IgM Binding Research Articles

Treatment‐responsive primary autoimmune cerebellar ataxia in a patient with IgG and IgM anticerebellar antibodies

Primary autoimmune cerebellar ataxia (PACA) in the absence of another triggering disease represents an emerging category of neurological illness. We report such a case whose ataxia was markedly responsive to plasma exchange. We analyzed patient serum for the presence of IgM or IgG anticerebellar neuronal antibodies. Case presentation: rat cerebellar slice cultures incubated with patient sera were studied for IgG and IgM antibody uptake, intracellular binding, and neuronal death. Patient serum was evaluated for anti-myelin associated glycoprotein (anti-MAG) and associated anti-glycolipid antibodies. Antibodies were taken up by viable cerebellar neurons and bound to intracellular antigens. Uptake and predominantly nuclear binding of IgG were seen in granule cells whereas cytoplasmic binding of IgM was observed predominantly in Purkinje cells. Intracellular antibody accumulation was not accompanied by neuronal death, consistent with the patient's excellent clinical response to plasma exchange. Anti-MAG or other associated anti-glycolipid antibodies were not detected. PACA may be associated with both IgG and IgM antibodies reactive with cerebellar neuronal antigens. Our patient's response to plasma exchange supports a role for antineuronal antibodies in disease pathogenesis and emphasizes the need for rapid diagnosis and treatment.

Effects of early nutrition and sanitary conditions on antibody levels in early and later life of broiler chickens

Immune maturation of broiler chickens may be affected by management, such as early life feeding strategy (early versus delayed nutrition) or by low or high sanitary conditions (LSC versus HSC). We compared systemic maternal (MAb), natural (NAb), natural auto- (NAAb), and antigen specific antibody (SpAb) levels (IgM, IgY) between broilers (n = 48 per treatment) that received early (EN) or delayed nutrition for 72 h (DN) housed in either low (LSC) or high sanitary conditions (HSC) between 7 and 35 d of age. We found minimal interactions between feeding strategy and sanitary conditions. At 7 d of age, broilers receiving EN compared with DN, had elevated levels of IgM binding keyhole limpet hemocyanin (KLH), phosphoryl-conjugated ovalbumin (PC-OVA), and muramyl dipeptide (MDP), whereas effects of feeding strategy diminished at later ages. In LSC compared with HSC broilers, levels of NAb agglutinating RRBC and sheep red blood cells (SRBC) were already elevated from 14 d of age onwards. At 33 d of age, antibody levels (NAb, NAAb, anti-LPS, anti-MDP) were all elevated in LSC, compared with HSC broilers, for both IgM and IgY, but not IgM against KLH. Western blotting revealed different binding patterns of NAAb against chicken liver homogenate, which may indicate that the NAAb repertoire is affected by antigenic pressure. Our data suggest that antibody levels are affected for an important part by environmental conditions (feeding strategy and sanitary conditions), but minimally by their interaction. However, it remains to be further studied whether the enhanced levels of antibodies as initiated by EN and LSC contribute to enhanced resistance to infectious diseases.

Performance of molecular and serologic tests for the diagnosis of scrub typhus.

Diagnosis of scrub typhus, caused by the bacterium Orientia tsutsugamushi, is challenging because of the overlap of its non-specific symptoms with other infections coupled with the lack of sufficient data on the performance of diagnostic tests. Early diagnosis of scrub typhus is crucial to improve outcomes and this study evaluates the diagnostic performance of various tests. The present study aims at assessing the accuracy of various rapid diagnostic tests, serologic tests, and nucleic acid amplification methods on well-characterized patient samples. Adult patients with acute febrile illness and manifestations suggestive of scrub typhus confirmed by positive PCR in the blood, eschar or tissue were characterized as cases. Patients with acute febrile illness and a confirmed alternate etiology such as culture-confirmed typhoid, smear/PCR positive for malaria, PCR/NS1 antigen positive for dengue, PCR positive for influenza, PCR/MAT positive for leptospirosis, PCR positive for spotted fever were characterized as controls with other infections. The healthy controls consisted of subjects from the same geographic region. We performed the following tests on blood samples for scrub typhus and calculated the sensitivity, specificity, positive predictive value, and negative predictive value: (1) Quantitative real time PCR using 47kDa gene (qPCR); (2) Conventional PCR using 56kDa gene (cPCR); (3) Loop-mediated isothermal amplification assay (LAMP assay); (4) Immunofluorescence assay (IFA); (5) Enzyme-linked immunosorbent assay (ELISA); (6) Weil-Felix test(WF test); and (7) Immunochromatographic Rapid Diagnostic Test (RDT).Among the 316 participants, 158 had confirmed scrub typhus (cases) and 158 were controls. ELISA and RDT detecting Orientia tsutsugamushi specific IgM antibodies had excellent discriminative potential with sensitivities and specificities of 92%, 94% and 92%, 92% respectively. The sensitivity and specificity of IFA were found to be 95% and 74% respectively. IgM serology had a false positivity rate of 8% with other acute febrile illnesses such as dengue, leptospirosis and spotted fever due to the nonspecific binding of the pentavalent IgM. LAMP assay had 91.7% sensitivity and 77.2% specificity while qPCR provided excellent sensitivity (97%) and perfect specificity. In conclusion, ELISA and RDT detecting Orientia tsutsugamushi specific IgM antibodies have excellent sensitivity and specificity while the accuracy of IFA is suboptimal for the diagnosis of scrub typhus. Given its perfect specificity and superior sensitivity, qPCR is preferred for diagnostic confirmation in reference laboratories particularly for diagnosis of early disease with less than 7 days duration. This study provides a comprehensive evaluation of all currently available diagnostic tests for scrub typhus.

