IgG Subclasses Research Articles

Autoantibody mediated deficiency of IL-36-receptor antagonist in a subset of patients with psoriasis and psoriatic arthritis

ObjectivePsoriatic arthritis (PsA) is known as a seronegative form of spondylarthropathy. The interleukin-36 cytokine family may have a major role in disease pathogenesis and particularly the related cutaneous manifestations. In light of our recent observations on (transient) autoantibody phenotypes neutralizing endogenous anti-inflammatory receptor antagonists (progranulin, IL-1Ra) in different inflammatory conditions, we set out to investigate the potential role of such antibodies targeting IL-36 cytokine family members in PsA and psoriasis without arthritic manifestations (Pso). MethodsIn the present study we screened for hypothetic autoantibodies against the anti-inflammatory mediators IL-36 receptor antagonist (IL-36Ra) and anti-inflammatory IL-38 in PsA, Pso and inflammatory and healthy controls. Serum samples of patients with PsA (n = 254), Pso (n = 100), systemic lupus erythematosus (SLE, n = 50), rheumatoid arthritis (RA, n = 100), ulcerative colitis (UC, n = 50), Crohn´s disease (CD, n = 50), and healthy controls (n = 237) were screened for autoantibodies against IL-36Ra and IL-38 as well as IL-36Ra levels by ELISA. Biochemical analysis for immune complexes and atypic protein isoforms as well as IL-36 signaling reporter assays were performed. ResultsAnti-IL-36Ra antibodies were detected in five out of 100 (5.0 %) patients with Pso, in 12 of 254 (4.72 %) patients with PsA and in one of 50 (2 %) patients with CD, but in none of the other investigated inflammatory or healthy controls. The IL-36Ra autoantibodies belonged to the IgG1 subclass and their titers ranged between 1:200 to 1:1600. They resulted in immune-complex formation, depletion of serum IL-36Ra levels and were functional in terms of facilitating unrestricted IL-36 signaling. ConclusionIL-36Ra autoantibodies were found in subgroups of patients with Pso and PsA and may drive respective pathology.

Rational use of immunoglobulins (IVIgs and SCIgs) in secondary antibody deficiencies.

Immunoglobulins for intravenous use (IVIgs) and subcutaneous use (SCIgs) can prevent recurrent and severe infections in patients with secondary antibody deficiencies that are frequently linked to haematological/oncological malignancies as well as other clinical conditions and their respective treatments. Even so, as IVIgs and SCIgs are costly and their supply is limited, their clinical use must be optimised. The aim of this position paper is to provide structured practical guidance on the optimal use of IVIgs and SCIgs in secondary antibody deficiencies, particularly in haematological and oncological practice. The authors agree that the occurrence of severe infections is a prerequisite for the use of IVIgs. Serum IgG levels in general as well as IgG subclass levels can be additional indicators of whether a patient could benefit from IVIgs. Responsiveness to vaccines can help to identify immunodeficiency. Patients with chronic lymphocytic leukaemia or multiple myeloma who are receiving respective treatment, especially B-cell depletion therapy, but also some patients with autoimmune diseases are prone to antibody deficiencies and need IVIgs. For the optimal use of IVIgs and to maximise their potential benefit, the indication must be individually assessed for each patient. As a primary treatment goal, the authors define a sufficient prophylaxis of severe infections, which can be supported by normalising IgG levels. If the initiated treatment is insufficient or linked to intolerable adverse reactions, switching the product within the class of IVIgs or changing to a different batch of the same product can be considered. Pausing treatment can also be considered if there are no infections, which happens more frequently in summer, but treatment needs to be resumed once infections return. These structured recommendations for IVIg treatment in patients with secondary antibody deficiency may provide guidance for clinical practice and therefore help to allocate IVIgs to those who will benefit the most, without overusing valuable resources.

IgG Subtype Response against Virulence-Associated Protein A in Foals Naturally Infected with Rhodococcus equi.

