Investigating surface proteins and antibody combinations for detecting circulating tumor cells of various sarcomas.

Circulating tumor cells (CTCs) have gathered attention as a biomarker for carcinomas. However, CTCs in sarcomas have received little attention. In this work, we investigated cell surface proteins and antibody combinations for immunofluorescence detection of sarcoma CTCs. A microfluidic device that combines filtration and immunoaffinity using gangliosides 2 and cell surface vimentin (CSV) antibodies was employed to capture CTCs. For CTC detection, antibodies against cytokeratins 7 and 8 (CK), pan-cytokeratin (panCK), or a combination of panCK and CSV were used. Thirty-nine blood samples were collected from 21 patients of various sarcoma subtypes. In the independent samples study, samples were subjected to one of three antibody combination choices. Significant difference in CTC enumeration was found between CK and panCK + CSV, and between panCK and panCK + CSV. Upon stratification of CK+ samples, those of metastatic disease had a higher CTC number than those of localized disease. In the paired samples study involving cytokeratin-positive sarcoma subtypes, using panCK antibody detected more CTCs than CK. Similarly, for osteosarcoma, using panCK + CSV combination resulted in a higher CTC count than panCK. This study emphasized deliberate selection of cell surface proteins for sarcoma CTC detection and subtype stratification for studying cancers as heterogeneous as sarcomas.

Clinical significance of anti-mutated citrullinated vimentin antibodies in rheumatoid arthritis patients.

BACKGROUND: Anti-mutated citrullinated vimentin (MCV) antibodies have recently been recommended as a better arthritis diagnostic marker. OBJECTIVES: To investigate the association between anti-MCV antibodies and the clinical, functional, and radiographic characteristics of rheumatoid arthritis (RA) patients. METHODS: This case-control study was conducted on 40 RA patients and 40 healthy subjects. All patients were subjected to an assessment of disease using the 28-joint DAS (DAS28) and Clinical Disease Activity Index (CDAI), function by HAQ-DI, physical activity by International Physical Activity Questionnaire (IPAQ), fatigue by Functional Assessment of Chronic Illness Therapy (FACIT), serological tests as well as anti-MCV Abs measurement. A plain X-ray of both hands and wrists was done. RESULTS: The anti-MCV Abs level was significantly higher in RA patients than in healthy controls (P< 0.001). The anti-MCV Abs had a significant positive correlation with DAS, CDAI, HAQ, RF, Anti-CCP, and CRP (P= 0.006, 0.013, 0.005, < 0.001, < 0.001and 0.041 respectively) and a significant negative correlation with FACIT (p= 0.007). Positive anti-MCV RA patients had significantly higher erosions, JSN, and a total sharp score. CONCLUSIONS: Anti-MCV Abs may contribute to poor physical activity and more fatigue in RA patients beyond their established role in disease activity and erosion.

Anti-mutated citrullinated vimentin antibodies are increased in IPF patients.

INTRO: An increased prevalence of serum anti-MCV antibody is observed in the serum of patients with idiopathic pulmonary fibrosis (IPF) but the clinical relevance of these antibodies is unknown. METHODS: Patients from our center with a diagnosis of IPF according to the 2018 ATS/ERS/JRS/ALAT guidelines and at least one anti-MCV assay available were selected. All patients were part of the prospective cohort European IPF registry and selected between 03/2010 and 03/2018. We constituted two groups of patients according to the anti-MCV status at baseline to compare their characteristics at baseline and the evolution of lung function, survival and/or transplantation status. RESULTS: Anti-MCV data were available for 101 patients, of whom 86 had complete clinical data available. Twenty-nine (34 %) patients had a positive anti-MCV assay (MCV+), at a low level in most patients (29 UI/mL [IQR 25-40]), and 57 (66 %) patients a negative assay (MCV-). MCV+ patients were 20 men and 9 women, with a median age of 73 years [IQR 67-78]. MCV- patients were 49 men and 8 women with a median age of 72 years [IQR 64-77]. Sixty-two (75 %) patients were ex-smokers and 5 (6 %) were active smokers. Median cumulative tobacco smoke exposure was 22.5 (15.0-38.6) and was similar in both groups. Lung function test results and HRCT pattern distribution was similar in both groups at baseline. The median duration of follow-up was 3.5 years [IQR 2.1-5.0]. Lung function decline was similar in both groups. During the study period, 31 (36 %) patients died or have been transplanted with no difference in transplant-free survival status between the two groups. CONCLUSION: Low level anti-MCV autoimmunity was prevalent in IPF patients.

