies and meta-analyses are needed to explore the effect of pocket irrigation and other risk factors.

Breast Augmentation with Microtextured Anatomical Implants in 653 Women: Indications and Risk of Rotation.

BACKGROUND: Anatomical implants provide a wide range of options in terms of implant dimensions for breast augmentation. Nevertheless, many surgeons choose not to use anatomical implants due to the risk of rotation malposition and because their advantages over round implants are not clearly defined. METHODS: A retrospective review of medical records was performed on all women who underwent breast augmentation or implant exchange with microtextured anatomical implants from 2012 to 2019 in a single private clinic. The authors focused on the outcomes of a subgroup of women with glandular ptosis and nipple placement below the inframammary fold who underwent breast augmentation with anatomical implants. Furthermore, the incidence and risk factors for implant rotation were analyzed. RESULTS: In total, 653 women underwent primary breast augmentation (n = 529) or implant exchange (n = 124) with anatomical implants. The median follow-up period was 2.7 years (interquartile range, 1.6 to 3.9 years). The incidence of implant rotation was 14 (2.6 percent) in the primary augmentation group and four (3.2 percent) in the implant exchange group. Implant rotation was not associated with type of surgery (p = 0.76), implant projection (p = 0.23), or implant height (p = 0.48). The authors successfully used anatomical implants to elevate the nipple in 92.9 percent of the women with glandular ptosis without using a mastopexy. CONCLUSIONS: The study results indicate that the rotation risk with microtextured implants is similar to that with macrotextured implants. Furthermore, the authors found that high-projection anatomical implants can be used as an alternative to augmentation-mastopexy in women with glandular ptosis. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Allergen-specific IgG+ memory B cells are temporally linked to IgE memory responses.

BACKGROUND: IgE is the least abundant immunoglobulin and tightly regulated, and IgE-producing B cells are rare. The cellular origin and evolution of IgE responses are poorly understood. OBJECTIVE: The cellular and clonal origin of IgE memory responses following mucosal allergen exposure by sublingual immunotherapy (SLIT) were investigated. METHODS: In a randomized double-blind, placebo-controlled, time course SLIT study, PBMCs and nasal biopsy samples were collected from 40 adults with seasonal allergic rhinitis at baseline and at 4, 8, 16, 28, and 52 weeks. RNA was extracted from PBMCs, sorted B cells, and nasal biopsy samples for heavy chain variable gene repertoire sequencing. Moreover, mAbs were derived from single B-cell transcriptomes. RESULTS: Combining heavy chain variable gene repertoire sequencing and single-cell transcriptomics yielded direct evidence of a parallel boost of 2 clonally and functionally related B-cell subsets of short-lived IgE+ plasmablasts and IgG+ memory B cells. Mucosal grass pollen allergen exposure by SLIT resulted in highly diverse IgE and IgGE repertoires. These were extensively mutated and appeared relatively stable as per heavy chain isotype, somatic hypermutations, and clonal composition. Single IgGE+ memory B-cell and IgE+ preplasmablast transcriptomes encoded antibodies that were specific for major grass pollen allergens and able to elicit basophil activation at very low allergen concentrations. CONCLUSION: For the first time, we have shown that on mucosal allergen exposure, human IgE memory resides in allergen-specific IgG+ memory B cells. These cells rapidly switch isotype, expand into short-lived IgE+ plasmablasts, and serve as a potential target for therapeutic intervention.

Diverse and highly cross-reactive T-cell responses in ragweed allergic patients independent of geographical region.

