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Contains fulltext : 290702.pdf (Publisher’s version ) (Open Access) Innate immune cells experience long lasting metabolic and epigenetic changes after an encounter with specific stimuli. This facilitates enhanced immune responses upon secondary exposition to both the same and unrelated pathogens, a process termed trained immunity. Trained immunity-based vaccines (TIbV) are vaccines able to induce innate immune memory, thus conferring heterologous protection against a broad range of pathogens. While trained immunity has been well documented in the context of infections and multiple immune-mediated diseases, the role of innate immune memory and its contribution to the initiation and maintenance of chronic allergic diseases remains poorly understood. Over the last years, different studies attempting to uncover the role of trained immunity in allergy have emerged. Exposition to environmental factors impacting allergy development such as allergens or viruses induces the reprogramming of innate immune cells to acquire a more pro-inflammatory phenotype in the context of asthma or food allergy. Several studies have convincingly demonstrated that prevention of viral infections using TIbV contributes to reduce wheezing attacks in children, which represent a high-risk factor for asthma development later in life. Innate immune cells trained with specific stimuli might also acquire anti-inflammatory features and promote tolerance, which may have important implications for chronic inflammatory diseases such as allergies. Recent findings showed that allergoid-mannan conjugates, which are next generation vaccines for allergen-specific immunotherapy (AIT), are able to reprogram monocytes into tolerogenic dendritic cells by mechanisms depending on metabolic and epigenetic rewiring. A better understanding of the underlying mechanisms of trained immunity in allergy will pave the way for the design of novel trained immunity-based allergen vaccines as potential alternative strategies for the prevention and treatment of allergic diseases. 01 februari 2023

To discuss recently discovered mechanisms of action of some bacterial vaccines that may account for their clinical benefit in the prevention of recurrent wheezing and asthma exacerbations in infants and early childhood.Trained immunity has been shown to confer innate immune cells with a quite long-term nonspecific protection against a broad spectrum of pathogens. Inducers of trained immunity include some bacterial vaccines. Trained immunity-based vaccines (TIbV) of bacterial origin have the capability to induce nonspecific responses to a variety of pathogens, including respiratory viruses, in addition to their nominal bacterial antigens. Clinical data, from epidemiological surveys to well designed randomized clinical trials, indicate that TIbV formulated with bacteria prevent respiratory tract infections of viral cause, such as those associated with recurrent wheezing or asthma exacerbation, in children. Administration of these vaccines by the mucosal route may be important for their outcome in respiratory infections.Mucosal bacterial immunotherapy, including certain TIbV, confer protection against a broad spectrum of pathogens, such as viruses, through a mechanism mediated by trained immunity. Clinical studies on the use of these preparations against recurrent wheezing reflect these mechanistic effects. These findings open a new avenue for the development of new strategies for this condition.

The most important peach fruit allergen is Pru p 3, followed by Pru p 1, Pru p 4, and Pru p 7. We aimed to assess their role in subjects with peach fruit-induced allergy (anaphylaxis and OAS) and compare skin prick tests (SPT) vs. specific immunoglobulin E (sIgE) for predicting anaphylaxis. We also selected a control group. SPT included prevalent inhalant and plant food allergens plus peach peel extract. The sIgE to Pru p 1, Pru p 3, Pru p 4, and Pru p 7 were quantified. Compared with controls (n = 42), cases (n = 41) were younger (P = 0.003), more frequently female (P < 0.05) and had higher SPT positivity to peach peel (44% vs. 2.4%, P < 0.0001). There were significant differences in sensitization to several pollens: Olea europaea, Artemisia vulgaris, Prunus persica, Platanus acerifolia (all P < 0.001); and fruits: apple (P < 0.04), peanut (P < 0.002), tomato (P < 0.005), and melon (P < 0.05). Pru p 3 sIgE was detected in 61% of all cases (85% anaphylaxis and 38% OAS; P < 0.01 each) and 5% of controls (P < 0.001). Pru p 4 sIgE was present in 19% of cases and 7% of controls. The sIgE to Pru p 1 and Pru p 7 were not found. The odds ratio to predict anaphylaxis for peach peel SPT was 113 (confidence interval [CI], 20-613; P < 0.0001); for sIgE to Pru p 3, 22 (CI, 5.3-93; P < 0.0001); and for SPT positivity to selected plant food allergens, 5 (CI, 1-19; P < 0.05). In our study group, SPT with peel peach extract was a better predictor of anaphylaxis than Pru p 3 sIgE or other variables considered. The role of sIgE to Pru p 1, Pru p 4, and Pru p 7 seemed negligible.

The authors thank the patients and their families for participating in this study. They are grateful to the members of the David Sancho laboratory, especially to Carlos del Fresno, for helpful critical discussion. Rationale: Recurrent wheezing in children represents a severe public health concern. Wheezing attacks (WA), mainly associated with viral infections, lack effective preventive therapies. Objectives: To evaluate the efficacy and safety of mucosal sublingual immunotherapy based on whole inactivated bacteria (MV130) in preventing WA in children. Methods: A Phase 3 randomized, double-blind, placebo-controlled, parallel-group trial including a cohort of 120 children

The aim of this study was to compare airborne levels of Phl p 1 and Phl p 5, with Poaceae pollen concentrations inside and outside of the pollen season, and to evaluate their association with symptoms in grass allergic patients and the influence of climate and pollution. The Hirst and the Burkard Cyclone samplers were used for pollen and allergen quantification, respectively. The sampling period ran from 23 March 2009 to 27 July 2010. Twenty-three patients with seasonal allergic asthma and rhinitis used an electronic symptom card. The aerosol was extracted and quantified for Phl p 1 and Phl p 5 content. Descriptive statistics, non-parametric paired contrast of Wilcoxon, Spearman's correlations, and a categorical principal component analysis (CatPCA) were carried out. Significant variations in pollen, aeroallergen levels, pollen allergen potency, and symptoms score were observed in this study. Phl p 5 pollen allergen potency was higher at the beginning of the 2010 grass pollen season. Presence of Phl p 1 outside the pollen season with positive O3 correlation was clinically relevant. 45.5% of the variance was explained by two dimensions in the CatPCA analysis, showing the symptom relationships dissociated in two dimensions. In the first one, the more important relationship was with grass pollen grains concentration and Phl p 5 and to a lesser extent with Phl p 1 and levels of NO2 and O3, and in the second dimension, symptoms were associated with humidity and SO2. Clinically relevant out-season Phl p 1 was found with a positive O3 correlation. The effect of climate and pollution may have contributed to the higher seasonal allergic rhinitis symptom score recorded in 2009.

