Prioritized mass spectrometry increases the depth, sensitivity and data completeness of single-cell proteomics.

Major aims of single-cell proteomics include increasing the consistency, sensitivity and depth of protein quantification, especially for proteins and modifications of biological interest. Here, to simultaneously advance all these aims, we developed prioritized Single-Cell ProtEomics (pSCoPE). pSCoPE consistently analyzes thousands of prioritized peptides across all single cells (thus increasing data completeness) while maximizing instrument time spent analyzing identifiable peptides, thus increasing proteome depth. These strategies increased the sensitivity, data completeness and proteome coverage over twofold. The gains enabled quantifying protein variation in untreated and lipopolysaccharide-treated primary macrophages. Within each condition, proteins covaried within functional sets, including phagosome maturation and proton transport, similarly across both treatment conditions. This covariation is coupled to phenotypic variability in endocytic activity. pSCoPE also enabled quantifying proteolytic products, suggesting a gradient of cathepsin activities within a treatment condition. pSCoPE is freely available and widely applicable, especially for analyzing proteins of interest without sacrificing proteome coverage. Support for pSCoPE is available at http://scp.slavovlab.net/pSCoPE .

Carbohydrate fatty acid monosulphate: oil-in-water adjuvant enhances SARS-CoV-2 RBD nanoparticle-induced immunogenicity and protection in mice.

Development of SARS-CoV-2 vaccines that protect vulnerable populations is a public health priority. Here, we took a systematic and iterative approach by testing several adjuvants and SARS-CoV-2 antigens to identify a combination that elicits antibodies and protection in young and aged mice. While demonstrating superior immunogenicity to soluble receptor-binding domain (RBD), RBD displayed as a protein nanoparticle (RBD-NP) generated limited antibody responses. Comparison of multiple adjuvants including AddaVax, AddaS03, and AS01B in young and aged mice demonstrated that an oil-in-water emulsion containing carbohydrate fatty acid monosulphate derivative (CMS:O/W) most effectively enhanced RBD-NP-induced cross-neutralizing antibodies and protection across age groups. CMS:O/W enhanced antigen retention in the draining lymph node, induced injection site, and lymph node cytokines, with CMS inducing MyD88-dependent Th1 cytokine polarization. Furthermore, CMS and O/W synergistically induced chemokine production from human PBMCs. Overall, CMS:O/W adjuvant may enhance immunogenicity and protection of vulnerable populations against SARS-CoV-2 and other infectious pathogens.

Development of a TLR7/8 agonist adjuvant formulation to overcome early life hyporesponsiveness to DTaP vaccination.

Infection is the most common cause of mortality early in life, yet the broad potential of immunization is not fully realized in this vulnerable population. Most vaccines are administered during infancy and childhood, but in some cases the full benefit of vaccination is not realized in-part. New adjuvants are cardinal to further optimize current immunization approaches for early life. However, only a few classes of adjuvants are presently incorporated in vaccines approved for human use. Recent advances in the discovery and delivery of Toll-like receptor (TLR) agonist adjuvants have provided a new toolbox for vaccinologists. Prominent among these candidate adjuvants are synthetic small molecule TLR7/8 agonists. The development of an effective infant Bordetella pertussis vaccine is urgently required because of the resurgence of pertussis in many countries, contemporaneous to the switch from whole cell to acellular vaccines. In this context, TLR7/8 adjuvant based vaccine formulation strategies may be a promising tool to enhance and accelerate early life immunity by acellular B. pertussis vaccines. In the present study, we optimized (a) the formulation delivery system, (b) structure, and (c) immunologic activity of novel small molecule imidazoquinoline TLR7/8 adjuvants towards human infant leukocytes, including dendritic cells. Upon immunization of neonatal mice, this TLR7/8 adjuvant overcame neonatal hyporesponsiveness to acellular pertussis vaccination by driving a T helper (Th)1/Th17 biased T cell- and IgG2c-skewed humoral response to a licensed acellular vaccine (DTaP). This potent immunization strategy may represent a new paradigm for effective immunization against pertussis and other pathogens in early life.

CAF08 adjuvant enables single dose protection against respiratory syncytial virus infection in murine newborns.

