IgG Fraction Research Articles

Igome Graphs Suggest the Changes in the IgM and IgG Repertoires in Antiphospholipid Syndrome Are in Part Idiotypically Defined

Antiphospholipid Syndrome (APS) is characterized by the persistence of high-affinity antiphospholipid antibodies, manifesting clinically as pregnancy complications, thrombosis, or neurological symptoms. This study explores the changes in the IgM and IgG antibody repertoires in APS patients using igome graphs, which represent antibody repertoires through peptide libraries selected from phage display on the complete immunoglobulin fraction of the serum. Serum samples from healthy women and APS patients were processed to isolate IgM and IgG fractions, which were then used for phage selection and next-generation sequencing. Peptide sequences were analyzed using graph representation, clustering, and spectral embedding to identify significant differences between the repertoires. The study revealed that while IgM repertoires in APS patients exhibit a loss of public reactivities, the IgG repertoires do not follow this trend. Three orthogonal sequence motifs were identified in the IgM and IgG repertoires, suggesting potential idiotypic interactions. These findings highlight the intricate mechanisms of antibody selection and cross-reactivity in APS, offering insights into potential therapeutic approaches. The study underscores the importance of system-level analysis in understanding immune mechanisms and suggests that idiotypic interactions, though complex, could play a crucial role in APS pathogenesis.

Isolation of antibodies to hypochlorite-modified low-density lipoproteins from human serum and study of their specificity

BACKGROUND: Modified low-density lipoproteins have immunogenic properties and induce the production of antibodies. In this case, HOCl promotes the formation of subsequent active halogen-containing compounds interacting with proteins and lipid parts of low-density lipoproteins, which leads to their modification and the production of antibodies to them. AIM: The aim of this work is to isolate antibodies to hypochlorite modified low-density lipoproteins from human blood sera and study their specificity. MATERIALS AND METHODS: Malondialdehyde, acetic anhydride and sodium hypochlorite were used to obtain modified low-density lipoproteins. IgG antibodies to hypochlorite modified low-density lipoproteins were isolated by affinity chromatography. The total IgG fraction antibodies was previously isolated from human blood serum using MabSelect Xtra. The specific antibodies to hypochlorite modified low-density lipoproteins were isolated from this IgG pool by affinity chromatography. CNBr-Sepharose 4B conjugated with human serum albumin modified with NaOCl was used as a sorbent. The specificity of antibodies against hypochlorite modified low-density lipoproteins was tested using a competitive enzyme-linked immunosorbent assay. The competitors were hypochlorite modified low-density lipoproteins, acetic anhydride modified low-density lipoproteins and malondialdehyde modified low-density lipoproteins in concentrations (1–250 μg/ml). RESULTS: IgG antibodies against hypochlorite-modified proteins that interact with hypochlorite modified low-density lipoproteins were detected in human blood. According to ELISA date the binding of the isolated antibodies to hypochlorite modified low-density lipoproteins was almost completely inhibited only by appropriately modified low-density lipoproteins, that is, hypochlorite modified low-density lipoproteins, but not native low-density lipoproteins or acetic anhydride modified low-density lipoproteins. Malondialdehyde modified low-density lipoproteins also showed some competitive activity, but much weaker than hypochlorite modified low-density lipoproteins. Hypochlorite modified low-density lipoproteins itself and, to a lesser extent, malondialdehyde modified low-density lipoproteins competed for binding with antibodies of human serum to hypochlorite modified low-density lipoproteins. Acetic anhydride modified low-density lipoproteins and native low-density lipoproteins did not reduce the efficiency of antibody binding to their antigen. CONCLUSIONS: Hypochlorite modified low-density lipoproteins forms epitopes independent of other low-density lipoproteins modifications studied. These epitopes are responsible for the formation of specific antibodies.

