Anti-hinge Antibodies Research Articles

Impact of anti-human IgG hinge peptide antibodies on identification of patients with early seronegative rheumatoid arthritis.

The early diagnosis of seronegative rheumatoid arthritis (SNRA), characterised by the absence of rheumatoid factor and anti-citrullinated antibody, involves a greater challenge compared to seropositive RA (SPRA). This study aimed to assess the discriminatory potential of anti-human IgG hinge antibodies (AHAs) for patients with early SNRA. DMARDs-naive patients with SPRA (n=43), SNRA (n=21), and non-RA (n=49), with disease duration < 2 years, were included. Antigens comprised IgG1 or IgG4 F(ab')2 cleaved by pepsin or MMP-3 and their hinge peptide analogues. Eight IgG anti-hinge antibodies (AHAs) against these antigens were measured in sera from the patients and 58 healthy controls (HCs) using ELISA. Serum CRP and MMP-3 levels, and clinical disease activity index (CDAI), were obtained from medical records. The area under the curve (AUC) obtained from logistic regression and receiver operating characteristic curve analyses were used as a discriminant indicator. The levels of the IgG AHAs were as follows: SPRA≥SNRA≈non-RA>HC. None of the AHAs were effective in discriminating SNRA from non-RA. However, the combination of MMP-3 and AHAs against IgG4 hinge peptide analogues demonstrated the utility (AUC=0.94). Furthermore, combination of MMP-3, AHAs against IgG1 hinge peptide analogues and CDAI maximally exerted discriminatory power (AUC=0.997). Specific AHAs in combination with MMP-3 and CDAI are potentially useful to discriminate SNRA from non-RA.

Levels and in vitro functional effects of circulating anti-hinge antibodies in melanoma patients receiving the immune checkpoint inhibitor pembrolizumab.

The efficacy of PD-1 monoclonals such as pembrolizumab can be modulated by the signals delivered via their Fc region. Tumour/inflammation associated proteases can generate F(ab')2 fragments of therapeutic monoclonals, and subsequent recognition of F(ab')2 epitopes by circulating anti-hinge antibodies (AHA) can then, potentially, link F(ab')2 binding to the target antigen with novel Fc signalling. Although elevated in inflammatory diseases, AHA levels in cancer patients have not been investigated and functional studies utilising the full repertoire of AHA present in sera have been limited. AHA levels in pembrolizumab treated melanoma patients (n = 23) were therefore compared to those of normal donors and adalimumab treated patients. A subset of melanoma patients and the majority of adalimumab patients had elevated levels of AHA reactive with F(ab')2 fragments of IgG4 anti-PD-1 monoclonals (nivolumab, pembrolizumab) and IgG1 therapeutic monoclonals (rituximab, adalimumab). Survival analysis was restricted by the small patient numbers but those melanoma patients with the highest levels (>75% percentile, n = 5) of pembrolizumab-F(ab')2 reactive AHA had significantly better overall survival post pembrolizumab treatment (p = 0.039). In vitro functional studies demonstrated that the presence of AHA+ sera restored the neutrophil activating capacity of pembrolizumab to its F(ab')2 fragment. Neither pembrolizumab nor its F(ab')2 fragments can induce NK cell or complement dependent cytotoxicity (CDC). However, AHA+ sera in combination with pembrolizumab-F(ab')2 provided Fc regions that could activate NK cells. The ability of AHA+ sera to restore CDC activity was more restricted and observed using only one pembrolizumab and one adalimumab patient serum in combination with rituximab- F(ab')2. This study reports the presence of elevated AHA levels in pembrolizumab treated melanoma patients and highlight the potential for AHA to provide additional Fc signaling. The issue of whether tumour associated proteolysis of PD-1 mAbs and subsequent AHA recognition impacts on treatment efficacy requires further study.

Impairment of IgG Fc functions promotes tumor progression and suppresses NK cell antitumor actions

Natural killer (NK) cells mediate antibody dependent cytotoxic killing of cancer cells via cross-linking FcγR on NK cells with IgG-Fc. Studies have shown that the single-hinge cleaved IgGs (scIgGs) have dysfunctional Fc and failed engagement with FcγRs on immune cells. However, little is known about how scIgGs impact on antitumor immunity in the tumor microenvironment. In this study, we revealed a significant association of tumor scIgGs with tumor progression and poor outcomes of breast cancer patients (n = 547). Using multiple mouse tumor models, we demonstrated that tumor scIgGs reduced NK cell cytotoxic activities and resulted in aggressive tumor progression. We further showed that an anti-hinge specific monoclonal antibody (AHA) rescued the dysfunctional Fc in scIgGs by providing a functional Fc and restored NK cell cytotoxic activity. These findings point to a novel immunotherapeutic strategy to enhance Fc engagement with FcγRs for activation of anticancer immunity.

