TCDD Inhibition of IgG1 Production in Experimental Autoimmune Encephalomyelitis (EAE) and In Vitro

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) is a ligand for the aryl hydrocarbon receptor (AhR). TCDD is well-characterized to produce immunotoxicity, including suppression of antibody production. Previously we showed that TCDD inhibited myelin oligodendrocyte glycoprotein (MOG) peptide-specific IgG and attenuated disease in experimental autoimmune encephalomyelitis (EAE) model in mice. Thus, the purpose of this study was to characterize the effects of TCDD on IgG subclasses in EAE and in vitro and assess effects in B cells derived from various tissues. TCDD modestly suppressed intracellular IgG expression in splenocytes (SPLC), but not bone marrow (BM) or lymph node (LN) cells. To further understand TCDD’s effects on IgG, we utilized LPS and LPS + IL-4 in vitro to stimulate IgG3 and IgG1 production, respectively. TCDD preferentially suppressed IgG1+ cell surface expression, especially in SPLC. However, TCDD was able to suppress IgG1 and IgG3 secretion from SPLC and B cells, but not BM cells. Lastly, we revisited the EAE model and determined that TCDD suppressed MOG-specific IgG1 production. Together these data show that the IgG1 subclass of IgG is a sensitive target of suppression by TCDD. Part of the pathophysiology of EAE involves production of pathogenic antibodies that can recruit cytolytic cells to destroy MOG-expressing cells that comprise myelin, so inhibition of IgG1 likely contributes to TCDD’s EAE disease attenuation.

Isolation and immunogenicity of extracted outer membrane vesicles from Pseudomonas aeruginosa under antibiotics treatment conditions

Background and Objectives: Different types of antibiotics have been indicated to enhance the secretion of OMVs from Pseudomonas aeruginosa. We aimed to investigate the effect of meropenem and amikacin antibiotics on inducing the secre- tion of OMVs and immunologic features in P. aeruginosa. Materials and Methods: The OMVs were prepared from P. aeruginosa under hypervesiculation condition (treatment with amikacin and meropenem), and extraction was carried out by the sequential ultracentrifugation. Physicochemical features of extracted OMVs were evaluated by electron microscopy and SDS-PAGE. To quantify antibody synthesis and function after immunization with OMV, we used ELISA, serum bactericidal activity, and opsonophagocytosis. Production of cytokines from splenocytes of immunized mice was measured with ELISA. Results: Specific-antibody IgG production, particularly IgG1 subclass, increased in mice primed with hypervesiculation-de- rived OMVs compared to normal condition-derived OMVs. Serum bactericidal activity and opsonophagocytosis of secreted antibody was enhanced in mice primed with hypervesiculation-derived OMVs. Investigation of cytokine production showed the upregulation of IL-8, IL-12, IL-17, and TNF-α and downregulation of IL-10. Conclusion: Based on our findings, OMVs production can be increased by treating P. aeruginosa with amikacin and mero- penem antibiotics. Moreover, hypervesiculation-derived OMV scan possibly activate the humoral and cellular immune re- sponse more than normal OMVs.

IgG4-Mediated Neurologic Autoimmunities

Background and ObjectivesDescribe the unique functions of immunoglobulin G4 (IgG4) in IgG4-neurologic disorders (IgG4-ND) and explain why, in contrast to their IgG1-counterparts, they respond poorly to intravenous immune globulin (IVIg) but effectively to anti–B cell therapies.MethodsThe IgG4 structure and isotype switch, B cells and plasmablasts relevant to IgG4 production, and IgG4-induced disruption of the targeted antigens are reviewed and compared with IgG1-mediated autoimmune ND, where IVIg inhibits IgG1-triggered inflammatory effects.ResultsThe main IgG4-ND include muscle-specific kinase myasthenia; nodal/paranodal chronic inflammatory demyelinating polyradiculoneuropathy with antibodies to neurofascin-155, contactin-1/caspr-1, or pan-neurofascins; antileucine-rich, glioma-inactivated-1 and contactin-associated protein-like 2 associated-limbic encephalitis, Morvan syndrome, or neuromyotonia; and anti-IgLON5 disorder. The IgG4, because of its unique structural features in the hinge region, has noninflammatory properties being functionally monovalent and bispecific, unable to engage in cross-linking and internalization of the targeted antigen. In contrast to IgG1 subclass which is bivalent and monospecific, IgG4 does not activate complement and cannot bind to inhibitory Fcγ receptor (FcγRIIb) to activate cellular and complement-mediated immune responses, the key functions inhibited by IVIg. Because IVIg contains only 0.7%–2.6% IgG4, its idiotypes are of IgG1 subclass and cannot effectively neutralize IgG4 or sufficiently enhance IgG4 catabolism by saturating FcRn. In contrast, rituximab, by targeting memory B cells and IgG4-producing CD20-positive short-lived plasma cells, induces long-lasting clinical benefits.DiscussionRituximab is the preferred treatment in IgG4-ND patients with severe disease by effectively targeting the production of pathogenic IgG-4 antibodies. In contrast, IVIG is ineffective because it inhibits immunoinflammatory functions irrelevant to the mechanistic effects of IgG4 and contains IgG-1 idiotypes that cannot sufficiently neutralize or possibly catabolize IgG4. Controlled studies with anti-CD19/20 monoclonals that also activate FcγRIIb may be more promising in treating IgG4-ND.