Respiratory syncytial virus is a leading cause of morbidity and mortality in children, due in part to their distinct immune system, characterized by impaired induction of Th 1 immunity. Here we show application of cationic adjuvant formulation CAF08, a liposomal vaccine formulation tailored to induce Th 1 immunity in early life via synergistic engagement of Toll-like Receptor 7/8 and the C-type lectin receptor Mincle. We apply quantitative phosphoproteomics to human dendritic cells and reveal a role for Protein Kinase C-δ for enhanced Th1 cytokine production in neonatal dendritic cells and identify signaling events resulting in antigen cross-presentation. In a murine in vivo model a single immunization at birth with CAF08-adjuvanted RSV pre-fusion antigen protects newborn mice from RSV infection by induction of antigen-specific CD8+ T-cells and Th1 cells. Overall, we describe a pediatric adjuvant formulation and characterize its mechanism of action providing a promising avenue for development of early life vaccines against RSV and other respiratory viral pathogens.

mRNA booster vaccination protects aged mice against the SARS-CoV-2 Omicron variant.

The SARS-CoV-2 Omicron variant evades vaccine-induced immunity. While a booster dose of ancestral mRNA vaccines effectively elicits neutralizing antibodies against variants, its efficacy against Omicron in older adults, who are at the greatest risk of severe disease, is not fully elucidated. Here, we evaluate multiple longitudinal immunization regimens of mRNA BNT162b2 to assess the effects of a booster dose provided >8 months after the primary immunization series across the murine lifespan, including in aged 21-month-old mice. Boosting dramatically enhances humoral and cell-mediated responses with evidence of Omicron cross-recognition. Furthermore, while younger mice are protected without a booster dose, boosting provides sterilizing immunity against Omicron-induced lung infection in aged 21-month-old mice. Correlational analyses reveal that neutralizing activity against Omicron is strongly associated with protection. Overall, our findings indicate age-dependent vaccine efficacy and demonstrate the potential benefit of mRNA booster immunization to protect vulnerable older populations against SARS-CoV-2 variants.

Shaping Neonatal Immunization by Tuning the Delivery of Synergistic Adjuvants via Nanocarriers.

Adjuvanted nanocarrier-based vaccines hold substantial potential for applications in novel early-life immunization strategies. Here, via mouse and human age-specific in vitro modeling, we identified the combination of a small-molecule STING agonist (2'3'-cyclic GMP-AMP, cGAMP) and a TLR7/8 agonist (CL075) to drive the synergistic activation of neonatal dendritic cells and precision CD4 T-helper (Th) cell expansion via the IL-12/IFNγ axis. We further demonstrate that the vaccination of neonatal mice with quadrivalent influenza recombinant hemagglutinin (rHA) and an admixture of two polymersome (PS) nanocarriers separately encapsulating cGAMP (cGAMP-PS) and CL075 (CL075-PS) drove robust Th1 bias, high frequency of T follicular helper (TFH) cells, and germinal center (GC) B cells along with the IgG2c-skewed humoral response in vivo. Dual-loaded cGAMP/CL075-PSs did not outperform admixed cGAMP-PS and CL075-PS in vivo. These data validate an optimally designed adjuvantation system via age-selected small-molecule synergy and a multicomponent nanocarrier formulation as an effective approach to induce type 1 immune responses in early life.

Ocrelizumab during pregnancy and lactation: Rationale and design of the MINORE and SOPRANINO studies in women with MS and their infants.