Elevated Fab glycosylation of anti-hinge antibodies

Objective Rheumatoid arthritis (RA) is characterized by systemic inflammation and the presence of anti-citrullinated protein antibodies (ACPAs), which contain remarkably high levels of Fab glycosylation. Anti-hinge antibodies (AHAs) recognize immunoglobulin G (IgG) hinge neoepitopes exposed following cleavage by inflammation-associated proteases, and are also frequently observed in RA, and at higher levels compared to healthy controls (HCs). Here, we investigated AHA specificity and levels of Fab glycosylation as potential immunological markers for RA. Method AHA serum levels, specificity, and Fab glycosylation were determined for the IgG1/4-hinge cleaved by matrix metalloproteinase-3, cathepsin G, pepsin, or IdeS, using enzyme-linked immunosorbent assay and lectin affinity chromatography, in patients with early active RA (n = 69) and HCs (n = 97). Results AHA reactivity was detected for all hinge neoepitopes in both RA patients and HCs. Reactivity against CatG-IgG1-F(ab´)2s and pepsin-IgG4-F(ab´)2s was more prevalent in RA. Moreover, all AHA responses showed increased Fab glycosylation levels in both RA patients and HCs. Conclusions AHA responses are characterized by elevated levels of Fab glycosylation and highly specific neoepitope recognition, not just in RA patients but also in HCs. These results suggest that extensive Fab glycosylation may develop in response to an inflammatory proteolytic microenvironment, but is not restricted to RA.

Adalimumab Immunogenicity Is Negatively Correlated with Anti-Hinge Antibody Levels in Patients with Rheumatoid Arthritis.

Patients with rheumatoid arthritis (RA) are frequently treated with anti-tumor necrosis factor-α immunoglobulin therapy but develop neutralizing antibodies against these drugs, necessitating therapeutic monitoring of drug concentrations and anti-drug antibodies. Patients with RA have multiple factors related to their autoimmune disposition that interfere with conventionally used methods to detect anti-drug antibodies. Currently deployed analytical methods have significant limitations that hinder clinical interpretation and/or routine use, and no method can detect immunogenicity and drug levels simultaneously to provide clinically meaningful recommendations. Given these limitations, the objective of this study was to identify sources of and associations with assay interference in patients with RA. We designed a modular immunogenicity and drug concentration detection technology to identify the factors that interfere with the detection of adalimumab and anti-adalimumab antibodies in a cohort of 206 patients with RA. Patients were included from the University of Pittsburgh Rheumatoid Arthritis Comparative Effectiveness Research registry. In this cohort, we analyzed clinical and plasma factors associated with anti-adalimumab and anti-hinge antibodies. A novel flow cytometry-based assay was developed and validated that simultaneously measures adalimumab and anti-adalimumab antibody concentrations, overcoming many of the interference factors that are limitations of conventional assays, including anti-fragment crystallizable (Fc) and anti-hinge antibodies. C-reactive protein (P = 0.035), Disease Activity Score-28 (DAS28) score (P = 0.002), and disease activity category (P = 0.009) were significantly associated with anti-adalimumab antibodies but not with anti-hinge antibodies (P > 0.05). Anti-hinge antibodies were inversely associated with drug-neutralizing antibodies (P = 0.002). In patients with RA, anti-hinge antibodies may have a potential protective effect against the development of anti-adalimumab antibodies. SIGNIFICANCE STATEMENT: Using a novel cytometric assay that simultaneously measures drug and anti-drug antibodies, we overcame many interferences that hinder the clinical interpretation of adalimumab immunogenicity testing. Our investigation in patients with RA demonstrated that immunogenicity impaired the pharmacological action of adalimumab via analysis of RA disease severity markers. We also demonstrate that patients with anti-hinge antibodies had lower anti-adalimumab antibody levels and decreased drug neutralization. Our results suggest that anti-hinge antibodies can predict adalimumab immunogenicity before the start of therapy.

