Plasma-Derived Hemopexin as a Candidate Therapeutic Agent for Acute Vaso-Occlusion in Sickle Cell Disease: Preclinical Evidence.

People living with sickle cell disease (SCD) face intermittent acute pain episodes due to vaso-occlusion primarily treated palliatively with opioids. Hemolysis of sickle erythrocytes promotes release of heme, which activates inflammatory cell adhesion proteins on endothelial cells and circulating cells, promoting vaso-occlusion. In this study, plasma-derived hemopexin inhibited heme-mediated cellular externalization of P-selectin and von Willebrand factor, and expression of IL-8, VCAM-1, and heme oxygenase-1 in cultured endothelial cells in a dose-responsive manner. In the Townes SCD mouse model, intravenous injection of free hemoglobin induced vascular stasis (vaso-occlusion) in nearly 40% of subcutaneous blood vessels visualized in a dorsal skin-fold chamber. Hemopexin administered intravenously prevented or relieved stasis in a dose-dependent manner. Hemopexin showed parallel activity in relieving vascular stasis induced by hypoxia-reoxygenation. Repeated IV administration of hemopexin was well tolerated in rats and non-human primates with no adverse findings that could be attributed to human hemopexin. Hemopexin had a half-life in wild-type mice, rats, and non-human primates of 80-102 h, whereas a reduced half-life of hemopexin in Townes SCD mice was observed due to ongoing hemolysis. These data have led to a Phase 1 clinical trial of hemopexin in adults with SCD, which is currently ongoing.

Airway inflammation and dysbiosis in antibody deficiency despite the presence of IgG.

BACKGROUND: Patients with antibody deficiency suffer chronic respiratory symptoms, recurrent exacerbations, and progressive airways disease despite systemic replacement of IgG. Little is known about the respiratory tract biology of these patients. OBJECTIVE: We sought to measure immunoglobulin levels, inflammatory cytokines, and mediators of tissue damage in serum and sputum from patients with antibody deficiency and healthy controls; to analyze the respiratory microbiome in the same cohorts. METHODS: We obtained paired sputum and serum samples from 31 immunocompetent subjects and 67 antibody-deficient patients, the latter divided on computed tomography scan appearance into "abnormal airways" (bronchiectasis or airway thickening) or "normal airways." We measured inflammatory cytokines, immunoglobulin levels, neutrophil elastase, matrix-metalloproteinase-9, urea, albumin, and total protein levels using standard assays. We used V3-V4 region 16S sequencing for microbiome analysis. RESULTS: Immunodeficient patients had markedly reduced IgA in sputum but higher concentrations of IgG compared with healthy controls. Inflammatory cytokines and tissue damage markers were higher in immunodeficient patients, who also exhibited dysbiosis with overrepresentation of pathogenic taxa and significantly reduced alpha diversity compared with immunocompetent individuals. These differences were seen regardless of airway morphology. Sputum matrix-metalloproteinase-9 and elastase correlated inversely with alpha diversity in the antibody-deficient group, as did sputum IgG, which correlated positively with several inflammatory markers, even after correction for albumin levels. CONCLUSIONS: Patients with antibody deficiency, even with normal lung imaging, exhibit inflammation and dysbiosis in their airways despite higher levels of IgG compared with healthy controls.

Increased Respiratory Viral Detection and Symptom Burden Among Patients with Primary Antibody Deficiency: Results from the BIPAD Study.