BACKGROUND: Most disease-modifying therapies (DMTs) approved for the treatment of multiple sclerosis (MS) are not recommended during pregnancy, and discouraged while breastfeeding. However, discontinuation of some DMTs before pregnancy can leave women vulnerable to MS relapses. Although available data on ocrelizumab suggest no increased risk in terms of pregnancy or neonatal outcomes, it is unknown whether ocrelizumab transfers across the placenta or is absorbed through breastmilk; and if so, whether infant B cell development, immune responses or growth and development are affected. This manuscript describes two studies designed to address these uncertainties. METHODS/DESIGN: MINORE and SOPRANINO are multicentre open-label studies. MINORE, which addresses placental transfer, will recruit 44 women with MS or clinically isolated syndrome (CIS) exposed to ocrelizumab between 6 months before the last menstrual period (LMP) to the end of the first trimester. It will evaluate pharmacodynamic effects of potential in utero exposure through the proportion of infants with B cell numbers below lower limit of normal (LLN) at week 6 of life (primary endpoint); as well as through vaccine-induced antibody responses (reflecting B cell function) during the first year of life. Placental transfer will be assessed through measurement of ocrelizumab concentrations in paired samples at delivery (maternal blood as well as umbilical cord blood), and infant serum at week 6 of life. SOPRANINO, which evaluates breastmilk transfer, will recruit 20 women with MS or CIS who resume or initiate ocrelizumab treatment while breastfeeding. The effect of potential exposure through breastmilk will be assessed through the proportion of infants with B cell levels below LLN at 30 days after the mother's first post-partum ocrelizumab infusion (co-primary endpoint). Infant exposure via breastmilk will be assessed through ocrelizumab average daily infant dose in breastmilk over 60 days after the same infusion (co-primary endpoint). Vaccine-induced responses will be measured as in MINORE. Both studies will also measure infant growth and development over the first year of life and safety outcomes in both mothers and infants. All analyses will be descriptive, under an estimand framework. DISCUSSION: Both studies are designed to mimic real-world clinical practice. Treatment decisions for ocrelizumab are independent from study participation; as such, these studies will recruit women who decide, along with their physicians, to continue their pregnancies despite potential in utero exposure (for MINORE); or to breastfeed while under ocrelizumab treatment (for SOPRANINO). MINORE is the first prospective study to measure placental transfer of any DMT in MS, and to perform comprehensive assessments in infants and mothers. Results may inform the optimal contraception period for women treated with ocrelizumab who are planning a pregnancy. Similarly, SOPRANINO is the first prospective study to measure pharmacodynamic effects of ocrelizumab in breastfed infants in addition to pharmacokinetic parameters in breastmilk. SOPRANINO may establish whether breastfeeding is safe for infants whose mothers received treatment with ocrelizumab. CONCLUSION: By collecting detailed pharmacokinetic, pharmacodynamic and safety information, MINORE and SOPRANINO will contribute to understanding the risk/benefit of ocrelizumab in pregnant and lactating women with MS.

Normal B-cell ranges in infants: A systematic review and meta-analysis.

BACKGROUND: During the first year of life, B-cell level is a valuable indicator of whether external factors, such as exposure to B-cell-depleting therapies, have an adverse impact on immune system development. However, there are no standard reference ranges of B-cell levels in healthy infants by age. OBJECTIVE: Our aim was to estimate the normal range of B-cell levels in infants, by age, during the first year of life by pooling data from published studies. METHODS: Studies reporting B-cell levels measured by using flow cytometry and CD19 markers in healthy infants were identified via a systematic literature review. Quality and feasibility assessments determined suitability for inclusion in meta-analyses by age group and/or continuous age. Means and normal ranges (2.5th-97.5th percentile) were estimated for absolute and percentage B-cell levels. Sensitivity analyses assessed the impact of various assumptions. RESULTS: Of the 37 relevant studies identified, 28 were included in at least 1 meta-analysis. The means and normal ranges of B-cell levels were found to be 707 cells/µL in cord blood (range 123-2324 cells/µL), 508 cells/µL in infants aged 0 to 1 month (range 132-1369 cells/µL), 1493 cells/µL in infants aged 1 to 6 months (range 416-3877 cells/µL), and 1474 cells/µL in infants older than 6 months (range 416-3805 cells/µL). The continuous age model showed that B-cell levels peaked at week 26. Trends were similar for the percentage B-cell estimates and in sensitivity analyses. CONCLUSION: These meta-analyses provide the first normal reference ranges for B-cell levels in infants, by week of age, during the first year of life.

An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

An aluminum hydroxide:CpG adjuvant enhances protection elicited by a SARS-CoV-2 receptor binding domain vaccine in aged mice.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic, especially in low- and middle-income countries. Although vaccines against SARS-CoV-2 based on mRNA and adenoviral vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are required to meet global demand. Protein subunit vaccines formulated with appropriate adjuvants represent an approach to address this urgent need. The receptor binding domain (RBD) is a key target of SARS-CoV-2 neutralizing antibodies but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists alone or formulated with aluminum hydroxide (AH) and benchmarked them against AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that an AH and CpG adjuvant formulation (AH:CpG) produced an 80-fold increase in anti-RBD neutralizing antibody titers in both age groups relative to AH alone and protected aged mice from the SARS-CoV-2 challenge. The AH:CpG-adjuvanted RBD vaccine elicited neutralizing antibodies against both wild-type SARS-CoV-2 and the B.1.351 (beta) variant at serum concentrations comparable to those induced by the licensed Pfizer-BioNTech BNT162b2 mRNA vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and enhanced cytokine and chemokine production in human mononuclear cells of younger and older adults. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups.