'Non-criteria antiphospholipid antibodies': bridging the gap between seropositive and seronegative antiphospholipid syndrome.

OBJECTIVE: We aimed to analyse the prevalence of non-criteria anti-phospholipid (aPL) antibodies and their role in the diagnosis, treatment and prognosis in a cohort of patients with clinical features consistent with a diagnosis of antiphospholipid syndrome (APS), but persistently negative for criteria aPL - anti-cardiolipin antibodies (aCL), anti-ß2-glycoprotein I antibodies (aß2-GPI) and lupus anticoagulant (LA) - named seronegative APS (SN-APS). METHODS: Sera from SN-APS patients were tested for aCL by TLC-immunostaining, anti-vimentin/cardiolipin (aVim/CL) and anti-phosphatidylserine/prothrombin (anti-PS/PT) by ELISA. Control groups of our study were APS patients and healthy controls. RESULTS: We enrolled 114 consecutive SN-APS patients, 69 (60.5%) resulted positive for at least one non-criteria test in two occasions 12 weeks apart. Among the persistently positive patients to these tests, 97% resulted positive for aCL by TLC-immunostaining, 52.3% for aVim/CL and 17.4% for aPS/PT. SN-APS patients with double positivity (aCL by TLC-immunostaining and aVim/CL) showed a likelihood positive ratio of 8 to present mixed thrombotic and obstetrical features. Among SN-APS patients tested positive, after the therapeutic changes, three cases of recurrent thrombosis were observed [median follow-up 41 months (IQR 39.5)]. Twenty pregnancies were recorded in 17 SN-APS patients after the detection of unconventional aPL and 12 of them (60%) experienced a good outcome under conventional treatment for APS. CONCLUSIONS: This is the largest monocentric study demonstrating that aCL tested by TLC-immunostaining and aVim/CL can detect aPL positivity in SN-APS. It may encourage clinicians to monitor and provide adequate targeted therapy, which improve SN-APS prognosis.

Anti-vimentin/cardiolipin IgA in the anti-phospholipid syndrome: A new tool for 'seronegative' diagnosis.

Anti-phospholipid syndrome (APS) is a systemic autoimmune disorder defined by the simultaneous presence of vascular clinical events, pregnancy morbidity and anti-phospholipid antibodies (aPL). In clinical practice, it is possible to find patients with APS who are persistently negative for the routine aPL tests (seronegative APS; SN-APS). Recently, the identification of aPL immunoglobulin (Ig)A and/or anti-ß2-glycoprotein-I (ß2-GPI) IgA was shown to represent a further test in SN-APS patients. In this study we analyzed the presence of anti-vimentin/cardiolipin (aVim/CL) IgA in a large cohort of patients with SN-APS, evaluating their possible association with clinical manifestations of the syndrome. This study includes 60 consecutive SN-APS patients, 30 patients with APS and 40 healthy donors. aVim/CL IgA were detected by enzyme-linked immunosorbent assay (ELISA). Results show that 12 of 30 APS patients (40%) and 16 of 60 SN-APS patients (26.7%) resulted positive for aVim/CL IgA. Interestingly, SN-APS patients who tested positive for aVim/CL IgA showed a higher prevalence of arterial thrombosis (p = 0.017, likelihood positive ratio = 5.7). This study demonstrates for the first time, to our knowledge, the presence of aVim/CL IgA in sera of patients with APS. In particular, they revealed a potential usefulness in identification of a significant proportion of SN-APS patients. Moreover, as patients tested positive for aVim/CL IgA reported a high likelihood ratio to have the clinical features of APS, this test may be considered a suitable approach in the clinical evaluation of SN-APS.

New laboratory criteria of the autoimmune inflammation in pulmonary sarcoidosis and tuberculosis.

