Recombinant ADAMTS13 for Immune Thrombotic Thrombocytopenic Purpura.

In patients with immune thrombotic thrombocytopenic purpura (iTTP), autoantibodies against the metalloprotease ADAMTS13 lead to catastrophic microvascular thrombosis. However, the potential benefits of recombinant human ADAMTS13 (rADAMTS13) in patients with iTTP remain unknown. Here, we report the clinical use of rADAMTS13, which resulted in the rapid suppression of disease activity and complete recovery in a critically ill patient whose condition had proved to be refractory to all available treatments. We also show that rADAMTS13 causes immune complex formation, which saturates the autoantibody and may promote its clearance. Our data support the role of rADAMTS13 as a novel adjunctive therapy in patients with iTTP.

A link between circulating immune complexes and acute kidney injury in human visceral leishmaniasis.

Visceral leishmaniasis (VL) is an infectious disease caused by Leishmania infantum. Clinically, VL evolves with systemic impairment, immunosuppression and hyperactivation with hypergammaglobulinemia. Although renal involvement has been recognized, a dearth of understanding about the underlying mechanisms driving acute kidney injury (AKI) in VL remains. We aimed to evaluate the involvement of immunoglobulins (Igs) and immune complexes (CIC) in the occurrence of AKI in VL patients. Fourteen VL patients were evaluated between early treatment and 12 months post-treatment (mpt). Anti-Leishmania Igs, CIC, cystatin C, C3a and C5a were assessed and correlated with AKI markers. Interestingly, high levels of CIC were observed in VL patients up to 6 mpt. Concomitantly, twelve patients met the criteria for AKI, while high levels of cystatin C were observed up to 6 mpt. Plasmatic cystatin C was positively correlated with CIC and Igs. Moreover, C5a was correlated with cystatin C, CIC and Igs. We did not identify any correlation between amphotericin B use and kidney function markers in VL patients, although this association needs to be further explored in subsequent studies. Our data reinforce the presence of an important renal function impairment during VL, suggesting the involvement of Igs, CIC, and C5a in this clinical condition.

Preliminary results on long-term follow-up of systemic sclerosis patients under extracorporeal photopheresis.

BACKGROUND: Extracorporeal photopheresis (ECP) has been considered for treatment of patients with systemic sclerosis (SSc). OBJECTIVES: To study the 12-month effects of ECP on laboratory parameters and evaluate the SSc-related long-term survival. METHODS: 59 SSc patients who had received at least 6 ECP cycles were included. Lab parameters were assessed at baseline (ECP naïve), after 6 months, and after 12 months. 20-year follow-up data were collected for all patients. RESULTS: 31 (59/52.5%) patients presented with elevated serum III procollagen (sPIIINP) levels at baseline which significantly declined after 6- and 12-month ECP. Total lymphocyte counts as well as circulating immune complexes (CICs) significantly decreased after 12-months ECP. On long-term follow-up, patients had received a median of 37.5 (6-167) ECP cycles over a median period of 64 (6-281) months. 20-year follow-up revealed only 8 (59/13.6%) SSc-related deaths and 51 (59/86.4%) survivors. CONCLUSIONS: One-year ECP induces changes in lab parameters, such as sPIIINP, CICs, and lymphocyte counts, which have previously been implicated in the pathogenesis of SSc. More importantly, our data reveal, for the first time, that ECP-treated SSc patients appear to have extremely favorable 20-year survival rates compared to other SSc cohorts reported in the literature.

Analysis and Study on Epidemiological Features and Prognosis of Nephrotic Syndrome in Xinjiang and Heilongjiang.

