Immunoglobulin G Fraction Research Articles

Misleading antigenic von Willebrand factor levels in acquired von Willebrand syndrome secondary to monoclonal gammopathy of undetermined significance.

In the diagnosis and treatment of acquired von Willebrand syndrome (AVWS), von Willebrand factor (VWF) antigen levels (VWF:Ag) are helpful for quantifying blood VWF-protein levels. Most clinical laboratories measure VWF:Ag by latex immunoassay (LIA), but underlying diseases of AVWS may influence LIA results. A 60year-old AVWS patient with immunoglobulin G (IgG) kappa-type monoclonal gammopathy of undetermined significance (MGUS) showed reduced VWF activity but normal levels of VWF:Ag. His VWF multimers were broadly decreased, which represented a large discrepancy with VWF:Ag. To investigate the mechanism of this discrepancy, we measured the patient's plasma VWF:Ag by in-house enzyme-linked immunosorbent assay (ELISA) and LIA. We also purified the IgG fraction from the patient's serum and measured VWF:Ag in VWF-deficient plasma supplemented with this fraction. VWF:Ag measured by in-house ELISA (VWF:AgELISA) was much lower than that measured by LIA (VWF:AgLIA), which indicated reduced VWF-protein volume in blood. Indeed, VWF:Ag was detected by LIA in VWF-deficient plasma spiked with a patient-derived IgG fraction. These results suggest that LIA detected a non-specific immunoreaction and overestimated the patient's VWF:AgLIA. Clinicians should be aware that underlying diseases of AVWS could influence the LIA system, and interpret VWF:Ag cautiously.

Immunomodulatory and anti-inflammatory properties of immunoglobulin G antibodies.

Antibodies provide an essential layer of protection from infection and reinfection with microbial pathogens. An impaired ability to produce antibodies results in immunodeficiency and necessitates the constant substitution with pooled serum antibodies from healthy donors. Among the five antibody isotypes in humans and mice, immunoglobulin G (IgG) antibodies are the most potent anti-microbial antibody isotype due to their long half-life, their ability to penetrate almost all tissues and due to their ability to trigger a wide variety of effector functions. Of note, individuals suffering from IgG deficiency frequently produce self-reactive antibodies, suggesting that a normal serum IgG level also may contribute to maintaining self-tolerance. Indeed, the substitution of immunodeficient patients with pooled serum IgG fractions from healthy donors, also referred to as intravenous immunoglobulin G (IVIg) therapy, not only protects the patient from infection but also diminishes autoantibody induced pathology, providing more direct evidence that IgG antibodies play an active role in maintaining tolerance during the steady state and during resolution of inflammation. The aim of this review is to discuss different conceptual models that may explain how serum IgG or IVIg can contribute to maintaining a balanced immune response. We will focus on pathways depending on the IgG fragment crystallizable (Fc) as pre-clinical data in various mouse model systems as well as human clinical data have demonstrated that the IgG Fc-domain recapitulates the ability of intact IVIg with respect to its ability to trigger resolution of inflammation. We will further discuss how the findings already have or are in the process of being translated to novel therapeutic approaches to substitute IVIg in treating autoimmune inflammation.

Serum from half of patients with immune thrombocytopenia trigger macrophage phagocytosis of platelets.

Humoral antiplatelet factors, such as autoantibodies, are thought to primarily clear platelets by triggering macrophage phagocytosis in immune thrombocytopenia (ITP). However, there are few studies characterizing the capacity and mechanisms of humoral factor-triggered macrophage phagocytosis of platelets using specimens from patients with ITP. Here, we assessed sera from a cohort of 24 patients with ITP for the capacity to trigger macrophage phagocytosis of normal donor platelets and characterized the contribution of humoral factors to phagocytosis. Sera that produced a phagocytosis magnitude greater than a normal human serum mean+ 2 standard deviations were considered phagocytosis-positive. Overall, 42% (8/19) of MHC I alloantibody-negative ITP sera were phagocytosis-positive. The indirect monoclonal antibody immobilization of platelet antigens assay was used to detect immunoglobulin G (IgG) autoantibodies to glycoproteins (GP)IIb/IIIa, GPIb/IX, and GPIa/IIa. Autoantibody-positive sera triggered a higher mean magnitude of phagocytosis than autoantibody-negative sera. Phagocytosis correlated inversely with platelet counts among autoantibody-positive patients but not among autoantibody-negative patients. Select phagocytosis-positive sera were separated into IgG-purified and -depleted fractions via protein G and reassessed for phagocytosis. Phagocytosis was largely retained in the purified IgG fractions. In addition, we assessed serum concentrations of C-reactive protein, serum amyloid P, and pentraxin 3 as potential phagocytosis modulators. Pentraxin 3 concentrations correlated inversely with platelet counts among patients positive for autoantibodies. Taken together, sera from approximately half of the patients with ITP studied triggered macrophage phagocytosis of platelets beyond a normal level. An important role for antiplatelet autoantibodies in phagocytosis is supported; a role for pentraxins such as pentraxin 3 may be suggested.