Natural history of chronic idiopathic neutropenia of the adult

Chronic idiopathic neutropenia (CIN) is a rare benign condition caused by an immune attack against neutrophils, either primary or in the context of other autoimmune conditions, lymphoproliferative syndromes, and inborn errors of immunity. In this single-center prospective study, 131 adult CIN patients were enrolled (median age 55 years, range: 20–93). At baseline, 56% had anti-neutrophil autoantibodies and 31% had autoimmune comorbidities. Over a median 3-year follow-up, the rate of grade ≥ 2 infections was 42%, with 10% grade ≥ 3, irrespective of neutrophil counts, demographics, and anti-neutrophil antibodies positivity, and G-CSF was used in 6 patients only. No malignant evolution nor deaths were observed. Bone marrow evaluation showed a large granular lymphocyte (LGL) infiltrate in 52%, mostly polyclonal, and hypocellularity in 31% of cases. Immune-histochemistry highlighted deposits of IgG, IgM, and complement fractions C3 and C4d in most cases. Interestingly, 19% of tested patients displayed somatic mutations of myeloid genes with an association with age. In conclusion, adult CIN appears to be a benign condition without life-threatening infections, yet deserving an extensive hematologic evaluation including bone marrow assessment to inform the differential diagnosis.

Balantidium Coli Colitis Revealing a Common Variable Immunodeficiency: A Case Report

Balantidium Coli is a large protozoan parasite, measuring up to 150 micrometers, initially identified in 1857 and later renamed in 1861-1862. It is an animal parasite, with its primary reservoir being pigs (domestic pigs/wild boars), but it can also be found in other animal species such as dogs, cats, hyenas, buffaloes, camels, monkeys, and others. Humans are an accidental host, as they represent a dead-end in the parasitic life cycle. Infection by this parasite is typically asymptomatic or presents with mild symptoms. It becomes pathogenic only in cases of primary or secondary immunodeficiency, where it typically manifests as colitis; however, urinary tract involvement is also possible. In our case, Balantidium Coli infection presented multiple challenges. It is not a well-known parasite in our region, particularly since pig farming is not practiced in Morocco. The parasite was found in both the urine and stool of the patient, who exhibited clear signs of infection which is not usual with this parasite. This led to the "late" discovery of Common Variable Immunodeficiency (CVID) in the patient, a primary immunodeficiency typically diagnosed before the age of 4 and characterized by a deficiency in immunoglobulin production, particularly affecting the IgA and IgG fractions.

Expanding anti-venom strategies: Camelid polyclonal antibodies with high capacity to recognize snake venom

Camelid immunoglobulins represent a unique facet of antibody biology, challenging conventional understandings of antibody diversification. IgG2 and IgG3 in particular are composed solely of heavy chains and exhibit a reduced molecular weight (90 kDa); their elongated complementarity determining region (CDR) loops play a pivotal role in their functioning, delving deep into enzyme active sites with precision. Serum therapy stands as the primary venom-specific treatment for snakebite envenomation, harnessing purified antibodies available in diverse forms such as whole IgG, monovalent fragment antibody (Fab), or divalent fragment antibody F (ab')2. This investigation looks into the intricacies of IgGs derived from camelid serum previously immunized with crotamine and crotoxin, toxins predominantly in Crotalus durissus venom, exploring their recognition capacity, specificity, and cross-reactivity to snake venoms and its toxins. Initially, IgG purification employed affinity chromatography via protein A and G columns to segregate conventional antibodies (IgG1) from heavy chain antibodies (IgG2 and IgG3) of camelid isotypes sourced from Lama glama serum. Subsequent electrophoretic analysis (SDS-PAGE) revealed distinct bands corresponding to molecular weight profiles of IgG's fractions representing isotypes in Lama glama serum. ELISA cross-reactivity assays demonstrated all three IgG isotypes' ability to recognize the tested venoms. Notably, IgG1 exhibited the lowest interactivity in analyses involving bothropic and crotalic venoms. However, IgG2 and IgG3 displayed notable cross-reactivity, particularly with crotalic venoms and toxins, albeit with exceptions such as PLA2-CB, showing reduced reactivity, and C. atrox, where IgGs exhibited insignificant reactivity. In Western blot assays, IgG2 and IgG3 exhibited recognition of proteins within molecular weight (≈15 kDa) of C. d. collilineatus to C. d. terrificus, with some interaction observed even with bothropic proteins despite lower reactivity. These findings underscore the potential of camelid heavy-chain antibodies, suggesting Lama glama IgGs as prospective candidates for a novel class of serum therapies. However, further investigations are imperative to ascertain their suitability for serum therapy applications.