BACKGROUND: Patients with primary antibody deficiency (PAD) are at increased risk of respiratory tract infections, but our understanding of their nature and consequences remains limited. OBJECTIVE: To define the symptomatic and microbial burden of upper airway infection in adults with PAD relative to age-matched controls. METHODS: Prospective 12-month observational study consisting of a daily upper and lower airway symptom score alongside fortnightly nasal swab with molecular detection of 19 pathogen targets. RESULTS: A total of 44 patients and 42 controls (including 34 household pairs) were recruited, providing more than 22,500 days of symptom scores and 1,496 nasal swabs. Swab and questionnaire compliance exceeded 70%. At enrollment, 64% of patients received prophylactic antibiotics, with a 34% prevalence of bronchiectasis. On average, patients with PAD experienced symptomatic respiratory exacerbations every 6 days compared with 6 weeks for controls, associated with significant impairment of respiratory-specific quality-of-life scores. Viral detections were associated with worsening of symptom scores from a participant's baseline. Patients with PAD had increased odds ratio (OR) for pathogen detection, particularly viral (OR, 2.73; 95% CI, 2.09-3.57), specifically human rhinovirus (OR, 3.60; 95% CI, 2.53-5.13) and parainfluenza (OR, 3.06; 95% CI, 1.25-7.50). Haemophilus influenzae and Streptococcus pneumoniae were also more frequent in PAD. Young child exposure, IgM deficiency, and presence of bronchiectasis were independent risk factors for viral detection. Prophylactic antibiotic use was associated with a lower risk of bacterial detection by PCR. CONCLUSIONS: Patients with PAD have a significant respiratory symptom burden associated with increased viral infection frequency despite immunoglobulin replacement and prophylactic antibiotic use. This highlights a clear need for future therapeutic trials in the population with PAD, and informs future study design.

Topical application of human-derived Ig isotypes for the control of acute respiratory infection evaluated in a human CD89-expressing mouse model.

Recurrent and persistent airway infections remain prevalent in patients with primary immunodeficiency (PID), despite restoration of serum immunoglobulin levels by intravenous or subcutaneous plasma-derived IgG. We investigated the effectiveness of different human Ig isotype preparations to protect mice against influenza when delivered directly to the respiratory mucosa. Four polyvalent Ig preparations from pooled plasma were compared: IgG, monomeric IgA (mIgA), polymeric IgA-containing IgM (IgAM) and IgAM associated with the secretory component (SIgAM). To evaluate these preparations, a transgenic mouse expressing human FcαRI/CD89 within the myeloid lineage was created. CD89 was expressed on all myeloid cells in the lung and blood except eosinophils, reflecting human CD89 expression. Intranasal administration of IgA-containing preparations was less effective than IgG in reducing pulmonary viral titres after infection of mice with A/California/7/09 (Cal7) or the antigenically distant A/Puerto Rico/8/34 (PR8) viruses. However, IgA reduced weight loss and inflammatory mediator expression. Both IgG and IgA protected mice from a lethal dose of PR8 virus and for mIgA, this effect was partially CD89 dependent. Our data support the beneficial effect of topically applied Ig purified from pooled human plasma for controlling circulating and non-circulating influenza virus infections. This may be important for reducing morbidity in PID patients.

Topical application of nebulized human IgG, IgA and IgAM in the lungs of rats and non-human primates.

BACKGROUND: Recurrent and persistent infections are known to affect airways of patients with Primary Immunodeficiency despite appropriate replacement immunoglobulin serum levels. Interestingly, patients with Chronic Obstructive Pulmonary Disease or with non-CF bronchiectasis also show similar susceptibility to such infections. This may be due to the limited availability of immunoglobulins from the systemic circulation in the conductive airways, resulting in local immunodeficiency. Topical application of nebulized plasma-derived immunoglobulins may represent a means to address this deficiency. In this study, we assessed the feasibility of nebulizing plasma-derived immunoglobulins and delivering them into the airways of rats and non-human primates. METHODS: Distinct human plasma-derived immunoglobulin isotype preparations were nebulized with an investigational eFlow® nebulizer and analyzed in vitro or deposited into animals. Biochemical and immunohistological analysis of nebulized immunoglobulins were then performed. Lastly, efficacy of topically applied human plasma-derived immunoglobulins was assessed in an acute Streptococcus pneumoniae respiratory infection in mice. RESULTS: Characteristics of the resulting aerosols were comparable between preparations, even when using solutions with elevated viscosity. Neither the structural integrity nor the biological function of nebulized immunoglobulins were compromised by the nebulization process. In animal studies, immunoglobulins levels were assessed in plasma, broncho-alveolar lavages (BAL) and on lung sections of rats and non-human primates in samples collected up to 72 h following application. Nebulized immunoglobulins were detectable over 48 h in the BAL samples and up to 72 h on lung sections. Immunoglobulins recovered from BAL fluid up to 24 h after inhalation remained structurally and functionally intact. Importantly, topical application of human plasma-derived immunoglobulin G into the airways of mice offered significant protection against acute pneumococcal pneumonia. CONCLUSION: Taken together our data demonstrate the feasibility of topically applying plasma-derived immunoglobulins into the lungs using a nebulized liquid formulation. Moreover, topically administered human plasma-derived immunoglobulins prevented acute respiratory infection.

