Macrophage migration inhibitory factor (MIF) inhibitor iSO-1 promotes staphylococcal protein A-induced osteogenic differentiation by inhibiting NF-κB signaling pathway.

BACKGROUND: Osteomyelitis is among the most difficult to treat diseases in the field of orthopedics, and there is a lack of effective treatment modalities. Exploring the mechanisms of its development is beneficial for finding molecular targets for treatment. Increasing evidence suggests that macrophage migration inhibitory factor (MIF), as a proinflammatory mediator, is not only involved in various pathophysiological processes of inflammation but also plays an important role in osteogenic differentiation, while its specific regulatory mechanism in osteomyelitis remains unclear. METHODS: In the present study, staphylococcal protein A (SPA)-treated rat bone marrow mesenchymal stem cells (rBMSCs) were used to construct cell models of osteomyelitis. Rat and cell models of osteomyelitis were used to validate the expression levels of MIF, and to further explore the regulatory mechanisms of the MIF inhibitor methyl ester of (S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (iSO-1) and MIF knockdown on cell model of osteomyelitis toward osteogenic differentiation. RESULTS: We found that the expression level of MIF was upregulated in rat and cell models of osteomyelitis and subsequently demonstrated by the GSE30119 dataset that the expression level of MIF was also significantly upregulated in patients with osteomyelitis. Furthermore, SPA promotes MIF expression in rBMSCs while inhibiting the expression of osteogenic-related genes such as Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN) and collagen type-1 (COL-1) through activation of the nuclear factor kappa-B (NF-κB) pathway. In vivo, we further demonstrated that local injection of iSO-1 significantly increased the osteogenic activity in rat model of osteomyelitis. Importantly, we also demonstrated that MIF knockdown and the MIF inhibitor iSO-1 reversed the SPA-mediated inhibition of osteogenic differentiation of rBMSCs by inhibiting the activation of the NF-κB pathway, as evidenced by the upregulation of osteogenic-related gene expression and enhanced bone mineralization. CONCLUSION: ISO-1 and MIF knockdown can reverse the SPA-mediated inhibition of osteogenic differentiation in the rBMSCs model of osteomyelitis by inhibiting the NF-κB signaling pathway, providing a potential target for the treatment of osteomyelitis.

Staphylococcal Protein A Induces Leukocyte Necrosis by Complexing with Human Immunoglobulins.

One of the defining features of Staphylococcus aureus is its ability to evade and impair the human immune response through expression of staphylococcal protein A (SpA). Herein, we describe a previously unknown mechanism by which SpA can form toxic immune complexes when in the presence of human serum, which leads to the loss of human leukocytes. Further, we demonstrate that these toxic complexes are formed specifically through SpA's interaction with intact human IgG and that, in the presence of purified IgG Fab and Fc fragments, SpA shows no such toxicity. The mechanism of action of this toxicity appears to be one mediated by necrosis and not by apoptosis, as previously hypothesized, with up to 90% of human B cells rapidly becoming necrotic following stimulation with SpA-IgG complexes. This phenomenon depends on the immunoglobulin binding capacity of SpA, as a nonbinding mutant of SpA did not induce necrosis. Importantly, immune sera raised against SpA had the capacity to significantly reduce the observed toxicity. An unprecedented toxic effect of SpA-IgG complexes on monocytes was also observed, suggesting the existence of a novel mechanism independent from the interaction of SpA with the B cell receptor. Together, these data implicate SpA in inducing indiscriminate leukocyte toxicity upon formation of complexes with IgG and highlight the requirement for vaccination strategies to inhibit this mechanism. IMPORTANCE Staphylococcus aureus is one of the largest health care threats faced by humankind, with a reported mortality rate within the United States greater than that of HIV/AIDS, tuberculosis, and viral hepatitis combined. One of the defining features of S. aureus as a human pathogen is its ability to evade and impair the human immune response through expression of staphylococcal protein A. Herein, we show that SpA induces necrosis in various immune cells by complexing with human immunoglobulins. Vaccination of mice with a nontoxigenic SpA mutant induced sera capable of inhibiting this mechanism. These observations shed new light on the toxic mechanisms of this key staphylococcal virulence factor and on protective modalities of SpA-based vaccination.