Sarcoidosis and tuberculosis have many clinical and laboratory similarities, which allowed researchers to assume the presence of common pathogenetic mechanisms in the development of both diseases. Recently, much attention has been paid to investigate the autoimmune origins in these pathologies. The aim of this study is to find out the characteristics of the autoinflammatory immune response in sarcoidosis and tuberculosis. In patients with sarcoidosis (n = 93), tuberculosis (n = 28), and in healthy donors (n = 40), the serum anti-MCV concentration was measured by ELISA, and B cell subpopulations were analyzed by flow cytometry. Based on the results obtained, the formula ([B-naïve%]\[B-memory%]) * ([B-CD38%] + [B-CD5%]) / [anti-MCV] was described. The increase in the calculated index by more than 5 units with a sensitivity of 80.00% and a specificity of 93.10% (AUC = 0.926) suggest the presence of the autoimmune component, which is more typical for sarcoidosis, rather than tuberculosis patients and may serve as a diagnostic criterion.

In vitro elimination of autoreactive B cells from rheumatoid arthritis patients by universal chimeric antigen receptor T cells.

OBJECTIVES: Autoreactive B cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA), and B cell-depleting therapies using an antibodies, such as rituximab, have been suggested to be effective in RA treatment. However, transient B cell depletion with rituximab is associated with significant safety challenges related to global suppression of the immune system and thus increases the risks of infection and cancer development. To address selective and persistent issues associated with RA therapy, we developed a customised therapeutic strategy employing universal antifluorescein isothiocyanate (FITC) chimeric antigen receptor T cells (CAR-T cells) combined with FITC-labelled antigenic peptide epitopes to eliminate autoreactive B cell subsets recognising these antigens in RA. METHODS: For a proof-of-concept study, four citrullinated peptide epitopes derived from citrullinated autoantigens, namely, citrullinated vimentin, citrullinated type II collagen, citrullinated fibrinogen and tenascin-C, and a cyclocitrulline peptide-1 were selected as ligands for targeting autoreactive B cells; Engineered T cells expressing a fixed anti-FITC CAR were constructed and applied as a universal CAR-T cell system to specifically eliminate these protein-specific autoreactive B cells via recognition of the aforementioned FITC-labelled autoantigenic peptide epitopes. RESULTS: We demonstrated that anti-FITC CAR-T cells could be specifically redirected and kill hybridoma cells generated by immunisation with antigenic peptides, and autoreactive B cell subsets from RA patients via recognition of corresponding FITC-labelled citrullinated peptide epitopes. Additionally, the cytotoxicity of the CAR-T cells was dependent on the presence of the peptides and occurred in a dose-dependent manner. CONCLUSIONS: The approach described here provides a direction for precise, customised approaches to treat RA and can likely be applied to other systemic autoimmune diseases.

Effects of a gestational level of estradiol on cellular transition, migration, and inflammation in cervical epithelial and stromal cells.

PROBLEM: Estrogen (E2) is one of the main steroid hormones associated with pregnancy and parturition. High levels of E2 increase uterine contractions, promote fetal membrane weakening, and induce degradation of the cervical extracellular matrix (ECM). Current evidence supports the role of E2 in epithelial-to-mesenchymal transition (EMT) and inflammation in different cell types; however, its effects on the cellular components of the cervix are still unknown. METHOD OF STUDY: In this study, we assessed the effects of gestational levels of E2 in: (a) the cellular transition of endocervical epithelial cells (EEC) and cervical stromal cells (CSC) in vitro using immunocytochemical staining and Western blot analyses for EMT markers (cytokeratin-18, E-cadherin, N-cadherin, SNAIL, and vimentin); (b) cell migration using in vitro scratch assays; (c) inflammatory cytokine (interleukin 1ß and TNF-α) and MMP9 production under untreated and lipopolysaccharide (LPS)-treated conditions using immunoassays. RESULTS: E2 treatment and co-treatment with LPS as a proxy for infection maintained the metastate of EEC (expression of both cytokeratin and vimentin) and the mesenchymal state of CSC. E2 delayed wound healing, which mimics the tissue remodeling process, in EEC and CSC. E2 led to persistently elevated levels of vimentin throughout the EEC wound healing process. E2 did not affect inflammatory cytokine production by EEC and CSC but increased MMP9 production by EEC. CONCLUSION: Collectively, these results show that third trimester levels of E2 may permit localized inflammation, increase MMP-9 production, and cause an EMT-mediated impairment of the remodeling process in the cervix in vitro. These data suggest a potential contribution of E2 in cervical ripening.

Simultaneous testing of immunological sensitization to multiple antigens in sarcoidosis reveals an association with inorganic antigens specifically related to a fibrotic phenotype.