Robust and Specific Secretory IgA Against SARS-CoV-2 Detected in Human Milk.

SummaryThe SARS-CoV-2 immune response in human milk has not yet been examined, although protecting infants and young children from COVID-19 is critical for limiting community transmission and preventing serious illness and death. Here, milk samples from eight COVID-19-recovered and seven COVID-19-suspected donors were tested for antibody (Ab) binding to the SARS-CoV-2 Spike protein. All samples exhibited significant specific IgA reactivity to the full Spike, whereas 80% exhibited significant IgA and secretory (s)Ab binding to the Receptor-Binding Domain (RBD). Additionally, 67% samples exhibited IgG and/or IgM binding to RBD. IgA and sAb titers were highly correlated, indicating most IgA to be sIgA. Overall, these data indicate that a robust sIgA-dominant SARS-CoV-2 Ab response in human milk after infection should be expected in a significant majority of individuals. Further research is highly warranted to determine Ab functionality and the potential for exploiting extracted milk sIgA for therapeutic use.

Plasticity of the human IgM repertoire in response to long-term spaceflight.

Immune dysregulation is among the main adverse outcomes of spaceflight. Despite the crucial role of the antibody repertoire in host protection, the effects of spaceflight on the human antibody repertoire are unknown. Consequently, using high-throughput sequencing, we examined the IgM repertoire of five cosmonauts 25days before launch, after 64±11 and 129±20days spent on the International Space Station (ISS), and at 1, 7, and 30days after landing. This is the first study of this kind in humans. Our data revealed that the IgM repertoire of the cosmonauts was different from that of control subjects (n=4) prior to launch and that two out the five analyzed cosmonauts presented significant changes in their IgM repertoire during the mission. These modifications persisted up to 30days after landing, likely affected the specificities of IgM binding sites, correlated with changes in the V(D)J recombination process responsible for creating antibody genes, and coincided with a higher stress response. These data confirm that the immune system of approximately half of the astronauts who spent 6months on the ISS is sensitive to spaceflight conditions, and reveal individual responses indicating that personalized approaches should be implemented during future deep-space exploration missions that will be of unprecedented durations.

Binding of human serum proteins to Plasmodium falciparum-infected erythrocytes and its association with malaria clinical presentation

BackgroundThe pathogenesis of Plasmodium falciparum malaria is related to the ability of parasite‑infected erythrocytes (IEs) to adhere to the vascular endothelium (cytoadhesion/sequestration) or to surrounding uninfected erythrocytes (rosetting). Both processes are mediated by the expression of members of the clonally variant PfEMP1 parasite protein family on the surface of the IEs. Recent evidence obtained with laboratory-adapted clones indicates that P. falciparum can exploit human serum factors, such as IgM and α2-macroglobulin (α2M), to increase the avidity of PfEMP1-mediated binding to erythrocyte receptors, as well as to evade host PfEMP1-specific immune responses. It has remained unclear whether PfEMP1 variants present in field isolates share these characteristics, and whether they are associated with clinical malaria severity. These issues were investigated here.MethodsChildren 1–12 years reporting with P. falciparum malaria to Hohoe Municipal Hospital, Ghana were enrolled in the study. Parasites from children with uncomplicated (UM) and severe malaria (SM) were collected. Binding of α2M and IgM from non-immune individuals to erythrocytes infected by P. falciparum isolates from 34 children (UM and SM) were analysed by flow cytometry. Rosetting in the presence of IgM or α2M was also evaluated. Experimental results were analysed according to the clinical presentation of the patients.ResultsClinical data from 108 children classified as UM (n = 54) and SM cases (n = 54) were analysed. Prostration, severe malaria anaemia, and hyperparasitaemia were the most frequent complications. Three children were diagnosed with cerebral malaria, and one child died. Parasite isolates from UM (n = 14) and SM (n = 20) children were analysed. Most of the field isolates bound non-immune IgM (33/34), whereas the α2M-binding was less common (23/34). Binding of both non-immune IgM and α2M was higher but not significant in IEs from children with SM than from children with UM. In combination, IgM and α2M supported rosette formation at levels similar to that observed in the presence of 10% human serum.ConclusionsThe results support the hypothesis that binding of non-immune IgM and/or α2M to IEs facilitates rosette formation and perhaps contributes to P. falciparum malaria severity.