Long Noncoding RNA FAM83H-AS1 Modulates SpA-Inhibited Osteogenic Differentiation in Human Bone Mesenchymal Stem Cells.

Osteomyelitis, an infection of the bone and bone marrow, imposes a heavy burden on public health care systems owing to its progressive bone destruction and sequestration. Human bone mesenchymal stem cells (hBMSCs) play a key role in the process of bone formation, and mounting evidence has confirmed that long noncoding RNAs (lncRNAs) are involved in hBMSC osteogenic differentiation. Nevertheless, the exact function and molecular mechanism of lncRNAs in osteogenic differentiation during osteomyelitis development remain to be explored. In this study, hBMSCs were treated with staphylococcal protein A (SpA) during osteogenic differentiation induction to mimic osteomyelitis in vitro The results of lncRNA microarray analysis revealed that FAM83H-AS1 presented the lowest expression among the significantly downregulated lncRNAs. Functionally, ectopic expression of FAM83H-AS1 contributed to osteogenic differentiation of SpA-induced hBMSCs. Additionally, our findings revealed that FAM83H-AS1 negatively regulated microRNA 541-3p (miR-541-3p), and WNT3A was validated as a target gene of miR-541-3p. Mechanically, FAM83H-AS1 elevated WNT3A expression by competitively binding with miR-541-3p. Lastly, it was demonstrated that FAM83H-AS1/miR-541-3p/WNT3A ameliorated SpA-mediated inhibition of the osteogenic differentiation of hBMSCs, which provided a novel therapeutic strategy for patients with osteomyelitis.

Staphylococcus aureus Protein A induces osteoclastogenesis via the NF­κB signaling pathway.

Staphylococcus aureus (S. aureus) is the most common organism causing osteomyelitis, and Staphylococcus aureus protein A (SpA) is an important virulence factor anchored in its cell wall. However, the precise mechanisms underlying the bone loss caused by SpA have not been well understood. The present study aimed to investigate the effect of SpA on osteoclast differentiation, and the probable mechanism was investigated. Raw264.7 cells were treated with SpA in the absence or presence of receptor­activated (NF)­κB ligand for 5 days, and morphological and biochemical assays were used to assess osteoclastogenesis and explore the underlying mechanisms. Data demonstrated that SpA induced osteoclast differentiation and promoted bone resorption in a dose­dependent manner in the absence or presence of RANKL. In addition, the expression of osteoclast­specific genes, such as the tartrate resistant acid phosphatase, matrix metalloproteinase­9, cathepsin K, calcitonin receptors and d2 isoform of the vacuolar ATPase Vo domain, were enhanced by SpA. Furthermore, the SpA­induced osteoclast differentiation was associated with the degradation of inhibitor of κB­α, phosphorylation of NF­κB p65 and increased expression of nuclear factor of activated T­cells. However, by treatment with JSH­23, an NF­κB inhibitor, the formation of osteoclast­like cells and resorption pits was significantly reduced, and the expression of osteoclast­specific genes was also inhibited. Collectively, in the present study SpA induced osteoclast differentiation, promoted bone resorption, and the NF­κB signaling pathway was involved in this process.

[Recombinant Legionella pneumophila flagella protein A (rflaA) induces the secretion of IL-6 and IL-1ß in RAW264.7 cells in vitro].