Abstract 516: N-glycan moiety of IgG-Fc region is a prognostic biomarker for advancednon-small cell lung cancertreated with immune checkpoint inhibitors

Abstract Background: Although it has been known that N-glycan moiety in immunoglobulin G (IgG) fragment crystallizable (Fc) region, which affects antigen dependent cellular cytotoxicity (ADCC) activity is different between healthy individuals and cancer patients, the impact of N-glycan structure on cancer treatment is unknown. Here, we evaluated the putative ADCC activity by the N-glycan moiety of IgG-Fc using serum samples and analyzed its association with clinical benefits from immune checkpoint inhibitors (ICIs) in patients with advanced non-small cell lung cancer (NSCLC). Patients and Methods: Serum samples from healthy volunteers (HVs) were obtained from Tohoku Medical Megabank and chronic obstructive pulmonary disease (COPD) patients were enrolled as a smoking-related non-cancer disease cohort. Serum samples from advanced NSCLC patients receiving ICIs until disease progression or unacceptable toxicity were collected prior treatment. Three IgG fractions (area 1, 2 and 3) corresponding to three peaks identified by affinity chromatography using TSKgel FcR-IIIA-NPR column (Tosoh, Japan) were quantified and the putative ADCC activity was estimated based on the ratio of area 3, which associates with highest ADCC activity, to the whole area (area 3 (%)). Mann-Whitney U-test, Kaplan-Meier methods and the log-rank test were conducted for statistical analyses using JMP Pro software (ver. 14.0). Results: Forty-two COPD and ninty-six NSCLC patients were registered in the study between Jan 2016 and May 2021 at Wakayama Medical University. Characteristics of the patients were as follows: median age, 70 (range, 49-91); male. 78%; smoker, 79%; previous treatment ≥1, 77%; performance status 0-1, 82%; stage IV, 69%; squamous/non-squamous/unknown, 29/69/2%; nivolumab/pembrolizumab/atezolizumab, 46/42/12%. The objective response rate was 25.0% (24/96). The median progression free survival (PFS) and median overall survival (OS) was 72 days (95%CI, 49 to 127) and 310 days (95% CI, 216 to 453), respectively. The area 3 (%) was significantly lower in NSCLC patients than both HVs and COPD patients (P=0.0002 and p<0.0001, respectively) and no significant difference was observed between HVs and COPD samples. When time-to-event analysis was conducted by dividing into two groups in NSCLC patients at the median of the area 3 (%), OS was significantly longer in patient with higher ratio than those with lower one in both entire cohort (median OS, 952 vs 482; 95% CI, 453 days-not reached (NR) and 233-744 days; p=0.0236) and 2nd line subset (median OS, 952 vs 292; 95% CI, 453 days-NR and 120-692 days; p=0.001). Evaluation of IgG fraction was not associated with tumor response or PFS in this study cohort. Conclusion: Our results suggest that evaluation of the N-glycan moiety of IgG-Fc in peripheral blood has a potential as a prognostic biomarker in advanced NSCLC patients treated with ICIs. Citation Format: Jun Oyanagi, Yasuhiro Koh, Yasuyuki Akiyama, Atsushi Morimoto, Koichi Sato, Shunsuke Teraoka, Daichi Fujimoto, Nahomi Tokudome, Atsushi Hayata, Yuichi Ozawa, Hiroaki Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. N-glycan moiety of IgG-Fc region is a prognostic biomarker for advancednon-small cell lung cancertreated with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 516.