BACKGROUNDS: The pathogenesis of nephrotic syndrome (NS) is complex, and there are differences between regions. This study attempted to collect clinicopathological data of patients diagnosed with NS in Xinjiang and Heilongjiang in the past 2 years, so as to explore the onset features of NS and treatment and prognosis of patients in the two regions. METHODS: Clinical data of 375 patients diagnosed with NS using renal biopsy in Xinjiang and Heilongjiang from March 2019 to March 2021 were collected. Clinical data of patients before treatment were collected, and the chi-square test was utilized to compare the differences in the sex distribution of two groups. The U test was utilized to compare abnormal distribution continuous data between two groups, such as age, hemoglobin, plasma albumin, proteinuria, and triglycerides. Independent sample t-test was utilized to compare normal distribution continuous data between two groups, such as serum total protein, serum creatinine, blood urea nitrogen, glomerular filtration rate, and total cholesterol. The independent sample t-test was also used to compare the immunoglobulin levels and complement levels between the two groups after treatment, including IgA, IgG, IgM, C3, and C4. Kaplan-Meier method was used to analyze and plot the cumulative curves of complete remission rate and partial remission rate. RESULTS: For 275 NS patients from Xinjiang, the male-to-female ratio was 0.81 : 1. For 84 patients from Heilongjiang, the male-to-female ratio was 1.05 : 1. The onset ages of patients in Xinjiang and Heilongjiang were 22-45 years old and 22-47 years old, respectively. Respectively, there were 221 cases (80.36%) and 66 cases (78.57%) of primary NS in Xinjiang and Heilongjiang. There were 54 cases (19.64%) and 18 cases (21.43%) of secondary NS in Xinjiang and Heilongjiang, respectively. There was no statistically significant difference in cause distribution between the two regions (p = 0.756). After treatment, immunoglobulin levels (IgA (p = 0.009), IgG (p = 0.002), IgM (p < 0.001)) and complement C3 (p < 0.001) and C4 (p < 0.001) levels in Xinjiang and Heilongjiang were statistically significant. 129 cases in Xinjiang (46.91%) and 55 cases in Heilongjiang (65.48%) were treated with glucocorticoid (GC) combined with immunosuppressive therapy, respectively. After receiving treatment, 67 (24.36%) of 275 patients in Xinjiang achieved complete remission, 166 (60.36%) achieved partial remission, 22 (26.19%) of 84 patients in Heilongjiang achieved complete remission, and 56 (66.67%) achieved partial remission, and there was no statistically significant difference in remission rate between the two regions (p = 0.159). Patients in Xinjiang and Heilongjiang achieved complete remission at an average of 10.34 weeks (9.98-10.70) and 9.95 weeks (9.26-10.65), respectively. There was no significant difference in complete remission rates between the two regions (p = 0.663). Patients in Xinjiang and Heilongjiang achieved partial remission at an average of 8.76 weeks (8.38-9.14) and 7.99 weeks (7.33-8.65), respectively. There was no significant difference in the partial remission rate between the two regions (p = 0.065). CONCLUSION: The causes of NS in Xinjiang and Heilongjiang were similar. After treatment, there were differences in immunoglobulin levels (IgA, IgG, IgM) and complement levels (C3, C4) in the two regions. The main treatment methods used in the two regions were GC combined with immunosuppressive therapy. The prognosis of patients in the two regions was similar. The complete remission rate and partial remission rate after treatment in the two regions were similar, and the average time required to achieve complete remission and partial remission was also similar.

Immunoglobulin G Immune Complexes May Contribute to Neutrophil Activation in the Course of Severe Coronavirus Disease 2019.

Severe coronavirus disease 2019 (COVID-19) is associated with an overactive inflammatory response mediated by macrophages. Here, we analyzed the phenotype and function of neutrophils in patients with COVID-19. We found that neutrophils from patients with severe COVID-19 express high levels of CD11b and CD66b, spontaneously produce CXCL8 and CCL2, and show a strong association with platelets. Production of CXCL8 correlated with plasma concentrations of lactate dehydrogenase and D-dimer. Whole blood assays revealed that neutrophils from patients with severe COVID-19 show a clear association with immunoglobulin G (IgG) immune complexes. Moreover, we found that sera from patients with severe disease contain high levels of immune complexes and activate neutrophils through a mechanism partially dependent on FcγRII (CD32). Interestingly, when integrated in immune complexes, anti-severe acute respiratory syndrome coronavirus 2 IgG antibodies from patients with severe COVID-19 displayed a higher proinflammatory profile compared with antibodies from patients with mild disease. Our study suggests that IgG immune complexes might promote the acquisition of an inflammatory signature by neutrophils, worsening the course of COVID-19.