Objective To investigate the effect of recombinant Legionella pneumophila flagella protein A (rflaA) on the secretion of interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) by RAW264.7 macrophage and the possible mechanism. Methods RAW264.7 cells were treated with 0.000, 0.125, 0.250, 0.500, 1.000, 2.000, 4.000 and 8.000 µg/mL rflaA to determine the EC50 of rflaA using CCK-8 assay. Secretion of IL-6 and IL-1ß were measured by ELISA at 24, 36 and 48 hours after treatment of the cells with 0.04, 0.08 and 0.16 µg/mL rflaA. At 6, 12, 24, 36 and 48 hours after treatment of the cells with 0.04, 0.08 and 0.16 µg/mL rflaA, the expressions of IL-6, IL-1ß, NOD-like receptor protein 3 (NLRP3) and caspase-1 mRNAs were detected by quantitative real-time PCR, and the expressions of NLRP3 and caspase-1 proteins were tested by Western blotting. Results RflaA enhanced the expressions of IL-6 and IL-1ß, and the higher concentration of rflaA was more potential. The expressions of IL-6 and IL-1ß reached peak when the cells were treated with 0.16 µg/mL rflaA for 36 hours. Treatment of RAW264.7 cells with rflaA promoted the expressions of IL-6 and IL-1ß, NLRP3 and caspase-1 mRNA, and 0.16 µg/mL rflaA was the most potential at 12 hours after treatment. Expressions of NLRP3 and caspase-1 protein increased after treatment with rflaA, and 0.16 µg/mL rflaA induced the highest expression of both proteins at 24 hours after treatment. Conclusion RflaA could enhance the secretion of IL-6 and IL-1ß by promoting the expressions of NLRP3 and caspase-1 in RAW264.7 cells.

CcpA Affects Infectivity of Staphylococcus aureus in a Hyperglycemic Environment.

Many bacteria regulate the expression of virulence factors via carbon catabolite responsive elements. In Gram-positive bacteria, the predominant mediator of carbon catabolite repression is the catabolite control protein A (CcpA). Hyperglycemia is a widespread disorder that predisposes individuals to an array of symptoms and an increased risk of infections. In hyperglycemic individuals, the bacterium Staphylococcus aureus causes serious, life-threatening infections. The importance of CcpA in regulating carbon catabolite repression in S. aureus suggests it may be important for infections in hyperglycemic individuals. To test this suggestion, hyperglycemic non-obese diabetic (NOD; blood glucose level ≥20 mM) mice were challenged with the mouse pathogenic S. aureus strain Newman and the isogenic ccpA deletion mutant (MST14), and the effects on infectivity were determined. Diabetic NOD mice challenged with the ccpA deletion mutant enhanced the symptoms of infection in an acute murine pneumonia model relative to the parental strain. Interestingly, when diabetic NOD mice were used in footpad or catheter infection models, infectivity of the ccpA mutant decreased relative to the parental strain. These differences greatly diminished when normoglycemic NOD mice (blood glucose level ≤ 10 mM) were used. These data suggest that CcpA is important for infectivity of S. aureus in hyperglycemic individuals.

Toxicity and Pharmacokinetic Profile for Single-Dose Injection of ABY-029: a Fluorescent Anti-EGFR Synthetic Affibody Molecule for Human Use.

PURPOSE: ABY-029, a synthetic Affibody peptide, Z03115-Cys, labeled with a near-infrared fluorophore, IRDye® 800CW, targeting epidermal growth factor receptor (EGFR) has been produced under good manufacturing practices for a US Food and Drug Administration-approved first-in-use human study during surgical resection of glioma, as well as other tumors. Here, the pharmacology, phototoxicity, receptor activity, and biodistribution studies of ABY-029 were completed in rats, prior to the intended human use. PROCEDURES: Male and female Sprague Dawley rats were administered a single intravenous dose of varying concentrations (0, 245, 2449, and 24,490 µg/kg corresponding to 10×, 100×, and 1000× an equivalent human microdose level) of ABY-029 and observed for up to 14 days. Histopathological assessment of organs and tissues, clinical chemistry, and hematology were performed. In addition, pharmacokinetic clearance and biodistribution of ABY-029 were studied in subgroups of the animals. Phototoxicity and ABY-029 binding to human and rat EGFR were assessed in cell culture and on immobilized receptors, respectively. RESULTS: Histopathological assessment and hematological and clinical chemistry analysis demonstrated that single-dose ABY-029 produced no pathological evidence of toxicity at any dose level. No phototoxicity was observed in EGFR-positive and EGFR-negative glioma cell lines. Binding strength and pharmacokinetics of the anti-EGFR Affibody molecules were retained after labeling with the dye. CONCLUSION: Based on the successful safety profile of ABY-029, the 1000× human microdose 24.5 mg/kg was identified as the no observed adverse effect level following intravenous administration. Conserved binding strength and no observed light toxicity also demonstrated ABY-029 safety for human use.