Background Allergoid–mannan conjugates are novel vaccines for allergen-specific immunotherapy being currently assayed in phase 2 clinical trials. Allergoid–mannan conjugates target dendritic cells (DCs) and generate functional forkhead box P3 (FOXP3)-positive Treg cells, but their capacity to reprogram monocyte differentiation remains unknown. Objective We studied whether allergoid–mannan conjugates could reprogram monocyte differentiation into tolerogenic DCs and the underlying molecular mechanisms. Methods Monocytes from nonatopic and allergic subjects were differentiated into DCs under conventional protocols in the absence or presence of allergoid–mannan conjugates. ELISA, real-time quantitative PCR, coculture, flow cytometry, and suppression assay were performed. Metabolic and epigenetic techniques were also used. Results Monocyte differentiation from nonatopic and allergic subjects into DCs in the presence of allergoid–mannan conjugates yields stable tolerogenic DCs. Lipopolysaccharide-stimulated mannan-tolDCs show a significantly lower cytokine production, lower TNF-α/IL-10 ratio, and higher expression of the tolerogenic molecules PDL1, IDO, SOCS1, SOCS3, and IL10; and they induce higher numbers of functional FOXP3+ Treg cells than conventional DC counterparts. Mannan-tolDCs shift glucose metabolism from Warburg effect and lactate production to mitochondrial oxidative phosphorylation. They also display epigenetic reprogramming involving specific histone marks within tolerogenic loci and lower expression levels of histone deacetylase genes. Mannan-tolDCs significantly increase the expression of the anti-inflammatory miRNA-146a/b and decrease proinflammatory miRNA-155. Conclusions Allergoid–mannan conjugates reprogram monocyte differentiation into stable tolerogenic DCs via epigenetic and metabolic reprogramming. Our findings shed light on the novel mechanisms by which allergoid–mannan conjugates might contribute to allergen tolerance induction during allergen-specific immunotherapy.

BACKGROUND: Polymerized allergens conjugated to non-oxidized mannan (PM-allergoids) are novel vaccines targeting dendritic cells (DCs). Previous experimental data indicate that PM-allergoids are readily taken up by DCs and induce Treg cells. This first-in-human study was aimed to evaluate safety and to find the optimal dose of house dust mite PM-allergoid (PM-HDM) administered subcutaneously (SC) or sublingually (SL). METHODS: In a randomized, double-blind, double-dummy, placebo-controlled trial, 196 subjects received placebo or PM-HDM at 500, 1000, 3000, or 5000 mannan-conjugated therapeutic units (mTU)/mL in 9-arm groups for 4 months. All subjects received 5 SC doses (0.5 ml each) every 30 days plus 0.2 ml SL daily. The primary efficacy outcome was the improvement of titrated nasal provocation tests (NPT) with D. pteronyssinus at baseline and at the end of the study. All adverse events and reactions were recorded and assessed. Secondary outcomes were the combination of symptom and medication scores (CSMS) and serological markers. RESULTS: No moderate or severe adverse reactions were reported. Subjects improving the NPT after treatment ranged from 45% to 62% in active SC, 44% to 61% in active SL and 16% in placebo groups. Statistical differences between placebo and active groups were all significant above 500 mTU, being the highest with 3000 mTU SL (p = 0.004) and 5000 mTU SC (p = 0.011). CSMS improvement over placebo reached 70% (p < 0.001) in active 3000 mTU SC and 40% (p = 0.015) in 5000 mTU SL groups. CONCLUSIONS: PM-HDM immunotherapy was safe and successful in achieving primary and secondary clinical outcomes in SC and SL at either 3000 or 5000 mTU/ml.

Summary MV130 is an inactivated polybacterial mucosal vaccine that confers protection to patients against recurrent respiratory infections, including those of viral etiology. However, its mechanism of action remains poorly understood. Here, we find that intranasal prophylaxis with MV130 modulates the lung immune landscape and provides long-term heterologous protection against viral respiratory infections in mice. Intranasal administration of MV130 provides protection against systemic candidiasis in wild-type and Rag1-deficient mice lacking functional lymphocytes, indicative of innate immune-mediated protection. Moreover, pharmacological inhibition of trained immunity with metformin abrogates the protection conferred by MV130 against influenza A virus respiratory infection. MV130 induces reprogramming of both mouse bone marrow progenitor cells and in vitro human monocytes, promoting an enhanced cytokine production that relies on a metabolic shift. Our results unveil that the mucosal administration of a fully inactivated bacterial vaccine provides protection against viral infections by a mechanism associated with the induction of trained immunity., Graphical abstract, Highlights • MV130 reduces morbi-mortality in mouse models of viral respiratory infection • MV130 modulates mouse immunity complying with key features of trained immunity • MV130 promotes reprogramming of mouse bone marrow progenitors • MV130 induces trained immunity in human monocytes, Brandi et al. investigate the mechanism of action of an inactivated polybacterial mucosal immunomodulator, MV130, which confers protection in patients suffering from recurrent respiratory infections. They show that MV130 induces trained immunity both in mouse bone marrow progenitors, resulting in protection from viral infection, and in human monocytes in vitro.

ObjectivesSeveral studies have described peach tree (PT) as an occupational allergen. The aim of this work was to assess the effect ofPrunus persica9 (Pru p 9), a recently identified allergen from PT pollen, in exposed workers.MethodsThe study included people who reported respiratory symptoms after handling PT in orchards during the flowering period (Blanca village, Murcia region, south-east Spain). After completing a detailed questionnaire, participants underwent skin prick test (SPT) and nasal provocation test (NPT). The IgE response was analysed by SDS-PAGE and immunoblotting assays.ResultsA total of 21 cases were included (mean age 45 years; 57% women). Most were polysensitised to common pollens, although one person was sensitised only to PT pollen. All cases had a positive SPT to this pollen, and 43% also to Pru p 9. All participants reported having rhinitis, and six participants reported having also asthma. Immunoblotting showed a heterogeneous IgE pattern for several proteins, with Pru p 9 recognised in nine cases. Most participants sensitised to PT pollen and Pru p 9 had positive NPTs, while those who were not sensitised to Pru p 9 tested negative.ConclusionsWe demonstrate for the first time that Pru p 9, an allergen from PT pollen, can induce respiratory symptoms following occupational exposure. This must be considered a relevant allergen when people working with PT cultivars develop respiratory symptoms.