Immunopathological analysis of the expression of glomerular exostosin 1 and exostosin 2 in Japanese patients with lupus nephritis.

Exostosin 1 and exostosin 2 (EXT1/EXT2) on glomerular basement membrane (GBM) were recently reported as novel putative antigens in secondary membranous nephropathy with autoimmune disease. However, the clinical significance of glomerular EXT1/EXT2 remains elusive in patients with lupus nephritis (LN). The immunofluorescence staining pattern of glomerular EXT1/EXT2 is also undetermined in membranous LN (MLN) or proliferative LN (PLN). We cross-sectionally analyzed patients with MLN (pure class V, n = 11) and PLN (class III, IV, and mixed class III/IV + V, n = 22) who underwent renal biopsies between 2010 and 2020 at Showa University Hospital. Glomerular EXT1/EXT2 expressions were evaluated by immunofluorescence. T-helper (Th) cell-related serum inflammatory cytokines were measured using enzyme-linked immunosorbent assay. The positivity for both EXT1/EXT2 was higher in patients with MLN than PLN (90.9% vs 63.6%, P = 0.212). MLN showed global and bright granular EXT1/EXT2 expressions along GBM, while PLN showed segmental and moderate expressions on GBM. Additionally, glomerular EXT1/EXT2 positivity was not associated with the degree of proteinuria or renal function in MLN and PLN patients, but the levels of serum anti-dsDNA antibody and circulating immune complexes were lower in patients with EXT1/EXT2-positive MLN than EXT1/EXT2-negative PLN. Moreover, serum complement levels and IL-4/IFN-γ ratios were elevated in EXT1/EXT2-positive MLN than EXT1/EXT2-negative PLN. Collectively, immunofluorescence staining for glomerular EXT1/EXT2 had characteristic patterns between MLN and PLN. Glomerular EXT1/EXT2 expressions tended to be high in Th2-dominant MLN patients without severe hypocomplementemia and elevated autoantibodies. Thus, EXT1/EXT2 might be involved in the unique developmental mechanism of MLN.

Heat-Inactivation of Human Serum Destroys C1 Inhibitor, Pro-motes Immune Complex Formation, and Improves Human T Cell Function.

Heat-inactivation of sera is used to reduce possible disturbing effects of complement factors in cell-culture experiments, but it is controversially discussed whether this procedure is appropriate or could be neglected. Here, we report a strong impact of heat-inactivation of human sera on the activation and effector functions of human CD4+ T cells. While T cells cultured with native sera were characterized by a higher proliferation rate and higher expression of CD28, heat-inactivated sera shaped T cells towards on-blast formation, higher cytokine secretion (interferon γ, tumor necrosis factor, and interleukin-17), stronger CD69 and PD-1 expression, and increased metabolic activity. Heat-inactivated sera contained reduced amounts of complement factors and regulators like C1 inhibitor, but increased concentrations of circulating immune complexes. Substitution of C1 inhibitor reduced the beneficial effect of heat-inactivation in terms of cytokine release, whereas surface-molecule expression was affected by the addition of complex forming anti-C1q antibody. Our data clearly demonstrate a beneficial effect of heat-inactivation of human sera for T cell experiments but indicate that beside complement regulators and immune complexes other components might be relevant. Beyond that, this study further underpins the strong impact of the complement system on T cell function.

Glomerulonephritis with severe nephrotic syndrome induced by immune complexes composed of galactose-deficient IgA1 in primary Sjögren's syndrome: a case report.