Oral Passive Immunization With Plasma-Derived Polyreactive Secretory-Like IgA/M Partially Protects Mice Against Experimental Salmonellosis.

Secretory immunoglobulins have a critical role in defense of the gastrointestinal tract and are known to act by preventing bacterial acquisition. A stringent murine model of bacterial infection with Salmonella enterica Typhimurium was used to examine protection mediated by oral passive immunization with human plasma-derived polyreactive IgA and IgM antibodies (Abs) reconstituted as secretory-like immunoglobulins (SCIgA/M). This reagent has been shown to trigger Salmonella agglutination and to limit the entry of bacterium into intestinal Peyer's patches via immune exclusion. We now demonstrate that upon administration into ligated intestinal loops, SCIgA/M properly anchors in the mucus and is protected from degradation to a better extent that IgA/M or IgG. Moreover, prophylactic oral administration of SCIgA/M before intragastric infection of mice with a virulent strain of S. enterica Typhimurium allows to protect infected animals, as reflected by reduced colonization of both mucosal and systemic compartments, and conserved integrity of intestinal tissues. In comparison with IgA/M or IgG administration, SCIgA/M provided the highest degree of protection. Moreover, such protective efficacy is also observed after therapeutic oral delivery of SCIgA/M. Either prophylactic or therapeutic treatment with passively delivered SCIgA/M ensured survival of up to 50% of infected mice, while untreated animals all died. Our findings unravel the potential of oral passive immunization with plasma-derived polyreactive SCIgA/M Abs to fight gastrointestinal infections.

Reconstituted high-density lipoprotein modulates activation of human leukocytes.

An anti-inflammatory effect of reconstituted High Density Lipoprotein (rHDL) has been demonstrated in atherosclerosis and in sepsis models. An increase of adhesion molecules as well as tissue factor expression on endothelial cells in response to inflammatory or danger signals are attenuated by the treatment with rHDL. Here we show the inhibitory effect of rHDL on the activation of human leukocytes in a whole blood assay as well as on monocyte-derived human dendritic cells (DC). Multiplex analysis of human whole blood showed that phytohaemagglutinin (PHA)-induced secretion of the cytokines IL-1ß, IL-1RA, IL-2R, IL-6, IL-7, IL-12(p40), IL-15 and IFN-α was inhibited. Furthermore, an inhibitory effect on the production of the chemokines CCL-2, CCL-4, CCL-5, CXCL-9 and CXCL-10 was observed. Activation of granulocytes and CD14+ monocytes by PHA is inhibited dose-dependently by rHDL shown as decreased up-regulation of ICAM-1 surface expression. In addition, we found a strong inhibitory effect of rHDL on toll-like receptor 2 (TLR2)- and TLR4-mediated maturation of DC. Treatment of DC with rHDL prevented the up-regulation of cell surface molecules CD80, CD83 and CD86 and it inhibited the TLR-driven activation of inflammatory transcription factor NF-κB. These findings suggest that rHDL prevents activation of crucial cellular players of cellular immunity and could therefore be a useful reagent to impede inflammation as well as the link between innate and adaptive immunity.

Intravenous immunglobulin binds beta amyloid and modifies its aggregation, neurotoxicity and microglial phagocytosis in vitro.