Background Polymerized allergoids conjugated to mannan (PM) are suitable vaccines for allergen‐specific immunotherapy (AIT). Alum remains the most widely used adjuvant in AIT, but its way of action is not completely elucidated. The better understanding of the mechanisms underlying alum adjuvanticity could help to improve AIT vaccine formulations. Objective We sought to investigate the potential influence of alum in the tolerogenic properties imprinted by PM at the molecular level. Methods Flow cytometry, ELISAs, cocultures, intracellular staining and suppression assays were performed to assess alum and PM effects in human dendritic cells (DCs). BALB/c mice were immunized with PM alone or adsorbed to alum. Allergen‐specific antibodies, splenocyte cytokine production and splenic forkhead box P3 (FOXP3)+ regulatory T (Treg) cells were quantified. Metabolic and immune pathways were also studied in human DCs. Results Alum decreases PD‐L1 expression and IL‐10 production induced by PM in human DCs and increases pro‐inflammatory cytokine production. Alum impairs PM‐induced functional FOXP3+ Treg cells and promotes Th1/Th2/Th17 responses. Subcutaneous immunization of mice with PM plus alum inhibits in vivo induction of Treg cells promoted by PM without altering the capacity to induce functional allergen‐specific blocking antibodies. Alum inhibits mTOR activation and alters metabolic reprogramming by shifting glycolytic pathways and inhibiting reactive oxygen species (ROS) production in PM‐activated DCs, impairing their capacity to generate functional Treg cells. Conclusion We uncover novel mechanisms by which alum impairs the tolerogenic properties induced by PM, which might well contribute to improve the formulation of novel vaccines for AIT., This study investigated the potential influence of alum in the tolerogenic properties imprinted by PM. Alum decreased expression of PD‐L1 and production of IL‐10, IL‐6 and lactate, increased IL‐23 release and inhibited mTOR activation in PM‐activated DCs. Alum suppressed PM‐induced functional FOXP3+ Treg cells and promoted Th1/Th2/Th17 responses.

Viral outbreaks have become significant threats to global human public health. New emerging viruses, pathogen mutations, and even the progressive loss of efficacy in some existing vaccines are behind this problem, which is amplified by the rapid virus spread given the ease of current mobility. Taking into account that these outbreaks arise in the absence of conventional effective vaccines, alternative approaches based on trained (innate) immunity are being considered. This immunity is dependent on a functional reprogramming of innate immune cells, leading to an enhanced nonspecific response towards different pathogens, including viruses. Trained immunity-based vaccines (TIbVs), defined as vaccine formulations containing trained immunity inducers, could be used during viral outbreaks to confer non-specific protection but also to enhance adaptive specific immune responses. In this chapter, we aim to illustrate how TIbVs could tackle the above-mentioned situations derived from viral outbreaks, reviewing the potential of available TIbVs in such urgent situations with a special mention to COVID-19.

Peach tree allergens are present in fruit, pollen, branches, and leaves, and can induce systemic, respiratory, cutaneous, and gastrointestinal symptoms. We studied the capacity of peach fruit/Pru p 1, Pru p 3, Pru p 4, Pru p 7 and peach pollen/Pru p 9 for inducing symptoms following oral or respiratory exposure in a large group of subjects. We included 716 adults (aged 21 to 83 y.o.) exposed to peach tree pollen and fruit intake in the study population. Participants completed a questionnaire and were skin tested with a panel of inhalant and food allergens, including peach tree pollen, Pru p 9 and peach fruit skin extract. Immunoglobulin E antibodies (SIgE) to Pru p 1, Pru p 3, Pru p 4 and Pru p 7 were quantified. Sensitised subjects underwent oral food challenge with peach fruit and nasal provocation test with peach tree pollen and Pru p 9. The prevalence of sensitisation to peach fruit was 5% and most of these had SIgE to Pru p 3, with a very low proportion to Pru p 4 SIgE and no SIgE to Pru p 1 and Pru p 7. In only 1.8%, anaphylaxis was the clinical entity induced. Cases with positive skin tests to peach and SIgE to Pru p 3 presented a good tolerance after oral challenge with peach fruit. The prevalence of skin sensitisation to peach tree pollen was 22%, with almost half recognising Pru p 9. This induced respiratory symptoms in those evaluated by nasal provocation. In a large population group exposed to peach fruit and peach tree pollen, most individuals were tolerant, even in those with SIgE to Pru p 3. A positive response to Pru p 9 was associated with respiratory allergy.

Recent clinical observations indicate that bacterial vaccines induce cross-protection against infections produced by different microorganisms. MV130, a polyvalent bacterial sublingual preparation designed to prevent recurrent respiratory infectious diseases, reduces the infection rate in patients with recurrent respiratory tract infections. On the other hand, mesenchymal stem cells (MSCs) are key cell components that contribute to the maintenance of tissue homeostasis and exert both immunostimulatory and immunosuppressive functions. Herein, we study the effects of MV130 in human MSC functionality as a potential mechanism that contributes to its clinical benefits. We provide evidence that during MV130 sublingual immunization of mice, resident oral mucosa MSCs can take up MV130 components and their numbers remain unchanged after vaccination, in contrast to granulocytes that are recruited from extramucosal tissues. MSCs treated in vitro with MV130 show an increased viability without affecting their differentiation potential. In the short-term, MSC treatment with MV130 induces higher leukocyte recruitment and T cell expansion. In contrast, once T-cell activation is initiated, MV130 stimulation induces an up-regulated expression of immunosuppressor factors in MSCs. Accordingly, MV130-primed MSCs reduce T lymphocyte proliferation, induce the differentiation of dendritic cells with immunosuppressive features and favor M2-like macrophage polarization, thus counterbalancing the immune response. In addition, MSCs trained with MV130 undergo functional changes, enhancing their immunomodulatory response to a secondary stimulus. Finally, we show that MSCs are able to uptake, process and retain a reservoir of the TLR ligands derived from MV130 digestion which can be subsequently transferred to dendritic cells, an additional feature that also may be associated to trained immunity.