Factors Associated with Neutralizing Antibody Responses following 2-Dose and 3rd Booster Monovalent COVID-19 Vaccination in Japanese People Living with HIV.

People living with HIV (PLWH) could be at risk of blunted immune responses to COVID-19 vaccination. We investigated factors associated with neutralizing antibody (NAb) responses against SARS-CoV-2 and variants of concern (VOCs), following two-dose and third booster monovalent COVID-19 mRNA vaccination in Japanese PLWH. NAb titers were assessed in polyclonal IgG fractions by lentiviral-based pseudovirus assays. Overall, NAb titers against Wuhan, following two-dose vaccination, were assessed in 82 PLWH on treatment, whereby 17/82 (20.73%) were classified as low-NAb participants. Within the low-NAb participants, the third booster vaccination enhanced NAb titers against Wuhan and VOCs, albeit to a significantly lower magnitude than the rest. In the multivariate analysis, NAb titers against Wuhan after two-dose vaccination correlated with age and days since vaccination, but not with CD4+ count, CD4+/CD8+ ratio, and plasma high-sensitivity C-Reactive protein (hsCRP). Interestingly, an extended analysis within age subgroups revealed NAb titers to correlate positively with the CD4+ count and negatively with plasma hsCRP in younger, but not older, participants. In conclusion, a third booster vaccination substantially enhances NAb titers, but the benefit may be suboptimal in subpopulations of PLWH exhibiting low titers at baseline. Considering clinical and immune parameters could provide a nuanced understanding of factors associated with vaccine responses in PLWH.

Intramuscular vaccination against SARS-CoV-2 transiently induces neutralizing IgG rather than IgA in the saliva.

The mucosal immunity is crucial for restricting SARS-CoV-2 at its entry site. Intramuscularly applied vaccines against SARS-CoV-2 stimulate high levels of neutralizing Abs in serum, but the impact of these intramuscular vaccinations on features of mucosal immunity is less clear. Here, we analyzed kinetic and functional properties of anti-SARS-CoV-2 Abs in the saliva after vaccination with BNT162b2. We analyzed a total of 24 healthy donors longitudinally for up to 16 months. We found that specific IgG appeared in the saliva after the second vaccination, declined thereafter and reappeared after the third vaccination. Adjusting serum and saliva for the same IgG concentration revealed a strong correlation between the reactivity in these two compartments. Reactivity to VoCs correlated strongly as seen by ELISAs against RBD variants and by live-virus neutralizing assays against replication-competent viruses. For further functional analysis, we purified IgG and IgA from serum and saliva. In vaccinated donors we found neutralizing activity towards authentic virus in the IgG, but not in the IgA fraction of the saliva. In contrast, IgA with neutralizing activity appeared in the saliva only after breakthrough infection. In serum, we found neutralizing activity in both the IgA and IgG fractions. Together, we show that intramuscular mRNA vaccination transiently induces a mucosal immunity that is mediated by IgG and thus differs from the mucosal immunity after infection. Waning of specific mucosal IgG might be linked to susceptibility for breakthrough infection.

Anti-Candida Antibodies of Patients with Invasive Candidiasis Inhibit Growth, Alter Cell Wall Structure, and Kill Candida albicans In Vitro

Invasive candidiasis (IC), caused by Candida yeasts, particularly Candida albicans, poses a significant threat with high mortality rates. Diagnosis is challenging due to Candida's common presence in human microbiota. To address this, our research group developed an immunofluorescence assay detecting Candida albicans Germ Tube Antibodies (CAGTA) in IC patients. CAGTA, indicative of invasive processes, is associated with a lower mortality rate in ICU patients. Based on this premise, this study aims to provide results regarding the lack of knowledge about the potential activity of CAGTA against invasive infections in humans caused by the fungus Candida albicans. Therefore, in order to characterize the activity of CAGTA produced by patients with IC, we used sera from 29 patients with IC caused by either C. albicans or non-albicans Candida species. Whole serum IgG antibodies were fractionated into anti-blastospores, CAGTA-enriched, and purified CAGTA and the assessments included XTT colorimetric assays for metabolic activity, CFU counts for viability, and microscopy for growth, viability, and morphological analysis. The CAGTA-enriched IgG fraction significantly reduced the metabolic activity and viability of C. albicans compared to anti-blastospores. Purified CAGTA altered germ tube cell wall surfaces, as revealed by electron microscopy, and exhibited fungicidal properties by DiBAC fluorescent staining. In conclusion, antibodies in response to invasive candidiasis have antifungal activity against Candida albicans, influencing metabolic activity, viability, and cell wall structure, leading to cell death. These findings suggest the potential utility of CAGTA as diagnostic markers and support the possibility of developing immunization protocols against Candida infections.