Coronavirus Disease 2019-Related Alterations of Total and Anti-Spike IgG Glycosylation in Relation to Age and Anti-Spike IgG Titer.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been affecting the world since January 2020 and has caused millions of deaths. To gain a better insight into molecular changes underlying the COVID-19 disease, we investigated here the N-glycosylation of three immunoglobulin G (IgG) fractions isolated from plasma of 35 severe COVID-19 patients, namely total IgG1, total IgG2, and anti-Spike IgG, by means of MALDI-TOF-MS. All analyses were performed at the glycopeptide level to assure subclass- and site-specific information. For each COVID-19 patient, the analyses included three blood withdrawals at different time-points of hospitalization, which allowed profiling longitudinal alterations in IgG glycosylation. The COVID-19 patients presented altered IgG N-glycosylation profiles in all investigated IgG fractions. The most pronounced COVID-19-related changes were observed in the glycosylation profiles of antigen-specific anti-Spike IgG1. Anti-Spike IgG1 fucosylation and galactosylation showed the strongest variation during the disease course, with the difference in anti-Spike IgG1 fucosylation being significantly correlated with patients’ age. Decreases in anti-Spike IgG1 galactosylation and sialylation in the course of the disease were found to be significantly correlated with the difference in anti-Spike IgG plasma concentration. The present findings suggest that patients’ age and anti-S IgG abundance might influence IgG N-glycosylation alterations occurring in COVID-19.

Production of an in-House Rabbit Anti-Camel Immunoglobulin G (IgG) Conjugated With Horseradish Peroxidase (HRP) For Use in Immunoblots

The present study was aimed to develop anti-camel immunoglobulin G (IgG) conjugate. Camel serum was obtained from healthy camels and the IgG was extracted from this serum using the commercially available IgG purification kits. The electrophoretic profile of camel IgG showed four bands of molecular weight 66, 50, 44 and 33 kDa. Two rabbits were immunised with the purified camel IgG for production of rabbit anti-camel IgG. IgG fraction from serum collected from the immunised rabbits at the end of the immunisation protocol was purified using the commercially available IgG purification kits. The purified IgG tested for reactivity using ELISA against camel serum and then coupled to horseradish peroxidase. The produced conjugate then tested for reactivity using western immunoblotting against serum from camels infected with Haemonchus longistipes and normal camel serum. The conjugate was able to react with camel serum and was able to detect three components of H. longistipes of molecular weight 126, 76 and 18 kDa.

Abstract P014: Clinical impact of evaluating serum IgG fractions in advanced non-small cell lung cancer treated with immune checkpoint inhibitors

Abstract Background: Structure of N-linked glycan in immunoglobulin G (IgG) fragment crystallizable (Fc) region is associated with antigen-dependent cellular cytotoxicity (ADCC) activity. Although it has been known that N-glycan moiety is different between healthy individuals and cancer patients, the relationship between the N-glycan structure and treatment response in cancer patients is unknown. Here, we evaluated the putative ADCC activity by the N-glycan moiety of IgG-Fc using peripheral blood and analyzed its association with clinical benefits from immune checkpoint inhibitors (ICIs) in patients with advanced non-small cell lung cancer (NSCLC). Patients and Methods: Advanced NSCLC patients received nivolumab, pembrolizumab or atezolizumab treatment until disease progression or unacceptable toxicity. Serum samples were collected at baseline. Tumor responses were classified into complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) based on RECIST v1.1. Durable clinical benefit (DCB) was defined as patients whose response was lasting over 6 months. Three IgG fractions (area 1, 2 and 3) corresponding to three peaks identified by affinity chromatography using TSKgel FcR-IIIA-NPR column (Tosoh, Japan) were quantified and the putative ADCC activity was estimated based on the ratio of area 3, which associates with highest ADCC activity, to the whole area. All statistical analyses were carried out using JMP Pro software (ver. 14.0). Mann-Whitney U-test was conducted. Time to event analysis was conducted using Kaplan-Meier methods and the log-rank test. Results: Ninety-seven patients were registered in the study between Jan 2016 and Nov 2018 at Wakayama Medical University. Characteristics of the patients were as follows: median age, 70 (range, 49-91); male. 78%; smoker, 79%; previous treatment ≥1, 77%; performance status 0-1, 82%; stage III/IV, 31/69%; squamous/non-squamous/unknown, 29/69/2%; nivolumab/pembrolizumab/atezolizumab, 46/42/12%. The objective response rate was 25.0% (24/96) and the disease control rate was 55.2% (53/96). The DCB rate was 45.4% (44/96). The median progression free survival (PFS) was 72 days (95%CI, 49 to 127) and median overall survival (OS) was 310 days (95% CI, 229 to 475). When time-to-event analysis was conducted by dividing into two groups at the median of the ratio of area 3 to the whole area, OS was significantly longer in patient with higher ratio than those with lower one in both entire cohort (median OS, 952 vs 482; 95% CI, 482 days-not reached and 233-744 days; log rank p=0.0292) and 2nd line cohort (median OS, 952 vs 292; 95% CI, 453 days-not reached and 120-692 days; log rank p=0.0010). Evaluation of IgG fraction was not associated with tumor response or PFS in this study cohort. Conclusion: Our results suggest that evaluation of the N-glycan moiety of IgG-Fc in peripheral blood has a potential as a prognostic biomarker in advanced NSCLC patients treated with ICIs. Citation Format: Jun Oyanagi, Yasuhiro Koh, Yasuyuki Akiyama, Atsushi Morimoto, Koichi Sato, Shunsuke Teraoka, Daichi Fujimoto, Nahomi Tokudome, Atsushi Hayata, Yuichi Ozawa, Hiroaki Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Clinical impact of evaluating serum IgG fractions in advanced non-small cell lung cancer treated with immune checkpoint inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P014.