Staphylococcus aureus Protein A Disrupts Immunity Mediated by Long-Lived Plasma Cells.

Infection with Staphylococcus aureus does not induce long-lived protective immunity for reasons that are not completely understood. Human and murine vaccine studies support a role for Abs in protecting against recurring infections, but S. aureus modulates the B cell response through expression of staphylococcus protein A (SpA), a surface protein that drives polyclonal B cell expansion and induces cell death in the absence of costimulation. In this murine study, we show that SpA altered the fate of plasmablasts and plasma cells (PCs) by enhancing the short-lived extrafollicular response and reducing the pool of bone marrow (BM)-resident long-lived PCs. The absence of long-lived PCs was associated with a rapid decline in Ag-specific class-switched Ab. In contrast, when previously inoculated mice were challenged with an isogenic SpA-deficient S. aureus mutant, cells proliferated in the BM survival niches and sustained long-term Ab titers. The effects of SpA on PC fate were limited to the secondary response, because Ab levels and the formation of B cell memory occurred normally during the primary response in mice inoculated with wild-type or SpA-deficient S. aureus mutant. Thus, failure to establish long-term protective Ab titers against S. aureus was not a consequence of diminished formation of B cell memory; instead, SpA reduced the proliferative capacity of PCs that entered the BM, diminishing the number of cells in the long-lived pool.

Silencing of Long Non-Coding RNA NONHSAT009968 Ameliorates the Staphylococcal Protein A-Inhibited Osteogenic Differentiation in Human Bone Mesenchymal Stem Cells.

BACKGROUND/AIMS: Osteomyelitis is defined as an inflammation of the bones and bone marrow. The inflammatory microenvironment attenuates the osteogenic differentiation capacity of stem cells and inhibits osteoblast-mediated bone formation, leading to net bone loss. However, the whole expression profile, function and side effect of long non-coding RNAs (lncRNAs) on osteogenic differentiation of stem cells in an inflammatory microenvironment of osteomyelitis are not known. METHODS: In the present study, human bone mesenchymal stem cells (hBMSCs) were treated with different concentrations of Staphylococcal protein A (SpA) to trigger an inflammatory microenvironment in vitro to partly duplicate the inflammatory microenvironment of osteomyelitis, which was confirmed using ELISA for detecting the inflammatory cytokines. The complete expression profiles of lncRNAs and mRNA during osteogenic differentiation of hBMSCs in an inflammatory microenvironment triggered by SpA were analyzed using a lncRNA microarray. LncRNA expression levels were verified by quantitative reverse transcription PCR analysis (qRT-PCR). The expression of NONHSAT009968 in hB-MSCs was silenced by infection with lentivirus expressing NONHSAT009968-shRNA. The expression of Runx2, OCN, OPN, COL1A1, and alkaline phosphatase (ALP) activity was detected by western blot. Alizarin red staining and ALP activity detection were carried out. RESULTS: The results of ELISA showed that SpA treatment induced secretion of inflammatory cytokines IL-1A, IL-6, and TNFA. The results of alizarin red staining and ALP detection showed that SpA treatment suppressed the osteogenic differentiation of hBMSCs. A total of 2033 lncRNAs were found with aberrant expression in SpA-treated hBMSCs compared to controls. Among these lncRNAs, 641 were down-regulated and 1392 were up-regulated. Based on the results of qRT-PCR, lncRNA NONHSAT009968 was chosen for further investigation. The results of alizarin red staining, ALP activity detection, and western blot detection of Runx2, OCN, OPN, COL1A1, and ALP indicated that NONHSAT009968 silencing ameliorates SpA-inhibited osteogenic differentiation in hBMSCs. CONCLUSION: Our present study provides a basis for future analyses of the role of lncRNAs in osteoblastic differentiation in an inflammatory environment triggered by SpA, and lncRNA NONHSAT009968 might be a new target for promoting osteoblast formation.