Recurrent urinary tract infections (RUTIs) and recurrent vulvovaginal candidiasis (RVVCs) represent major healthcare problems with high socio-economic impact worldwide. Antibiotic and antifungal prophylaxis remain the gold standard treatments for RUTIs and RVVCs, contributing to the massive rise of antimicrobial resistance, microbiota alterations and co-infections. Therefore, the development of novel vaccine strategies for these infections are sorely needed. The sublingual heat-inactivated polyvalent bacterial vaccine MV140 shows clinical efficacy for the prevention of RUTIs and promotes Th1/Th17 and IL-10 immune responses. V132 is a sublingual preparation of heat-inactivated Candida albicans developed against RVVCs. A vaccine formulation combining both MV140 and V132 might well represent a suitable approach for concomitant genitourinary tract infections (GUTIs), but detailed mechanistic preclinical studies are still needed. Herein, we showed that the combination of MV140 and V132 imprints human dendritic cells (DCs) with the capacity to polarize potent IFN-γ– and IL-17A–producing T cells and FOXP3+ regulatory T (Treg) cells. MV140/V132 activates mitogen-activated protein kinases (MAPK)-, nuclear factor-κB (NF-κB)- and mammalian target of rapamycin (mTOR)-mediated signaling pathways in human DCs. MV140/V132 also promotes metabolic and epigenetic reprogramming in human DCs, which are key molecular mechanisms involved in the induction of innate trained immunity. Splenocytes from mice sublingually immunized with MV140/V132 display enhanced proliferative responses of CD4+ T cells not only upon in vitro stimulation with the related antigens contained in the vaccine formulation but also upon stimulation with phytohaemagglutinin. Additionally, in vivo sublingual immunization with MV140/V132 induces the generation of IgG and IgA antibodies against all the components contained in the vaccine formulation. We uncover immunological mechanisms underlying the potential mode of action of a combination of MV140 and V132 as a novel promising trained immunity-based vaccine (TIbV) for GUTIs.

BACKGROUND Aedes aegypti and Dermatophagoides pteronyssinus contain important allergens including cross-reactive tropomyosins. However, the functional and clinical relevance of their cross-reactivity is still debated. OBJECTIVE To analyse the humoral and cellular cross-reactivity of recombinant Aed a 10.01, Aed a 10.02 and Der p 10. METHODS Sera from 15 Austrian house dust mite-allergic, Der p 10-sensitized individuals were tested for IgE reactivity to recombinant tropomyosins in ELISA, inhibition ELISA and basophil activation tests. BALB/c mice were immunized with Aed a 10.01 or Aed a 10.02, and their sera were assessed for reactivity to all tropomyosins. Splenocytes were stimulated with all tropomyosins and synthetic peptides representing the amino acid sequence of Aed a 10.01. RESULTS IgE antibodies of Der p 10-sensitized patients cross-reacted with both tropomyosins from A. aegypti. Aed a 10.01 was a more potent inhibitor of IgE binding to Der p 10 and a stronger activator of basophils sensitized with Der p 10-specific IgE than Aed a 10.02. Murine antibodies raised against Aed a 10.01 and Aed a 10.02 cross-reacted with Der p 10. Aed a 10.01-specific antibody showed stronger cross-reactivity with Der p 10 than Aed a 10.02-specific antibody. Splenocytes from both groups of mice proliferated similarly to all tropomyosins. Five cross-reactive T cell-activating regions were identified. CONCLUSION AND CLINICAL RELEVANCE Tropomyosins from D. pteronyssinus and A. aegypti show humoral and cellular cross-reactivity, involving 5 potential T cell-activating regions. The more pronounced cross-reactivity of Aed a 10.01 and Der p 10 matched the higher sequence similarity of both proteins.

Allergen-specific immunotherapy (AIT) is the single disease-modifying treatment for allergy. Clinical trials show AIT to be safe and effective for many patients; however, it still faces problems related to efficacy, safety, long treatment duration and low patient adherence. There has been intensive research to develop alternative strategies, including novel administration routes, adjuvants or hypoallergenic molecules. Promising results are reported for some of them, but clinical progress is still moderate. Allergoids conjugated to nonoxidized mannan from Saccharomyces cerevisiae have emerged as a novel concept of vaccine targeting dendritic cells (DCs). Preclinical human and animal models demonstrated that allergoids conjugated to mannan enhance allergen uptake, promote healthy responses to allergens by inducing Th1 and T regulatory (Treg) cells, and show clinical efficacy in veterinary medicine. Dose-finding phase II clinical trials in humans are currently ongoing. We review the current stage of allergoids conjugated to mannan as next generation vaccines for AIT.

Recurrent respiratory tract infections (RRTIs) are the first leading cause of community-and nosocomial-acquired infections. Antibiotics remain the mainstay of treatment, enhancing the potential to develop antibiotic resistances. Therefore, the development of new alternative approaches to prevent and treat RRTIs is highly demanded. Daily sublingual administration of the whole heat-inactivated polybacterial preparation (PBP) MV130 significantly reduced the rate of respiratory infections in RRTIs patients, however, the immunological mechanisms of action remain unknown. Herein, we study the capacity of MV130 to immunomodulate the function of human dendritic cells (DCs) as a potential mechanism that contribute to the clinical benefits. We demonstrate that DCs from RRTIs patients and healthy controls display similar ex vivo immunological responses to MV130. By combining systems biology and functional immunological approaches we show that MV130 promotes the generation of Th1/Th17 responses via receptor-interacting serine/threonine-protein kinase-2 (RIPK2)-and myeloid-differentiation primary-response gene88 (MyD88)-mediated signalling pathways under the control of IL-10. In vivo BALB/c mice sublingually immunized with MV130 display potent systemic Th1/Th17 and IL-10 responses against related and unrelated antigens. We elucidate immunological mechanisms underlying the potential way of action of MV130, which might help to design alternative treatments in other clinical conditions with high risk of recurrent infections. This work was supported by grant IPT-2012-0639-090000 from INNPACTO and MINECO, Spain to Inmunotek S.L. The authors' laboratories are supported by grants SAF2014-52706-R to O.P. from MINECO, Spain, and SAF2016-79040R to D.S. from MINECO and European Fund for Regional Development, Spain. O.P. is a Ramon y Cajal Scholar funded by MINECO and the European Social Fund. L.C. is a recipient of a European Respiratory Society Fellowship (RESPIRE2-2013-3708). We thank to Juan Lopez-Relano and Sarai Martinez-Cano for excellent technical assistance with mice experiments. Sí