Rhodococcus equi is an intracellular bacterium that causes suppurative pneumonia in foals. T-helper (Th) 1 cells play an important role in the protective response against R. equi. In mice and humans, the directionality of IgG switching reflects the polarization of Th-cell responses, but this has not been fully elucidated in horses. In this 4-year study, we classified R. equi-infected foals into surviving and non-surviving group and investigated differences in IgG subclass response to virulence-associated protein A, the main virulence factor of R. equi, between the groups. IgGa, IgGb, and IgG(T) titers were significantly higher in the non-surviving group compared with the surviving group. The titers of IgGa and IgG(T), IgGb and IgG(T), and IgGa and IgGb, respectively, were positively correlated, and the IgG(T)/IgGb ratio in the non-surviving group was significantly higher than that in the surviving group. The IgG(T) titer tended to increase more than the IgGa and IgGb titers in the non-surviving group compared with the surviving group. Our findings suggest that the IgG(T) bias in IgG subclass responses reflects the immune status, which exacerbates R. equi infection.

A Third COVID-19 Vaccine Dose in Kidney Transplant Recipients Induces Antibody Response to Vaccine and Omicron Variants but Shows Limited Ig Subclass Switching.

This study addresses the challenges faced by kidney transplant recipients (KTRs) in mounting effective immune responses against COVID-19. By evaluating the antibody responses to a third dose of monovalent mRNA COVID-19 vaccine and its effectiveness against Omicron subvariants (BA.1 and BA.5), this study reveals significant reductions in both binding and neutralizing antibodies in KTRs compared to healthy controls. The research highlights altered IgG subclass switching and heterogeneous responses within the KTR population. Reduced recognition of variants, coupled with differences in IgG subclasses, decreases both the quality and quantity of protective antibodies after vaccination in KTRs. These findings underscore the need for tailored vaccination strategies for immunosuppressed populations such as KTRs. Alternative formulations and doses of COVID-19 vaccines should be considered for people with severely compromised immune systems, as more frequent vaccinations may not significantly improve the response, especially regarding neutralizing antibodies.

IgG subclass shifts occurring at acute exacerbations in autoimmune nodopathies.

Autoimmune nodopathy associated with anti-contactin1 (CNTN1) IgG4 antibodies frequently manifests as acute axonal degeneration in addition to detachment of the paranodal myelin loops. The acute destruction of myelinated nerve fibers does not match the function of IgG4, which cannot activate the complement pathway. IgG subclass switching from IgG1 or IgG3 to IgG4 has been observed in some patients with autoimmune diseases associated with IgG4 throughout their disease course. Serial changes in IgG subclasses, clinico-neurophysiological features, and nerve and renal pathology were reviewed in three patients with anti-CNTN1-associated autoimmune nodopathy and one patient with anti-contactin-associated protein1 (Caspr1) autoimmune nodopathy. All four patients had predominantly IgG4 autoantibodies, whereas they showed evidence of acute axonal degeneration. The IgG1 subclass was present in all patients at their progressing stage but then disappeared at follow-up. Nerve pathology in the patients with anti-CNTN1 and anti-Caspr1 autoimmune nodopathies showed both structural changes in the paranodes and evidence of acute axonal degeneration. Renal biopsy specimens from two patients with membranous glomerulonephritis and anti-CNTN1 autoimmune nodopathy showed deposition of IgG1 and complement on the glomerular basement membrane, as well as IgG4. In patients with autoimmune nodopathies associated with anti-CNTN1 and anti-Caspr1 IgG4 antibodies, IgG1 subclass autoantibodies were present at their acute exacerbations and might have contributed to the axonal degeneration and glomerular injury. IgG1 disappeared with the cessation of disease progression, which indicates that the IgG1 subclass is a possible biomarker of disease activity.

An Integrated Signaling Threshold Initiates IgG Response toward Virus-like Immunogens.

Class-switched neutralizing Ab (nAb) production is rapidly induced upon many viral infections. However, due to the presence of multiple components in virions, the precise biochemical and biophysical signals from viral infections that initiate nAb responses remain inadequately defined. Using a reductionist system of synthetic virus-like structures, in this study, we show that a foreign protein on a virion-sized liposome can serve as a stand-alone danger signal to initiate class-switched nAb responses without T cell help or TLR but requires CD19. Introduction of internal nucleic acids (iNAs) obviates the need for CD19, lowers the epitope density (ED) required to elicit the Ab response, and transforms these structures into highly potent immunogens that rival conventional virus-like particles in their ability to elicit strong Ag-specific IgG. As early as day 5 after immunization, structures harboring iNAs and decorated with just a few molecules of surface Ag at doses as low as 100 ng induced all IgG subclasses of Ab in mice and reproduced the IgG2a/2c restriction that is long observed in live viral infections. These findings reveal a shared mechanism for the nAb response in mice. High ED is capable but not necessary for driving Ab secretion. Instead, even a few molecules of surface Ag, when combined with nucleic acids within these structures, can trigger strong IgG production. As a result, the signaling threshold for induction of IgG in individual B cells is set by dual signals originating from both ED on the surface and the presence of iNAs within viral particulate immunogens.