Organic and inorganic antigens were studied simultaneously in the same cohort of sarcoidosis patients to investigate whether correlations between clinical characteristics and immunological sensitization could reveal new phenotypes. Sensitization to antigens of mycobacteria, Propionibacterium acnes catalase and vimentin was investigated in 201 sarcoidosis and 51 obstructive sleep apnoea patients, serving as control group. Sensitization to aluminium, beryllium, silica and zirconium was also studied in 105 of the sarcoidosis patients and in 24 of the controls. A significantly higher percentage of sarcoidosis patients (27·6%) than controls (4·2%) had an immunological response to metals or silica (P = 0·014). A higher percentage of these sarcoidosis patients showed fibrosis on chest X-ray 5 years after the diagnosis (69·2 versus 30·3%, P = 0·016). No significant differences in mycobacterial or vimentin enzyme-linked immunospot (ELISPOT) assay results were observed between sarcoidosis and control patients. A significantly lower percentage of sarcoidosis patients (3·5%) than control patients (15·7%) had a positive ELISPOT for P. acnes catalase (P = 0·003). However, sarcoidosis patients sensitized to P. acnes catalase were more likely to have skin involvement, while sarcoidosis patients sensitized to mycobacterial antigens were more likely to have cardiac involvement. Our study suggests a more prominent role for inorganic triggers in sarcoidosis pathogenesis than previously thought. Immunological sensitization to inorganic antigens was associated with development of fibrotic sarcoidosis. No association was found between sensitization to bacterial antigens or vimentin and sarcoidosis in Dutch patients. However, our data suggest that trigger-related phenotypes can exist in the heterogeneous population of sarcoidosis patients.

Cysteinyl leukotriene D4 (LTD4) promotes airway epithelial cell inflammation and remodelling.

OBJECTIVE: Cysteinyl leukotrienes (CysLTs), a group of inflammatory lipid mediators, are found elevated in obese-asthmatic patients. Leukotriene D4 (LTD4), a representative CysLT, is implicated in promoting lung inflammation and remodelling in allergic asthma, but its role in non-allergic asthma, especially in obese-asthmatic patients, is not known. Here, using primary human small airway epithelial cells (SAECs) we have investigated the mechanism of LTD4-induced inflammation and remodelling and assessed high proneness of obese mice to develop asthma upon challenge with allergen ovalbumin (OVA). METHODS: Primary human small airway epithelial cells (SAECs) were stimulated with different concentrations of LTD4 for different time intervals and various inflammatory markers were measured through cytokine array, membrane-based ELISA and Western blotting. An air-liquid interface (ALI) model of SAECs was used to study the effects of LTD4-induced remodelling in SAECs using Western blotting, H&E staining and PAS staining. Further, OVA-based murine model was used to examine the propensity of high-fat diet (HFD)-fed obese mice to develop asthma symptoms by studying the infiltration of inflammatory cells (assessed by bronchioalveolar lavage (BAL) cytology) and airway remodelling (assessed by histopathology) upon allergen exposure. RESULTS: The human primary small airway epithelial cells (SAECs) treated with LTD4 showed significant alterations in the levels of inflammatory markers such as GM-CSF, TNF-α, IL-1ß, EGF and eotaxin in dose- and time-dependent manner. Further, LTD4 enhanced the activation of inflammasomes as evidenced by increased levels of NALP3, cleaved caspase-1 and IL-1ß. LTD4 also enhanced inflammation by increasing the expression of COX-2 in SAECs. The airway remodelling markers Vimentin and Muc5AC were found elevated in ALI culture of SAECs when stimulated with LTD4, as it also increased TGF-ß levels and activation of Smad2/3 phosphorylation in SAECs. Last, sensitization and challenge of HFD-fed obese mice with OVA showed increased infiltration of inflammatory cells in BAL and enhanced levels of remodeling phenotypes like loss of cilia, mucus cell metaplasia and collagen deposition in mice lung tissues. CONCLUSION: The results suggest that LTD4 could induce inflammatory response in human airway epithelial cell by activating NALP3 inflammasome. LTD4 could further promote airway epithelial cells' remodelling through TGF-ß/smad2/3-mediated pathway. Our in vivo results suggested that obesity predisposed the OVA challenged mice to develop lung inflammation and remodelling akin to asthma-like phenotypes during obesity.