We have recently reported that grass pollen allergoids conjugated with nonoxidized mannan of Saccharomyces cerevisae using glutaraldehyde results in a novel hypoallergenic mannan-allergen complex with improved properties for allergen vaccination. Using this approach, human dendritic cells show a better allergen uptake and cytokine profile production (higher IL-10/IL-4 ratio) for therapeutic purposes. Here we aim to address whether a similar approach can be extended to dogs using canine dendritic cells. Six healthy Spanish Greyhound dogs were used as blood donors to obtain canine dendritic cells (DC) derived from peripheral blood monocytes. Allergens from Dermatophagoides farinae mite were polymerized and conjugated with nonoxidized mannan. Nuclear magnetic resonance (NMR), gel electrophoresis (SDS-PAGE), immunoblotting and IgE-ELISA inhibition studies were conducted to evaluate the main characteristics of the allergoid obtained. Mannan-allergen conjugate and controls were assayed in vitro for canine DC uptake and production of IL-4 and IL-10. The results indicate that the conjugation of D. farinae allergens with nonoxidized mannan was feasible using glutaraldehyde. The resulting product was a polymerized structure showing a high molecular weight as detected by NMR and SDS-PAGE analysis. The mannan-allergen conjugate was hypoallergenic with a reduced reactivity with specific dog IgE. An increase in both allergen uptake and IL-10/IL-4 ratio was obtained when canine DCs were incubated with the mannan-allergen conjugate, as compared with the control allergen preparations (unmodified D. farinae allergens and oxidized mannan-allergen conjugate). We conclude that hypoallergenic D. farinae allergens coupled to nonoxidized mannan is a novel allergen preparation suitable for canine allergy immunotherapy targeting dendritic cells.

Recurrent urinary tract infections (RUTIs) are one of the most common bacterial infectious diseases, especially in women. Antibiotics remain the mainstay of treatment, but their overuse is associated with antibiotic-resistant infections and deleterious effects in the microbiota. Therefore, alternative approaches are fully demanded. Sublingual immunization with MV140 (Uromune), a polyvalent bacterial preparation (PBP) of whole heat-inactivated bacteria, demonstrated clinical efficacy for the treatment of RUTIs, but the involved immunological mechanisms remain unknown. Herein, we demonstrated that MV140 endorses human dendritic cells (DCs) with the capacity to generate Th1/Th17 and IL-10-producing T cells by mechanisms depending on spleen tyrosine kinase (Syk)- and myeloid differentiation primary response gene 88 (MyD88)-mediated pathways. MV140-induced activation of nuclear factor κB (NF-κB) and p38 in human DCs is essential for the generated Th1/Th17 and IL-10 immune responses whereas c-Jun N-terminal Kinase (JNK) and extracellular-signal regulated kinase (ERK) contribute to Th1 and IL-10 responses, respectively. Sublingual immunization of BALB/c mice with MV140 also induces potent systemic Th1/Th17 and IL-10 responses in vivo. We uncover immunological mechanisms underlying the way of action of MV140, which might well also contribute to understand the rational use of specific PBPs in other clinical conditions with potential high risk of recurrent infections.

The oral mucosa is a first line of defense against pathogenic organisms and yet tolerates food antigens and resident bacteria. Mucosal epithelial cells are emerging as important regulators of innate and adaptive immune responses. However, the contribution of oral epithelial cells (OECs) determining oral immunity is understudied. Here, we evaluated the ability of H413 and TR146 cells, two OEC lines derived from human oral squamous cell carcinomas, and primary OECs to modulate immune responses to a cocktail of Gram+ and Gram- bacteria known as MV130. OECs expressed CD40 constitutively and class II major histocompatibility complex (MHC II) molecules when stimulated with IFNγ, but not CD80 or CD86. Dendritic cells (DCs) treated with bacteria in co-culture with OECs did not fully mature, as judged by the expression of MHC II, CD80 and CD86, and barely released IL-12 and TNFα, compared to control DCs. Furthermore, in the presence of OECs, DCs were unable to stimulate allogenic naive CD4 T cells to produce IFNγ and TNFα. Similarly, OECs in culture with total CD4 T cells or Th1 cells stimulated with anti-CD3 and anti-CD28 antibodies abrogated CD25 and CD69 expression, T cell proliferation and the release of IFNγ and TNFα. The inhibition on T cell activation by OECs was cell-contact dependent, TGFβ independent and largely irreversible. Overall, this behavior of OECs is likely key to avoid immune system over-reaction against resident bacteria.

Background: The mosquito Aedes aegypti is a potential source of important clinically relevant allergens. However, the allergenicity and cross-reactivity of most of these has not been fully described. Methods: Natural wild-type mosquito tropomyosin was purified by size exclusion and anionic-exchange chromatography from an A. aegypti extract. Further characterization was accomplished by MALDI-TOF/TOF. Two recombinant variants of tropomyosin were obtained by expression in Escherichia coli. Specific IgE measurement by ELISA and skin tests for mosquito extract were performed in 12 patients with asthma or allergy rhinitis residing on the Caribbean island of Martinique. Cross-reactivity between natural A. aegypti tropomyosin and recombinant tropomyosins from A. aegypti, house dust mite, shrimp and Ascaris lumbricoides was analyzed by ELISA competition. Results: Four variants of natural tropomyosin were purified. A band of 32 kDa in SDS-PAGE representing 2 tropomyosin variants (Aed a 10.0101 and Aed a 10.0201) reacted with specific IgE of 4 of the 12 (33%) allergic patients and with rabbit polyclonal anti-shrimp tropomyosin. A high degree of cross-reactivity (60-70%) was detected between natural mosquito tropomyosin and Blo t 10, Der p 10 and Lit v 1, and a lower degree with Asc l 3 from A. lumbricoides (A. aegypti tropomyosin; however, rAed a 10.0201 showed a low inhibitory capacity. Conclusion: Tropomyosin is a new IgE-binding protein from A. aegypti. Two of the 4 variants identified showed different degree of cross-reactivity with tropomyosins from other arthropods. The potential allergenic role of each variant should be further investigated.