On the role of antibody affinity and avidity in the IgE-mediated allergic response.

Type I hypersensitivity, also known as classical allergy, is mediated via allergen-specific IgE antibodies bound to type I FcR (FcεRI) on the surface of mast cells and basophils upon cross-linking by allergens. This IgE-mediated cellular activation may be blocked by allergen-specific IgG through multiple mechanisms, including direct neutralization of the allergen or engagement of the inhibitory receptor FcγRIIb which blocks IgE signal transduction. In addition, co-engagement of FcεRI and FcγRIIb by IgE-IgG-allergen immune complexes causes down regulation of receptor-bound IgE, resulting in desensitization of the cells. Both, activation of FcεRI by allergen-specific IgE and engagement of FcγRIIb by allergen-specific IgG are driven by allergen-binding. Here we delineate the distinct roles of antibody affinity versus avidity in driving these processes and discuss the role of IgG subclasses in inhibiting basophil and mast cell activation.

Immunological characterization of IgG subclass deficiency reveals decreased Tregs and increased circulating costimulatory and regulatory immune checkpoints.

Immunoglobulin G subclass deficiencies (IgGsd) comprise a wide clinical spectrum from no symptoms to repeated respiratory infections and risk for the development of lung damage. Our aims were to investigate whether the immunological phenotype of IgGsd patients on and off immunoglobulin replacement therapy (IgRT) was reflected in the clinical features of IgGsd. Thirty patients with IgGsd were included in this prospective study of 18 months of IgRT, followed by 7-18 months of IgRT discontinuation. Blood samples were collected when patients were on and off IgRT and compared with samples from 34 cross-sectional healthy controls. An in-depth lymphocyte phenotyping was performed by flow cytometry and plasma levels of immune checkpoints were assessed. IgG3 subclass deficiency was most common. Patients with IgGsd had decreased levels of activated T cells and B cells and plasma levels of negative immune checkpoint molecules correlated negatively with T cell and B cell activation. The decreased T cell activation level was unaffected by IgRT, while the B cell activation was partly restored. Of note, decreased levels of activated regulatory T cells (Tregs) were found in IgGsd patients and was partly restored during IgRT. The profile of comorbidities did not associate with Treg levels. IgGsd is associated with decreased B cell and T cell activation including Tregs, and increased plasma levels of negative immune checkpoint molecules. The consequence of reduced activated Tregs in IgGsd remains unclear. Decreased immune cell activation was partly restored during IgRT, demonstrating that IgRT may contribute to improved immune function in patients with IgGsd.

Human cerebrospinal fluid monoclonal CASPR2 autoantibodies induce changes in electrophysiology, functional MRI, and behavior in rodent models

Anti-contactin associated protein receptor 2 (CASPR2) encephalitis is a severe autoimmune encephalitis with a variable clinical phenotype including behavioral abnormalities, cognitive decline, epileptic seizures, peripheral nerve hyperexcitability and neuropathic pain. The detailed mechanisms of how CASPR2 autoantibodies lead to synaptic dysfunction and clinical symptoms are largely unknown. Aiming for analyses from the molecular to the clinical level, we isolated antibody-secreting cells from the cerebrospinal fluid of two patients with CASPR2 encephalitis. From these we cloned four anti-CASPR2 human monoclonal autoantibodies (mAbs) with strong binding to brain and peripheral nerves. All were highly hypermutated and mainly of the IgG4 subclass. Mutagenesis studies determined selective binding to the discoidin domain of CASPR2. Surface plasmon resonance revealed affinities with dissociation constants KD in the pico- to nanomolar range. CASPR2 mAbs interrupted the interaction of CASPR2 with its binding partner contactin 2 in vitro and were internalized after binding to CASPR2-expressing cells. Electrophysiological recordings of rat hippocampal slices after stereotactic injection of CASPR2 mAbs showed characteristic afterpotentials following electrical stimulation. In vivo experiments with intracerebroventricular administration of human CASPR2 mAbs into mice and rats showed EEG-recorded brain hyperexcitability but no spontaneous recurrent seizures. Behavioral assessment of infused mice showed a subtle clinical phenotype, mainly affecting sociability. Mouse brain MRI exhibited markedly reduced resting-state functional connectivity without short-term structural changes. Together, the experimental data support the direct pathogenicity of CASPR2 autoantibodies. The minimally invasive EEG and MRI techniques applied here may serve as novel objective, quantifiable tools for improved animal models, in particular for subtle neuropsychiatric phenotypes or repeated measurements.