IgG anti-hinge antibodies against IgG4 F(ab\u2019)2 fragments generated using pepsin are useful diagnostic markers for rheumatoid arthritis: implications of the possible roles of metalloproteinases and IgG subclasses in generating immunogenic hinge epitopes

BackgroundPepsin agglutinators, discovered over 50 years ago, have been recently referred to as anti-hinge antibodies (AHAs) because of their reaction with the IgG hinge epitope. AHAs have different reactivity for each hinge epitope generated by each protease that cleaves the hinge region at different sites. Moreover, AHAs have different reactivity against different hinge epitopes derived from each IgG subclass even when the same protease is used. Since the expression of matrix metalloproteinase-3 (MMP-3) is enhanced in rheumatoid arthritis (RA), AHA production could also be increased. The purpose of this study was to determine whether the levels of AHAs against IgG hinge epitopes produced by MMP-3 are elevated in RA.MethodsThe serum levels of IgG or IgA AHAs against the IgG1/IgG4 F(ab’)2 fragments, generated by either MMP-3 or pepsin, were measured using ELISA in 111 patients with RA and 81 healthy controls (HC). Receiver operating characteristic (ROC) analysis was used for obtaining optimal cutoff values and cutoff values indicating high specificity (> 95%) of the AHA. The targeted epitope of a specific AHA was investigated through inhibition ELISA.ResultsSeven AHAs were statistically higher in RA patients than in HC, except IgG AHA against IgG1 F(ab’)2, which was generated by MMP-3 proteolytic cleavage. The areas under the ROC curve were 0.66–0.80, although the sensitivities at high specificity were low (5.4–24.3%). The cumulative number of positive AHAs in each individual was statistically higher in RA patients than in HC, suggesting the extreme extent of AHA repertoires in RA. Inhibition studies revealed that IgG AHAs against IgG4 F(ab’)2 fragments generated by pepsin cross-reacted with IgG1 F(ab’)2 fragments generated by pepsin. Multivariate logistic regression analysis identified the IgG AHA against IgG4 F(ab’)2 fragments generated by pepsin as an independent variable for RA diagnosis, even in RA patients who were negative for both RF and ACPA (odds ratio 1.18, 95% CI 1.06–1.32; P = 0.003). Additional experiments using non-RA patients finally strengthened the diagnostic utility.ConclusionIn RA patients, we observed diversification and amplification of AHA repertoires and diagnostic utility of the specific AHA against IgG4 F(ab’)2 fragments generated by pepsin but not MMP-3.

An update on research advances in rheumatoid arthritis: from clinic to basic science

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterised by breach of self-tolerance and production of autoantibodies, particularly rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA). Many events can lead to loss of tolerance in RA and they include genetic, environmental and epigenetic factors as well as post-translational modifications. Current therapies in RA tend to treat symptoms or slow down the disease progression but no cure exists at the moment. This review provides a summary of the main recent research advances regarding new therapies in RA and new studies about the effect of genetic, environment and epigenetics in this autoimmune disease. Moreover, it summarises the present knowledge about the role of “new” autoantibodies in RA focussing on anti-carbamylated protein antibodies (anti-CarP), anti-hinge antibodies, anti-acetylated protein antibodies and anti-malondialdehyde-acetaldehyde adducts (MAA) antibody. Finally, a brief summary on the recent role of Fab N-glycosylation is reported.

Molecular characterization of human anti-hinge antibodies derived from single-cell cloning of normal human B cells

Anti-hinge antibodies (AHAs) are an autoantibody subclass that, following proteolytic cleavage, recognize cryptic epitopes exposed in the hinge regions of immunoglobulins (Igs) and do not bind to the intact Ig counterpart. AHAs have been postulated to exacerbate chronic inflammatory disorders such as inflammatory bowel disease and rheumatoid arthritis. On the other hand, AHAs may protect against invasive microbial pathogens and cancer. However, despite more than 50 years of study, the origin and specific B cell compartments that express AHAs remain elusive. Recent research on serum AHAs suggests that they arise during an active immune response, in contrast to previous proposals that they derive from the preexisting immune repertoire in the absence of antigenic stimuli. We report here the isolation and characterization of AHAs from memory B cells, although anti-hinge-reactive B cells were also detected in the naive B cell compartment. IgG AHAs cloned from a single human donor exhibited restricted specificity for protease-cleaved F(ab')2 fragments and did not bind the intact IgG counterpart. The cloned IgG-specific AHA-variable regions were mutated from germ line-derived sequences and displayed a high sequence variability, confirming that these AHAs underwent class-switch recombination and somatic hypermutation. Consistent with previous studies of serum AHAs, several of these clones recognized a linear, peptide-like epitope, but one clone was unique in recognizing a conformational epitope. All cloned AHAs could restore immune effector functions to proteolytically generated F(ab')2 fragments. Our results confirm that a diverse set of epitope-specific AHAs can be isolated from a single human donor.