BACKGROUND: Ragweed frequently causes seasonal allergies in North America and Europe. In the United States, several related ragweed species with diverse geographical distribution cause allergic symptoms. Cross-reactivity towards related ragweed species of IgE and treatment-induced IgG4 has been demonstrated previously. However, less is known about the underlying T-cell cross-reactivity. METHODS: The allergen content of ragweed extracts was determined by mass spectrometry and related to T-cell epitopes of Amb a allergens (group 1, 3, 4, 5, 8 and 11) in 20 American ragweed allergic patients determined by FluoroSpot and proliferation assays. T-cell responses to 50 frequently recognized Amb a-derived T-cell epitopes and homologous peptides from western ragweed (Amb p), giant ragweed (Amb t) and mugwort (Art v) were investigated in an additional 11 American and 14 Slovakian ragweed allergic donors. RESULTS: Ragweed extracts contained all known allergens and isoallergens thereof. Donor T-cell responses were diverse and directed against all Amb a 1 isoallergens and to most minor allergens investigated. Similar response patterns were seen in American and Slovakia donors. Several epitopes were cross-reactive between isoallergens and ragweed species, some even including mugwort. T-cell cross-reactivity generally correlated with allergen sequence homology. CONCLUSION: T-cell epitopes of multiple allergens/isoallergens are involved in the diverse T-cell responses in ragweed allergic individuals. T-cell lines were highly cross-reactive to epitopes of related ragweed species without any apparent geographical response bias. These data support that different ragweed species can be considered an allergen homology group with Amb a as the representative species regarding diagnosis as well as allergy immunotherapy.

Immunoproteomic analysis of house dust mite antigens reveals distinct classes of dominant T cell antigens according to function and serological reactivity.

BACKGROUND: House dust mite (HDM) allergens are a common cause of allergy and allergic asthma. A comprehensive analysis of proteins targeted by T cells, which are implicated in the development and regulation of allergic disease independent of their antibody reactivity, is still lacking. OBJECTIVE: To comprehensively analyse the HDM-derived protein targets of T cell responses in HDM-allergic individuals, and investigate their correlation with IgE/IgG responses and protein function. METHODS: Proteomic analysis (liquid chromatography-tandem mass spectrometry) of HDM extracts identified 90 distinct protein clusters, corresponding to 29 known allergens and 61 novel proteins. Peripheral blood mononuclear cells (PBMC) from 20 HDM-allergic individuals were stimulated with HDM extracts and assayed with a set of ~2500 peptides derived from these 90 protein clusters and predicted to bind the most common HLA class II types. 2D immunoblots were made in parallel to elucidate IgE and IgG reactivity, and putative function analyses were performed in silico according to Gene Ontology annotations. RESULTS: Analysis of T cell reactivity revealed a large number of T cell epitopes. Overall response magnitude and frequency was comparable for known and novel proteins, with 15 antigens (nine of which were novel) dominating the total T cell response. Most of the known allergens that were dominant at the T cell level were also IgE reactive, as expected, while few novel dominant T cell antigens were IgE reactive. Among known allergens, hydrolase activity and detectable IgE/IgG reactivity are strongly correlated, while no protein function correlates with immunogenicity of novel proteins. A total of 106 epitopes accounted for half of the total T cell response, underlining the heterogeneity of T cell responses to HDM allergens. CONCLUSIONS AND CLINICAL RELEVANCE: Herein, we define the T cell targets for both known allergens and novel proteins, which may inform future diagnostics and immunotherapeutics for allergy to HDM.

Grass pollen immunotherapy: where are we now.

During allergen immunotherapy (AIT), the allergic patient is exposed to the disease-inducing antigens (allergens) in order to induce clinical and immunological tolerance and obtain disease modification. Large trials of grass AIT with highly standardized subcutaneous and sublingual tablet vaccines have been conducted to document the clinical effect. Induction of blocking antibodies as well as changes in the balance between T-cell phenotypes, including induction of regulatory T-cell subtypes, have been demonstrated for both treatment types. These observations increase the understanding of the immunological mechanism behind the clinical effect and may make it possible to use the immunological changes as biomarkers of clinical effect. The current review describes the recent mechanistic findings for subcutaneous immunotherapy and sublingual immunotherapy/tablet treatment and discusses how the observed immunological changes translate into a scientific foundation for the observed clinical effects of grass pollen immunotherapy and lead to new treatment strategies for grass AIT.