735 - A case series of dupilumab for vitiligo and alopecia areata in the setting of AD

Abstract Introduction/Background Dupilumab is a human monoclonal antibody of the IgG4 subclass and binds to the IL-4R alpha subunit, causing downstream inhibition of IL-4 and IL-13 signaling and thus downregulating the T-helper-2 (Th2) cytokine response.1,2,3 Approved by the Food and Drug Administration in 2017, dupilumab is FDA-approved to treat atopic dermatitis (moderate-to-severe), asthma (moderate-to-severe), chronic rhinosinusitis with nasal polyposis, and eosinophilic ­esophagitis. Vitiligo is a common autoimmune depigmenting skin disorder that is prevalent in 1.38% of US adults, and 2.16% in adolescents in the US, with a range of 0.4-2% in most populations.4,5,6 It occurs when the immune system of the body attacks melanocytes, skin cells, that produce melanin, and has been linked to the Koebner phenomenon which occurs when trauma induces lesions, with events including scratching as would be noted in AD.7 AD has also been linked to Vitiligo, especially in children under the age of 12 years.8,9,10 There are many available therapies for vitiligo, topical, systemic, phototherapy, and surgical types being most commonly used.11,12,13 Objectives To address specific features of AA/ Vitiligo/ AD overlap that would support benefit of dupilumab prescribing. Methods An IRB-exempt review was conducted of patient charts for individuals who received dupilumab who had alopecia areata or vitiligo associated with AD. Results Six patients with AD and alopecia universalis (AU), and one patient with AD, vitiligo, and alopecia areata (AA) were identified for review. The patients treated for AU included a 5 year-old Asian female, a 12 year-old white female, a 14 year-old African American male, A 15 year-old Hispanic female, a 26 year-old African American female, and a 78 year-old Hispanic female. The five year-old regrew hair for 4 months but had rapid loss when forced to discontinue due to insurance reasons. The 12 year-old female who had AU for 8 years and had no regrowth. The 15 year-old, 26 year-old, and 78 year-old had rapid regrowth of hair in addition to AD improvement, however, the 15 year-old required addition of an oral JAK inhibitor to retain hair growth. The vitiligo/AA patient is a 61 year-old female patient who had 50% BSA confetti lesion vitiligo affecting the chest, back, abdomen, arms, and legs. She had rapid disease stabilization (2 months) and at 1 year had 90% facial and 70% extremity repigmentation with dupilumab and topical 1.5% ruxolitinib. The same patient had 40% scalp hair loss which fully resolved upon repigmentation of the scalp. Response was noted rapidly but plateaued at 12 months. Conclusions The overlap of AD with AA and Vitiligo points to a shared pathogenesis of the conditions. One of the likely reasons for this is the Koebner phenomenon, which has been poorly characterized. We hypothesize that the Koebner phenomenon is triggered through an IL-4/ IL-13 mechanism, i.e. for specific individuals IL-4 and IL-13 hyper-reactivity can be a Koebner-based trigger. Additionally, AA overlaps with AD is poorly characterized. However, the linkage has been recognized in recent AAD guidelines addressing AD comorbidities.14 We hypothesize that IL-4/ IL-13 hyper-reactivity in the skin can act similarly in AU. There is in AA an overlap of TH1-CXCL9/10 expression and interferon gamma overexpression in addition to TH2- including IL-13 overexpression.15 Therefore, the blockade of IL-4/ IL-13 may be effective through multiple mechanisms of activity. There is already notable Phase 2a data supporting dupilumab usage in AA.16 Our experience demonstrates sustained hair growth in half of patients treated. AA/ Vitiligo overlap appears to respond well to dupilumab. This is supported by a recent case report demonstrating benefit of dupilumab in programmed cell death inhibitor-1 induced vitiligo with associated refractory pruritus.17 Liu et al have observed in vitro that rising IL-4 levels were linked to increased vitiligo risk.18 On the other hand, some reports of vitiligo after initiating dupilumab therapy do exist, with some new-onset and worsening described. These cases are limited but bear consideration. In our patient, disease stabilization was noted in a generalized confetti-vitiligo patient, but a topical JAK inhibitor was used adjunctively.19,20,21,22,23 Given the need for long-term maintenance, the safety of dupilumab is an attractive feature. Therefore, further exploration of dupilumab therapy for vitiligo/AA/AD, vitiligo/AD/Koebner+, and AU/ AD is needed. This is particularly important to address in patients under the age of 12 years who currently have no approved systemic medications for vitiligo and AU.24 When AU, vitiligo, and combinations of the two are comorbid with AD, there is an expectation of circulating IL-4/13 elevations and localized IL-4/13 elevation that support a potential role for dupilumab therapy in these conditions. Given that AD is associated with vitiligo of childhood, and severe alopecia areata is linked to AD, systemic therapy would be necessary in these individuals.