Case Report: Prominent Brainstem Involvement in Two Patients With Anti-CASPR2 Antibody-Associated Autoimmune Encephalitis

Anti-contactin-associated protein-like 2 (CASPR2) antibody-associated autoimmune encephalitis is commonly characterized by limbic encephalitis with clinical symptoms of mental and behavior disorders, cognitive impairment, deterioration of memory, and epilepsy. The classical lesions reported are located at the medial temporal lobe or hippocampus, whereas prominent brainstem lesions have not been addressed to date. Herein, we reported two patients mimicking progressive brainstem infarction with severe neurological manifestations. On brain magnetic resonance imaging (MRI), prominent brainstem lesions were noted, although multifocal lesions were also shown in the juxtacortical and subcortical white matters, basal ganglia, hippocampus, and cerebellar hemisphere. Unexpectedly and interestingly, both cases had detectable CASPR2 antibodies in sera, and an exclusive IgG1 subclass was documented in the further analysis. They were treated effectively with aggressive immunosuppressive therapies including corticosteroids, intravenous immunoglobulin G, and rituximab, with the first case achieving a rapid remission and the other undergoing a slow but gradual improvement. To the best of our knowledge, this is the first report on prominent brainstem involvement with definite MRI lesions in anti-CASPR2 antibody-associated autoimmune encephalitis, which helps to expand the clinical spectrum of this rare autoimmune disease and update the lesion patterns in the CNS.

Can Antinuclear Antibodies (ANA) be Monoclonal?

Background. Nuclear staining by immunofluorescence in a kidney biopsy is often seen in patients with positive antinuclear antibodies (ANA) in the serum. These ANA are usually polyclonal, but herein we report 9 cases with an unusual finding of monoclonal nuclear staining by immunofluorescence on kidney biopsy. Case Presentation. Nine cases with predominant stain for kappa or lambda light chain were identified by searching the renal pathology laboratory database for the past 10 years. All cases had positive stain for only kappa (six cases) or lambda (three cases) light chain in the nuclei. Eight out of nine cases had positive nuclear IgG stain, and one case had positive nuclear IgA stain. Among cases with positive nuclear IgG staining, six cases were positive for IgG1 subclass, one case was positive for IgG2 subclass, and one case was positive for IgG3 subclass. All patients with positive IgG nuclear stain, who had testing for ANA, had positive ANA. Patients with positive IgG1 subclass did not have monoclonal protein in the serum or urine, but the patient with positive IgG2 subclass and lambda light chain stain in the nuclei had IgG lambda monoclonal gammopathy. Conclusions. We identified a new unique pattern of nuclear stain by immunofluorescence in kidney biopsies that suggests the presence of monoclonal ANA. Workup for underlying monoclonal gammopathy is warranted in such patients.