Alum:CpG adjuvant enables SARS-CoV-2 RBD-induced protection in aged mice and synergistic activation of human elder type 1 immunity.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic especially for low- and middle-income countries. While vaccines against SARS-CoV-2 based on mRNA and adenoviral-vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are needed to meet global demand. In this context, protein subunit vaccines formulated with appropriate adjuvants represent a promising approach to address this urgent need. Receptor-binding domain (RBD) is a key target of neutralizing antibodies (Abs) but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists, including those activating STING, TLR3, TLR4 and TLR9, alone or formulated with aluminum hydroxide (AH), and benchmarked them to AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that the AH and CpG adjuvant formulation (AH:CpG) demonstrated the highest enhancement of anti-RBD neutralizing Ab titers in both age groups (∼80-fold over AH), and protected aged mice from the SARS-CoV-2 challenge. Notably, AH:CpG-adjuvanted RBD vaccine elicited neutralizing Abs against both wild-type SARS-CoV-2 and B.1.351 variant at serum concentrations comparable to those induced by the authorized mRNA BNT162b2 vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and synergistically enhanced cytokine and chemokine production in human young adult and elderly mononuclear cells. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups. ONE SENTENCE SUMMARY: Alum and CpG enhance SARS-CoV-2 RBD protective immunity, variant neutralization in aged mice and Th1-polarizing cytokine production by human elder leukocytes.

Immunobiology of Carbohydrates: Implications for Novel Vaccine and Adjuvant Design Against Infectious Diseases.

Carbohydrates are ubiquitous molecules expressed on the surface of nearly all living cells, and their interaction with carbohydrate-binding proteins is critical to many immunobiological processes. Carbohydrates are utilized as antigens in many licensed vaccines against bacterial pathogens. More recently, they have also been considered as adjuvants. Interestingly, unlike other types of vaccines, adjuvants have improved immune response to carbohydrate-based vaccine in humans only in a few cases. Furthermore, despite the discovery of many new adjuvants in the last years, aluminum salts, when needed, remain the only authorized adjuvant for carbohydrate-based vaccines. In this review, we highlight historical and recent advances on the use of glycans either as vaccine antigens or adjuvants, and we review the use of currently available adjuvants to improve the efficacy of carbohydrate-based vaccines. A better understanding of the mechanism of carbohydrate interaction with innate and adaptive immune cells will benefit the design of a new generation of glycan-based vaccines and of immunomodulators to fight both longstanding and emerging diseases.

Inflammasomes within Hyperactive Murine Dendritic Cells Stimulate Long-Lived T Cell-Mediated Anti-tumor Immunity.

Central to anti-tumor immunity are dendritic cells (DCs), which stimulate long-lived protective T cell responses. Recent studies have demonstrated that DCs can achieve a state of hyperactivation, which is associated with inflammasome activities within living cells. Herein, we report that hyperactive DCs have an enhanced ability to migrate to draining lymph nodes and stimulate potent cytotoxic T lymphocyte (CTL) responses. This enhanced migratory activity is dependent on the chemokine receptor CCR7 and is associated with a unique transcriptional program that is not observed in conventionally activated or pyroptotic DCs. We show that hyperactivating stimuli are uniquely capable of inducing durable CTL-mediated anti-tumor immunity against tumors that are sensitive or resistant to PD-1 inhibition. These protective responses are intrinsic to the cDC1 subset of DCs, depend on the inflammasome-dependent cytokine IL-1ß, and enable tumor lysates to serve as immunogens. If these activities are verified in humans, hyperactive DCs may impact immunotherapy.

Cellular and molecular mechanisms of antifungal innate immunity at epithelial barriers: The role of C-type lectin receptors.