9 p.-8 fig., Immunotherapy for treating IgE-mediated allergies requires high doses of the corresponding allergen. This may result in undesired side effects and, to avoid them, hypoallergenic allergens (allergoids) polymerized with glutaraldehyde are commonly used. Targeting allergoids to dendritic cells to enhance cell uptake may result in a more effective immunotherapy.Allergoids coupled to yeast mannan, as source of polymannoses, would be suitable for this purpose, since mannose-binding receptors are expressed on these cells.Conventional conjugation procedures of mannan to proteins use oxidized mannan to release reactive aldehydes able to bind to free amino groups in the protein; yet, allergoids lack these latter because their previous treatment with glutaraldehyde.The aim of this study was to obtain allergoids conjugated to mannan by an alternative approach based on just glutaraldehyde treatment, taking advantage of the mannoprotein bound to the polymannose backbone. Allergoid-mannan glycoconjugates were produced in a single step by treating with glutaraldehyde a defined mixture of allergens derived from Phleum pratense grass pollen and native mannan (nonoxidized) from Saccharomyces cerevisae. Analytical and structural studies, including 2D-DOSY and 1H-13C HSQC nuclear magnetic resonance spectra, demonstrated the feasibility of such an approach. The glycoconjugates obtained were polymers of high molecular weight showing a higher stability than the native allergen or the conventional allergoid without mannan. The allergoid-mannan glycoconjugates were hypoallergenic as detected by the IgE reactivity with sera from grass allergic patients, even with lower reactivity than conventional allergoid without mannan. Thus, stable hypoallergenic allergoids conjugated to mannan suitable for using in immunotherapy can be achieved using glutaraldehyde. In contrast to mannan oxidation, the glutaraldehyde approach allows to preserve mannoses with their native geometry, which may be functionally important for its receptor-mediated recognition., This work was supported by grant IDI-20110410 from the Centre for the Development of Industrial Technology (CDTIMINECO, Spain)

Background Polymerized allergoids coupled to nonoxidized mannan (PM‐allergoids) may represent novel vaccines targeting dendritic cells (DCs). PM‐allergoids are better captured by DCs than native allergens and favor Th1/Treg cell responses upon subcutaneous injection. Herein we have studied in mice the in vivo immunogenicity of PM‐allergoids administered sublingually in comparison with native allergens. Methods Three immunization protocols (4‐8 weeks long) were used in Balb/c mice. Serum antibody levels were tested by ELISA. Cell responses (proliferation, cytokines, and Tregs) were assayed by flow cytometry in spleen and lymph nodes (LNs). Allergen uptake was measured by flow cytometry in myeloid sublingual cells. Results A quick antibody response and higher IgG2a/IgE ratio were observed with PM‐allergoids. Moreover, stronger specific proliferative responses were seen in both submandibular LNs and spleen cells assayed in vitro. This was accompanied by a higher IFNγ/IL‐4 ratio with a quick IL‐10 production by submandibular LN cells. An increase in CD4+ CD25high FOXP3+ Treg cells was detected in LNs and spleen of mice treated with PM‐allergoids. These allergoids were better captured than native allergens by antigen‐presenting (CD45+ MHC‐II +) cells obtained from the sublingual mucosa, including DCs (CD11b+) and macrophages (CD64+). Importantly, all the differential effects induced by PM‐allergoids were abolished when using oxidized instead of nonoxidized PM‐allergoids. Conclusion Our results demonstrate for the first time that PM‐allergoids administered through the sublingual route promote the generation of Th1 and FOXP3+ Treg cells in a greater extent than native allergens by mechanisms that might well involve their better uptake by oral antigen‐presenting cells.

Background Saliva and muscle derived mosquito allergens have been purified and characterized. However, the complete set of allergens remains to be elucidated. In this study, we identified and characterized IgE-binding proteins from the mosquito species A. aegypti. Methods Serum was obtained from 15 allergic individuals with asthma and/or rhinitis and sensitized to mosquito. IgE-binding was determined by ELISA. Total proteins from freeze dried bodies of A. aegypti were extracted and IgE reactive proteins were identified by 2D-gel electrophoresis, followed by Western blot with pooled or individual sera. IgE-reactive spots were further characterized by mass spectrometry. Results Twenty five IgE-reactive spots were identified, corresponding to 10 different proteins, some of which appeared as different variants or isoforms. Heat shock cognate 70 (HSC-70) and Tropomyosin showed IgE reactivity with 60% of the sera, Lysosomal aspartic protease and “AAEL006070-PA” (Uniprot: Q177P3) with 40% and the other proteins with less than 33.3% of the sera. Different variants, or isoforms of Tropomyosin, Arginine or Creatine kinase, Glyceraldehyde-3-phosphate dehydrogenase (GPDH), Calcium-binding protein and Phosphoglycerate mutase were also identified. The mixture of three allergens (Aed a 6, Aed a 8 and Aed a 10) seems to identify more than 80% of A. aegypti sensitized individuals, indicating that these allergens should be considered when designing of improved mosquito allergy diagnostic tools. Conclusions The newly identified allergens may play a role in the pathophysiology of mosquito allergy in the Tropics, and some of them might be important arthropod-related proteins involved in cross-reactivity between A. aegypti and other allergenic arthropods. This article is protected by copyright. All rights reserved.

Background Cross-reactivity between Aedes aegypti and mites, cockroaches, and shrimp has been previously suggested, but the involved molecular components have not been fully described. Objective To evaluate the cross-reactivity between A aegypti and other arthropods. Methods Thirty-four serum samples from patients with asthma and/or allergic rhinitis were selected, and specific IgE to A aegypti , Dermatophagoides pteronyssinus , Dermatophagoides farinae , Blomia tropicalis , Periplaneta americana . and Litopenaeus vannamei was measured by enzyme-linked immunosorbent assay. Cross-reactivity was investigated using pooled serum samples from allergic patients, allergenic extracts, and the recombinant tropomyosins (Aed a 10.0201, Der p 10, Blo t 10, Lit v 1, and Per a 7). Four IgE reactive bands were further characterized by matrix-assisted laser desorption/ionization tandem time of flight. Results Frequency of positive IgE reactivity was 82.35% to at least one mite species, 64.7% to A aegypti , 29.4% to P americana , and 23.5% to L vannamei . The highest IgE cross-reactivity was seen between A aegypti and D pteronyssinus (96.6%) followed by L vannamei (95.4%), B tropicalis (84.4%), and P americana (75.4%). Recombinant tropomyosins from mites, cockroach, or shrimp inhibited the IgE reactivity to the mosquito at a lower extent than the extracts from these arthropods. Several bands of A aegypti cross-reacted with arthropod extracts, and 4 of them were identified as odorant binding protein, mitochondrial cytochrome C, peptidyl-prolyl cis-trans isomerase, and protein with hypothetical magnesium ion binding function. Conclusion We identified 4 novel cross-reactive allergens in A aegypti allergenic extract. These molecules could influence the manifestation of allergy to environmental allergens in the tropics.