Factors associated with IgG levels in adults with IgG subclass deficiency

Background Factors associated with IgG levels in adults with IgG subclass deficiency (IgGSD) are incompletely understood. We studied adults with IgGSD with subnormal IgG1 only, subnormal IgG1/IgG3, or subnormal IgG3 only without other subnormal IgG subclasses, IgA, or IgM. We compiled: age; sex; autoimmune condition(s) (AC); atopy; IgG, IgG subclasses, IgA, IgM; IgGsum (IgG1 + IgG2 + IgG3 + IgG4); and D (percentage difference between IgGsum and IgG). We compared attributes of patients with/without subnormal IgG (< 7.00 g/L; subnormal IgG1 subclass groups only) and analyzed IgGsum and IgG relationships. We performed backward stepwise regressions on IgG using independent variables IgG subclasses, age, and sex and on D using independent variables age and sex. Results There were 39 patients with subnormal IgG1 only (89.7% women), 53 with subnormal IgG1/IgG3 (88.7% women), and 115 with subnormal IgG3 only (91.3% women). Fifteen patients (38.5%) and 32 patients (60.4%) in the respective subnormal IgG1 subclass groups had subnormal IgG. Attributes of patients with/without IgG < 7.00 g/L were similar, except that AC prevalence was lower in patients with subnormal IgG1 only and IgG < 7.00 g/L than ≥ 7.00 g/L (p = 0.0484). Mean/median IgG1 and IgG2 were significantly lower in patients with IgG < 7.00 g/L in both subnormal IgG1 subclass groups (p < 0.0001, all comparisons). Regressions on IgG in three subclass groups revealed positive associations with IgG1 and IgG2 (p < 0.0001 each association). Regressions on D revealed no significant association. IgG1 percentages of IgGsum were lower and IgG2 percentages were higher in patients with subnormal IgG1 subclass levels than subnormal IgG3 only (p < 0.0001 all comparisons). Conclusions We conclude that both IgG1 and IgG2 are major determinants of IgG in patients with subnormal IgG1, combined subnormal IgG1/IgG3, or subnormal IgG3 and that in patients with subnormal IgG1 or combined subnormal IgG1/IgG3, median IgG2 levels are significantly lower in those with IgG < 7.00 g/L than those with IgG ≥ 7.00 g/L.

Detection of antibodies to erythropoietin-based drugs: is it possible to create the universal test system?

Our aim was to compare different immobilized erythropoietin (EPO) preparations for their ability to detect anti-EPO IgG antibodies in blood sera of EPO-treated patients with ELISA technique. 294 serum samples of the patients treated with erythropoietin were analyzed. 127 serum samples of patients who did not receive recombinant human EPO (rhEPO) were studied for comparative analysis. ELISA assay was performed, and different rhEPO drugs were immobilized on the anti-EPO monoclonal antibody-coated plates. Horseradish peroxidase-conjugated mouse monoclonal antibodies to human IgG, IgG1, and IgG4 was used for detection. The following drugs were studied: recombinant human erythropoietin rhEPO-beta (Shandong Kexing Bioproducts), European standard of erythropoietin BRP 3, commercial drugs Aranesp (Amgen Europe B.V.), Mircera (F. Hoffmann-La Roche Ltd.), Eprex (Johnson & Johnson LLC), Eralfon (Pharmaceutical Company Sotex). The sensitivity of the method was expressed as a positivity index (IP). IP calculated as the ratio of OD from tested sera to OD at the cut-off levels. The latter was assumed as a mean OD±SD for serum samples from EPO-naive patients. The results were evaluated as positive with IP > 1.1, and negative at IP < 0.9. Results in the range of 0.9 ≤ IP < 1.1 were considered as unidentified. Among the 294 samples, 32 specimens were evaluated as positive or unidentified for total IgG anti-EPO antibodies. The unidentified samples were detected in 1.0-1.7% of all cases. IgG1 subclass antibodies were found in 50-56.3% of patients and IgG4 subclass antibodies, in 43.850% of the patients. Mann—Whitney test showed a significant difference between the test samples compared to control group for all the ELISA modifications (p = 0.001). The Kruskal—Wallis test did not show significant differences between the IP results obtained with any of five immobilized EPO drugs (p = 0.05). The correlation quotient of IP was in the range of 0.99-0.96 for total IgG and > 0.98 for two subclasses of antibodies. Linear regression coefficients were close to one, thus indicating absence of significant differences in the sensitivity of the compared methods. This study indicate the opportunity of using the similar test systems to determine anti-EPO antibodies in the patients treated with various rhEPO drugs. Therefore, it is possible to develop a universal commercial test system to this purpose.