Antibody reactivity with new antigens revealed in multi-transgenic triple knockout pigs may cause early loss of pig kidneys in baboons.

Recent advances in gene editing technology have enabled the production of multi-knockout (KO) and transgenic pigs in order to overcome immunologic barriers in xenotransplantation (XTx). However, the genetic manipulations required to produce these changes may have the unintended consequence of producing or revealing neoantigens reactive with natural antibodies present in baboons. In this study, we examined whether the neoantigens that develop in multi-transgenic (mTg) GalT, Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH), β-1,4-N-acetyl-galactosaminyl transferase 2 (B4) KO pigs can cause rejection of xenografts in baboons. Five baboons that had <35% cytotoxicity against GalT-KO peripheral blood mononuclear cells (PBMCs) in a pre-screening assay received pig kidneys and vascularized thymic grafts (VT+K) from multi-transgenic hCD47, human thrombomodulin (hTBM), human endothelial protein C receptor (EPCR) with/without hCD46 and hCD55 with GalT-KO/NeuGC-KO/B4-KO (mTg Tri-KO) swine. In order to further examine the effects of anti-donor non-Gal natural antibody (nAb), anti-pig preformed IgM and IgG nAb binding against the GalT-KO PBMCs was compared with the donor-type PBMCs using donor pretransplant sera as well as 5 additional naïve baboon sera by flow cytometric analysis. Five baboons that received VT+K grafts had stable renal function in the first 11days (serum creatinine<1.5mg/dL). Two of the five baboons had higher binding of preformed IgG to mTg Tri-KO PBMCs than to GalT-KO PBMCs (mTg Tri-KO>GalT-KO), and they rejected their grafts at POD 20. In contrast, the other three baboons demonstrated either mTg Tri-KO=GalT-KO or mTg Tri-KO<GalT-KO, and they maintained renal function 43, 52, and 154days without rejection. Among 10 baboon sera, two had less antibody binding against PBMCs that were syngeneic to the mTg Tri-KO than against GalT-KO PBMCs (mTg Tri-KO<GalT-KO); three had similar binding to mTg Tri-KO and GalT-KO PBMCs (mTg Tri-KO=GalT-KO); and five had higher binding to m Tg Tri-KO than to GalT-KO PBMCs (mTg Tri-KO>GalT-KO). These data suggest that neoantigens associated with mTg Tri-KO promote acute xenograft rejection in a pig-to-baboon VT+K XTx model. The screening assays may be useful to select "safe" recipients to receive mTg Tri-KO kidneys.