BACKGROUND: Primary Sjögren's syndrome (pSS) is an auto-immune disease characterized by sialadenitis and dacryoadenitis with lymphoplasmacytic cell infiltration. In pSS, not only sicca symptoms, but also extra-glandular involvement induced by immune abnormalities based on pSS occurs. Renal involvement is one such important life-threatening extra-glandular involvement. Although the aberrant glycosylated IgA in pSS as a product of over-activated B cells is a risk factor of renal involvement, its association has not been clarified. Here we report a case of glomerulonephritis (GN) induced by immune complexes (IC) composed of galactose-deficient IgA1 (Gd-IgA1) in a patient with pSS. CASE PRESENTATION: A 48-year-old Japanese woman with pSS was admitted to our hospital because of a two-month history of nephrotic syndrome. Seven years before she had been diagnosed with pSS from keratoconjunctivitis sicca, elevation of serum anti-Ro/SSA antibody titer and lymphoplasmacytic cell infiltration around salivary ducts of the small salivary glands. Renal biopsy revealed diffuse bubbling appearance in glomerular basement membrane (GBM) with scarce mesangial proliferation. Immunofluorescence showed granular IgA, C3 and Gd-IgA1 staining of GBM. Light chain staining showed no monoclonality. Electron microscopy showed electron dense deposits mainly in the intra-membranous and paramesangial areas and slightly in the subepithelial area. Additional serum analysis confirmed elevation of Gd-IgA1 (13.5 µg/mL), which was comparable with that seen in IgA nephropathy, and qualitative enzyme-linked immunosorbent assay of IgA-containing circulating immune complex (IgA-CIC) was positive. Thus, we diagnosed GN induced by IC composed of Gd-IgA1. Furthermore, retrospectively performed immunofluorescence of the small salivary gland evaluated at the diagnosis of pSS showed positive Gd-IgA1 staining of infiltrating lymphoplasmacytic cells. Therefore, we concluded that Gd-IgA1 produced by over-activated B cells in pSS formed circulating IC and thereby induced GN. After induction therapy with high dose prednisolone and mycophenolate mofetil, the nephrotic syndrome remitted within 3 weeks, the serum Gd-IgA1 level decreased to the normal range (3.8 µg/mL), and serum IgA-CIC disappeared in the 6th month after induction therapy. CONCLUSIONS: Our findings clearly demonstrate an association between aberrant glycosylated IgA and the renal involvement seen in pSS, thereby helping to clarify the renal significance of aberrant glycosylated IgA in pSS.

Similarities and differences between the immunopathogenesis of COVID-19-related pediatric multisystem inflammatory syndrome and Kawasaki disease.

Multisystem inflammatory syndrome associated with the SARS-CoV-2 pandemic has recently been described in children (MIS-C), partially overlapping with Kawasaki disease (KD). We hypothesized that (a) MIS-C and prepandemic KD cytokine profiles may be unique and justify the clinical differences observed, and (b) SARS-CoV-2-specific immune complexes (ICs) may explain the immunopathology of MIS-C. Seventy-four children were included: 14 with MIS-C, 9 patients positive for SARS-CoV-2 by PCR without MIS-C (COVID), 14 with prepandemic KD, and 37 healthy controls (HCs). Thirty-four circulating cytokines were quantified in pretreatment serum or plasma samples and the presence of circulating SARS-CoV-2 ICs was evaluated in MIS-C patients. Compared with HCs, the MIS-C and KD groups showed most cytokines to be significantly elevated, with IFN-γ-induced response markers (including IFN-γ, IL-18, and IP-10) and inflammatory monocyte activation markers (including MCP-1, IL-1α, and IL-1RA) being the main triggers of inflammation. In linear discriminant analysis, MIS-C and KD profiles overlapped; however, a subgroup of MIS-C patients (MIS-Cplus) differentiated from the remaining MIS-C patients in IFN-γ, IL-18, GM-CSF, RANTES, IP-10, IL-1α, and SDF-1 and incipient signs of macrophage activation syndrome. Circulating SARS-CoV-2 ICs were not detected in MIS-C patients. Our findings suggest a major role for IFN-γ in the pathogenesis of MIS-C, which may be relevant for therapeutic management.