Insufficient Anthrax Lethal Toxin Neutralization Is Associated with Antibody Subclass and Domain Specificity in the Plasma of Anthrax-Vaccinated Individuals

Anthrax vaccine adsorbed (AVA) is a significant line of defense against bioterrorist attack from Bacillus anthracis spores. However, in a subset of individuals, this vaccine may produce a suboptimal quantity of anti-protective antigen (PA), antibodies that are poorly neutralizing, and/or antibody titers that wane over time, necessitating annual boosters. To study individuals with such poor responses, we examine the properties of anti-PA in a subset of vaccinated individuals that make significant quantities of antibody but are still unable to neutralize toxin. In this cohort, characterized by poorly neutralizing antibody, we find that increased IgG4 to IgG1 subclass ratios, low antibody avidity, and insufficient antibody targeting domain 4 associate with improper neutralization. Thus, future vaccines and vaccination schedules should be formulated to improve these deficiencies.

Cytokine Profiles and Antibody Response Associated to Choclo Orthohantavirus Infection

BackgroundNew World Hantaviruses (NWHs) are the etiological agent underlying hantavirus cardiopulmonary syndrome (HCPS), a severe respiratory disease with high mortality rates in humans. In Panama, infections with Choclo Orthohantavirus (CHOV) cause a much milder illness characterized by higher seroprevalence and lower mortality rates. To date, the cytokine profiles and antibody responses associated with this milder form of HCPS have not been defined. Therefore, in this study, we examined immune serological profiles associated with CHOV infections.MethodsFor this retrospective study, sera from fifteen individuals with acute CHOV-induced HCPS, were analyzed alongside sera from fifteen convalescent phase individuals and thirty-three asymptomatic, CHOV-seropositive individuals. Cytokine profiles were analyzed by multiplex immunoassay. Antibody subclasses, binding, and neutralization against CHOV-glycoprotein (CHOV-GP) were evaluated by ELISA, and flow cytometry.ResultsHigh titers of IFNγ, IL-4, IL-8, and IL-10 serum cytokines were found in the acute individuals. Elevated IL-4 serum levels were found in convalescent and asymptomatic seropositive individuals. High titers of IgG1 subclass were observed across the three cohorts analyzed. Neutralizing antibody response against CHOV-GP was detectable in few acute individuals but was strong in both convalescent and asymptomatic seropositive individuals.ConclusionA Th1/Th2 cytokine signature is characteristic during acute mild HCPS caused by CHOV infection. High expression of Th2 and IL-8 cytokines are correlated with clinical parameters in acute mild HCPS. In addition, a strong IL-4 signature is associated with different cohorts, including asymptomatic individuals. Furthermore, asymptomatic individuals presented high titers of neutralizing antibodies.

Immunogenicity study of a Streptococcus suis autogenous vaccine in preparturient sows and evaluation of passive maternal immunity in piglets

Background Streptococcus suis is an important pathogen that causes severe diseases mostly in weaned piglets. Only available vaccines in the field are those composed of killed bacteria (bacterins) but data about their effectiveness are missing. We report here a field study on the immunological response induced by an autogenous vaccine applied in pre-parturient sows. Using a farm with recurrent S. suis serotype 7 problems, the study was divided in three experiments: (I) Sows received the vaccine at 7 and 3 weeks pre-farrowing. (II) Replacement gilts introduced to the herd received the vaccine at 4 and 7 weeks after their entry in quarantine and a boost 3 weeks pre-farrowing. (III) Gilts from experiment II received another boost 3 weeks pre-farrowing at their 3rd/4th parity. Levels, isotype profile and opsonophagocytosis capacity of the serum antibodies induced by vaccination were evaluated in sows and maternal immunity in piglets. Results In sows (I), the vaccine induced a slight, albeit significant, increase in anti-S. suis total antibodies after 2 doses when compare to basal levels already present in the animals. These antibodies showed a high opsonic capacity in vitro, highlighting their potential protective capacity. A gilt vaccination program of 3 doses (II) resulted in a significant increase in anti-S. suis total antibodies. Levels of maternal immunity transferred to piglets were high at 7 days of age, but rapidly decreased by 18 days of age. A gilt vaccination program ensued a higher transfer of maternal immunity in piglets compared to control animals; nevertheless duration was not improved at 18 day-old piglets. The vaccine response in both gilts and sows was mainly composed of IgG1 subclass, which was also the main Ig transferred to piglets. IgG2 subclass was also found in piglets, but its level was not increased by vaccination. Finally, a recall IgG1 response was induced by another boost vaccination at 3rd/4th parity (III), indicating that the vaccine induced the establishment of a lasting memory response in the herd. Conclusions Overall, an optimal gilt/sow vaccination program might result in increased antibody responses; nevertheless duration of maternal immunity would not last long enough to protect post-weaned piglets.