BAFF neutralization impairs the autoantibody-mediated clearance of dead adipocytes and aggravates obesity-induced insulin resistance.

B cell-activating factor (BAFF) is a critical TNF-family cytokine that regulates homeostasis and peripheral tolerance of B2 cells. BAFF overproduction promotes autoantibody generation and autoimmune diseases. During obesity, BAFF is predominantly produced by white adipose tissue (WAT), and IgG autoantibodies against adipocytes are identified in the WAT of obese humans. However, it remains to be determined if the autoantibodies formed during obesity affect WAT remodeling and systemic insulin resistance. Here, we show that IgG autoantibodies are generated in high-fat diet (HFD)-induced obese mice that bind to apoptotic adipocytes and promote their phagocytosis by macrophages. Next, using murine models of obesity in which the gonadal WAT undergoes remodeling, we found that BAFF neutralization depleted IgG autoantibodies, increased the number of dead adipocytes, and exacerbated WAT inflammation and insulin resistance. RNA sequencing of the stromal vascular fraction from the WAT revealed decreased expression of immunoglobulin light-chain and heavy-chain variable genes suggesting a decreased repertoire of B cells after BAFF neutralization. Further, the B cell activation and the phagocytosis pathways were impaired in the WAT of BAFF-neutralized mice. In vitro, plasma IgG fractions from BAFF-neutralized mice reduced the phagocytic clearance of apoptotic adipocytes. Altogether, our study suggests that IgG autoantibodies developed during obesity, at least in part, dampens exacerbated WAT inflammation and systemic insulin resistance.

Oral immunization with Shigella sonnei WRSs2 and WRSs3 vaccine strains elicits systemic and mucosal antibodies with functional anti-microbial activity.

Shigella causes bacillary dysentery and is responsible for a high burden of disease globally. Several studies have emphasized the value of functional antibody activity to understand Shigella immunity and correlates of protection. The anti-microbial function of local (mucosal) antibodies and their contribution to preventing Shigella infection remain unknown. The goal of this study was to identify the functional humoral immune effectors elicited by two Shigella sonnei live oral vaccine candidates, WRSs2 and WRSs3. Complement-dependent bactericidal [serum bactericidal antibody (SBA)/bactericidal antibody (BA)] and opsonophagocytic killing antibody (OPKA) activity were determined in sera and stool extracts as indicators of systemic and local anti-microbial immunity. High levels of SBA/BA and OPKA were detected in serum as well as in fecal extracts from volunteers who received a single dose of WRSs2 and WRSs3. Functional antibody activity peaked on days 10 and 14 post-vaccination in fecal and serum samples, respectively. Bactericidal and OPKA titers were closely associated. Peak fold rises in functional antibody titers in serum and fecal extracts were also associated. Antibody activity interrogated in IgG and IgA purified from stool fractions identified IgG as the primary driver of mucosal bactericidal and OPKA activity, with minimal functional activity of IgA alone, highlighting an underappreciated role for IgG in bacterial clearance in the mucosa. The combination of IgG and IgA in equal proportions enhanced bactericidal and OPKA titers hinting at a co-operative or synergistic action. Our findings provide insight into the functional anti-microbial capacity of vaccine-induced mucosal IgG and IgA and propose an operative local humoral effector of protective immunity.IMPORTANCEThere is an urgent need for a safe, effective, and affordable vaccine against Shigella. Understanding the immunological underpinning of Shigella infection and the make-up of protective immunity is critical to achieve the best approach to prevent illness caused by this mucosal pathogen. We measured the complement-dependent bactericidal and opsonophagocytic antibody killing in serum and stool extracts from adult volunteers vaccinated with Shigella sonnei live oral vaccine candidates WRSs2 and WRSs3. For the first time, we detected functional antibody responses in stool samples that were correlated with those in sera. Using purified stool IgA and IgG fractions, we found that functional activity was mediated by IgG, with some help from IgA. These findings provide insight into the functional anti-microbial capacity of vaccine-induced mucosal IgG and IgA and support future studies to identify potential markers of protective mucosal immunity.