Antibody‐mediated platelet activation in COVID‐19: A coincidence or a new mechanism of the dysregulated coagulation system?

Antibody‐mediated platelet activation in COVID‐19: A coincidence or a new mechanism of the dysregulated coagulation system?

Platelet Activation by Antiphospholipid Antibodies Depends on Epitope Specificity and is Prevented by mTOR Inhibitors.

Antiphospholipid antibodies (aPL) have been reported to activate platelets. This is considered to be one of the pathogenic properties of aPL. Even though aPL heterogeneity is quite well established, little is known, if the ability to activate platelets is common to all aPL or depends on antigen specificity. To further study this issue, we analyzed the ability of three human monoclonal aPL with distinctly different antigenic specificities to activate platelets in vitro. The results obtained with human monoclonal aPL were validated with immunoglobulin G (IgG) fractions obtained from patients with antiphospholipid syndrome (APS). A co-factor-independent human monoclonal anticardiolipin aPL had no discernible effect on human platelets. Two monoclonal aPL reactive against β2 glycoprotein I (β2GPI) induced platelet aggregation, integrin αIIbβ3 activation and P-selectin surface expression. These data could be confirmed with patient IgG fractions which could only induce aggregation, if they had anti-β2GPI activity. Anti-β2GPI aPL-induced platelet activation depended on interaction of aPL with the low affinity Fcγ-receptor IIa on the platelet surface. It was completely abolished by pretreatment of platelet-rich plasma with the mechanistic target of rapamycin (mTOR) inhibitors rapamycin or everolimus. This extends previous data showing that mTOR is involved in signaling of anti-β2GPI in monocytes and endothelial cells. In conclusion, anti-β2GPI aPL activate platelets while co-factor-independent anticardiolipin aPL have no effect. mTOR is involved in this signaling process which has implications beyond APS, because so far the role of mTOR signaling in platelets is incompletely explored and requires further study.

Prevention of Dominant IgG Adsorption on Nanocarriers in IgG-Enriched Blood Plasma by Clusterin Precoating.

Nanocarriers for medical applications must work reliably within organisms, independent of the individual differences in the blood proteome. Variation in the blood proteome, such as immunoglobulin levels, is a result of environmental, nutrition, and constitution conditions. This variation, however, should not influence the behavior of nanocarriers in biological media. The composition of the protein corona is investigated to understand the influence varying immunoglobulin levels in the blood plasma have on the interactions with nanocarriers. Specifically, the composition of the nanocarriers' coronas is analyzed after incubation in plasma with normal or elevated immunoglobulin G (IgG) levels, and cellular uptake is monitored in cell lines containing different immunoglobulin receptors. Here, it is reported that upon doubling the IgG concentration in plasma, the IgG fraction in the protein corona increases by a factor of 40 independent of the nanocarrier material. This results in a significant increase in uptake in cells exhibiting IgG binding receptors. Furthermore, precoating nanocarriers with clusterin successfully prevents dominant IgG‐adsorption and additionally reduces cellular internalization, after incubation with IgG‐enriched plasma. Therefore, precoating nanocarriers may be utilized as a powerful method to reduce the influence of individual variations in blood composition on the protein corona.