Beyond citrullination: other post-translational protein modifications in rheumatoid arthritis.

The presence of autoantibodies is one of the hallmarks of rheumatoid arthritis (RA). In the past few decades, rheumatoid factors (autoantibodies that recognize the Fc-tail of immunoglobulins) as well as anti-citrullinated protein antibodies (ACPAs) have been studied intensively. ACPAs recognize post-translationally modified proteins in which the amino acid arginine has been converted into a citrulline. More recently, other autoantibody systems recognizing post-translationally modified proteins have also gained attention, including autoantibodies recognizing fragmented immunoglobulin (anti-hinge antibodies), autoantibodies recognizing acetylated proteins and autoantibodies recognizing proteins that are modified by adducts formed under oxidative stress. In particular, detailed insights have been obtained on the presence and properties of autoantibodies recognizing carbamylated proteins, commonly called anti-carbamylated protein (anti-CarP) antibodies. In this Review, we summarize the current knowledge relating to these emerging autoantibodies that recognize post-translationally modified proteins identified in RA, with an emphasis on anti-CarP antibodies.

Anti-Hinge Antibodies Recognize IgG Subclass- and Protease-Restricted Neoepitopes.

Anti-hinge Abs (AHAs) target neoepitopes exposed after proteolytic cleavage of IgG. In this study, we explored the diversity of protease- and IgG subclass-restricted AHAs and their potential as immunological markers in healthy donors (HDs) and patients with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). AHA reactivity against IgG-degrading enzyme of Streptococcus pyogenes (IdeS)- or pepsin-generated F(ab')2 fragments of all four human IgG subclasses was determined. AHA reactivity against one or more out of eight F(ab')2 targets was found in 68% (68 of 100) of HDs, 69% (68 of 99) of SLE patients, and 81% (79 of 97) of RA patients. Specific recognition of hinge epitopes was dependent on IgG subclass and protease used to create the F(ab')2 targets, as confirmed by inhibition experiments with F(ab')2 fragments and hinge peptides. Reactivity against IdeS-generated F(ab')2 targets was found most frequently, whereas reactivity against pepsin-generated F(ab')2 targets better discriminated between RA and HDs or SLE, with significantly higher AHA levels against IgG1/3/4. In contrast, AHA levels against pepsin-cleaved IgG2 were comparable. No reactivity against IdeS-generated IgG2-F(ab')2s was detected. The most discriminatory AHA reactivity in RA was against pepsin-cleaved IgG4, with a 35% prevalence, ≥5.8-fold higher than in HDs/SLE, and significantly higher levels (p < 0.0001). Cross-reactivity for F(ab')2s generated from different IgG subclasses was only observed for subclasses having homologous F(ab')2 C termini (IgG1/3/4). For IgG2, two pepsin cleavage sites were identified; anti-hinge reactivity was restricted to only one of these. In conclusion, AHAs specifically recognize IgG subclass- and protease-restricted hinge neoepitopes. Their protease-restricted specificity suggests that different AHA responses developed under distinct inflammatory or infectious conditions and may be markers of, and participants in, such processes.

A peptide immunization approach to counteract a Staphylococcus aureus protease defense against host immunity