Bone marrow (BM) cells fractioned in Percoll gradients yield a low-density fraction (Fr3) highly enriched in suppressor activity. Previously, it has been demonstrated that BM associated suppressor activity was mediated by early myeloid cells, through a mechanism dependent on endogenous IFNγ and nitric oxide production after bacterial stimuli, e.g. lipopolysaccharide (LPS). However, the mechanism(s) through which the IFNγ is produced in BM has not yet been fully elucidated. Therefore, in the present study we investigated the involvement of IL-12, IL-18 and IFNβ on the production of IFNγ and nitric oxide in cultures of BM Fr3 cells, and characterized the IFNγ-producing cells, in response to LPS. The results show that both IL-12 and IFNβ, but not IL-18, are involved on IFNγ production. However, only IFNβ appears to be critical on nitric oxide production. Furthermore, we found that cells of the Thy1.2+CD3+ phenotype produce IFNγ and are tightly involved on nitric oxide production by BM Fr3 cells. In conclusion, IFNβ appears to be critical on IFNγ- and nitric oxide production by BM cells in response to LPS, through a mechanism that is dependent on Thy1.2+CD3+ IFNγ-producing cells.

Allergen specific immunotherapy (ASIT) and environmental control are the only etiologic treatments of allergic rhino-conjunctivitis, asthma and atopic dermatitis. The clinical benefit of ASIT relies on the selection of the patients and the identification and administration of the allergen, or allergens. Different routes of administration have been investigated, including subcutaneous, intradermal, epicutaneous, sublingual, inhaled, or intra-lymphatic. While subcutaneous and sublingual allergen specific immunotherapy may require from 3 to 5 years of treatment, clinical efficacy with intra-lymphatic treatment can be achieved after 3 injections. The most severe side effect of ASIT is anaphylaxis. Novel approaches are being investigated to reduce the allergenicity of immunotherapy vaccines, maintaining immunogenicity. Peptide immunotherapy has been directed mostly against autoimmune diseases, but the use of synthetic peptides for ASIT is a promising field in basic science, applied immunology and in clinical development. Short synthetic peptides bear allergen-specific CD4 T-cell epitopes which induce tolerance by stimulating regulatory (Treg) and Th1 cells. In the present patent review, we describe new trends in allergen immunotherapy using peptides, which, from a clinical point of view, are promising.

Scaling techniques were used to analyze the fractal nature of colonies of 15 cell lines growing in vitro as well as of 16 types of tumor developing in vivo. All cell colonies were found to exhibit exactly the same growth dynamics—which correspond to the molecular beam epitaxy (MBE) universality class. MBE dynamics are characterized by 1), a linear growth rate, 2), the constraint of cell proliferation to the colony/tumor border, and 3), surface diffusion of cells at the growing edge. These characteristics were experimentally verified in the studied colonies. That these should show MBE dynamics is in strong contrast with the currently established concept of tumor growth: the kinetics of this type of proliferation rules out exponential or Gompertzian growth. Rather, a clear linear growth regime is followed. The importance of new cell movements—cell diffusion at the tumor border—lies in the fact that tumor growth must be conceived as a competition for space between the tumor and the host, and not for nutrients or other factors. Strong experimental evidence is presented for 16 types of tumor, the growth of which cell surface diffusion may be the main mechanism responsible in vivo. These results explain most of the clinical and biological features of colonies and tumors, offer new theoretical frameworks, and challenge the wisdom of some current clinical strategies.

Background: Madrid has a short but intensive grass pollen season, in which 79% of the total grass pollen load is released from the middle of May to the middle of June. The main objectives of this study were to quantify Trisetum paniceum (wild oats) aeroallergen in the atmosphere in Madrid from February to December 1996 and to correlate the aeroallergen concentrations with grass pollen counts. Methods: Two different samplers were used to assess allergen exposure; a Burkard spore trap was used to collect pollen grains and a high-volume air sampler to collect airborne particles. A total of 182 air filters were collected and extracted in 1 ml of PBS and analysed by ELISA inhibition, using pooled sera from highly allergic individuals. Results:T. paniceum aeroallergens were detected not only during the grass pollen season, but also before and after. Wild oat allergens had two main peaks of 1 and 1.9 µg/m3, occurring in late May and July, respectively. The time series analysis established the existence of lags between the two main variables pollen counts and aeroallergen activity. Analysis of the data by the Spearman rank test and linear regression showed a weak correlation between grass allergenic activity and grass pollen counts (Spearman’s rho = 0.29). Data obtained from time series analysis demonstrated that grass allergenic activity correlated strongly with current and 5-week-old grass pollen grain counts (r2 = 0.73). Conclusions: Wild oats allergenic activity was detected during the entire year and not only during the pollen season. This fact is an important aspect to be considered in the clinical follow-up and treatment of grass pollen-sensitised patients in Madrid.

Mucosal immunity is the first line of defence of the organism against several pathogens and, at the same time, it is of critical importance in allergic diseases. Oral vaccines have been developed with the aim of enhancing the immune response to pathogens and for the treatment of allergic diseases. One of the major issues concerning oral vaccines is the use of oral adjuvants which could facilitate antigen presentation with the consequent induction of an effective immune response. The present review consists of an analysis, point by point, of the different patents that have been presented in the last 12 months in the different agencies: European (EP), US, and World Intellectual Property Organization (WIPO) and a general analysis of the future developments and trends in this emerging area.

Adoptive immunotherapy with cyclophosphamide (Cy) increases the host resistance against tumor growth. The precise mechanism(s) by which this therapy enhances tumor suppression is unclear. Cy induces the development of early myeloid cells that may be strongly antiproliferative through NO production. These cells are similar to the natural suppressor cells found in normal bone marrow with a potential antitumor effect. Here we have addressed whether the development of NO-producing cells may be involved in this tumor resistance in Cy-treated mice. The results show a synergism between Cy treatment and tumor-specific lymphocytes transferred systemically (i.v.) or locally (Winn’s assay) that results in a strong tumor suppression. Inhibition of NO production by NG-monomethyl-l-arginine at the site of tumor inoculation results in a loss of the protection achieved by the combined therapy. Cy-treated mice develop splenic early myeloid (CD11b, Gr-1, CD31 (ER-MP12), ER-MP20, ER-MP54) cells producing large amounts of NO upon T cell-derived signals (IFN-γ plus CD40 ligation) able to inhibit tumor cell growth in vitro. Early myeloid cells (ER-MP54+) and cells expressing inducible NO synthase are increased at the site of tumor challenge in mice treated with the combined therapy, but not in those treated with Cy or immune cell transfer alone. Thus, Cy induces the expansion of early myeloid cells, inhibiting tumor cell growth by a mechanism involving NO. Both the recruitment and the activation of these myeloid cells at the site of tumor challenge appear to be dependent on the presence of tumor-specific lymphocytes.