Humans are constantly exposed to fungi, either in the form of commensals at epithelial barriers or as inhaled spores. Innate immune cells play a pivotal role in maintaining commensal relationships and preventing skin, mucosal, or systemic fungal infections due to the expression of pattern recognition receptors that recognize fungal cell wall components and modulate both their activation status and the ensuing adaptive immune response. Commensal fungi also play a critical role in the modulation of homeostasis and disease susceptibility at epithelial barriers. This review will outline cellular and molecular mechanisms of anti-fungal innate immunity focusing on C-type lectin receptors and their relevance in the context of host-fungi interactions at skin and mucosal surfaces in murine experimental models as well as patients susceptible to fungal infections.

Vascular Endothelium in Neonatal Sepsis: Basic Mechanisms and Translational Opportunities.

Neonatal sepsis remains a major health issue worldwide, especially for low-birth weight and premature infants, with a high risk of death and devastating sequelae. Apart from antibiotics and supportive care, there is an unmet need for adjunctive treatments to improve the outcomes of neonatal sepsis. Strong and long-standing research on adult patients has shown that vascular endothelium is a key player in the pathophysiology of sepsis and sepsis-associated organ failure, through a direct interaction with pathogens, leukocytes, platelets, and the effect of soluble circulating mediators, in part produced by endothelial cells themselves. Despite abundant evidence that the neonatal immune response to sepsis is distinct from that of adults, comparable knowledge on neonatal vascular endothelium is much more limited. Neonatal endothelial cells express lower amounts of adhesion molecules compared to adult ones, and present a reduced capacity to neutralize reactive oxygen species. Conversely, available evidence on biomarkers of endothelial damage in neonates is not as robust as in adult patients, and endothelium-targeted therapeutic opportunities for neonatal sepsis are almost unexplored. Here, we summarize current knowledge on the structure of neonatal vascular endothelium, its interactions with neonatal immune system and possible endothelium-targeted diagnostic and therapeutic tools for neonatal sepsis. Furthermore, we outline areas of basic and translational research worthy of further study, to shed light on the role of vascular endothelium in the context of neonatal sepsis.

Superantigenic Activation of Human Cardiac Mast Cells.

B cell superantigens, also called immunoglobulin superantigens, bind to the variable regions of either the heavy or light chain of immunoglobulins mirroring the lymphocyte-activating properties of classical T cell superantigens. Protein A of Staphylococcus aureus, protein L of Peptostreptococcus magnus, and gp120 of HIV are typical immunoglobulin superantigens. Mast cells are immune cells expressing the high-affinity receptor for IgE (FcεRI) and are strategically located in the human heart, where they play a role in several cardiometabolic diseases. Here, we investigated whether immunoglobulin superantigens induced the activation of human heart mast cells (HHMCs). Protein A induced the de novo synthesis of cysteinyl leukotriene C4 (LTC4) from HHMCs through the interaction with IgE VH3+ bound to FcεRI. Protein L stimulated the production of prostaglandin D2 (PGD2) from HHMCs through the interaction with κ light chains of IgE. HIV glycoprotein gp120 induced the release of preformed (histamine) and de novo synthesized mediators, such as cysteinyl leukotriene C4 (LTC4), angiogenic (VEGF-A), and lymphangiogenic (VEGF-C) factors by interacting with the VH3 region of IgE. Collectively, our data indicate that bacterial and viral immunoglobulin superantigens can interact with different regions of IgE bound to FcεRI to induce the release of proinflammatory, angiogenic, and lymphangiogenic factors from human cardiac mast cells.

Innate Immune Modulation by GM-CSF and IL-3 in Health and Disease.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and inteleukin-3 (IL-3) have long been known as mediators of emergency myelopoiesis, but recent evidence has highlighted their critical role in modulating innate immune effector functions in mice and humans. This new wealth of knowledge has uncovered novel aspects of the pathogenesis of a range of disorders, including infectious, neoplastic, autoimmune, allergic and cardiovascular diseases. Consequently, GM-CSF and IL-3 are now being investigated as therapeutic targets for some of these disorders, and some phase I/II clinical trials are already showing promising results. There is also pre-clinical and clinical evidence that GM-CSF can be an effective immunostimulatory agent when being combined with anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA-4) in patients with metastatic melanoma as well as in novel cancer immunotherapy approaches. Finally, GM-CSF and to a lesser extent IL-3 play a critical role in experimental models of trained immunity by acting not only on bone marrow precursors but also directly on mature myeloid cells. Altogether, characterizing GM-CSF and IL-3 as central mediators of innate immune activation is poised to open new therapeutic avenues for several immune-mediated disorders and define their potential in the context of immunotherapies.