Staphylococcus aureus Protein A Mediates Interspecies Interactions at the Cell Surface of Pseudomonas aeruginosa.

UNLABELLED: While considerable research has focused on the properties of individual bacteria, relatively little is known about how microbial interspecies interactions alter bacterial behaviors and pathogenesis. Staphylococcus aureus frequently coinfects with other pathogens in a range of different infectious diseases. For example, coinfection by S. aureus with Pseudomonas aeruginosa occurs commonly in people with cystic fibrosis and is associated with higher lung disease morbidity and mortality. S. aureus secretes numerous exoproducts that are known to interact with host tissues, influencing inflammatory responses. The abundantly secreted S. aureus staphylococcal protein A (SpA) binds a range of human glycoproteins, immunoglobulins, and other molecules, with diverse effects on the host, including inhibition of phagocytosis of S. aureus cells. However, the potential effects of SpA and other S. aureus exoproducts on coinfecting bacteria have not been explored. Here, we show that S. aureus-secreted products, including SpA, significantly alter two behaviors associated with persistent infection. We found that SpA inhibited biofilm formation by specific P. aeruginosa clinical isolates, and it also inhibited phagocytosis by neutrophils of all isolates tested. Our results indicate that these effects were mediated by binding to at least two P. aeruginosa cell surface structures-type IV pili and the exopolysaccharide Psl-that confer attachment to surfaces and to other bacterial cells. Thus, we found that the role of a well-studied S. aureus exoproduct, SpA, extends well beyond interactions with the host immune system. Secreted SpA alters multiple persistence-associated behaviors of another common microbial community member, likely influencing cocolonization and coinfection with other microbes. IMPORTANCE: Bacteria rarely exist in isolation, whether on human tissues or in the environment, and they frequently coinfect with other microbes. However, relatively little is known about how microbial interspecies interactions alter bacterial behaviors and pathogenesis. We identified a novel interaction between two bacterial species that frequently infect together-Staphylococcus aureus and Pseudomonas aeruginosa We show that the S. aureus-secreted protein staphylococcal protein A (SpA), which is well-known for interacting with host targets, also binds to specific P. aeruginosa cell surface molecules and alters two persistence-associated P. aeruginosa behaviors: biofilm formation and uptake by host immune cells. Because S. aureus frequently precedes P. aeruginosa in chronic infections, these findings reveal how microbial community interactions can impact persistence and host interactions during coinfections.

Adjuvant effect of B domain of staphyloccocal protein A displayed on the surface of hepatitis B virus capsid.

The hepatitis B virus (HBV) capsid-based recombinant particles, which display both major hydrophilic region of HBV surface antigen (HBV-MHR) and B domain of Staphylococcal protein A (SPAB ), were produced using Escherichia coli as expression host. SPAB was used as an adjuvant to elicit the immune response to HBV-MHR, and its adjuvant effect in the immunized mice was estimated with varying the position and amount of SPAB on the HBV capsid particles. Compared to the emulsified aluminum gel (alum gel) that is a currently commercialized vaccine adjuvant, SPAB caused the significantly higher level of anti-HBV immunoglobulin G (IgG) titer and seroconversion rate, and notably SPAB at the most surface-exposed position on the recombinant particle led to the highest immune response. Moreover, SPAB caused much lower ratio of IgG1 to IgG2a compared to alum gel, indicating that helper T-cell 1-mediated immune response (responsible for cytotoxic T-cell stimulation) is relatively more stimulated by SPAB , unlike alum gel that mainly stimulates helper T-cell 2-mediated immune response (responsible for B-cell stimulation). Although HBV-MHR and HBV capsid particle were used as proof-of-concept in this study, SPAB can be used as a highly effective adjuvant with other disease-specific antigens on the surface of other virus-like particles to produce various recombinant vaccines with high potency.