NEW WORLD MONKEYS AS MODELS FOR TESTING TRIPLE-KNOCKOUT (TKO) PIG RED BLOOD CELL OR SKIN TRANSPLANTS

Background: TKO pigs are an optimal source of organs for transplantation into human recipients, who do not commonly have high levels of preformed antibodies against TKO pig cells. However, Old World monkeys (OWMs), e.g., baboons, do have antibodies directed to TKO pig cells, associated with significant serum cytotoxicity. New World monkeys (NWMs), e.g., capuchins, have low or no antibodies to TKO pig cells. Our aims were (i) to measure anti-pig IgM/IgG binding and (ii) complement-dependent cytotoxicity (CDC) to GTKO and TKO pig cells using sera from humans, baboons, and capuchins, and (iii) to measure the efficacy of anti-thymocyte globulin (ATG) and Rituximab in these three species. Methods: (i) Sera from humans (n=9), baboons (n=6), and capuchins (n=9) were tested by flow cytometry for binding of IgM and IgG to peripheral blood mononuclear cells (PBMCs) from GTKO and TKO pigs. Binding intensity was presented as relative geometric mean (rGM). (ii) The sera (final concentration 25%) were also tested for CDC to the same pig cells by flow cytometry. (iii) The effect of ATG and Rituximab on human (n=2), baboon (n=2), and capuchin (n=3) PBMCs or B cells deletion was measured by flow cytometry. Results: Binding of serum IgM/IgG to pig cells (Figure1) Mean serum anti-GTKO IgM and IgG binding were both significantly lower in baboons than in humans and capuchins (p<0.01). Mean baboon anti-TKO IgM binding was significantly higher than in humans and capuchins (p<0.01). Mean capuchin anti-TKO IgG was significantly lower than in humans and baboons (p<0.01). Serum CDC to pig cells (Figure1) Mean serum CDC of GTKO PBMCs did not differ between the three species. Baboon serum CDC of TKO PBMCs was significantly greater than in humans and capuchins (p<0.01), but there was no difference between humans and capuchins. The CDC of ATG and Rituximab on PBMCs and B cells (Figure2) ATG (25-50µg/ml) depleted fewer PBMCs in capuchins and baboons than in humans, but the depletion in capuchins was not significantly different from that in baboons, except at high concentration (100µg/ml). In contrast, the effect of Rituximab in depleting B cells in capuchins was very limited, and significantly less than in humans and baboons (p<0.05 or 0.01). Conclusions: When TKO pig organs are transplanted, OWMs, e.g., baboons, would be less than ideal as recipients as they do not mimic the human immune response. NWMs, e.g., capuchins, are to be preferred. However, their small size and the reduced effect of ATG, and, particularly, Rituximab, limit their use as recipients of organs, though they would be ideal for studies of TKO pig red blood cell transfusion or skin transplantation, where immunosuppressive therapy is not required.Work on xenotransplantation at the University of Alabama at Birmingham is supported in part by NIH NIAID U19 grant AI090959 and in part by a grant to UAB from United Therapeutics.

Evidence for GTKO/β4GalNT2KO Pigs as the Preferred Organ-source for Old World Nonhuman Primates as a Preclinical Model of Xenotransplantation.

Background.Triple-knockout (TKO) pigs (in which expression of the 3 known pig carbohydrate xenoantigens has been deleted) are likely to be an optimal source of organs for transplantation into human recipients, many of whom do not have natural antibodies against TKO pig cells. However, old world monkeys, for example, baboons, have natural antibodies directed to TKO cells (to a “fourth” xenoantigen that is exposed after TKO).Methods.We measured (1) anti-pig IgM/IgG binding, and (2) complement-dependent cytotoxicity (CDC), by flow cytometry to α1,3-galactosyltransfearse gene-knockout (GTKO), GTKO/β4GalNT2KO (that do not express the “fourth” xenoantigen), and TKO pig peripheral blood mononuclear cells (PBMCs) using 72 baboon sera (30 specific pathogen-free [SPF], and 42 non-SPF baboons).Results.Mean IgM antibody binding to GTKO/β4GalNT2KO pig PBMCs was significantly lower than to GTKO or TKO pig PBMCs (P < 0.01). Mean IgG antibody binding to GTKO/β4GalNT2KO pig PBMCs was significantly lower than to TKO PBMCs (P < 0.01). Mean CDC of GTKO/β4GalNT2KO pig PBMCs was significantly lower than of GTKO or TKO pig PBMCs (P < 0.01). SPF baboon serum IgM and IgG binding to, and CDC of, GTKO/β4GalNT2KO or TKO PBMCs were significantly lower than non-SPF baboon sera (P < 0.01).Conclusions.Although TKO pigs form the basis for proposed clinical trials of xenotransplantation, it is difficult to identify baboons with a low or negative CDC to TKO pigs. For pig-to-baboon organ transplantation, the use of GTKO/β4GalNT2KO pigs would be preferable. The use of SPF baboons as recipients might be a minor advantage.

Effect of intravenous immunoglobulin (IVIg) on primate complement-dependent cytotoxicity of genetically engineered pig cells: relevance to clinical xenotransplantation

Triple-knockout (TKO) pigs may be ideal sources of organs for clinical xenotransplantation because many humans have no preformed antibody to TKO pig cells. Intravenous immunoglobulin (IVIg) is widely used for severe infection or the treatment/prevention of antibody-mediated rejection in allotransplantation. Anti-pig antibodies in IVIg could be harmful in clinical xenotransplantation. It is unknown whether anti-TKO pig antibodies are present in IVIg. The main aim of this study was to investigate in vitro whether IVIg contains anti-TKO pig antibodies with cytotoxic effect to pig cells. Undiluted pooled human serum (HS) and five different commercial preparations of IVIg were tested for IgM and IgG binding to red blood cells (RBCs) from wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and TKO pigs by flow cytometry. Complement-dependent lysis of IVIg against these pig pRBCs was measured by hemolytic assay. Pooled HS and 4 of 5 IVIg commercial preparations contained anti-pig IgG that bound to WT and GTKO pRBCs, but not to TKO pRBCs. One preparation of IVIg contained antibodies that bound to TKO pRBCs, but there was no cytotoxicity of IVIg to TKO pRBCs. The results suggest that IVIg administration to human recipients of TKO pig grafts would be safe. However, the specific preparation of IVIg would need to be screened before its administration.