Novel antibody-antibiotic conjugate eliminates intracellular S. aureus.

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

Novel staphylococcal glycosyltransferases SdgA and SdgB mediate immunogenicity and protection of virulence-associated cell wall proteins.

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.

Rozrolimupab, a mixture of 25 recombinant human monoclonal RhD antibodies, in the treatment of primary immune thrombocytopenia.

Rozrolimupab, a recombinant mixture of 25 fully human RhD-specific monoclonal antibodies, represents a new class of recombinant human antibody mixtures. In a phase 1 or 2 dose escalation study, RhD(+) patients (61 subjects) with primary immune thrombocytopenia received a single intravenous dose of rozrolimupab ranging from 75 to 300 µg/kg. The primary outcome was the occurrence of adverse events. The principal secondary outcome was the effect on platelet levels 7 days after the treatment. The most common adverse events were headache and pyrexia, mostly mild, and reported in 20% and 13% of the patients, respectively, without dose relationship. Rozrolimupab caused an expected transient reduction of hemoglobin concentration in the majority of the patients. At the dose of 300 µg/kg platelet responses, defined as platelet count ≥ 30 × 10(9)/L and an increase in platelet count by > 20 × 10(9)/L from baseline were observed after 72 hours and persisted for at least 7 days in 8 of 13 patients (62%). Platelet responses were observed within 24 hours in 23% of patients and lasted for a median of 14 days. Rozrolimupab was well tolerated and elicited rapid platelet responses in patients with immune thrombocytopenia and may be a useful alternative to plasma-derived products. This trial is registered at www.clinicaltrials.gov as #NCT00718692.

Limits for antibody affinity maturation and repertoire diversification in hypervaccinated humans.

The immune system is known to generate a diverse panel of high-affinity Abs by adaptively improving the recognition of pathogens during ongoing immune responses. In this study, we report the biological limits for Ag-driven affinity maturation and repertoire diversification by analyzing Ab repertoires in two adult volunteers after each of three consecutive booster vaccinations with tetanus toxoid. Maturation of on-rates and off-rates occurred independently, indicating a kinetically controlled affinity maturation process. The third vaccination induced no significant changes in the distribution of somatic mutations and binding rate constants implying that the limits for affinity maturation and repertoire diversification had been reached. These fully matured Ab repertoires remained similar in size, genetically diverse, and dynamic. Somatic mutations and kinetic rate constants showed normal and log-normal distribution profiles, respectively. Mean values can therefore be considered as biological constants defining the observed boundaries. At physiological temperature, affinity maturation peaked at k(on) = 1.6 × 10(4) M(-1) s(-1) and k(off) = 1.7 × 10(-4) s(-1) leading to a maximum mean affinity of K(D) = 1.0 × 10(-9) M. At ambient temperature, the average affinity increased to K(D) = 3.4 × 10(-10) M mainly due to slower off-rates. This experimentally determined set of constants can be used as a benchmark for analysis of the maturation level of human Abs and Ab responses.

Assessing energetic contributions to binding from a disordered region in a protein-protein interaction .

Many functional proteins are at least partially disordered prior to binding. Although the structural transitions upon binding of disordered protein regions can influence the affinity and specificity of protein complexes, their precise energetic contributions to binding are unknown. Here, we use a model protein-protein interaction system in which a locally disordered region has been modified by directed evolution to quantitatively assess the thermodynamic and structural contributions to binding of disorder-to-order transitions. Through X-ray structure determination of the protein binding partners before and after complex formation and isothermal titration calorimetry of the interactions, we observe a correlation between protein ordering and binding affinity for complexes along this affinity maturation pathway. Additionally, we show that discrepancies between observed and calculated heat capacities based on buried surface area changes in the protein complexes can be explained largely by heat capacity changes that would result solely from folding the locally disordered region. Previously developed algorithms for predicting binding energies of protein-protein interactions, however, are unable to correctly model the energetic contributions of the structural transitions in our model system. While this highlights the shortcomings of current computational methods in modeling conformational flexibility, it suggests that the experimental methods used here could provide training sets of molecular interactions for improving these algorithms and further rationalizing molecular recognition in protein-protein interactions.