Distinct patterns of auto-reactive antibodies associated with organ-specific immune-related adverse events.

ClinicalTrials.gov identifier: NCT03391869.

Coagulation Parameters in Post-Covid-19 Condition in Relation to Various Titers of Anti-SARS-CoV-2 IgG in Blood Plasma.

Post-COVID-19 condition is thought to affect 10-20% of people at least 3 months after a diagnosis of COVID-19 and two months of symptoms. Post-COVID-19 condition presents itself with many clinical effects with varying degrees of severity ranging from a mild cough to a life-threatening coagulopathy. Our study aimed to identify a relationship between the titers of anti-SARS-CoV-2 IgG and anticoagulation parameters: antithrombin III (ATIII), protein C (PC) and thrombomodulin (TM). Blood plasma was collected from healthy donors aged 25-45 who had recovered from COVID-19 3-6 months ago and their titers of anti-SARS-CoV-2 IgG and ATIII, PC, and TM were measured. We found that concentrations and activities of key anticoagulation parameters (ATIII, PC, and TM) measured in donor plasma during the post-COVID-19 varied in relation to the titers of anti-SARS-CoV-2 IgG. While we identified a dysfunction of anticoagulation parameters in patients with post-COVID-19, we aim to explore the subpopulation antibody IgG fraction directly using in vivo and in vitro experiments with the possibility to contribute to the development of treatment options for post-COVID-19 conditions.

Activity of domain III-specific antibodies in early convalescence: A case study

The Dengue virus complex (DENV), formed by four serotypes, constitutes the most important arbovirus affecting humans. The structural domain III of their envelope protein (DIII) elicits strongly neutralizing serotype-specific antibodies. Contrasting results have been obtained regarding their role in the serum neutralizing activity of infected patients. We used a DENV immune serum from a secondary infection to examine the impact of characterizing the anti-DIII antibody response after affinity purification with recombinant DIII proteins to eliminate potential interferences from the interactions with human plasma proteins and other anti-DENV antibodies. Total anti-DENV IgG repertoire and anti-DIIIE antibodies were compared in functionality. In early convalescence, reactivity of anti-DIII antibodies is serotype specific and exhibits the strongest reactivity with infecting serotypes. Purification of anti-DIII antibodies emphasizes the reactivity profile as compared to total IgG fraction and serum. Serotype-specificity of the virus neutralization activity correlated with the apparent kD of the binding to recombinant DIIIs.

NMOSD IgG Impact Retinal Cells in Murine Retinal Explants.

Neuromyelitis optica spectrum disorders (NMOSD) are chronic inflammatory diseases of the central nervous system, characterized by autoantibodies against aquaporin-4. The symptoms primarily involve severe optic neuritis and longitudinally extensive transverse myelitis. Although the disease progression is typically relapse-dependent, recent studies revealed retinal neuroaxonal degeneration unrelated to relapse activity, potentially due to anti-aquaporin-4-positive antibodies interacting with retinal glial cells such as Müller cells. In this exploratory study, we analysed the response of mouse retinal explants to NMOSD immunoglobulins (IgG). Mouse retinal explants were treated with purified IgG from patient or control sera for one and three days. We characterized tissue response patterns through morphological changes, chemokine secretion, and complement expression. Mouse retinal explants exhibited a basic proinflammatory response ex vivo, modified by IgG addition. NMOSD IgG, unlike control IgG, increased gliosis and decreased chemokine release (CCL2, CCL3, CCL4, and CXCL-10). Complement component expression by retinal cells remained unaltered by either IgG fraction. We conclude that human NMOSD IgG can possibly bind in the mouse retina, altering the local cellular environment. This intraretinal stress may contribute to retinal degeneration independent of relapse activity in NMOSD, suggesting a primary retinopathy.