Objective: The existence of Platanus pollinosis is not generally accepted despite the production of very large quantities of airborne Platanus pollen in many cities of the United States and Europe. The aim of this study was to investigate if Platanus pollen really contributes to the symptoms of the patients with pollinosis in the Madrid area. Methods: We carried out systematic skin prick testing with Platanus pollen extract on 47 patients seen in our allergy center with spring-summer pollinosis symptoms. Each patient maintained symptom score diaries before, during, and after the Platanus pollination season. The average symptom scores were calculated and compared with the Platanus pollen counts. Measurements of specific IgE by ELISA and immunoblotting also were performed in each patient. Results: The Platanus skin tests were positive in 33 of the 39 patients first seen with seasonal symptoms during Platanus pollen season and only in three of the eight patients without symptoms during Platanus exposure (Fisher's exact test; p Platanus -positive skin test patients also had a positive ELISA result. Furthermore, the average 24-hour rhinitis symptom scores of the 39 patients first seen with seasonal symptoms during March through April showed significant correlation with Platanus pollen counts ( r s = 0.57, p Platanus pollinosis. Conclusions: Platanus pollen is an important cause of pollinosis in Madrid. A protein with a molecular weight of 17 kd appeared to be its major allergen.

Background Allergen immunotherapy (AIT) is the only curative treatment for allergy. AIT faces pitfalls related to efficacy, security, duration, and patient compliance. Novel vaccines overcoming such inconveniences are in demand. Objectives We sought to study the immunologic mechanisms of action for novel vaccines targeting dendritic cells (DCs) generated by coupling glutaraldehyde-polymerized grass pollen allergoids to nonoxidized mannan (PM) compared with glutaraldehyde-polymerized allergoids (P) or native grass pollen extracts (N). Methods Skin prick tests and basophil activation tests with N, P, or PM were performed in patients with grass pollen allergy. IgE-blocking experiments, flow cytometry, confocal microscopy, cocultures, suppression assays, real-time quantitative PCR, ELISAs, and ELISpot assays were performed to assess allergen capture by human DCs and T-cell responses. BALB/c mice were immunized with PM, N, or P. Antibody levels, cytokine production by splenocytes, and splenic forkhead box P3 (FOXP3) + regulatory T (Treg) cells were quantified. Experiments with oxidized PM were also performed. Results PM displays in vivo hypoallergenicity, induces potent blocking antibodies, and is captured by human DCs much more efficiently than N or P by mechanisms depending on mannose receptor– and dendritic cell–specific intercellular adhesion molecule 3–grabbing nonintegrin–mediated internalization. PM endorses human DCs to generate functional FOXP3 + Treg cells through programmed death ligand 1. Immunization of mice with PM induces a shift to nonallergic responses and increases the frequency of splenic FOXP3 + Treg cells. Mild oxidation impairs these effects in human subjects and mice, demonstrating the essential role of preserving the carbohydrate structure of mannan. Conclusions Allergoids conjugated to nonoxidized mannan represent suitable vaccines for AIT. Our findings might also be of the utmost relevance to development of therapeutic interventions in other immune tolerance–related diseases.

Natural suppressor (NS) activity is mediated by cells (NS cells) of bone marrow origin with ability to suppress nonspecifically proliferative responses of lymphocytes. Here we show that pharmacologic concentrations (10(-6)-10(-8) M) of glucocorticoids (GC) greatly inhibit NS activity, as detected by coculturing bone marrow and spleen cells stimulated with B cell (LPS) or T cell (concanavalin A) mitogens. Progesterone antagonizes GC-mediated inhibition of NS activity, suggesting that GC were acting through a receptor-dependent mechanism. A prior treatment of NS cells with GC (10(-5) M) has no effect on the NS activity mediated by these cells. GC are required in culture during the first 24 hr of the suppressor assay. Addition of low amounts of IFN-gamma to GC-treated cultures fully reverses NS cell-mediated suppression. IL-2 produces a reversion as well, while addition of IL-3 or IL-4 does not prevent the GC effect. Neutralizing anti-IFN-gamma antibodies, but not anti-IL-2 or anti-TGF-beta, greatly inhibit NS activity in absence of GC. Taken together, these results indicate that GC inhibit NS activity by impairing endogenous cytokine production required to obtain successful NS cell activation, and not by acting directly on NS cells (i.e., inhibiting the secretion of putative NS factors). Among the cytokines involved in NS cell activation, IFN-gamma appears to be critical, since its addition readily overrides the GC effect and its neutralization results in strong inhibition of NS activity.

SUMMARYAntibodies recognizing anionic phospholipids have been described in systemic lupus erythematosus (SLE) and other autoimmune diseases. Recent studies have shown that some of these antibodies may recognize a cardiolipin-binding protein (apolipoprotein H) rather than phospholipids. A similar possibility is conceivable for other cardiolipin-binding proteins that are targets of autoantibodies. In this study we have addressed whether this might be the case for histones, a set of highly cationic and widely distributed proteins that react in a well known autoantibody system. Our results indicate that: (i) histones bind to anionic phospholipids (cardiolipin and phosphatidylserine) with high avidity, but not to zwitterionic phospholipids (phosphatidylcholine); (ii) monoclonal and polyclonal antihistone antibodies recognize histones bound to cardiolipin; (iii) the addition of histones to serum samples containing antihistone antibodies often enhances their anticardiolipin reactivity. In addition, we have found that antihistone-producing hybridomas derived from MKL-lpr mice may show anticardiolipin activity due to the presence of histones in the cell culture supernatants with the resultant formation of immune complexes. Taken together, the results suggest a potential role for histones in the anticardiolipin activity detected in sera containing antihistone antibodies. These histone-phospholipid interactions should be taken into account when evaluating the pathogenic effects of antihistone antibodies or other autoantibodies reacting with nuclear components (e.g. nucleosomes) containing histones.