Kinetic, affinity, and diversity limits of human polyclonal antibody responses against tetanus toxoid.

Due to technical limitations, little knowledge exists on the composition of Ag-specific polyclonal Ab responses. Hence, we here present a molecular analysis of two representative human Ab repertoires isolated by using a novel single-cell cloning approach. The observed genetic diversity among tetanus toxoid-specific plasma cells indicate that human polyclonal repertoires are limited to the order of 100 B cell clones and hypermutated variants thereof. Affinity and kinetic binding constants are log-normally distributed, and median values are close to the proposed affinity ceilings for positive selection. Abs varied a million-fold in affinity but were restricted in their off-rates with an upper limit of 2 x 10(-3) s(-1). Identification of Abs of high affinity without hypermutations in combination with a modest effect of hypermutations on observed affinity increases indicate that Abs selected from the naive repertoire are not only of low affinity but cover a relatively large span in affinity, reaching into the subnanomolar range.

Extensive restrictions in the VH sequence usage of the human antibody response against the Rhesus D antigen.

Anti-Rhesus D immunoglobulin purified from human sera is used as a prophylactic reagent in Rhesus D negative women at risk of alloimmunization during pregnancy. We are currently developing a Rhesus D antigen-specific recombinant polyclonal antibody drug lead for replacing the existing blood derived-products. By analyzing the RhD-specific antibody VH repertoires from eight alloimmunized women we found, in agreement with previous studies, a strong preference for the VH 3-33 "superspecies" gene segments which encompasses the IGHV3-30-3*01, IGHV3-30*18, and IGHV3-33*01 VH alleles. Even more extensive genetic restriction was observed among five donors, which produced antibodies of identical V-D-J usage and CDR3 loop length and joining regions of similar amino acid composition. In addition, we find a high degree of sequence relatedness to previously isolated anti-Rhesus D antibodies. Such close homology in VH domains indicates that significant structural restrictions are operating in the selection of antibodies recognizing RhD as seen for T cell receptors. Moreover, some VH domains were isolated in their germline configuration indicating that anti-RhD antibodies of relatively high affinity are present in the naïve antibody repertoire of Rhesus negative individuals which offers an explanation for the strong and clinically significant immunogenicity of the Rhesus D.

Reduced susceptibility of recombinant polyclonal antibodies to inhibitory anti-variable domain antibody responses.

The immunogenicity of therapeutic Abs is a concern as anti-drug Abs may impact negatively on the pharmacodynamics and safety profile of Ab drugs. The factors governing induction of anti-drug Abs are not fully understood. In this study, we describe a model based on mouse-human chimeric Abs for the study of Ab immunogenicity in vivo. Six chimeric Abs containing human V regions and mouse C regions were generated from six human anti-Rhesus D Abs and the Ag-binding characteristics of the parental human Abs were retained. Analysis of the immune response toward the individual chimeric Abs revealed the induction of anti-variable domain Abs including anti-idiotypic Abs against some of these, thereby demonstrating the applicability of the model for studying anti-drug Ab responses in vivo. Immunization of BALB/c, C57, and outbred NMRI mice with a polyclonal composition consisting of all six chimeric Abs demonstrated that the immunogenicity of the individual Abs was haplotype dependent. Chimeric Abs, which were nonimmunogenic when administered individually, did not become immunogenic as part of the polyclonal composition, implying the absence of epitope spreading. Ex vivo Ab-binding studies established a clear correlation between the level of immunogenicity of the Abs comprised in the composition and the impact on the pharmacology of the Abs. These analyses demonstrate that under these conditions this polyclonal Ab composition was generally less susceptible to blocking Abs than the respective mAbs.