Pathogens that induce acute and chronic infections, as well as certain cancers, employ numerous strategies to thwart host cellular and humoral immune defenses. One proposed evasion mechanism against humoral immunity is a localized expression of extracellular proteases that cleave the IgG hinge and disable host IgG functions. Host immunity appears to be prepared to counter such a proteolytic tactic by providing a group of autoantibodies, denoted anti-hinge antibodies that specifically bind to cleaved IgGs and provide compensating functional restoration in vitro. These respective counter-measures highlight the complex interrelationships among pathogens and host immunity and suggested to us a possible means for therapeutic intervention. In this study, we combined an investigation of pathogen-mediated proteolysis of host IgGs with an immunization strategy to boost host anti-hinge antibodies. In a Staphylococcus aureus infection model using an artificial tissue cage (wiffle ball) implanted into rabbits, cleaved rabbit IgGs were detected in abundance in the abscesses of untreated animals early after infection. However, in animals previously immunized with peptide analogs of the cleaved IgG hinge to generate substantial anti-hinge antibody titers, S. aureus colony formation was markedly reduced compared to control animals or those similarly immunized with a scrambled peptide sequence. The results of this study demonstrate that extensive local proteolysis of IgGs occurs in a test abscess setting and that immunization to increase host anti-hinge antibodies provided substantial acute protection against bacterial growth.

Antibodies to IgG4 hinge can be found in rheumatoid arthritis patients during all stages of disease and may exacerbate chronic antibody-mediated inflammation.

In rheumatoid arthritis (RA), autoantibodies such as anti-citrullinated protein antibodies (ACPAs) develop in response to neoepitopes that are formed under conditions of chronic inflammation. These autoantibodies may subsequently be fragmented by inflammation-associated proteases, leading to the formation of F(ab')2 fragments. The hinge of F(ab')2 fragments can serve as a neoepitope, and so-called antihinge antibodies (AHAs) can be found in RA patients, which might modulate the function of (fragmented) autoantibodies. We undertook this study to investigate the presence and specificities of AHAs in different stages of RA and to study their function. The presence of AHAs was assessed by radioimmunoassay in healthy controls, blood donors who later developed RA, patients with arthralgia, patients with early RA, and patients with established RA. Specificity of the AHAs was analyzed with inhibition assays, and complement-activating ability was studied with a C4b deposition assay. Antibodies to IgG1 hinge, IgG2 hinge, and IgG4 hinge were detected in patients with established RA, with anti-IgG4 hinge antibodies being most specific (appearing in 1% of healthy controls, 3.8% of blood donors who later developed RA, 13% of arthralgia patients, 19% of early RA patients, and 16% of established RA patients). Anti-IgG4 hinge antibodies were subclass specific and were able to restore C4b deposition by IgG4 F(ab')2 fragments. In patients with arthralgia and patients with early RA, anti-IgG4 hinge antibodies were associated with rheumatoid factors and ACPAs. Anti-IgG4 hinge antibodies are present in RA patients and have low sensitivity but high specificity for RA. Since a significant proportion of ACPAs can be of the IgG4 subclass, the formation of anti-IgG4 hinge antibodies may represent one mechanism of ACPA-mediated inflammation.

Abstract A107: Elevated hinge cleavage of immunoglobulin G correlates with immune suppression and increased matrix metalloproteinases in breast tumor tissues