Proper definition of the set of autoantibody-targeted antigens relies on appropriate reference group selection.

Autoimmune diseases are frequently associated with autoantibodies. Recently, large sets of autoantibody-targeted antigens ("autoantigen-omes") of patient and control sera have been revealed, enabling autoantigen-omic approaches. However, statistical standards for defining such autoantigen-omes are lacking. The z-score indicates how many standard deviations an antigen reactivity of a given sample is from the mean reactivity of the corresponding antigen in a reference group. Hence, it is a common measure to define significantly positive reactivity in autoantigen profiling approaches. Here, we address the risk of biased analyses resulting from unbalanced selection of the reference group. Three study groups were selected. Patients-of-interest were chronic inflammatory demyelinating polyneuropathy (CIDP); controls were other neuropathies (ONP); and healthy controls (HC). Each serum was screened for significant autoantigen reactivity using HuProt™ protein arrays. We compared three possible selections of reference groups for statistical z-score calculations: method#1, the control groups (ONP + HC); method #2, all groups together; method #3, the respective other groups (e.g., CIDP + HC for the ONP autoantigen-ome). The method selection seriously affected the size of the autoantigen-omes. Method #1 introduced a bias favoring significantly more antigens per patient in the CIDP group (for z >4: 19 ±â€¯3 antigens) than in the control groups (ONP: 2 ±â€¯1; HC: 0 ±â€¯0). The more balanced methods #2 and #3 did not result in significant differences. This contribution may help to avoid interpretation biases and to develop guidelines for population studies revealing autoantigen-omes via high throughput studies such as protein microarrays, immunoprecipitation with mass spectrometry, or phage display assays.

Strongyloides-specific IgA, IgG and IgG immune complex profile in patients with pulmonary tuberculosis.

AIMS: To describe an anti-Strongyloides IgA, IgG and IgG immune complex antibody response profile in patients with pulmonary tuberculosis. METHODS AND RESULTS: Saliva and serum samples were collected from 100 individuals: group I, 50 apparently healthy individuals; and group II, 50 pulmonary tuberculosis patients. The IgA, IgG and IgG immune complex detection were carried out via an ELISA immunoenzymatic test. Optical density medians in saliva samples of IgA antibody (median of 7.21) and IgG-IC (median of 4.95) were significantly higher in tuberculosis group compared to control individuals (median IgA of 3.93 and IgG-IC of 2.38). CONCLUSION: This study presents antibody data to the field of pulmonary tuberculosis and strongyloidiasis coinfection, including saliva samples, and especially IgG immune complex detection.

Accurate detection of Zika virus IgG using a novel immune complex binding ELISA.

OBJECTIVES: Accurate serological assays are urgently needed to support public health responses to Zika virus (ZIKV) infection with its potential to cause foetal damage during pregnancy. Current flavivirus serology for ZIKV infections lacks specificity due to cross-reacting antibodies from closely related other flaviviruses. In this study, we evaluated novel serological tests for accurate ZIKV IgG detection. METHODS: Our ELISAs are based on immune complex binding. The high specificity is achieved by the simultaneous incubation of labelled ZIKV antigen and unlabelled flavivirus homolog protein competitors. Two assays were validated with a panel of 406 human samples from PCR-confirmed ZIKV patients collected in Brazil (n = 154), healthy blood donors and other infections from Brazil, Europe, Canada and Colombia (n = 252). RESULTS: The highest specificity (100% [252/252, 95% confidence interval (CI) 98.5-100.0]) was shown by the ZIKV ED3 ICB ELISA using the ED3 antigen of the ZIKV envelope. A similar test using the NS1 antigen (ZIKV NS1 ICB ELISA) was slightly less specific (92.1% [232/252, 95% CI 88.0-95.1]). The commercial Euroimmun ZIKV ELISA had a specificity of only 82.1% (207/252, 95% CI 76.8-86.7). Sensitivity was high (93-100%) from day 12 after onset of symptoms in all three tests. Seroprevalence of ZIKV IgG was analysed in 87 samples from Laos (Asia) confirming that the ED3 ELISA showed specific reactions in other populations. CONCLUSIONS: The novel ED3 ICB ELISA will be useful for ZIKV-specific IgG detection for seroepidemiological studies and serological diagnosis for case management in travellers and in countries where other flavivirus infections are co-circulating.