Intravenous Immunoglobulin (IVIG) has been proposed as a potential therapeutic for Alzheimer's disease (AD) and its efficacy is currently being tested in mild-to-moderate AD. Earlier studies reported the presence of anti-amyloid beta (Aß) antibodies in IVIG. These observations led to clinical studies investigating the potential role of IVIG as a therapeutic agent in AD. Also, IVIG is known to mediate beneficial effects in chronic inflammatory and autoimmune conditions by interfering with various pathological processes. Therefore, we investigated the effects of IVIG and purified polyclonal Aß-specific antibodies (pAbs-Aß) on aggregation, toxicity and phagocytosis of Aß in vitro, thus elucidating some of the potential mechanisms of action of IVIG in AD patients. We report that both IVIG and pAbs-Aß specifically bound to Aß and inhibited its aggregation in a dose-dependent manner as measured by Thioflavin T assay. Additionally, IVIG and the purified pAbs-Aß inhibited Aß-induced neurotoxicity in the SH-SY5Y human neuroblastoma cell line and prevented Aß binding to rat primary cortical neurons. Interestingly, IVIG and pAbs-Aß also increased the number of phagocytosing cells as well as the amount of phagocytosed fibrillar Aß by BV-2 microglia. Phagocytosis of Aß depended on receptor-mediated endocytosis and was accompanied by upregulation of CD11b expression. Importantly, we could also show that Privigen dose-dependently reversed Aß-mediated LTP inhibition in mouse hippocampal slices. Therefore, our in vitro results suggest that IVIG may have an impact on different processes involved in AD pathogenesis, thereby promoting further understanding of the effects of IVIG observed in clinical studies.

Cellular processes essential for African swine fever virus to infect and replicate in primary macrophages.

The macrophage (Mø) is an essential immune cell for innate immunity. Such cells are targeted by African swine fever virus (ASFV). The early phases of infection with ASFV have been previously characterized in non-leukocyte cells such as Vero cells. Here, we report on several additional key parameters that ASFV utilizes during the infection of primary Mø. Related to virus infection, we established that receptor-mediated endocytosis of the virus by Mø is not the exclusive means of entry to infect the host cells. Analysis of the ensuing processes identified divalent cation-dependent activities to be particularly important, relating to the virus requirement for microtubule assembly needed for endocytic and endosomal processing. Actin-dependent endocytosis and endocytic flux involving microtubule activity are also implicated, pointing to entry via phagocytosis. Subsequently, the virus avoids terminal degradation by circumventing mature lysosome activities, including autophagosome-lysosome delivery. Nevertheless, the replicative cycle is apparently dependent on certain lysosomal functions, i.e. activities sensitive to propylamine are essential for the virus, whereas vinblastine- and leupeptin-sensitive functions only partially influence viral replication. The present work has identified cellular processes essential for ASFV to infect and replicate in the macrophage. These findings will improve our understanding of the cellular pathways employed by viruses infecting immune scavenger cells.

Intravenous immunoglobulin contains a broad repertoire of anticarbohydrate antibodies that is not restricted to the IgG2 subclass.

BACKGROUND: Specificities for carbohydrate IgG antibodies, thought to be predominantly of the IgG2 subclass, have never been broadly examined in healthy human subjects. OBJECTIVE: To examine commercial intravenous immunoglobulin (IVIG) preparations for their ability to recognize a wide range of glycans and to determine the contribution of IgG2 to the binding pattern observed. METHODS: We used a glycan microarray to evaluate IVIG preparations and a control mix of similar proportions of human myeloma IgG1 and IgG2 for binding to 377 glycans, courtesy of the Consortium for Functional Glycomics Core H. Glycans recognized were categorized using public databases for their likely cellular sources. IgG2 was depleted from IVIG by using immunoaffinity chromatography, and depletion was confirmed by using nephelometry and surface plasmon resonance. RESULTS: Nearly half of the glycans bound IgG. Some of the glycans with the greatest antibody binding can be found in structures of human pathogenic bacteria (eg, Streptococcus pneumoniae, Mycobacterium tuberculosis, Vibrio cholera) and nonpathogenic bacteria, including LPS and lipoteichoic acid, capsular polysaccharides, and exopolysaccharides. Surprisingly, depletion of IgG2 had only a modest effect on anticarbohydrate recognition patterns compared with the starting IVIG preparation. Little to no binding activity was detected to human endogenous glycans, including tumor-associated antigens. CONCLUSIONS: This novel, comprehensive analysis provides evidence that IVIG contains a much wider range than previously appreciated of anticarbohydrate IgG antibodies, including those recognizing both pathogenic and non-pathogen-associated prokaryotic glycans.