Abstract Growing evidence indicates that the tumor microenvironment has suppressive immune system and immune evasion is emerging as one of the cancer hallmarks. Immunoglobulin G (IgG) is an important component of human adaptive immunity in removing pathological cells including tumor cells. Recently, we reported that the cancer therapeutic antibody trastuzumab, a humanized IgG1, can be cleaved at the hinge region by matrix metalloproteinases (MMPs) in vitro, and the hinge cleaved trastuzumab (scIgG-T) showed significant loss of immune effector functions such as antibody dependent cellular cytotoxicity (ADCC) and anti-tumor efficacy in vivo (Fan et al., Breast Cancer Research 2012). The objectives of this study are 1) to investigate if the Fc hinge cleavage of IgG occurs in tumor tissues of breast cancer patients; 2) to understand the relationship among IgG Fc hinge cleavage, MMP expression, and anticancer immunity. Methods: Surgically removed fresh tumor tissues (n=23, 11 with upfront surgery and 12 with neoadjuvant therapies) from breast cancer patients with informed consent (protocol # HSC-MS-10-0580) were snap frozen in liquid nitrogen and stored in a -80oC freezer until analysis. Normal breast tissues (n=20) were obtained from an outside vendor (Cureline Inc., CA) as snap frozen tissues. Peripheral blood mononuclear cells (PBMCs) were freshly prepared from patient blood samples collected before surgery and were preserved in liquid nitrogen storage tank until analysis. Hinge cleaved IgGs were quantified by ELISA and immuno-histochemistry (IHC) detection with a panel of anti-hinge specific antibodies developed at Johnson &amp; Johnson. MMP expression was profiled using a reverse phase protein array method (Ray Biotech Inc., CA) and populations of immune effector cells in patient PBMCs were determined by multi-color flow cytometry. Results: Levels of single hinge cleaved IgGs (scIgGs) were significantly higher in breast cancer tumor tissue than in normal breast tissue. The elevated Fc hinge cleavage of IgGs in breast tumor tissues was positively correlated with high levels of MMPs and expression of MMP-9 showed linear correlation (R=0.78) with the level of scIgGs in the tumor tissues. Notably, Fc hinge cleavage of IgGs in residual tumor tissues from patients treated with neoadjuvant therapies was similar to the levels in the normal breast tissues, while tumor tissues from patients with up-front surgery had significantly higher Fc hinge cleavage than both normal and neoadjuvant treated tumor tissues. In comparison with PBMCs from patients with the up-front surgery, PBMCs from patients with neoadjuvant therapies had an increased percentage of NK cells (CD56+) and immune cells with Fc gamma receptor IIa (CD32a) expression. In addition, percentages of immune effector cells with CD56+/CD16+ (Fc gamma receptor RIII), CD56+/CD64+ (Fc gamma receptor RI), and CD14+/CD16+ were also higher in PBMCs from patients treated with neoadjuvant therapies than those from patients with up-front surgery. Taken together, our results suggest that the reduced scIgG levels in neoadjuvant treated tumor tissue correlated with the better profiles of immune effector cells, while the increased Fc hinge cleavage in breast tumor tissue before therapeutic intervention is linked with immune suppression in the tumor microenvironment. Citation Format: Ningyan zhang, Anneliese Gonzalez, Hui Deng, Xuejun Fan, Luis Baez Vallecillo, songlin zhang, Randall Brezski, Emily Roberson, Robert Jorden, William Strohl, Zhiqiang An. Elevated hinge cleavage of immunoglobulin G correlates with immune suppression and increased matrix metalloproteinases in breast tumor tissues. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A107.

Antibodies to constant domains of therapeutic monoclonal antibodies: Anti-hinge antibodies in immunogenicity testing

Rheumatoid factors are antibodies directed against IgG that may confound immunogenicity testing for therapeutic monoclonal antibodies. We developed antigen-binding assays to monitor anti-drug-antibody (ADA) responses against infliximab and adalimumab using F(ab')2 fragments of the drug. This avoids possible detection of rheumatoid factor activity. During development of these assays, a number of sera from patients before treatment as well as several healthy control sera were tested positive. None of these sera contained antibodies specific for the therapeutic mAb. Instead, they were found to contain anti-hinge antibodies. We demonstrate that this aspecific antibody binding can be inhibited by adding F(ab')2 of intravenous immunoglobulin (IVIG), which consists of pooled polyclonal IgG derived from plasma. Using this protocol, anti-infliximab antibodies can be measured specifically without interference by anti-hinge antibodies.

The Cytoplasmic Tail Dileucine Motif LL572 Determines the Glycosylation Pattern of Membrane-type 1 Matrix Metalloproteinase

Membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP-14) drives fundamental physiological and pathological processes, due to its ability to process a broad spectrum of substrates. Because subtle changes in its activity can produce profound physiological effects, MT1-MMP is tightly regulated. Currently, many aspects of this regulation remain to be elucidated. It has recently been discovered that O-linked glycosylation defines the substrate spectrum of MT1-MMP. We hypothesized that a mutual interdependency exists between MT1-MMP trafficking and glycosylation. Lectin precipitation, metabolic labeling, enzymatic deglycosylation, and site-directed mutagenesis studies demonstrate that the LL(572) motif in the cytoplasmic tail of MT1-MMP influences the composition of the complex O-linked carbohydrates attached to the hinge region of the protein. This influence appears to be independent from major effects on cell surface trafficking. MT1-MMP undergoes extensive processing after its synthesis. The origins and the molecular characters of its multiple forms are incompletely understood. Here, we develop and present a model for the sequential, post-translational processing of MT1-MMP that defines stages in the post-synthetic pathway pursued by the protein.