Onset age is a risk factor for refractory pediatric IgA vasculitis: a retrospective cohort study.

BACKGROUND: Though outcome differences between children and adults with immunoglobulin A vasculitis (IgAV) has been well documented, it remains unclear if disease features in pediatric IgAV patients vary with onset age. We aimed to explore clinical features and prognosis of pediatric IgAV stratified by onset age. METHODS: We retrospectively reviewed records of patients under 18 years old diagnosed with IgAV from January 1999 to December 2018 in one tertiary medical center in Taiwan. Patients were grouped by onset age: ≤ 6 years old, 6-12 years old (> 6, ≤ 12), and 12-18 years old (> 12, < 18). Demographics, laboratory data, incidence of gastrointestinal, renal, and joint involvement, corticosteroid dependence, recurrence, and refractory disease were analyzed. Recurrence was defined as disease flare-up after complete remission and discontinuation of all medications for at least 3 months. Corticosteroid dependence was defined by more than 6 weeks of daily oral corticosteroid intake. Refractory disease was defined as not achieving complete remission 6 months after disease onset. Statistical analysis was performed using R software (v3.6.0). RESULTS: There were 484 IgAV patients, with an onset age of 6.10 (4.72-8.58) (median (IQR)) years old. There were 234 (48.3%) patients ≤6 years old, 210 (43.4%) 6-12 years old, and 40 (8.3%) 12-18 years old. One hundred and thirty (26.9%) patients had renal involvement, which was more frequent in older children (≤ 6 years old, 18.4%; 6-12 years old, 31.0%; 12-18 years old, 55.0%; p <  0.001). There were 361 patients (74.6%) with joint involvement; younger children were affected more frequently (≤ 6 years old, 82.1%; 6-12 years old, 71.9%; 12-18 years old, 45.0%; p <  0.001). Gastrointestinal involvement was present in 311 (64.3%) patients, showing no difference among age groups. There were 46 patients (9.5%) with recurrent IgA vasculitis, 136 (28.1%) with corticosteroid dependent and 76 (15.7%) with refractory disease. Corticosteroid dependence and refractory disease occurred more frequently as onset age increased (p <  0.001). CONCLUSION: Pediatric IgAV with different onset ages are associated with distinct clinical manifestations and outcomes. The risk of developing corticosteroid dependence, refractory disease and renal involvement increased with onset age.

Structural characteristics of circulating immune complexes in calves with bronchopneumonia: Impact on the quiescent leukocytes.

Calf bronchopneumonia is accompanied by increased level of circulating immune complexes (CIC), and we analysed size, and protein and lipid constituents of these CIC with an attempt to elucidate the connection between the CIC structural properties and their capacity to modulate leukocyte function. CIC of heathy calves (CICH) and calves with naturally occurring bronchopneumonia (CICD) were isolated by PEG precipitation and analysed by electrophoresis and chromatography. The predominant CIC proteins were IgG, albumin, and transferrin. Affinity isolated serum and CIC IgG coprecipitated several proteins, but only 75 and 80 kDa proteins bound CIC IgG, exclusively. 60 and 65 kDa proteins co-precipitated with CICD IgG, unlike CICH IgG. In both CICH and CICD, oleic acid-containing phospholipids predominated. In CICD, the content of oleic and vaccenic acid was higher than in CICH, while myristic, palmitic, stearic, linoleic and arachidonic acid showed lower content. Dynamic light scattering displayed difference in particle size distribution between CICH and CICD; 1280 nm large particles were present only in CICD. The effect of CICH and CICD on mononuclear cells (MNC) and granulocytes was analysed in vitro. CICH and CICD, with slight difference in intensity, stimulate MNC apoptosis, promote cell cycle arrest of unstimulated MNC, and cell cycle progression of PHA stimulated MNC. Both CIC reduced granulocyte apoptosis after 24 h while after 48 h this effect was detected for CICD only. These results indicate that structural differences of CICH and CICD might interfere with the CIC functional capacity, which we consider important for evaluation of CIC immunoregulatory function.