Autoantibodies against podocytic UCHL1 are associated with idiopathic nephrotic syndrome relapses and induce proteinuria in mice

Idiopathic steroid sensitive nephrotic syndrome (INS), the most frequent childhood nephropathy, is thought to be mediated by a circulating soluble factor that reversibly affects the renal protein sieving. The efficiency of rituximab therapy recently highlighted the involvement of B cells. Here we studied the involvement of a specific immunoglobulin G (IgG) in the disease. After plasma fractionation by size exclusion chromatography, a detachment of cultured podocyte was observed with one IgG-containing fraction from 47% patients in relapse, 9% of patients in remission and 0% of controls. Podocyte protein lysates were immunoprecipitated by IgG from those plasma fractions identifying a list of 41 podocyte proteins after proteomic analysis. Five podocyte targets were selected on statistical and biological criteria. Specific antibodies were tested and only anti-Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) IgG led to podocyte detachment. UCHL1 was mainly found inside the podocyte but also weakly expressed on podocyte cell surface. Incubation of either anti-UCHL1 IgG or plasma fractions with recombinant UCHL1 prevented podocyte detachment. Plasma levels of anti-UCHL1 IgG were significantly increased in relapsing INS patients compared to patients in remission and controls. Proteinuria correlated with anti-UCHL1 IgG level at various stages of the disease. Purified patient anti-UCHL1 antibodies induced proteinuria and podocyte foot effacement in mice. Altogether, these results identified UCHL1 as a target podocyte protein of autoantibodies in a set of relapsing patients and support a causative role of anti-UCHL1 autoantibodies in the development of INS.

The influence of immunoglobulin class G from blood plasma of patients with stroke on the activity of some parameters of hemostasis system.

Today, cardiovascular diseases are one of the main causes of disability of the population. Most of the illnesses, including stroke, are accompanied by the appearance immunoglobulin G (IgG) in the blood circulation. According to the literature sources and previous experiments, it is known that IgG made influence on the hemostasis system. Objectives of the investigation are pure enzymes: thrombin, factor X, proenzyme of protein C and prothrombin under the influence of IgG fraction that were separated from the plasma of patients with atherothrombotic and cardioembolic ischemic stroke and patients with other neurological diseases. IgG was separated by affinity chromatography. Disc electrophoresis was used to control antibodies' purity. The main goal of the experiment is to test the potential influence of all fractions of separated IgG on the process of hydrolysis of specific chromogenic substrates by the key factors of the hemostasis system such as thrombin, factor X and protein C. The appearance of IgG in the blood stream during the atherothrombotic and cardioembolic ischemic stroke and other neurological diseases was proved. Concentration of the IgG in plasma of patients with cardioembolic ischemic stroke was significantly higher compared with healthy donors, whereas the IgG in plasma of patients with atherothrombotic ischemic stroke and other neurological disorders was not significantly different. Their potential ability to influence the enzymes such as thrombin, factor X, proenzyme of protein C and prothrombin was shown. It was proved that the antibody fractions of all experimental groups significantly accelerate the process of splitting chromogenic substrate by thrombin but inhibited cleavage of a specific chromogenic substrate by protein C.

Immunoglobulin G elution in protein A chromatography employing the method of chromatofocusing for reducing the co-elution of impurities.

Purification processes for monoclonal Immunoglobulin G (IgG) typically employ protein A chromatography as a capture step to remove most of the impurities. One major concern of the post-protein A chromatography processes is the co-elution of some of the host cell proteins (HCPs) with IgG in the capture step. In this work, a novel method for IgG elution in protein A chromatography that reduces the co-elution of HCPs is presented where a two-step pH gradient is self-formed inside a protein A chromatography column. The complexities involved in using an internally produced pH gradient in a protein A chromatography column employing adsorbed buffering species are discussed though equation-based modeling. Under the conditions employed, ELISA assays show a 60% reduction in the HCPs co-eluting with the IgG fraction when using the method as compared to conventional protein A elution without affecting the IgG yield. Evidence is also obtained which indicates that the amount of leached protein A present in free solution in the purified product is reduced by the new method. Biotechnol. Bioeng. 2017;114: 154-162. © 2016 Wiley Periodicals, Inc.