Staphylococcal protein A-formulated immune complexes suppress enterotoxin-induced cellular responses in nasal polyps.

BACKGROUND: Recent studies have revealed that Staphylococcus aureus and its components participate in the pathogenesis of eosinophilic airway diseases, such as chronic rhinosinusitis with nasal polyps. OBJECTIVE: We sought to determine whether staphylococcal protein A (SpA) from S aureus regulated cellular responses in nasal polyps, especially when coupled to immunoglobulins in immune complexes (ICs). METHODS: Dispersed nasal polyp cells (DNPCs) or peripheral blood monocytes were cultured in vitro with SpA in the presence or absence of IgG, and IL-5, IL-13, IFN-γ, IL-17A, and IL-10 levels were measured in the supernatants. The effect of SpA exposure on staphylococcal enterotoxin B-induced cytokine production by DNPCs in the presence and absence of IgG, IgA, and autologous serum was also examined. RESULTS: Exposure to SpA induced DNPCs to produce significantly higher IL-10, IL-13, and IL-17A levels than DNPCs without SpA, although the magnitude of the IL-17A increase was less than that of IL-10 and IL-13. SpA induced IL-10 production mainly from adherent DNPCs, and this was significantly enhanced in the presence of IgG; similar results were observed in peripheral blood monocytes. IC formation between SpA and IgG (SpA-IgG ICs) was confirmed by using native polyacrylamide gel electrophoresis. SpA-IgG ICs, but not SpA alone, almost completely suppressed staphylococcal enterotoxin B-induced IL-5, IL-13, IFN-γ, and IL-17A production by DNPCs; similar inhibition was observed in DNPCs treated with SpA in the presence of either IgA or autologous serum. CONCLUSIONS: Our results suggest that SpA can regulate the pathogenesis of enterotoxin-induced inflammation in patients with chronic rhinosinusitis with nasal polyps through coupling to immunoglobulins.

Immune modulation by a tolerogenic myelin oligodendrocyte glycoprotein (MOG)10-60 containing fusion protein in the marmoset experimental autoimmune encephalomyelitis model.

Current therapies for multiple sclerosis (MS), a chronic autoimmune neuroinflammatory disease, mostly target general cell populations or immune molecules, which may lead to a compromised immune system. A more directed strategy would be to re-enforce tolerance of the autoaggressive T cells that drive tissue inflammation and injury. In this study, we have investigated whether the course of experimental autoimmune encephalomyelitis (EAE) in mice and marmosets can be altered by a potent tolerizing fusion protein. In addition, a multi-parameter immunological analysis was performed in marmosets to assess whether the treatment induces modulation of EAE-associated cellular and humoral immune reactions. The fusion protein, CTA1R9K-hMOG10-60-DD, contains a mutated cholera toxin A1 subunit (CTA1R9K), a dimer of the Ig binding D region of Staphylococcus aureus protein A (DD), and the human myelin oligodendrocyte glycoprotein (hMOG) sequence 10-60. We observed that intranasal application of CTA1R9K-hMOG10-60-DD seems to skew the immune response against myelin oligodendrocyte glycoprotein (MOG) towards a regulatory function. We show a reduced number of circulating macrophages, reduced MOG-induced expansion of mononuclear cells in peripheral blood, reduced MOG-induced production of interleukin (IL)-17A in spleen, increased MOG-induced production of IL-4 and IL-10 and an increased percentage of cells expressing programmed cell death-1 (PD-1) and CC chemokine receptor 4 (CCR4). Nevertheless, the treatment did not detectably change the EAE course and pathology. Thus, despite a detectable effect on relevant immune parameters, the fusion protein failed to influence the clinical and pathological outcome of disease. This result warrants further development and improvement of this specifically targeted tolerance inducing therapy.