Identification and characterization of antigen-specific CD4+ T cells targeting renally expressed antigens in human lupus nephritis with two independent methods.

In the search for anti-renal autoreactivity in human lupus nephritis, we stimulated blood-derived CD4+ T cells from patients with systemic lupus erythematosus with various kidney lysates. Although only minor responses were detectable, these experiments led to the development of a search algorithm that combined autoantibody association with human lupus nephritis and target gene expression in inflamed kidneys. Applying this algorithm, five potential T cell antigens were identified. Blood-derived CD4+ T cells were then stimulated with these antigens. The cells were magnetically enriched prior to measurement with flow cytometry to facilitate the detection of very rare autoantigen-specific cells. The detected responses were dominated by IFN-γ-producing CD4+ T cells. Additionally, IL-10-producing CD4+ T cells were found. In a next step, T cell reactivity to each single antigen was independently evaluated with T cell libraries and [3H]-thymidine incorporation assays. Here, Vimentin and Annexin A2 were identified as the main T cell targets. Finally, Vimentin reactive T cells were also found in the urine of three patients with active disease. Overall, our experiments show that antigen-specific CD4+ T cells targeting renally expressed antigens arise in human lupus nephritis and correlate with disease activity and are mainly of the Th1 subset.

Association of vimentin antibody and other non-HLA antibodies with treated antibody mediated rejection in heart transplant recipients.

Non-human leukocyte antigen (HLA) antibodies have been implicated in heart transplantation rejection. However, targets of non-HLA antibodies remain elusive. Here, we utilized a panel of multiplex beads-based assay to determine the specificity of non-HLA antibodies following heart transplantation. We utilized a selected cohort of recipients who did not have HLA donor specific antibodies, but were diagnosed with antibody mediated rejection and treated for antibody mediated rejection. We found the presence of vimentin antibody was associated with treated antibody mediated rejection. Our results suggest that, in heart transplant recipients who are suspected of AMR but in the absence of HLA donor specific antibodies, non-HLA antibodies should be examined.

Morphological Characteristics of the Action of Cationic Peptide Warnerin on Regeneration of the Connective Tissue around Implanted Teflon Catheters in Mice under Conditions of Immunosuppression.

Warnerin pretreatment of catheter segments subcutaneously implanted to mice under conditions of immunosuppression led to a significant increase in the number of neutrophils in the surrounding tissues on day 1; the number of fibroblasts tended to decrease by day 3. Immunohistochemical study showed the presence of T and B lymphocytes on day 3, but no positive reactions to vimentin and CD34 were observed during the first 2 days. These changes suggest that warnerin reduced the intensity of regeneration processes in tissues around the implant, which can be used for suppression of fibrosis.

Anti-modified citrullinated vimentin antibody: a novel biomarker associated with cardiac systolic dysfunction in patients with rheumatoid arthritis.

BACKGROUND: Studies have demonstrated that seropositive patients with rheumatoid arthritis (RA) are susceptible to cardiovascular diseases (CVDs). In this study, we aimed to determine the association of autoantibodies with the echocardiographic parameters of systolic and diastolic dysfunction in such patients. METHODS: In this cross-sectional study, we evaluated patients with RA who were referred to our clinic from October 2017 to August 2018. After the exclusion of patients with concomitant CVD, all patients underwent transthoracic echocardiography and measurement of plasma autoantibodies. Moreover, possible confounders-including medications, CVD risk factors, Framingham risk score, disease activity score-28, duration of disease, simple disease activity index, and functional status-were assessed. RESULTS: We studied 135 patients with RA (mean age = 52.3 years; 111 (82.2%) females). We had missing data rates of up to 8.9% for some characteristics. E velocity was inversely correlated with rheumatoid factor (P = 0.009). Furthermore, the plasma levels of anti-citrullinated protein and anti-modified citrullinated vimentin (anti-MCV) antibodies were negatively correlated with left ventricular ejection fraction (LVEF) (P = 0.019 and P<0.001, respectively). After an adjustment for possible confounders, the linear regression model demonstrated that the anti-MCV level and the patient's age are significant predictors of LVEF. The receiver operating characteristic curve showed that anti-MCV antibody titer≥547.5 (IU/mL) signifies reduced LVEF (<50%) with a sensitivity of 85.7% and specificity of 93% (C-statistic = 0.843). CONCLUSIONS: Our findings showed a significant inverse correlation between anti-MCV antibody titer and LVEF. These results indicate that the application of anti-MCV is promising for the screening and early detection of cardiac systolic dysfunction. Future prospective studies will determine its role.