Relative content detection of oligomannose modification of IgM heavy chain induced by TNP-antigen in an early vertebrate through nanoLC-MS/MS

N-glycan modification is reported to be important in regulating the structure and function of immunoglobulins in mammals. While, the study on teleost immunoglobulin glycosylation is still limitted. In this study, we constructed a TNP-antigen driven model, and detected the site-specific N-glycans of PBS-immunized and TNP-specific Oreochromis niloticus serum IgM through 18O-labeling and nanoLC-MS/MS. These methods are widely used for peptide enrichment and protein modification identification, but rarely used in detecting the level of N-glycosylation in teleost Igs that driven by specific antigen. The results revealed that there are four N-glycosylation sites in O.niloticus IgM heavy chain, namely, the Asn-315 site in the CH2 domain, the Asn-338 site in the CH3 domain, and the Asn-509 and Asn-551 sites in the CH4 domain, All of the four residues were efficiently N-glycosylated. After immunized with TNP-antigen, the signal strength of oligomannose in the TNP-specific IgM in primary mass spectrometry was significantly higher than that in the PBS-immunized IgM. Notably, the TNP-specific IgM had an Asn-509 site fully occupied with oligomannose, while only a small amount of oligomannose was found in the PBS-immunized IgM of this site. N-glycans in other sites were mainly complex-type with a low content of fucosylation and sialylated. The oligomannose in TNP-specific IgM was further verified to be essential for the binding of IgM and MBL. These results demonstrated that the TNP-antigen induced the site-specific oligomannose modification of O.niloticus IgM heavy chain, and played an important role in the interaction of IgM and MBL, which provided insights into the evolutionary understanding of the IgM oligomannose modification and function.

Selective inhibition of anti-MAG IgM autoantibody binding to myelin by an antigen-specific glycopolymer.

Anti‐myelin‐associated glycoprotein (MAG) neuropathy is a disabling autoimmune peripheral neuropathy that is caused by circulating monoclonal IgM autoantibodies directed against the human natural killer‐1 (HNK‐1) epitope. This carbohydrate epitope is highly expressed on adhesion molecules such as MAG, a glycoprotein present in myelinated nerves. We previously showed the therapeutic potential of the glycopolymer poly(phenyl disodium 3‐O‐sulfo‐β‐d‐glucopyranuronate)‐(1→3)‐β‐d‐galactopyranoside (PPSGG) in selectively neutralizing anti‐MAG IgM antibodies in an immunological mouse model and ex vivo with sera from anti‐MAG neuropathy patients. PPSGG is composed of a biodegradable backbone that multivalently presents a mimetic of the HNK‐1 epitope. In this study, we further explored the pharmacodynamic properties of the glycopolymer and its ability to inhibit the binding of anti‐MAG IgM to peripheral nerves. The polymer selectively bound anti‐MAG IgM autoantibodies and prevented the binding of patients’ anti‐MAG IgM antibodies to myelin of non‐human primate sciatic nerves. Upon PPSGG treatment, neither activation nor inhibition of human and murine peripheral blood mononuclear cells nor alteration of systemic inflammatory markers was observed in mice or ex vivo in human peripheral blood mononuclear cells. Intravenous injections of PPSGG to mice immunized against the HNK‐1 epitope removed anti‐MAG IgM antibodies within less than 1 hr, indicating a fast and efficient mechanism of action as compared to a B‐cell depletion with anti‐CD20. In conclusion, these observations corroborate the therapeutic potential of PPSGG for an antigen‐specific treatment of anti‐MAG neuropathy. Read the Editorial Highlight for this article on page 465.

Old World Monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (Triple-Knockout)

Triple-knockout (TKO) pigs (with added protective human transgenes) are likely to be optimal sources of organs for clinical organ xenotransplantation because many humans have minimal or no natural antibody to TKO pig cells. However, Old World monkeys (OWMs) have naturally-existing antibodies directed to TKO cells. We measured anti-pig IgM/IgG binding, and complement-dependent cytotoxicity to wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and TKO pig peripheral blood mononuclear cells (PBMCs) using sera from humans, several OWMs, and two New World monkeys (NWMs). Furthermore, we compared survival of GTKO (n = 5) and TKO (n = 3) pig kidneys in baboons. OWMs had significantly greater IgM binding and cytotoxicity to TKO PBMCs than humans or NWMs. Mean anti-TKO IgM was significantly higher in OWMs and significantly lower in NWMs than in humans. Cytotoxicity of OWM sera to TKO PBMCs was significantly greater than of human serum, but there was no significant difference between human and NWM sera. The median survival of TKO pig kidneys (4 days) in baboons was significantly shorter than that of GTKO kidneys (136 days) (p < 0.05). Even though considered ideal for clinical xenotransplantation, the presence of naturally-existing antibodies to TKO pig cells in OWMs complicates the transplantation of TKO pig kidneys in OWMs.