Bone Marrow Endothelial Cells Take Up Blood-Borne Immune Complexes via Fcγ Receptor IIb2 in an Erythropoietin-Dependent Manner.

Immune complexes (ICs) in blood are efficiently removed mainly by liver reticuloendothelial systems consisting of sinusoidal endothelial cells and Kupffer cells expressing FcγR. The bone marrow (BM) also has sinusoidal vasculatures, and sinusoidal BM endothelial cells (BMECs) bear unique function, including hematopoietic niches and traffic regulation of hematopoietic cells. In this study, we found that sinusoidal BMECs express FcγRIIb2, which is markedly increased in anemic conditions or by the administration of erythropoietin (Epo) in healthy mice. BMECs expressed Epo receptor (EpoR), and the Epo-induced increase in FcγRIIb2 expression was abolished in Epor-/- ::HG1-Epor transgenic mice, which lack EpoR in BMECs except for BM erythroblasts, suggesting the effect was directly mediated via EpoR on BMECs. Further, although BMECs hardly captured i.v.-injected soluble ICs in healthy mice, Epo administration induced a remarkable increase in the uptake of ICs in a FcγRIIb-dependent manner. Enhancement of the IC incorporation capacity by Epo was also observed in cultured BMECs in vitro, suggesting the direct effect of Epo on BMECs. Moreover, we found that i.v.-injected ICs in Epo-treated mice were more rapidly removed from the circulation than in PBS-treated mice. These results reveal a novel function of BMECs to efficiently remove circulating blood-borne ICs in an FcγRIIb2-mediated manner.

A Personalized Update on IgA Nephropathy: A New Vision and New Future Challenges.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world among patients undergoing renal biopsy. Approximately 30% of patients with IgAN develop end-stage kidney disease 20 years after renal biopsy. It is a glomerulopathy with a very broad clinical presentation, making it difficult to stratify and treat. IgAN is characterized by dysregulation of the immune system, which causes an abnormal synthesis of IgA1 that is deglycosylated causing its mesangial deposition. IgAN pathogenesis is incompletely understood; the current multi-hit hypothesis of IgAN pathogenesis does not explain the range of glomerular inflammation and renal injury associated with mesangial IgA deposition. Although associations between IgAN and glomerular and circulating markers of complement activation are established, the mechanism of complement activation and contribution to glomerular inflammation and injury are not defined. On the other hand, the renal-gut connection can also play an important role in the pathogenesis of IgAN with possible therapeutic implications. In order to standardize the histological findings, the Oxford Classification has allowed clarifying renal lesions that confer potential risk of progression. Currently, except for the blockade of the renin-angiotensin-aldosterone system, no other therapies are available in clinical setting for the treatment of IgAN, although the range of new drugs under investigation is extensive. The incorporation in the next trials of clinical parameters such as the amount of hematuria and histological lesions may allow more personalized therapeutic approaches. To summarize, in recent years, several important efforts have taken place in the understanding of IgAN, but still, further studies are warranted to elucidate the best therapeutic strategies according to the risk to improve the prognosis of this entity.

Classical complement activation on human erythrocytes in subjects with systemic lupus erythematosus and a history of autoimmune hemolytic anemia.