Pathogen-triggered activation of plasmacytoid dendritic cells induces IL-10-producing B cells in response to Staphylococcus aureus.

Induction of polyclonal B cell activation is a phenomenon observed in many types of infection, but its immunological relevance is unclear. In this study we show that staphylococcal protein A induces T cell-independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the V(H)3(+) BCR. We further demonstrate that Staphylococcus aureus strains with high surface protein A expression concomitantly trigger activation of human plasmacytoid dendritic cells (pDC). Sensitivity to chloroquine, cathepsin B inhibition, and a G-rich inhibitory oligodeoxynucleotide supports the involvement of TLR9 in this context. We then identify pDC as essential cellular mediators of B cell proliferation and Ig production in response to surface protein A-bearing S. aureus. The in vivo relevancy of these findings is confirmed in a human PBMC Nod/scid(Prkdc)/γc(-/-) mouse model. Finally, we demonstrate that co-operation of pDC and B cells enhances B cell-derived IL-10 production, a cytokine associated with immunosuppression and induction of IgG4, an isotype frequently dominating the IgG response to S. aureus. IL-10 release is partially dependent on TLR2-active lipoproteins, a hallmark of the Staphylococcus species. Collectively, our data suggest that S. aureus exploits pDC and TLR to establish B cell-mediated immune tolerance.

Half-life extension of a single-chain diabody by fusion to domain B of staphylococcal protein A.

Binding of a therapeutic protein to a long-circulating plasma protein can result in a strongly extended half-life. Among these plasma proteins, albumin and immunoglobulins are of special interest because of their exceptionally long half-life, which is to a great extent determined by recycling through the neonatal Fc receptor (FcRn). Many strategies have been established employing reversible binding to albumin, e.g. using an albumin-binding domain from streptococcal protein G. We show here that the half-life of a recombinant antibody molecule can also be prolonged by fusion to a single immunoglobulin-binding domain (IgBD) from staphylococcal protein A. This domain (domain B, SpA(B)) is composed of 56 amino acid residues and was fused to the C-terminus of a bispecific single-chain diabody (scDb). The scDb-SpA(B) fusion protein was produced in HEK293 cells and retained its antigen-binding activity as shown by enzyme-linked immunosorbent assay and flow cytometry. Furthermore, the fusion protein was capable of binding to human and mouse IgG in a pH-dependent manner. In mice, the terminal half-life of the fusion protein was improved from ∼1-2 h of the unmodified scDb to 11.8 h. Although the fusion protein did not reach the long half-life seen for IgG, our results established the applicability of a single bacterial IgBD for half-life extension purposes.

Serum killing of Ureaplasma parvum shows serovar-determined susceptibility for normal individuals and common variable immuno-deficiency patients.

BACKGROUND: Many Gram-negative bacteria, unlike Gram-positive, are directly lysed by complement. Ureaplasma can cause septic arthritis and meningitis in immunocompromised individuals and induce premature birth. Ureaplasma has no cell wall, cannot be Gram-stain classified and its serum susceptibility is unknown. METHODS: Survival of Ureaplasma serovars (SV) 1, 3, 6 and 14 (collectively Ureaplasma parvum) were measured following incubation with normal or immunoglobulin-deficient patient serum (relative to heat-inactivated controls). Blocking monoclonal anti-C1q antibody and depletion of calcium, immunoglobulins, or lectins were used to determine the complement pathway responsible for killing. RESULTS: Eighty-three percent of normal sera killed SV1, 67% killed SV6 and 25% killed SV14; greater killing correlating to strong immunoblot identification of anti-Ureaplasma antibodies; killing was abrogated following ProteinA removal of IgG1. All normal sera killed SV3 in a C1q-dependent fashion, irrespective of immunoblot identification of anti-Ureaplasma antibodies; SV3 killing was unaffected by total IgG removal by ProteinG, where complement activity was retained. Only one of four common variable immunodeficient (CVID) patient sera failed to kill SV3, despite profound IgM and IgG deficiency for all; however, killing of SV3 and SV1 was restored with therapeutic intravenous immunoglobulin therapy. CONCLUSIONS: Only the classical complement pathway mediated Ureaplasma-cidal activity, sometimes in the absence of observable immunoblot reactive bands.