Monomerization of dimeric IgG of intravenous immunoglobulin (IVIg) increases the antibody reactivity against intracellular antigens.

Intravenous immunoglobulin (IVIg) preparations are derived from pooled plasma from up to 60,000 healthy human donors and reflect the immunologic experience of the donor population. IVIg contains monomeric and dimeric IgG populations which are in a dynamic equilibrium depending on concentration, pH, temperature, donor pool size, time and stabilizers added in order to keep the portion of dimeric IgG below a certain level. In the present study, monomeric and dimeric fractions were isolated by size exclusion chromatography. The dimeric fractions, however, showed a dynamic instability and tended to dissociate. Both dimeric and monomeric IgG fractions were acid treated (pH 4) in order to dissociate the dimeric IgG. Western-blot analysis identified a sub-population of SDS resistant IgG dimers. Furthermore, the reactivities of the fractions were tested against a panel of self- and exo-antigens. There was a marked increase in activity of the dimeric compared to the monomeric IgG fraction against various intracellular self-antigens. Our data indicates that the increased reactivities of pH 4-treated fractions can mainly be attributed to dimer dissociation, as pH 4-treated monomers do not show significantly increased activities against a range of antigens.

Self-reactivity in the dimeric intravenous immunoglobulin fraction.

Therapeutic intravenous immunoglobulin (IVIg) preparations contain antibodies reflecting the cumulative antigen experience of the donor population. IVIg contains variable amounts of monomeric and dimeric IgG, but there is little information available on their comparative antibody specificities. We have isolated highly purified fractions of monomeric and dimeric IgG by size-exclusion chromatography. Following treatment of all fractions at pH4, analyses by immunodot and immunocytology on human cell lines showed a preferential recognition of autoantigens in the dimeric IgG fraction. Investigation of the HEp-2 cytoplasmic proteome by 2D-PAGE, Western blot, and subsequent identification of IVIg reactive spots by mass spectrometry (LC-MS/MS) showed that IVIg recognized only a restricted set of the total proteins. Similar experiments showed that more antigens were recognized by the dimeric IgG fraction, especially when the dissociated dimer fraction was used, as compared to its monomeric counterpart. These observations are consistent with idiotype-anti-idiotype masking of auto-specific Abs in the dimeric fraction of IVIg.

Intravenous immunoglobulin preparations contain anti-Siglec-8 autoantibodies.

BACKGROUND: Human intravenous immunoglobulin (IVIg) preparations are used for the treatment of autoimmune and allergic diseases. Natural autoantibodies are believed to contribute to IVIg-mediated anti-inflammatory effects. OBJECTIVE: To address the question of whether IVIg preparations contain anti-sialic acid-binding Ig-like lectin-8 (anti-Siglec-8) autoantibodies. METHODS: The presence of possible anti-Siglec-8 autoantibodies in IVIg preparations was first examined by functional eosinophil death and apoptosis assays. Specificity of IVIg effects was shown by depleting anti-Siglec-8 autoantibodies from IVIg. Binding of purified anti-Siglec-8 autoantibodies to recombinant Siglec-8 was demonstrated by an immunodot assay. RESULTS: IVIg exerts cytotoxic effects on purified human blood eosinophils. Both potency and efficacy of the IVIg-mediated eosinophil killing effect was enhanced by IL-5, granulocyte/macrophage colony-stimulating factor, IFN-gamma, TNF-alpha, and leptin. Similarly, inflammatory eosinophils obtained from patients suffering from the hypereosinophilic syndrome (HES) demonstrated increased Siglec-8 cytotoxic responses when compared with normal blood eosinophils. Pharmacologic blocking experiments indicated that the IVIg-mediated additional eosinophil death in the presence of cytokines is largely caspase-independent, but it depends on reactive oxygen species. Anti-Siglec-8 autoantibody-depleted IVIg failed to induce caspase-independent eosinophil death. CONCLUSION: IVIg preparations contain natural anti-Siglec-8 autoantibodies. CLINICAL IMPLICATIONS: Anti-Siglec-8 autoantibodies present in IVIg preparations may have therapeutic relevance in autoimmune and allergic diseases, respectively, such as Churg-Strauss syndrome.