INTRODUCTION: Autoimmune hemolytic anemia (AIHA) is a serious manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. In order to more fully understand the causative pathways, we utilized sera from subjects with SLE and active AIHA, or a history of AIHA, to evaluate the classical complement pathway, anti-erythrocyte antibodies, and immune complexes. METHODS: To evaluate antibody-mediated complement activation on the surface of erythrocytes, as occurs in AIHA, blood type O erythrocytes were incubated with sera from 19 subjects with SLE and a history of AIHA. Circulating anti-erythrocyte antibodies and immune complexes were measured with ELISA-based assays. RESULTS: In total, 90% of subjects with SLE and a history of AIHA, but not active clinical hemolysis, had measurable anti-erythrocyte antibodies. Of those with anti-erythrocyte antibody, 53% demonstrated complement opsonization on the erythrocyte surface >twofold above negative control and 29% generated the anaphylatoxin C5a. CONCLUSIONS: For subjects with SLE and a history of AIHA, the persistence of circulating anti-erythrocyte antibodies and resultant erythrocyte complement opsonization and anaphylatoxin generation suggests the possibility that these complement effectors contribute to chronic morbidity and risk of AIHA relapse.

Automated, Generic Reagent and Ultratargeted 2D-LC-MS/MS Enabling Quantification of Biotherapeutics and Soluble Targets down to pg/mL Range in Serum.

Mass spectrometry has recently emerged as a powerful analytical tool for the assessment of pharmacokinetics and biomarkers in drug development. Compared with ligand binding assays, a major advantage of mass spectrometry-based assays is that they are less dependent on high quality binding reagents, while a key limitation is the relatively lower sensitivity. To address the sensitivity issue, we have developed a generic reagent, ultratargeted two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) method which combines commercially available protein A affinity capture, targeted analyte isolation by 2D-LC, and targeted detection by multiple reaction monitoring (MRM). A targeted-2D-with-dilution configuration was designed to automate 2D-LC-MS/MS. This method was systematically evaluated using an anti-CD22 monoclonal antibody spiked into monkey and human serum, where lower limits of quantification (LLOQ) of 0.78 and 1.56 ng/mL were achieved, respectively. This represents an over 100-fold improvement in assay sensitivity compared to the conventional LC-MS/MS method. The performance of the method was further confirmed by analyzing another monoclonal antibody, bevacizumab, as well as a soluble antigen, circulating PD-L1. The results indicate that our method enables quantification of antibody therapeutics and antigen biomarkers in both clinical and nonclinical samples in the pg/mL to low ng/mL range. Protein A affinity capture was employed as a universal sample preparation procedure applicable to both full-length antibody therapeutics and antibody-antigen complexes. This novel method is also fully automated and proven to be highly robust for routine bioanalysis in drug development.

Functional effects of immune complexes formed between pembrolizumab and patient-generated anti-drug antibodies.

The PD-1-targeting IgG4 antibody pembrolizumab has significant anti-tumor activity in a proportion of stage IV melanoma patients. A subset of patients develop anti-drug antibodies (ADA) which can form immune complexes (IC) with pembrolizumab. Although IC can induce powerful, Fc-mediated, immune-regulatory effects, their functional impact during pembrolizumab treatment is unclear. The functional effects of IC generated in vitro using pembrolizumab and patient-derived ADA was, therefore, investigated. Screening identified a patient whose trough serum samples from three treatment cycles contained both ADA with neutralizing activity and low levels of pembrolizumab. This patient responded well to therapy over 2 years and had ongoing, infusion-related, hypersensitivity reactions despite the later absence of detectable ADA. The components of IC were mimicked by forming a complex of pembrolizumab by absorption onto a solid phase with or without subsequent exposure to the ADA+ patient sera. Complexes comprised of pembrolizumab alone significantly inhibited TLR4 (LPS)-driven IL-10 production by PBMC and stimulated the generation of reactive oxygen species by granulocytes. In contrast, soluble and solid-phase F(ab´)2 fragments of pembrolizumab had no effect demonstrating the requirement for cross-linked Fc regions. IC containing pembrolizumab and ADA could additionally induce complement and NK activation. The results of this study demonstrate that, when oligomerized, the Fc region of pembrolizumab alone can provide immuno-regulatory signals. Furthermore, IC containing both pembrolizumab and patient-generated ADA can induce additional signals. These Fc-mediated signals may modulate both hypersensitivity reactions and anti-tumor responses associated with pembrolizumab therapy.