The prophylactic effects of human IgG derived from sera containing high anti-PcrV titers against pneumonia-causing Pseudomonas aeruginosa

ABSTRACTThe PcrV cap structure of the type III secretory apparatus of Pseudomonas aeruginosa is a vaccine target. Human immunoglobulin G (IgG) molecules extracted from sera containing high or low anti-PcrV titers were tested for their effects against P. aeruginosa pneumonia in a mouse model. Among 198 volunteers, we selected the top 10 high anti-PcrV titer sera and the bottom 10 low anti-PcrV titer sera and extracted the IgG fraction from each serum sample. First, we examined the effects of the IgG against virulent P. aeruginosa. A lethal dose of P. aeruginosa premixed with saline, low titer human IgG, high titer human IgG, or rabbit-derived polyclonal anti-PcrV IgG was intratracheally administered into the lungs of mice, and their survival and lung inflammation were evaluated for 24 h. The high anti-PcrV titer human IgG had a prophylactic effect. Next, the prophylactic effects of intravenous administration of extracted and pooled high or low anti-PcrV titer human IgG were examined. Here, prophylactic intravenous administration of pooled high anti-PcrV titer human IgG, which showed binding capacity to P. aeruginosa PcrV, was more effective than the administration of its low titer pooled equivalent, and the measured physiological and inflammatory parameters correlated with the anti-PcrV titer levels. This result indirectly implies that high anti-PcrV titers in blood can help to protect against virulent P. aeruginosa infections. In addition, the IgG fractions from such high titer sera have potential to be a source of specific intravenous immunoglobulin products for passive vaccination against virulent P. aeruginosa infections.

Human IgG Antinuclear Antibodies Induce Pregnancy Loss in Mice by Increasing Immune Complex Deposition in Placental Tissue: In Vivo Study.

A threefold higher prevalence of antinuclear antibodies (ANA) has been reported in patients with recurrent pregnancy loss (RPL). Nevertheless, the role of ANA in reproductive failure is still unclear. The aim of this study was to investigate the role of ANA during early pregnancy in vivo. We used pregnant mice treated with immunoglobulin G (IgG) obtained from normal healthy subjects (NHS); ANA(+) sera of patients with RPL; and ANA(+) sera from women with uncomplicated pregnancies (HW). Placental immunohistochemical/immunofluorescence staining was performed to detect complement and immune complex deposition. ELISA was performed to evaluate complement levels. ANA(+) IgG from RPL women significantly increased embryo resorption rate, reduced C3, and increased C3a serum levels compared to NHS IgG or ANA(+) -HW IgG. Increased C3 deposition and increased immune complex staining in placental tissues from mice treated with ANA(+) -RPL IgG fraction compared to NHS- and ANA(+) -HW-IgG-treated mice were found. ANA(+) IgG injection in mice is able to induce fetal resorption and complement activation. The presence on placental tissues of immune complexes and complement fragments suggests the complement activation as a possible mechanism of placental damage.

Insights into the surface topology of polyhydroxyalkanoate synthase: self-assembly of functionalized inclusions.

The polyhydroxyalkanoate (PHA) synthase catalyzes the synthesis of PHA and remains attached to the hydrophobic PHA inclusions it creates. Although this feature is actively exploited to generate functionalized biobeads via protein engineering, little is known about the structure of the PHA synthase. Here, the surface topology of Ralstonia eutropha PHA synthase was probed to inform rational protein engineering toward the production of functionalized PHA beads. Surface-exposed residues were detected by conjugating biotin to inclusion-bound PHA synthase and identifying the biotin-conjugated lysine and cysteine residues using peptide fingerprinting analysis. The identified sites (K77, K90, K139, C382, C459, and K518) were investigated as insertion sites for the generation of new protein fusions. Insertions of FLAG epitopes into exposed sites K77, K90, K139, and K518 were tolerated, retaining >65 % of in vivo activity. Sites K90, K139, and K518 were also tested by insertion of the immunoglobulin G (IgG)-binding domain (ZZ), successfully producing PHA inclusions able to bind human IgG in vitro. Although simultaneous insertions of the ZZ domain into two sites was permissive, insertion at all three lysine sites inactivated the synthase. The K90/K139 double ZZ insertion had the optimum IgG-binding capacity of 16 mg IgG/g wet PHA beads and could selectively purify the IgG fraction from human serum. Overall, this study identified surface-exposed flexible regions of the PHA synthase which either tolerate protein/peptide insertions or are critical for protein function. This further elucidates the structure and function of PHA synthase and provides new opportunities for generating functionalized PHA biobeads.