The Detection of Serum IgMs to Thyroglobulin in Subacute Thyroiditis Suggests a Protective Role of IgMs in Thyroid Autoimmunity.

The role of serum immunoglobulin (Ig)Ms in autoimmune thyroid diseases is uncertain. We looked for IgMs to thyroglobulin (Tg) in patients with subacute thyroiditis (SAT), which is characterized by high serum Tg levels, the possible de novo appearance of IgGs to Tg (TgAb-IgGs), and no autoimmune sequelae. TgAb-IgMs and TgAb-IgGs were detected by binding to Tg using the enzyme-linked immunosorbent assay (ELISA). The upper reference limit of TgAb-IgMs and TgAb-IgGs was established in 40 normal subjects. We looked for TgAb-IgMs in 16 patients with SAT, 11 with Hashimoto's thyroiditis (HT), and 8 with Graves' disease (GD) who were all positive for TgAb-IgGs. IgM binding to bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), and glucagon in ELISA was measured. Inhibition of TgAb-IgMs binding to coated Tg was evaluated by preincubating serum samples or IgG-depleted samples with soluble Tg. TgAb-IgMs were positive in 10/16 patients with SAT, 2/11 with HT, and 1/8 with GD. TgAb-IgMs were higher in SAT (0.95; 0.42-1.13) (median; 25th-75th percentiles) than in HT (0.47; 0.45-0.51) and GD patients (0.35; 0.33-0.40) (P < .005 for both). IgM binding of SAT sera to BSA, KLH, and glucagon was significantly lower than Tg. Preincubation with soluble Tg reduced the binding of IgMs to coated Tg by 18.2% for serum samples and by 35.0% and 42.1% for 2 IgG-depleted samples. TgAb-IgM levels were inversely, although nonsignificantly, correlated with Tg concentrations. Tg leak associated with thyroid injury induces the production of specific TgAb-IgMs, which, in turn, increases the clearance of Tg and might prevent the establishment of a persistent thyroid autoimmune response.

Refining cell-based assay to detect MOG-IgG in patients with central nervous system inflammatory diseases

BackgroundGiven that the spectrum of myelin oligodendrocyte glycoprotein immunoglobulin G (MOG-IgG) associated disease is yet to be fully defined, development of sensitive and highly specific assays to identify MOG-IgG is crucial to precisely define the clinical phenotypes, disease courses and prognosis to describe the full spectrum of MOG-IgG associated diseases. Here, we aim to validate a new in-house live cell-based assay (CBA) for screening MOG-IgG in patients with central nervous system inflammatory diseases. MethodsWe generated a full length MOG transfected HEK293 stable cell line using pIRES2-eGFP vector. Sera from 355 patients with central nervous system inflammatory diseases and 25 healthy individuals were evaluated for MOG-IgG seropositivity using in-house cell-based immunofluorescence assay (CBA-IF). The specificity of IgG (H + L) and IgG1-Fc secondary antibodies as well as IgM binding were determined by cell-based flow cytometry (CBA-FACS). The optimal cut-offs for determining seropositivity in CBA-FACS were calculated and the concordance of CBA-IF score and CBA-FACS was studied. The results of our CBA-IF were compared with the Oxford CBA-IF. Results11.5% (41/355) of patients were seropositive for MOG-IgG and had clinical phenotypes that were within the known clinical spectrum of MOG-IgG associated diseases. No typical multiple sclerosis patients, aquaporin-4-IgG positive neuromyelitis optica spectrum disorder or healthy individuals were MOG-IgG seropositive. Using CBA-FACS, the anti-human IgG (H + L) was found to be comparable to IgG1-Fc antibody. No IgM binding was observed in all the samples tested. CBA-IF score and CBA-FACS yielded high correlation. The concordance of the NCC CBA-IF with the Oxford CBA-IF was 98%. ConclusionWe have developed MOG-IgG CBAs that have different characteristics and benefits but with high specificity and concordance. The complementary use of two methods and follow-up study with larger cohort will increase the clinical usefulness of MOG-IgG CBAs.