LGI1 encephalitis: potentially complement-activating anti-LGI1-IgG subclasses 1/2/3 are associated with the development of hippocampal sclerosis

Two-thirds of published patients with anti-leucine rich, glioma inactivated 1 (LGI1) encephalitis develop hippocampal sclerosis (HS). It is likely that this contributes to residual cognitive long-term deficits and the risk of epilepsy. Almost all patients harbor anti-LGI1-immunoglobulin G-(IgG-) subclass 4, which is considered a “benign”, non-destructive subclass. In contrast, neuropathological case studies have suggested that the classical complement cascade may contribute to mediotemporal cell death in patients with LGI1 antibodies. IgG subclasses 1, 2, or 3 are required to initiate this cascade. We hypothesized that patients with these anti-LGI1-IgG1/2/3 in addition to IgG4 have a higher risk of developing HS than patients with anti-LGI1-IgG4 alone. We retrospectively assessed all anti-LGI1 encephalitis patients from this center with anti-LGI1-IgG-subclass information and follow-up MRI available. Nine out of 20 patients had developed HS (45%). Volumetric FreeSurfer analysis confirmed the visual HS diagnoses. HS and a lower hippocampal volume were associated with anti-LGI1-IgG1/2/3. All six patients with this IgG subclass status developed HS. There was no association with older or younger age at onset, female sex, longer latency from disease onset to start of immunotherapy, less intense immunotherapy, higher serum titers of LGI1 antibodies, LGI1 antibodies in CSF or higher LGI1-specific antibody indices. There was no association between anti-LGI1-IgG1/2/3 status and neuropsychological performance, epilepsy, or general neurological performance. This confirms our hypothesis that anti-LGI1-IgG1/2/3 in serum puts patients at risk of developing HS. If these findings can be confirmed and clinically corroborated, patients with anti-LGI1-IgG1/2/3 might become candidates for anti-complement-directed immunological treatments.

Abstract Tu091: Mechanism of beta1-adrenergic receptor signaling by IgG3 subclass of autoantibodies

Autoimmune response to self-antigens results in autoantibodies (AAbs), wherein AAbs against extra cellular loop (ECL) 2 of β1-adrenergic receptor (β1AR) is known to underlie dilated cardiomyopathy (DCM). Contrarily, recent studies show that patients with β1AR AAbs belonging to the IgG3 subclass have beneficial outcomes. However, the signaling mechanisms that underlie the beneficial outcomes are not well understood. We have previously shown that IgG3(+) β1AR AAbs facilitates uniquely biased β1AR signaling in response to β-blocker compared to non-IgG3(+) β1AR AAbs. Since IgG3(+) β1AR AAbs are associated with favorable patient outcomes, it is critical to determine the mechanistic basis of the unique beneficial signaling pathway that may have therapeutic potential. HEK 293 cells stably expressing human β1AR (HEK-β1AR) was generated and treated with β1AR AAbs. Minimal changes in phosphorylation of β1AR was observed following IgG3(+) β1AR AAbs, while significant phosphorylation of β1ARs was observed with non-IgG3(+) β1AR AAbs. To dissect the diverse signaling responses mediated by IgG3(+) and non-IgG(+) β1AR AAbs, HEK-β1AR cells were treated with IgG3(+) or non-IgG3(+) β1AR AAbs. β1ARs were immunoprecipitated and subjected to mass-spectrometry analysis to identify unique co-immunoprecipitating proteins that could complex with receptor in presence of IgG3(+) or non-IgG3(+) β1AR AAbs. One of the proteins that was differentially recruited to the β1AR complex was Insulin Receptor Substrate 4 (IRS4). Traditionally, IRS4 is a signaling hub downstream of insulin/insulin growth factor receptors mediating glucose uptake. The differential interaction of IRS4 with β1AR in the presence of IgG3(+) vs. non-IgG3(+) β1AR AAbs reflects that IRS4 could be a key signaling regulator of uniquely biased signal transduction pathway in the presence of IgG3(+) vs. non-IgG3(+) β1AR AAbs. Mechanisms underlying this unique IRS4 mediated signaling modulation will be discussed.