The cerebrospinal fluid immunoglobulin transcriptome and proteome in neuromyelitis optica reveals central nervous system-specific B cell populations.

BackgroundNeuromyelitis optica (NMO) is a severe demyelinating disorder of the central nervous system (CNS) associated with the presence of an autoimmune antibody response (AQP4-IgG) against the water channel aquaporin-4 (AQP4). It remains unclear whether pathologic AQP4-IgG in the CNS is produced entirely by peripheral plasma cells or is generated in part by infiltrating B cells. To determine the overlap of AQP4-IgG idiotypes between the CNS and periphery, we compared the immunoglobulin G (IgG) transcriptome of cerebrospinal fluid (CSF) plasmablasts with the CSF and serum IgG proteomes in 7 AQP4-seropositive NMO patients following exacerbation.MethodsCSF variable region Ig heavy- (VH) and light-chain (VL) transcriptome libraries were generated for each patient from CSF plasmablasts by single cell sorting, reverse transcriptase polymerase chain reaction (RT-PCR), and DNA sequencing. Recombinant antibodies were generated from clonally expanded, paired VH and VL sequences and tested for AQP4-reactivity by cell-binding assay. CSF and serum IgG fractions were searched for sequences that matched their respective CSF IgG transcriptome. Matching peptides within the same patient’s CSF and serum IgG proteomes were also identified.ResultsIn each NMO patient, we recovered CSF IgG VH and VL sequences that matched germline-mutated IgG protein sequences from the patient’s CSF and serum IgG proteomes. Although a modest variation was observed between patients, the overlap between the transcriptome and proteome sequences was found primarily, but not exclusively, within the CSF. More than 50% of the CSF IgG transcriptome sequences were exclusively found in the CSF IgG proteome, whereas 28% were found in both the CSF and blood IgG proteome, and 18% were found exclusively in the blood proteome. A comparable distribution was noted when only AQP4-specific IgG clones were considered. Similarly, on average, only 50% of the CSF IgG proteome matched corresponding peptide sequences in the serum.ConclusionsDuring NMO exacerbations, a substantial fraction of the intrathecal Ig proteome is generated by an intrathecal B cell population composed of both novel and peripherally-derived clones. Intrathecal CSF B cell clones may contribute to NMO disease exacerbation and lesion formation and may be an important target for preventative therapies.

The intrinsic pathogenic role of autoantibodies to aquaporin 4 mediating spinal cord disease in a rat passive-transfer model

Neuromyelitis optica (NMO) is causally linked to autoantibodies (ABs) against aquaporin 4 (AQP4). Here, we focused on the pathogenic effects exclusively mediated by human ABs to AQP4 in vivo. We performed cell-free intrathecal (i.th.) passive transfer experiments in Lewis rats using purified patient NMO immunoglobulin G (IgG) and various recombinant human anti-AQP4 IgG-ABs via implanted i.th. catheters. Repetitive application of patient NMO IgG fractions and of recombinant human anti-AQP4 ABs induced signs of spinal cord disease. Magnetic resonance imaging (MRI) revealed longitudinal spinal cord lesions at the site of application of anti-AQP4 IgG. Somatosensory evoked potential amplitudes were reduced in symptomatic animals corroborating the observed functional impairment. Spinal cord histology showed specific IgG deposition in the grey and white matter in the affected areas. We did not find inflammatory cell infiltration nor activation of complement in spinal cord areas of immunoglobulin deposition. Moreover, destructive lesions showing axon or myelin damage and loss of astrocytes and oligodendrocytes were all absent. Immunoreactivity to AQP4 and to the excitatory amino acid transporter 2 (EAAT2) was markedly reduced whereas immunoreactivity to the astrocytic marker glial fibrillary acid protein (GFAP) was preserved. The expression of the NMDA-receptor NR1 subunit was downregulated in areas of IgG deposition possibly induced by sustained glutamatergic overexcitation. Disease signs and histopathology were reversible within weeks after stopping injections. We conclude that in vivo application of ABs directed at AQP 4 can induce a reversible spinal cord disease in recipient rats by inducing distinct histopathological abnormalities. These findings may be the experimental correlate of “penumbra-like” lesions recently reported in NMO patients adjacent to effector-mediated tissue damage.