Identification of Eimeria tenella sporozoite immunodominant mimotopes by random phage-display peptide libraries-a proof of concept study.

Coccidiosis, caused by parasites of numerous Eimeria species, has long been recognized as an economically significant disease in the chicken industry worldwide. The rise of anti-coccidian resistance has driven a search for other parasite management techniques. Recombinant antigen vaccination presents a highly feasible alternative. Properly identifying antigens that might trigger a potent immune response is one of the major obstacles to creating a viable genetically modified vaccine. This study evaluated a reverse immunology approach for the identification of B-cell epitopes. Antisera from rabbits and hens inoculated with whole-sporozoites of E. tenella were used to identify Western blot antigens. The rabbit IgG fraction from the anti-sporozoite serum exhibited the highest reactogenicity; consequently, it was purified and utilized to screen two random Phage-display peptide libraries (12 mer and c7c mer). After three panning rounds, 20 clones from each library were randomly selected, their nucleotide sequences acquired, and their reactivity to anti-sporozoite E. tenella serum assessed. The selected peptide clones inferred amino acid sequences matched numerous E. tenella proteins. The extracellular domain of the epidermal growth factor-like (EGF-like) repeats, and the thrombospondin type-I (TSP-1) repeats of E. tenella micronemal protein 4 (EtMIC4) matched with the c7c mer selected clones CNTGSPYEC (2/20) and CMSTGLSSC (1/20) respectively. The clone CSISSLTHC that matched with a conserved hypothetical protein of E. tenella was widely selected (3/20). Selected clones from the 12-mer phage display library AGHTTQFNSKTT (7/20), GPNSAFWAGSER (2/20) and HFAYWWNGVRGP (8/20) showed similarities with a cullin homolog, elongation factor-2 and beta-dynein chain a putative E. tenella protein, respectively. Four immunodominant clones were previously selected and used to immunize rabbits. By ELISA and Western blot, all rabbit anti-clone serums detected E. tenella native antigens. Thus, selected phagotopes contained recombinant E. tenella antigen peptides. Using antibodies against E. tenella sporozoites, this study demonstrated the feasibility of screening Phage-display random peptide libraries for true immunotopes. In addition, this study looked at an approach for finding novel candidates that could be used as an E. tenella recombinant epitope-based vaccine.

Repeated Traumatic Brain Injury is Associated with Neurotoxic Plasma Autoantibodies Directed against the Serotonin 2A and Alpha 1 Adrenergic Receptors.

Traumatic brain injury (TBI) was associated with increased plasma agonist autoantibodies targeting the serotonin 2A receptor. Repeated TBI exposure is associated with high risk for neurodegenerative and neuropsychiatric complications. Here we tested a hypothesis that repeated TBI is associated with plasma agonist autoantibodies targeting more than one kind of catecholamine G-protein coupled receptor. Protein-A affinity chromatography was used to isolate the IgG fraction of plasma in forty-two middle-aged and older adults who had experienced one or more TBI exposures. The Ig (1/40th dilution=7.5 ug/mL) were tested for neurotoxicity in mouse neuroblastoma cells using an acute neurite retraction assay indicative of Gq11/IP3/Ca2+ and RhoA/Rho kinase signaling pathways' activation. Three different linear synthetic peptides corresponding to the second extracellular loop of the alpha 1A, alpha 2A or serotonin 2A receptors were used as target antigen in different enzyme-linked immunoassays. The second extracellular loop receptor peptides themselves (alpha 1A, alpha 2A) or a fragment (serotonin 2A) were tested for ability to prevent Ig-induced neurite retraction. Patients who had experienced either repeated TBI (N=10) or a single TBI with a co-morbid autoimmune disease (N=5) were significantly more likely to harbor neurotoxic plasma autoantibodies targeting both alpha 1 adrenergic and serotonin 2A receptors vs. patients having only a single TBI. Ig-induced neurotoxicity was significantly prevented by co-incubation with either 850 nM prazosin (alpha 1 adrenergic receptor) and/or 500 nM M100907 (serotonin 2A receptor) antagonists. Alpha 1 adrenergic receptor and serotonin 2A receptor Ig immunoreactive level and titer were significantly increased in repeated TBI and single TBI/autoimmune patients (N=7-8) compared to age-matched TBI patients without neurotoxic plasma Ig (N=4). SN.8, a linear synthetic peptide corresponding to a conserved region of the second extracellular loop (ECL) of the serotonin 2A receptor completely prevented neurite retraction induced by repeated TBI plasma Ig. A repeated TBI patient harboring alpha adrenergic receptor AAB alone experienced prospective steep decline in cognitive function over two years. Repeated TBI and TBI with associated autoimmunity harbored more than one kind of neurotoxic catecholaminergic agonist GPCR autoantibody each associated with high risk for steep rate of cognitive decline. Specific immunoassays using the second extracellular receptor loop as target antigen are needed to detect each specific different GPCR autoantibody. A fragment of the second ECL of the serotonin 2A receptor (SN.8) neutralized Ig-induced neurotoxicity in repeated TBI or TBI with associated systemic autoimmunity.

