A bacterial etiology of rheumatoid arthritis (RA) has been suspected since the beginnings of modern germ theory. Recent studies implicate mucosal surfaces as sites of disease initiation. The common occurrence of periodontal dysbiosis in RA suggests that oral pathogens may trigger the production of disease-specific autoantibodies and arthritis in susceptible individuals. We used mass spectrometry to define the microbial composition and antigenic repertoire of gingival crevicular fluid in patients with periodontal disease and healthy controls. Periodontitis was characterized by the presence of citrullinated autoantigens that are primary immune targets in RA. The citrullinome in periodontitis mirrored patterns of hypercitrullination observed in the rheumatoid joint, implicating this mucosal site in RA pathogenesis. Proteomic signatures of several microbial species were detected in hypercitrullinated periodontitis samples. Among these, Aggregatibacter actinomycetemcomitans (Aa), but not other candidate pathogens, induced hypercitrullination in host neutrophils. We identified the pore-forming toxin leukotoxin-A (LtxA) as the molecular mechanism by which Aa triggers dysregulated activation of citrullinating enzymes in neutrophils, mimicking membranolytic pathways that sustain autoantigen citrullination in the RA joint. Moreover, LtxA induced changes in neutrophil morphology mimicking extracellular trap formation, thereby releasing the hypercitrullinated cargo. Exposure to leukotoxic Aa strains was confirmed in patients with RA and was associated with both anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF). The effect of HLA-DRB1 shared epitope alleles on autoantibody positivity was limited to RA patients that were exposed to Aa. These studies identify the periodontal pathogen Aa as a candidate bacterial trigger of autoimmunity in RA.

NETosis, an antimicrobial form of neutrophil cell death, is considered a primary source of citrullinated autoantigens in rheumatoid arthritis (RA) and immunogenic DNA in systemic lupus erythematosus (SLE). Activation of the citrullinating enzyme peptidylarginine deiminase type 4 (PAD4) is believed to be essential for neutrophil extracellular trap (NET) formation and NETosis. PAD4 is therefore viewed as a promising therapeutic target to inhibit the formation of NETs in both diseases. In this review, we examine the evidence for PAD4 activation during NETosis and provide experimental data to suggest that protein citrullination is not a universal feature of NETs. We delineate two distinct biological processes, leukotoxic hypercitrullination (LTH) and defective mitophagy, which have been erroneously classified as “NETosis.” While these NETosis mimics share morphological similarities with NETosis (i.e., extracellular DNA release), they are biologically distinct. As such, these processes can be readily classified by their stimuli, activation of distinct biochemical pathways, the presence of hypercitrullination, and antimicrobial effector function. NETosis is an antimicrobial form of cell death that is NADPH oxidase-dependent and not associated with hypercitrullination. In contrast, LTH is NADPH oxidase-independent and not bactericidal. Rather, LTH represents a bacterial strategy to achieve immune evasion. It is triggered by pore-forming pathways and equivalent signals that cumulate in calcium-dependent hyperactivation of PADs, protein hypercitrullination, and neutrophil death. The generation of citrullinated autoantigens in RA is likely driven by LTH, but not NETosis. Mitochondrial DNA (mtDNA) expulsion, the result of a constitutive defect in mitophagy, represents a second NETosis mimic. In the presence of interferon-α and immune complexes, this process can generate highly interferogenic oxidized mtDNA, which has previously been mistaken for NETosis in SLE. Distinguishing NETosis from LTH and defective mitophagy is paramount to understanding the role of neutrophil damage in immunity and the pathogenesis of human diseases. This provides a framework to design specific inhibitors of these distinct biological processes in human disease.

TP53 (tumor protein p53) is the most commonly mutated cancer driver gene, but drugs that target mutant tumor suppressor genes, such as TP53, are not yet available. Here, we describe the identification of an antibody highly specific to the most common TP53 mutation (R175H, in which arginine at position 175 is replaced with histidine) in complex with a common human leukocyte antigen–A (HLA-A) allele on the cell surface. We describe the structural basis of this specificity and its conversion into an immunotherapeutic agent: a bispecific single-chain diabody. Despite the extremely low p53 peptide-HLA complex density on the cancer cell surface, the bispecific antibody effectively activated T cells to lyse cancer cells that presented the neoantigen in vitro and in mice. This approach could in theory be used to target cancers containing mutations that are difficult to target in conventional ways.

Background Antibodies to citrullinated proteins (ACPAs) are a hallmark of rheumatoid arthritis (RA). Porphyromonas gingivalis peptidylarginine deiminase (PPAD) has been implicated in the initiation of RA by generating citrullinated neoantigens and due to its ability to autocitrullinate. Objectives To define the citrullination status and biology of PPAD in P gingivalis and to characterize the anti-PPAD antibody response in RA and associated periodontal disease (PD). Methods PPAD in P gingivalis cells and culture supernatant was analyzed by immunoblotting and mass spectrometry to detect citrullination. Recombinant PPAD (rPPAD), inactive mutant PPAD (rPPADC351S), and N-terminal truncated PPAD (rPPADNtx) were cloned and expressed inE coli. Patients with RA and healthy controls were assayed for IgG antibodies to citrullinated rPPAD and unmodified rPPADC351S by ELISA. Anti-PPAD antibodies were correlated with anti-CCP3 antibody levels, RA disease activity, and PD status. Results PPAD from P gingivalis is truncated at the N- and C-terminal domains and not citrullinated. Only when artificially expressed in E coli, full-length rPPAD, but not truncated (fully active) rPPADNtx, is autocitrullinated. Anti-PPAD antibodies show no heightened reactivity to citrullinated rPPAD, but are exclusively directed against the unmodified enzyme. Antibodies against PPAD do not correlate with anti-CCP levels and disease activity in RA. By contrast, anti-PPAD antibody levels are significantly decreased in RA patients with PD. Conclusions PPAD autocitrullination is not the underlying mechanism linking PD and RA. N-terminal processing protects PPAD from autocitrullination and enhances enzyme activity. Anti-PPAD antibodies may have a protective role for the development of PD in RA patients.

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Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.

Introduction Several months into the COVID-19 pandemic, safe and effective treatments against this global health disaster have yet to be identified. Clinical research trials around the world are underway testing a wide array of possible medications. In particular, the off-label use of hydroxychloroquine for COVID-19 prophylaxis and treatment has created many unprecedented challenges for the scientific community and the public. Areas covered We critically assessed major events from February – May 2020 that contributed to widespread use of hydroxychloroquine for the treatment and prophylaxis of COVID-19. We aimed to explore how opinions toward hydroxychloroquine may shift from early enthusiasm (based on in vitro and preliminary clinical data) to the hope for a miracle cure (through communication and promotion of questionable results) and, finally, to a rise of skepticism as more in-depth analyses are emerging. Expert opinion Mindful and rigorous acquisition of data, as well as its interpretation, are essential to an effective pandemic response. The rapid and premature promotion of results has had major implications for global crisis management, even creating distrust among the public. It is crucial for the medical and scientific community to incorporate the lessons learned from this situation.