Abstract We065: Sex Differences in Autoimmune Signatures After Myocardial Infarction in Mice

Introduction: Myocardial infarction (MI) is caused by ischemic injury to the left ventricle (LV), leading to an inflammatory response, scar formation, and loss of LV function. The role of the autoimmune (AI) response during chronic MI, such as B cell activation and autoantibody production, is not well understood. As AI disease is much more prevalent in females, we hypothesized that female mice are more susceptible to MI-induced development of AI signatures. Methods: Permanent MI was induced by left coronary artery ligation in adult male and female C57BL/6J mice for 7, 28, or 56 days. LV function was tracked using echocardiography (VEVO 3100). Cytokines were measured by qPCR. LV IgG and IgM antibody deposition was measured by immunofluorescence. Plasma IgG and IgM were measured by ELISA, and plasma autoantibodies (AABs) (IgG and IgM subclasses) were measured with an autoantigen (AAN) array. Results: In both sexes, MI led to progressive LV hypertrophy, dilation, and wall thinning. MI increased spleen mass in D7, 28, and 56 females, but decreased spleen mass in D28 and 56 males. LV infarct cytokines (IL-1β, IL-6, IL-18, IFN-γ, TNF, CCL2, IL-10) were increased at D7, while IL-6 and IL-18 remained elevated at D28 and 56 to a higher degree in females. Several cytokines in the remote LV were increased in females relative to males, including TNF, IL-6, CCL2, and IL-18. Splenic cytokines were also differentially expressed between sexes, with IL-1β, IL-12a, IL-10, IFN-γ, IL-18, CCL2, and IL-4 increased in females at several time points. Infarct IgG and IgM deposition occurred in both sexes, but to a higher degree in females. Remote IgG and IgM deposition occurred at D56 in both sexes. For plasma AABs, we detected 34 AANs against which IgG AABs were significantly elevated (p<0.05) at D56 in females vs 9 in males, and 30 IgM AABs vs 1 in males. Of note, anti-IL-6 IgM AABs were elevated in D56 females (9.8±0.8-fold), likely due to persistently elevated IL-6 in the female infarct and remote LV vs males. Total levels of plasma IgM were increased only in females at D56 (1.79±0.15-fold). Conclusions: MI promotes development of AI signatures which is exacerbated in females, including increased spleen mass, LV and splenic cytokines, LV infarct and remote IgG and IgM antibody deposition, increased plasma IgG and IgM AABs, and increased total levels of plasma IgM. Targeting the AI response may be a viable option for treating chronic MI outcomes, particularly in females.

Quantification of human intravenous immunoglobulin from plasma and in process samples by affinity chromatography

Advances in affinity chromatography now make it possible to analyze immunoglobulin G from plasma and its fractions with a simple chromatographic method. Ligands derived from camelid antibodies have been developed which have affinity to all 4 subclasses of human IgG without a cross reactivity to other immunoglobulins. The commercially available Capture Select FcXL is the basis for a simple method for direct quantification of immunoglobulin G from plasma or from fractions from cold ethanol precipitation. After direct injection of the sample into the column the unbound proteins are washed out with equilibration buffer and eluted with a pH-step. The elution the peak is integrated, and quantity is derived form a standard curve. The limit of detection with 40 µg/mL, and a linearity up to 250 µg/mL allows an analysis of samples ranging from 0.04 to 50 mg/mL using varying injection volume without further dilution and the two-wavelength detection. A full cycle is completed within five minutes. This method can serve as orthogonal method for in-process control but also for process development.