Evaluation of immunogenicity of enterobactin conjugate vaccine for the control of Escherichia coli mastitis in dairy cows

Mastitis is the most common disease of dairy cows that incurs severe economic losses to the dairy industry. Currently, environmental mastitis pathogens are a major problem for most dairy farms. A current commercially available E. coli vaccine does not prevent clinical mastitis and production losses, likely due to antibody accessibility and antigenic variation issues. Therefore, a novel vaccine that prevents clinical disease and production losses is critically needed. Recently a nutritional immunity approach, which restricts bacterial iron uptake by immunologically sequestering conserved iron-binding enterobactin (Ent), has been developed. The objective of this study was to evaluate the immunogenicity of the Keyhole Limpet Hemocyanin-Enterobactin (KLH-Ent) conjugate vaccine in dairy cows. Twelve pregnant Holstein dairy cows in their 1st - 3rd lactations were randomized to the control or vaccine group, with 6 cows per group. The vaccine group received 3 subcutaneous vaccinations of KLH-Ent with adjuvants at drying off (D0), 20 (D21) and 40 (D42) days after drying off. The control group was injected with phosphate-buffered saline (pH 7.4) mixed with the same adjuvants at the same time points. Vaccination effects were assessed over the study period until the end of the first month of lactation. The KLH-Ent vaccine did not cause any systemic adverse reactions or reduction in milk production. Compared with the control group, the vaccine elicited significantly higher levels of serum Ent-specific IgG at calving (C0) and 30 d post calving (C30), mainly its IgG2 fraction, which was significantly higher at D42, C0, C14 and C30 d, with no significant change in IgG1 levels. Milk Ent-specific IgG and IgG2 levels in the vaccine group were significantly higher on C30. Fecal microbial community structures were similar for both control and vaccine groups on the same day and shifted directionally along the sampling days. In conclusion, the KLH-Ent vaccine successfully triggered strong Ent-specific immune responses in dairy cows without significantly affecting the gut microbiota diversity and health. The results show that Ent conjugate vaccine is a promising nutritional immunity approach in control of E. coli mastitis in dairy cows.

Development of Negative Controls for Fc-C-Type Lectin Receptor Probes.