Disordered Antigens and Epitope Overlap Between Anti–Citrullinated Protein Antibodies and Rheumatoid Factor in Rheumatoid Arthritis

Anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) are commonly present in rheumatoid arthritis (RA) without a clear rationale for their coexistence. Moreover, autoantibodies develop against proteins with different posttranslational modifications and native proteins without obvious unifying characteristics of the antigens. We undertook this study to broadly evaluate autoantibody binding in seronegative and seropositive RA to identify novel features of reactivity. An array was created using a total of 172,828 native peptides, citrulline-containing peptides, and homocitrulline-containing peptides derived primarily from proteins citrullinated in the rheumatoid joint. IgG and IgM binding to peptides were compared between cyclic citrullinated peptide (CCP)-positive RF+, CCP+RF-, CCP-RF+, and CCP-RF- serum from RA patients (n = 48) and controls (n = 12). IgG-bound and endogenously citrullinated peptides were analyzed for amino acid patterns and predictors of intrinsic disorder, i.e., unstable 3-dimensional structure. Binding to IgG-derived peptides was specifically evaluated. Enzyme-linked immunosorbent assay confirmed key results. Broadly, CCP+RF+ patients had high citrulline-specific IgG binding to array peptides and CCP+RF- and CCP-RF+ patients had modest citrulline-specific IgG binding (median Z scores 3.02, 1.42, and 0.75, respectively; P < 0.0001). All RA groups had low homocitrulline-specific binding. CCP+RF+ patients had moderate IgG binding to native peptides (median Z score 2.38; P < 0.0001). The highest IgG binding was to citrulline-containing peptides, irrespective of protein identity, especially if citrulline was adjacent to glycine or serine, motifs also seen in endogenous citrullination in the rheumatoid joint. Highly bound peptides had multiple features predictive of disorder. IgG from CCP+RF+ patients targeted citrulline-containing IgG-derived peptides. Disordered antigens, which are frequently citrullinated, and common epitopes for ACPAs and RF are potentially unifying features for RA autoantibodies.

Ibrutinib Therapy Downregulates Toso, the Fcr for IgM, Expression in CLL Patients

Background: Toso, the FcR receptor (FcmR) for IgM, is a type I transmembrane protein belonging to the immunoglobulin gene superfamily. Toso is expressed in lymphocytes, and plays an important role in B cell development and survival. In normal B cells, Toso expression increases after B cell receptor (BCR) stimulation, and Toso enhances BCR signaling induced cell survival, NK-kB pathway activation, and BCL-xL expression. CLL B cells express high levels of Toso, especially in IgHV unmutated patients. Upon binding of IgM to Toso on CLL cells, the Toso-IgM complex is internalized and undergoes lysosomal degradation. Given the importance of BCR signaling in CLL pathogenesis and treatment, we performed a series of preclinical and correlative studies to examine the cross talk between Toso and BCR signaling, effects of Toso on CLL cell survival and how Toso is affected by BCR signaling inhibition with ibrutinib. Methods and Results: Toso gene expression (FAIM3) was analyzed in serial samples from eight CLL patients treated with ibrutinib plus rituximab using Affymetrics HG U133 plus 2.0 oligonucleotide arrays at baseline, and after 1 and 4 weeks of continuous ibrutinib-based therapy. We noted that the relative mean Toso gene expression declined during ibrutinib therapy (-0.26 ± 0.12 after 2 weeks and -0.3 ± 0.18 after 4 weeks). Accordingly, Toso surface expression on CLL cells, assessed by flow cytometry, also significantly decreased after 1 and 4 weeks of ibrutinib therapy (Figure 1). We next measured plasma levels of soluble Toso (sToso), which is encoded by an alternative spliced Toso transcript, in serial samples from 35 CLL ibrutinib treated patients using an enzyme-linked immunosorbent assay (ELISA). We noted that baseline sToso levels in CLL patients were significantly higher than in 6 normal controls, i.e. 70 ± 12.1 arbitrary units (AU, n=35) versus 18 ± 8 AU (n=6, p&lt;0.01). sToso levels significantly declined during ibrutinib therapy to 51.9 ± 7.5 AU (n=35, p&lt;0.01) after 1 week of treatment and further decrease in the subsequent time points to 11.9 ± 2.8 AU (p&lt;0.01) after 12 months of ibrutinib therapy. In addition, we found that patients with unmutated IgHV had higher sToso levels at baseline compared to patients with mutated IgHV (76.9 ± 10.8 AU (n=19) versus 45.4 ± 6.7 AU (mean ± SEM, n=11, p&lt;0.05)(Figure 1). Furthermore, time to normalization of sToso levels was longer in unmutated IgHV patients. We then interrogated effects of in vitro stimulation of Toso on CLL viability and BCR signaling-related chemokines CCL3 and CCL4, using mouse monoclonal antibodies (clone 2A5) or artificial human Fc Toso (hToso-Fc) for Toso triggering. After stimulation of CLL cells with 1 or 10 mg/mL of 2A5 antibody or hToso-FC for 24 hours, we noted significantly improved CLL cell survival and increased levels of CCL3 and CCL4 chemokine secretion, particularly in samples from IgHV unmutated patients. The mean relative CLL cell viability after Toso stimulation was 154.2 ± 23.6% compared to unstimulated controls for 2A5 and 140.9 ± 17.3% after hToso-Fc treatment (n=6, p&lt;0.05). Toso triggering induced very high levels of CCL3 and CCL4 secretion using 2A5 (3901 ± 464% of controls) or hToso-FC (6712 ± 1717% of controls, n=6, p&lt;0.05)(Figure 1). Conclusions: These studies demonstrate that ibrutinib therapy downregulates Toso (FAIM3) gene expression, as well as cell surface and soluble Toso expression. IgHV unmutated patients have higher levels of soluble Toso and the effect on Toso activation on viability and CCL3 and CCL4 secretion is more pronounced in this group of patients. These findings corroborate the close relation between BCR signaling and Toso in CLL. Disclosures Wierda: Cyclcel: Research Funding; Miragen: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Loxo Oncology Inc.: Research Funding; Janssen: Research Funding; Xencor: Research Funding; Acerta Pharma Inc: Research Funding; Pharmacyclics LLC: Research Funding; Genentech: Research Funding; AbbVie: Research Funding; GSK/Novartis: Research Funding; Gilead Sciences: Research Funding; Juno Therapeutics: Research Funding; KITE pharma: Research Funding; Sunesis: Research Funding. Burger:Janssen Pharmaceuticals: Consultancy, Honoraria; Aptose Biosciences, Inc: Research Funding; Gilead Sciences: Research Funding; Pharmacyclics, an AbbVie company: Research Funding; AstraZeneca: Honoraria; BeiGene: Research Funding.

Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.

Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation. Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.

Relationship of Free Fatty Acid and Natural Autoantibodies 2 Weeks Postpartum

Natural autoantibodies (NAAb) have been found in plasma and milk of dairy cows. Natural autoantibodies maintain homeostasis and may prevent infections. The objective of this study was to evaluate the relationship between concentration of plasma free fatty acid (FFA) and the levels of NAAb in plasma binding glutamate dehydrogenase (GD) and carbonic anhydrase (CA) in cows 2 weeks after calving. In total, 55 Holstein-Friesian dairy with 60-d dry period lengths and fed 2 early lactation diets (glucogenic or lipogenic). Blood was sampled at week 1 and 2 after calving. Result showed that from week 1 and 2 relative to calving, levels of IgG and IgM binding CA were positively related with concentration of FFA in plasma. Levels of IgM binding GD were negatively related with concentration of FFA in plasma. It is concluded that IgG and IgM binding CA in plasma might be an indicator for energy status and reflect metabolic status in dairy cows 2 weeks after calving.

Detection of Salmonid IgM Specific to the Piscine Orthoreovirus Outer Capsid Spike Protein Sigma 1 Using Lipid-Modified Antigens in a Bead-Based Antibody Detection Assay.

Bead-based multiplex immunoassays are promising tools for determination of the specific humoral immune response. In this study, we developed a multiplexed bead-based immunoassay for the detection of Atlantic salmon (Salmo salar) antibodies against Piscine orthoreovirus (PRV). Three different genotypes of PRV (PRV-1, PRV-2, and PRV-3) cause disease in farmed salmonids. The PRV outer capsid spike protein σ1 is predicted to be a host receptor binding protein and a target for neutralizing and protective antibodies. While recombinant σ1 performed poorly as an antigen to detect specific antibodies, N-terminal lipid modification of recombinant PRV-1 σ1 enabled sensitive detection of specific IgM in the bead-based assay. The specificity of anti-PRV-1 σ1 antibodies was confirmed by western blotting and pre-adsorption of plasma. Binding of non-specific IgM to beads coated with control antigens also increased after PRV infection, indicating a release of polyreactive antibodies. This non-specific binding was reduced by heat treatment of plasma. The same immunoassay also detected anti-PRV-3 σ1 antibodies from infected rainbow trout. In summary, a refined bead based immunoassay created by N-terminal lipid-modification of the PRV-1 σ1 antigen allowed sensitive detection of anti-PRV-1 and anti-PRV-3 antibodies from salmonids.