Antimicrobial peptide LL-37 and recombinant human mannose-binding lectin express distinct age- and pathogen-specific antimicrobial activity in human newborn cord blood in vitro.

Background: There is a need to prevent and treat infection in newborns. One approach is administration of antimicrobial proteins and peptides (APPs) such as LL-37, a membrane-active cathelicidin antimicrobial peptide, and mannose-binding lectin (MBL), a pattern-recognition protein that binds to microbial surface polysaccharides resulting in opsonization and complement activation. Low plasma/serum levels of LL-37 and of MBL have been correlated with infection and exogenous administration of these agents may enhance host defense. Methods: The antimicrobial activity of LL-37 (15 µg/ml) or rMBL (0.5, 2 and 10 µg/ml) was tested in hirudin-anticoagulated preterm and term human cord blood (N = 12-14) against Staphylococcus aureus (SA) USA 300 (2x10 4 CFU/ml), Staphylococcus epidermis (SE) 1457 (2x10 4 CFU/ml) and Candida albicans (CA) SC5314 (1x10 4 CFU/ml). After incubation (1, 45, or 180 min), CFUs were enumerated by plating blood onto agar plates. Supernatants were collected for measurement of MBL via ELISA. Results: Preterm cord blood demonstrated impaired endogenous killing capacity against SA and SE compared to term blood. Addition of LL-37 strongly enhanced antimicrobial/antifungal activity vs SA, SE and CA in term blood and SE and CA in preterm blood. By contrast, rMBL showed modest fungistatic activity vs CA in a sub-analysis of term newborns with high basal MBL levels. Baseline MBL levels varied within preterm and term cohorts with no correlation to gestational age. In summary, exogenous LL-37 demonstrated significant antimicrobial activity against SA, SE and CA in term and SE and CA in preterm human blood tested in vitro. rMBL demonstrated modest antifungal activity in term cord blood of individuals with high baseline MBL levels. Conclusions: To the extent that our in vitro results predict the effects of APPs in vivo, development of APPs for prevention and treatment of infection should take into account host age as well as the target pathogen.

Secreted Phospholipases A2 in Hereditary Angioedema With C1-Inhibitor Deficiency.

BACKGROUND: Hereditary angioedema (HAE) caused by deficiency (type I) or dysfunction (type II) of the C1 inhibitor protein (C1-INH-HAE) is a disabling, potentially fatal condition characterized by recurrent episodes of swelling. We have recently found that patients with C1-INH-HAE have increased plasma levels of vascular endothelial growth factors and angiopoietins (Angs), which have been associated with vascular permeability in several diseases. Among these and other factors, blood endothelial cells and vascular permeability can be modulated by extracellular or secreted phospholipases A2 (sPLA2s). OBJECTIVE: We sought to investigate the enzymatic activity and biological functions of sPLA2 in patients with C1-INH-HAE. METHODS: sPLA2s enzymatic activity was evaluated in the plasma from 109 adult patients with C1-INH-HAE and 68 healthy donors in symptom-free period and attacks. Plasma level of group IIA sPLA2 (hGIIA) protein was measured in selected samples. The effect of C1-INH-HAE plasma on endothelial permeability was examined in vitro using a vascular permeability assay. The role of hGIIA was determined using highly specific sPLA2 indole inhibitors. The effect of recombinant hGIIA on C1-INH activity was examined in vitro by functional assay. RESULTS: Plasma sPLA2 activity and hGIIA levels are increased in symptom-free C1-INH-HAE patients compared with controls. sPLA2 activity negatively correlates with C1-INH protein level and function. C1-INH-HAE plasma increases endothelial permeability in vitro, and this effect is partially reverted by a specific hGIIA enzymatic inhibitor. Finally, recombinant hGIIA inhibits C1-INH activity in vitro. CONCLUSION: sPLA2 enzymatic activity (likely attributable to hGIIA), which is increased in C1-INH-HAE patients, can promote vascular permeability and impairs C1-INH activity. Our results may pave the way for investigating the functions of sPLA2s (in particular, hGIIA) in the pathophysiology of C1-INH-HAE and may inform the development of new therapeutic targets.