F(ab′)2-containing immune complexes and naturally occurring anti-hinge antibodies together stimulate complement amplification in vitro and in vivo

F(ab′)2-containing immune complexes and naturally occurring anti-hinge antibodies together stimulate complement amplification in vitro and in vivo

Human F(ab′) 2-containing immune complexes together with anti-hinge natural antibodies stimulate complement amplification in vitro and in vivo

The systemic inflammatory response syndrome (SIRS) is triggered by C5a generation following an excessive complement amplification, but it has remained unclear how complement amplification is stimulated. It is known that neutrophilic elastase can cleave IgG to F(ab′) 2 and that F(ab′) 2-containing immune complexes (F(ab′) 2-IC) stimulate complement amplification together with an unidentified plasma factor. We show that absorption of plasma on F(ab′) 2 from human IgG removed this factor and prevented F(ab′) 2-IC from stimulating complement amplification. The required factor was purified from pooled whole human IgG (IVIG) as those naturally occurring antibodies (NAbs) that bind to F(ab′) 2, but not to intact IgG. These “anti-hinge NAbs” restored complement amplification by F(ab′) 2-IC in absorbed plasma. Anti-hinge NAbs must have formed secondary, rigidified IC from F(ab′) 2-IC, because the F(ab′) 2 fragments evidently captured dimeric C3b, known as a potent C3 convertase precursor. This process may also stimulate complement amplification in vivo, because plasma from septic patients at the onset of SIRS indeed contained F(ab′) 2 fragments. The concentrations of F(ab′) 2 and that of factor Bb, an unbiased measure of complement amplification, correlated linearly with that of released elastase. Moreover, the F(ab′) 2 fragments migrated on gelfiltration columns together with anti-hinge NAbs as ICs with MW of up to ∼750 kDa, as verified on plasma of each of the nine patients studied. These findings provide for the first time a plausible mechanism of how F(ab′) 2-containing immune complexes stimulate complement amplification together with anti-hinge NAbs. The same mechanism may contribute to complement overreaction at the onset of SIRS.

Immunoglobulin Induction Therapy in Renal Transplant Recipients: Effects on Immunoglobulin and Regulatory Antibody Levels

We have previously shown that high pretransplant regulatory autoantibodies are associated with better kidney graft outcome. To analyze the effect of intravenous immunoglobulin (IVIG) induction therapy on these regulatory antibodies, we performed a prospective randomized study in 50 renal transplant recipients who were randomly assigned to receive 7 × 10 g IVIG or 7 × 10 g IV albumin infusions. Basic immunosuppressive therapy consisted of tacrolimus/azathioprine ( n = 24) and tacrolimus/mycophenolate mofetil ( n = 26), respectively. ELISA was used to assess IgG-/IgA-anti-Fab, -anti-F(ab) 2 and -anti-hinge regulatory antibodies. IVIG induction therapy resulted in upregulation of serum IgG and IgA levels within the first 20 days posttransplant ( P = .001, IgG; P = .04, IgA), so that a significant IgG deficiency was found only in non-IVIG patients (day 10: IgG <6 g/L: 7/25 (28%) non-IVIG versus 0/25 IVIG patients; P = .005). As the IVIG charges contained all of the regulatory antibodies tested, intravenous administration of these antibodies explain the elevated IgG- and IgA-anti-F(ab) 2 antibody levels found in IVIG compared to non-IVIG patients on day 10 ( P = .005 and P = .04, respectively). Our data indicated that IVIG induction prevented severe IgG deficiency in the early posttransplant period but had no impact on severe infectious complications. IVIG induction enhanced immunoregulatory antibody levels early posttransplant, which might provide graft protective effects.

Influence of the cardiac myosin hinge region on contractile activity.

The participation of cardiac myosin hinge in contractility was investigated by in vitro motility and ATPase assays and by measurements of sarcomere shortening. The effect on contractile activity was analyzed using an antibody directed against a 20-amino acid peptide within the hinge region of myosin. This antibody bound specifically at the hinge at a distance of 55 nm from the S1/S2 junction, was specific to human, dog, and rat cardiac myosins, did not crossreact with gizzard or skeletal myosin, and had no effect on ATPase activity of purified S1 and myofibrils. However, it completely suppressed the movement of actin filaments in in vitro motility assays and reduced active shortening of sarcomeres of skinned cardiac myocytes by half. Suppression of motion by the anti-hinge antibody may reflect a mechanical constraint imposed by the antibody upon the mobility of the S2 region of myosin. The results suggest that the steps in the mechanochemical energy transduction can be separately influenced through S2.