Biomarkers of neural integrity and immunoglobulin genes influence neurodegeneration in Alzheimer's disease

Compelling evidence has been presented in favor of herpes simplex virus type 1 (HSV1) being one of the causative agents of Alzheimer's disease (AD). The success of HSV1 as a pathogen relates to its sophisticated strategies to evade host immunosurveillance. One strategy involves encoding a decoy Fcγ receptor (FcγR) that thwarts the Fcγ-mediated effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), a potent host immunosurveillance mechanism against virally infected cells. The decoy FcγR binds to antibodies of all IgG subclasses, except IgG3; therefore, IgG3 would be expected to play an important role in viral clearance by neutralization and ADCC, and thus contribute to protection from HSV1-spurred diseases. Previous studies have shown significant association between anti-HSV1 IgG3 antibodies and cortical thinning of the areas of the brain typically altered in AD and also targeted by HSV1. The aim of the present investigation was to determine whether GM (γ marker) 5 and GM 21 allotypes, hereditary allelic determinants expressed on IgG3, together with brain biomarkers of neural integrity, contributed to neurodegeneration—as measured by mini-mental state examination (MMSE) score—in patients with AD. Multiple regression analyses showed that the homozygous GM 5/5 genotype, preserved right hippocampus, and right insula thickness were associated with higher MMSE scores (p < 0.001), whereas the opposite pattern and GM 5/21 genotype were associated with worse clinical profiles. Influence of GM 5/21-expressing IgG3 antibodies on the ADCC of HSV1-infected neurons could, at least partially, explain these results.

Multiorgan involvement and circulating IgG1 predict hypocomplementaemia in IgG4-related disease

ObjectivesHypocomplementaemia is common in patients with IgG4-related disease (IgG4-RD). We aimed to determine the IgG4-RD features associated with hypocomplementaemia and investigate mechanisms of complement activation in this disease.MethodsWe performed a...

Anti-TNF therapy impairs both short- and long-term IgG responses after repeated vaccination.

Recently, it has been questioned whether vaccination of patients with inflammatory (auto)immune diseases under anti-tumor necrosis factor (TNF) treatment leads to impaired vaccine-induced immune responses and protection against breakthrough infections. However, the effects of TNF blockade on short- and long-term immune responses after repeated vaccination remain unclear. Vaccination studies have shown that initial short-term IgG antibodies (Abs) carry highly galactosylated and sialylated Fc glycans, whilst long-term IgG Abs have low levels of galactosylation and sialylation and are most likely generated by long-lived plasma cells (PCs) derived primarily from the germinal center (GC) response. Thus, IgG Fc glycosylation patterns may be applicable to distinguish short- and long-term vaccine responses after repeated vaccination under the influence of anti-TNF treatment. We used COVID-19 vaccination as a model to investigate vaccine-induced IgG subclass levels and Fc glycosylation patterns, B cell subsets, and effector functions of short- and long-term Ab responses after up to three vaccinations in patients on anti-TNF or other immunosuppressive treatments and in healthy individuals. Using TriNetX, a global healthcare database, we determined the risk of SARS-CoV-2 breakthrough infections in vaccinated patients treated with anti-TNF or other immunosuppressive drugs. Anti-TNF treatment reduced the long-term abundance of all anti-S IgG subclasses with low levels of galactosylation and sialylation. Re-activation of potential memory B cells initially generated highly galactosylated and sialylated IgG antibodies, which were progressively reduced after each booster dose in anti-TNF-treated patients, especially in the elderly. The reduced short- and long-term IgG (1) levels in anti-TNF-treated patients correlated with diminished functional activity and an increased risk for the development of COVID-19. The data suggest that anti-TNF treatment reduces both GC-dependent long-lived PCs and GC-dependent memory B cell-derived short-lived PCs, hence both the long- and short-term IgG subclass responses, respectively, after repeated vaccination. We propose that anti-TNF therapy, especially in the elderly, reduces the benefit of booster vaccination.

IPINeT Ped-unPAD Study: Goals, Design, and Preliminary Results.