Co-opting endogenous immunoglobulin for the regulation of inflammation and osteoclastogenesis in humans and mice.

OBJECTIVE: Cells of the monocytic lineage play fundamental roles in the regulation of health, ranging from the initiation and resolution of inflammation to bone homeostasis. In rheumatoid arthritis (RA), the inflamed synovium exhibits characteristic infiltration of macrophages along with local osteoclast maturation, which, together, drive chronic inflammation and downstream articular destruction. The aim of this study was to explore an entirely novel route of immunoglobulin-mediated regulation, involving simultaneous suppression of the inflammatory and erosive processes in the synovium. METHODS: Using in vivo and in vitro studies of human cells and a murine model of RA, the ability of staphylococcal protein A (SPA) to interact with and modulate cells of the monocytic lineage was tested. In addition, the efficacy of SPA as a therapeutic agent was evaluated in murine collagen-induced arthritis (CIA). RESULTS: SPA showed a capacity to appropriate circulating IgG, by generating small immunoglobulin complexes that interacted with monocytes, macrophages, and preosteoclasts. Formation of these complexes resulted in Fcγ receptor type I-dependent polarization of macrophages to a regulatory phenotype, rendering them unresponsive to activators such as interferon-γ. The antiinflammatory complexes also had the capacity to directly inhibit differentiation of preosteoclasts into osteoclasts in humans. Moreover, administration of SPA in the early stages of disease substantially alleviated the clinical and histologic erosive features of CIA in mice. CONCLUSION: These findings demonstrate the overarching utility of immunoglobulin complexes for the prevention and treatment of inflammatory diseases. The results shed light on the interface between immunoglobulin complex-mediated pathways, osteoclastogenesis, and associated pathologic processes. Thus, therapeutic agents designed to harness all of these properties may be an effective treatment for arthritis, by targeting both the innate inflammatory response and prodestructive pathways.

An immunoconjugate of anti-CD24 and Pseudomonas exotoxin selectively kills human colorectal tumors in mice.

BACKGROUND & AIMS: Effective and selective treatment options are needed for patients with colorectal cancer (CRC). The CD24 mucin-like glycoprotein is overexpressed in CRCs; monoclonal antibodies (mAbs) against CD24 inhibit tumor cell growth in vitro and in vivo. Based on the tumor-specific expression of CD24, we investigated the potential of anti-CD24 SWA11 mAb, to deliver a cytotoxic agent into CRC cells. METHODS: We conjugated SWA11 to a Pseudomonas exotoxin derivative (PE38) via an Fc-binding ZZ domain from Staphylococcal protein A (which binds the Fc domain of mouse IgG2a immunoglobulins) to generate the immunotoxin SWA11-ZZ-PE38; IgG-ZZ-PE38 was used as control. Human HT-29 and COLO320 (CD24-positive) and HCT116 (CD24-negative) CRC cell lines were assayed for immunotoxin binding, cytotoxicity, viability, and apoptosis. Toxicity and antitumor efficacy were tested in mice. RESULTS: The immunotoxin preserved the affinity and specificity of SWA11, bound and selectively killed CD24-expressing CRC cells via apoptosis. IC(50) values ranged from 20 to 50 ng/mL-several orders of magnitude lower than that of the mAb alone. The immunotoxins were not toxic to mice at the maximum dose of 0.75 mg/kg. Growth of HT-29 xenograft tumors was significantly reduced in mice given SWA11-ZZ-PE38 (by 78%) compared to untreated mice. CONCLUSIONS: Anti-CD24 SWA11 mAb can deliver a PE exotoxin derivative to CRC cells and cause them to undergo apoptosis, without toxicity to normal tissues. This immunotoxin might be developed as a therapeutic treatment for patients with CRC.

Protein A-mediated multicellular behavior in Staphylococcus aureus.

The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.