Fc-C-type lectin receptor (Fc-CTLRs) probes are soluble chimeric proteins constituted of the extracellular domain of a CTLR fused with the constant fraction (Fc) of the human IgG. These probes are useful tools to study the interaction of CTLRs with their ligands, with applications similar to those of antibodies, often in combination with widely available fluorescent antibodies targeting the Fc fragment (anti-hFc). In particular, Fc-Dectin-1 has been extensively used to study the accessibility of β-glucans at the surface of pathogenic fungi. However, there is no universal negative control for Fc-CTLRs, making the distinction of specific versus nonspecific binding difficult. We describe here 2 negative controls for Fc-CTLRs: a Fc-control constituting of only the Fc portion, and a Fc-Dectin-1 mutant predicted to be unable to bind β-glucans. Using these new probes, we found that while Fc-CTLRs exhibit virtually no nonspecific binding to Candida albicans yeasts, Aspergillus fumigatus resting spores strongly bind Fc-CTLRs in a nonspecific manner. Nevertheless, using the controls we describe here, we were able to demonstrate that A. fumigatus spores expose a low amount of β-glucan. Our data highlight the necessity of appropriate negative controls for experiments involving Fc-CTLRs probes. IMPORTANCE While Fc-CTLRs probes are useful tools to study the interaction of CTLRs with ligands, their use is limited by the lack of appropriate negative controls in assays involving fungi and potentially other pathogens. We have developed and characterized 2 negative controls for Fc-CTLRs assays: Fc-control and a Fc-Dectin-1 mutant. In this manuscript, we characterize the use of these negative controls with zymosan, a β-glucan containing particle, and 2 human pathogenic fungi, Candida albicans yeasts and Aspergillus fumigatus conidia. We show that A. fumigatus conidia nonspecifically bind Fc-CTLRs probes, demonstrating the need for appropriate negative controls in such assays.

Analyzing schizophrenia-related phenotypes in mice caused by autoantibodies against NRXN1α in schizophrenia

The molecular pathological mechanisms underlying schizophrenia remain unclear; however, genomic analysis has identified genes encoding important risk molecules. One such molecule is neurexin 1α (NRXN1α), a presynaptic cell adhesion molecule. In addition, novel autoantibodies that target the nervous system have been found in patients with encephalitis and neurological disorders. Some of these autoantibodies inhibit synaptic antigen molecules. Studies have examined the association between schizophrenia and autoimmunity; however, the pathological data remain unclear. Here, we identified a novel autoantibody against NRXN1α in patients with schizophrenia (n = 2.1%) in a Japanese cohort (n = 387). None of the healthy control participants (n = 362) were positive for anti-NRXN1α autoantibodies. Anti-NRXN1α autoantibodies isolated from patients with schizophrenia inhibited the molecular interaction between NRXN1α and Neuroligin 1 (NLGN1) and between NRXN1α and Neuroligin 2 (NLGN2). Additionally, these autoantibodies reduced the frequency of the miniature excitatory postsynaptic current in the frontal cortex of mice. Administration of anti-NRXN1α autoantibodies from patients with schizophrenia into the cerebrospinal fluid of mice reduced the number of spines/synapses in the frontal cortex and induced schizophrenia-related behaviors such as reduced cognition, impaired pre-pulse inhibition, and reduced social novelty preference. These changes were improved through the removal of anti-NRXN1α autoantibodies from the IgG fraction of patients with schizophrenia. These findings demonstrate that anti-NRXN1α autoantibodies transferred from patients with schizophrenia cause schizophrenia-related pathology in mice. Removal of anti-NRXN1α autoantibodies may be a therapeutic target for a subgroup of patients who are positive for these autoantibodies.

Intact Transition Epitope Mapping—Serological Inspection by Epitope EXtraction (ITEM—SIX)

Precision medicine requests accurate serological inspections to precisely stratify patients for targeted treatment. Intact transition epitope mapping analysis proved surrogate seroconversion of a model organism's serum when spiked with a monoclonal murine anti-Ovalbumin antibody (mAb) with epitope resolution. Isolation of the IgG fraction from blood serum applied two consecutive protein precipitation steps followed by ultrafiltration and resulted in an ESI-MS analysis-ready IgG preparation. For epitope mapping by epitope extraction, the Ovalbumin antigen was digested with trypsin. After desalting, the peptide mixture was added to the ESI-MS-ready IgG preparation from mAb-spiked serum and the solution was incubated to form an immune complex between the Ovalbumin-derived epitope peptide and the anti-Ovalbumin mAb. Then, the entire mixture of proteins and peptides was directly electrosprayed. Sorting of ions in the mass spectrometer's gas phase, dissociation of the immune complex ions by collision-induced dissociation, and recording of the epitope peptide ion that had been released from the immune complex proved the presence of the anti-Ovalbumin mAb in serum. Mass determination of the complex-released epitope peptide ion with isotope resolution is highly accurate, guaranteeing high specificity of this novel analysis approach, which is termed Intact Transition Epitope Mapping-Serological Inspections by Epitope EXtraction (ITEM-SIX).