Immunologic and functional evidence for anti-Siglec-9 autoantibodies in intravenous immunoglobulin preparations.

Human intravenous immunoglobulin (IVIg) preparations are increasingly used for the treatment of autoimmune diseases. Earlier work demonstrated the presence of autoantibodies against Fas in IVIg, suggesting that IVIg might be able to induce caspase-dependent cell death in Fas-sensitive cells. In this study, we demonstrate that sialic acid-binding Ig-like lectin 9 (Siglec) represents a surface molecule on neutrophils that is activated by IVIg, resulting in caspase-dependent and caspase-independent forms of cell death. Neutrophil death was mediated by naturally occurring anti-Siglec-9 autoantibodies present in IVIg. Moreover, the efficacy of IVIg-mediated neutrophil killing was enhanced by the proinflammatory cytokines granulocyte/macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma), and this additional cell death required reactive oxygen species (ROSs) but not caspases. Anti- Siglec-9 autoantibody-depleted IVIg failed to induce this caspase-independent neutrophil death. These findings contribute to our understanding of how IVIg preparations exert their immunoregulatory effects under pathologic conditions and may provide a possible explanation for the neutropenia that is sometimes seen in association with IVIg therapy.

Comparative analysis of antigen specificities in the monomeric and dimeric fractions of intravenous immunoglobulin.

Intravenous immunoglobulin (IVIG) preparations are derived from the pooled plasma of thousands of healthy donors and contain a complex mix of antibodies. Depending on the formulation, IVIG preparations contain variable amounts of monomeric and dimeric IgG. The biological and therapeutic significance of these IVIG fractions is still ill defined. Kinetic analysis of monomeric and dimeric IgG isolated by size-exclusion chromatography revealed a stable monomeric versus an unstable dimeric IgG fraction tending to dissociation. Biochemical analysis by 2D gel electrophoresis and isotype analysis showed no significant differences between the fractions. In contrast, comparative analysis by immunodot, ELISA, FACS, and immunohistology of monomeric and dimeric IgG fractions showed a preferential reactivity of the dimeric IgG on a variety of both self-antigens and exoantigens.

Porcine peripheral blood dendritic cells and natural interferon-producing cells.

Peripheral blood contains two major particular infrequent dendritic cells (DC) subsets linking the innate and specific immune system, the myeloid DC and plasmacytoid DC equivalent to the natural interferon-producing cells (NIPC). The functional characterization of these cells demands large volumes of blood, making a large animal model more appropriate and beneficial for certain studies. Here, two subsets of porcine blood mononuclear cells expressing swine workshop cluster 3 (SWC3, a SIRP family member), are described and compared to monocytes. The blood DC specialized in T-cell stimulation were major histocompatibility complex (MHC) class II+, CD80/86+, CD1+/-, CD4-, and in contrast to monocytes CD14-. A CD16- and a CD16+ subset could be discriminated. Granulocyte-macrophage colony-stimulating factor and interleukin-3 were survival factors for this DC subset, and culture induced an up-regulation of MHC class II and CD80/86. The second subset described, are porcine NIPC, typically CD4++, MHC class IIlow, CD80/86low, CD1-, CD8-/low, CD16-/low and CD45RA-/low. Porcine NIPC had high interleukin-3 binding capacity, and survived in response to this cytokine. Their unique function was strong interferon type I secretion after virus stimulation. Both subsets were endocytically active when freshly isolated, and down-regulated this activity after in vitro maturation. Taken together, the present report has delineated porcine blood DC and NIPC, permitting a more detailed understanding of innate immune defences, particularly in response to infections.