Background: An unclassified primary antibody deficiency (unPAD) is a widely heterogeneous clinical entity, recently identified within the spectrum of Inborn Errors of Immunity (IEIs). Since unPAD has been traditionally considered as a mild condition, it has incorrectly received little attention, resulting in the paucity of extensive and comparable studies describing its natural history. To address the gaps in characterizing, understanding, and managing pediatric unPAD patients, the Italian Primary Immunodeficiency Network (IPINet) Ped-unPAD study has recently been launched. Methods: Seventeen IPINeT Centers have expressed interest to participate, and data collection is still on-going. Hereby, we anticipate preliminary key issues emerging from the first 110 enrolled patients, attending three IPINet Centers. Results: A proportion of unPAD patients have experienced a severe infectious phenotype, which required hospitalization in a quarter of patients and antibiotic prophylaxis or Immunoglobulin Replacement Therapy in approximately 10% of patients. In this partial cohort, a mean follow-up (FU) of 5 years confirmed unPAD diagnosis in fifty percent of cases, with the remaining being reclassified as the Transient Hypogammaglobulinemia of Infancy (25%) and other IEIs (25%), such as a Common Variable Immunodeficiency, Selective IgA deficiency, Selective IgM deficiency, and IgG3 subclass deficiency. Conclusions: Despite a phenotype overlap at diagnosis, clinicians should be aware that unPAD is a mutable condition that deserves comprehensive evaluation and long-term monitoring to dissect the final diagnosis for optimal treatment.

Immunoinformatic approaches for ErpY-LemA chimeric protein design for use in leptospirosis control.

Currently, immunoinformatic approaches have shown promise in rapidly and cost-effectively identifying new antigens from the Leptospira proteome. Chimeric multiepitope proteins offer a strategy with significant potential for implementation in diagnosis and vaccines development. In this study, we detail the immunoinformatic analyses and design of a new recombinant chimeric protein constructed with epitopes identified from the sequences of ErpY-like and LemA proteins, previously identified as potential antigens for controlling leptospirosis. We expressed the chimeric protein using Escherichia coli heterologous systems, evaluated its antigenicity using serum from naturally infected patients, and its immunogenicity in mice as an animal model, with Freund as an adjuvant. The resulting recombinant chimeric protein, named rErpY-LemA, was successfully expressed and purified using a prokaryotic system, with an expected mass of 35kDa. Serologic assays using serum samples from naturally infected patients demonstrated recognition of the chimera protein by antibodies present in sera. Animals immunized with the chimera exhibited a significant IgG antibody response from the 7th day (P<0.001), persisting until day 49 of experimentation, with a titer of 1:12,800 (P<0.05). Notably, significant production of IgA, IgM, and IgG subclasses was observed in animals immunized with the chimera. These results highlight the promising role of immunoinformatics in rapidly identifying antigens and the potential of chimeric multiepitope proteins in developing effective strategies for leptospirosis control.

Proteinuria is a key to suspect autoimmune nodopathies.

Reports of patients who have autoimmune nodopathies concurrent with nephrotic syndrome are increasing. We investigated whether proteinuria could be a biomarker of autoimmune nodopathies. Qualitative urinalysis results were retrospectively obtained from 69 patients who were diagnosed with chronic inflammatory demyelinating polyneuropathy (CIDP) at a hospital in Japan. Proteinuria was graded as mild to severe (i.e., mild, 30-99; moderate, 100-299; severe, 300 mg/dL or more) according to the results of the urine dipstick test. Autoantibodies against the paranodal proteins contactin 1 (CNTN1), neurofascin 155 (NF155), and contactin-associated protein 1 (Caspr1) and the nodal protein neurofascin 186 (NF186) were measured, and the predominant IgG subclass was determined by enzyme-linked immunosorbent assay in sera from the 69 patients. Four patients (6%), five patients (7%), and one (1%) patient were positive for anti-CNTN1, anti-NF155, and anti-Caspr1 IgG4 antibodies, respectively. No patients had IgG4 antibodies against NF186. Proteinuria of mild or greater levels was found in three patients with anti-CNTN1 IgG4 and two patients with anti-NF155 IgG4 antibodies. The autoantibody-positive patients more frequently had proteinuria of mild or greater levels than the seronegative patients (p = 0.01). Proteinuria is a possible biomarker of autoimmune nodopathies associated with autoantibodies targeting CNTN1 or NF155. Urinalysis results should be carefully checked for quick differentiation of autoimmune nodopathies from CIDP. Patients who present with nephrotic syndrome should be tested for anti-CNTN1 IgG4 antibodies, and patients who exhibit mild proteinuria should be tested for anti-NF155 IgG4 antibodies.