Inhibition of FcγR-mediated phagocytosis by IVIg is independent of IgG-Fc sialylation and FcγRIIb in human macrophages

AbstractIn immune thrombocytopenia and warm autoimmune hemolytic anemia, circulating immunoglobulin G (IgG)-opsonized blood cells are cleared from the circulation by macrophages. Administration of intravenous immunoglobulin (IVIg) can prevent uptake, but the exact working mechanism is not known. The prevailing theory from murine studies, which states that Fc-sialylated IgG alters the balance between activating and inhibitory Fc-gamma receptors (FcγRs) by inducing upregulation of the inhibitory FcγRIIb on effector macrophages, is currently debated. We studied phagocytosis of IgG-opsonized blood cells in a human system, assessing the effect of IVIg and blocking anti-FcγR F(ab′)2 fragments on uptake by monocyte-derived macrophages (both M1 and M2 macrophages). Phagocytosis was remarkably sensitive to administration of IVIg, but unexpectedly, recombinant Fc-sialylated IgG or sialic acid-enriched IVIg were equally active as unsialylated IgG fractions in mediating this inhibition, independent of FcγRIIb expression. Instead, IVIg inhibited phagocytosis by direct blockade of FcγRs. IgG fractions enriched for IgG dimers with enhanced avidity for FcγRs showed increased inhibition compared with monomeric IgG fractions. Together, our data demonstrate that inhibition of IgG-mediated phagocytosis in human macrophages by IVIg is dependent on the capacity to directly bind FcγRs but is independent of FcγRIIb or sialylation of the Fc fragment in the human setting.

Angiotensin receptor type 1 and endothelin receptor type A on immune cells mediate migration and the expression of IL-8 and CCL18 when stimulated by autoantibodies from systemic sclerosis patients

IntroductionAgonistic autoantibodies (Aabs) against the angiotensin II receptor type 1 (AT1R) and the endothelin receptor type A (ETAR) have been identified in patients with systemic sclerosis (SSc). In our present study, we examined the expression of the AT1R and the ETAR in human immune cells and the pathological effects mediated through these receptors by their corresponding Aabs.MethodsProtein expression of AT1R and ETAR on peripheral blood mononuclear cells (PBMCs) from healthy individuals and SSc patients was analyzed using flow cytometry, and mRNA expression of both receptors in PBMCs from healthy donors was examined by real-time PCR. In addition, PBMCs from healthy donors were stimulated in vitro with affinity-purified immunoglobulin G (IgG) fractions from SSc patients positive for AT1R and ETAR Aabs, as well as with IgG from healthy donors serving as controls. Alterations in cell surface marker expression, cytokine secretion and chemotactic motility were analyzed using flow cytometry, enzyme-linked immunosorbent assays and chemotaxis assays, respectively. The results were correlated with the characteristics and clinical findings of the IgG donors.ResultsBoth AT1R and ETAR were expressed on PBMCs in humans. Protein expression of both receptors was decreased in SSc patients compared with that of healthy donors and declined during the course of disease. IgG fractions of SSc patients positive for AT1R and ETAR Aabs induced T-cell migration in an Aab level–dependent manner. Moreover, IgG of SSc patients stimulated PBMCs to produce more interleukin 8 (IL-8) and chemokine (C-C motif) ligand 18 (CCL18) than did the IgG of healthy donors. All effects were significantly reduced by selective AT1R and ETAR antagonists. Statistical analysis revealed an association of SSc-IgG induced high IL-8 concentrations with an early disease stage and of high CCL18 concentrations with lung fibrosis onset and vascular complications in the respective IgG donors.ConclusionIn our present study, we could demonstrate the expression of both AT1R and ETAR on human peripheral T cells, B cells and monocytes. The decreased receptor expression in SSc patients, the inflammatory and profibrotic effects upon Aab stimulation of PBMCs in vitro and the associations with clinical findings suggest a role for Aab-induced activation of immune cells mediated by the AT1R and the ETAR in the pathogenesis or even the onset of the disease.