Vimentin as a Multifaceted Player and Potential Therapeutic Target in Viral Infections.

Vimentin is an intermediate filament protein that plays key roles in integration of cytoskeletal functions, and therefore in basic cellular processes such as cell division and migration. Consequently, vimentin has complex implications in pathophysiology. Vimentin is required for a proper immune response, but it can also act as an autoantigen in autoimmune diseases or as a damage signal. Although vimentin is a predominantly cytoplasmic protein, it can also appear at extracellular locations, either in a secreted form or at the surface of numerous cell types, often in relation to cell activation, inflammation, injury or senescence. Cell surface targeting of vimentin appears to associate with the occurrence of certain posttranslational modifications, such as phosphorylation and/or oxidative damage. At the cell surface, vimentin can act as a receptor for bacterial and viral pathogens. Indeed, vimentin has been shown to play important roles in virus attachment and entry of severe acute respiratory syndrome-related coronavirus (SARS-CoV), dengue and encephalitis viruses, among others. Moreover, the presence of vimentin in specific virus-targeted cells and its induction by proinflammatory cytokines and tissue damage contribute to its implication in viral infection. Here, we recapitulate some of the pathophysiological implications of vimentin, including the involvement of cell surface vimentin in interaction with pathogens, with a special focus on its role as a cellular receptor or co-receptor for viruses. In addition, we provide a perspective on approaches to target vimentin, including antibodies or chemical agents that could modulate these interactions to potentially interfere with viral pathogenesis, which could be useful when multi-target antiviral strategies are needed.

Combination vaccine based on citrullinated vimentin and enolase peptides induces potent CD4-mediated anti-tumor responses.

BACKGROUND: Stress-induced post-translational modifications occur during autophagy and can result in generation of new epitopes and immune recognition. One such modification is the conversion of arginine to citrulline by peptidylarginine deiminase enzymes. METHODS: We used Human leukocyte antigen (HLA) transgenic mouse models to assess the immunogenicity of citrullinated peptide vaccine by cytokine Enzyme linked immunosorbant spot (ELISpot) assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma and ID8 ovarian models expressing either constitutive or interferon-gamma (IFNγ) inducible Major Histocompatibility Complex (MHC) class II (MHC-II) as represented by most human tumors. To determine the importance of CD4 T cells in tumor therapy, we analyzed the immune cell infiltrate into murine tumors using flow cytometry and performed therapy studies in the presence of CD4 and CD8 T cell depletion. We assessed the T cell repertoire to citrullinated peptides in ovarian cancer patients and healthy donors using flow cytometry. RESULTS: The combination of citrullinated vimentin and enolase peptides (Modi-1) stimulated strong CD4 T cell responses in mice. Responses resulted in a potent anti-tumor therapy against established tumors and generated immunological memory which protected against tumor rechallenge. Depletion of CD4, but not CD8 T cells, abrogated the primary anti-tumor response as well as the memory response to tumor rechallenge. This was further reinforced by successful tumor regression being associated with an increase in tumor-infiltrating CD4 T cells and a reduction in tumor-associated myeloid suppressor cells. The anti-tumor response also relied on direct CD4 T cell recognition as only tumors expressing MHC-II were rejected. A comparison of different Toll-like receptor (TLR)-stimulating adjuvants showed that Modi-1 induced strong Th1 responses when combined with granulocyte-macrophage colony-stimulating factor (GMCSF), TLR9/TLR4, TLR9, TLR3, TLR1/2 and TLR7 agonists. Direct linkage of the TLR1/2 agonist to the peptides allowed the vaccine dose to be reduced by 10-fold to 100-fold without loss of anti-tumor activity. Furthermore, a CD4 Th1 response to the citrullinated peptides was seen in ovarian cancer patients. CONCLUSIONS: Modi-1 citrullinated peptide vaccine induces potent CD4-mediated anti-tumor responses in mouse models and a CD4 T cell repertoire is present in ovarian cancer patients to the citrullinated peptides suggesting that Modi-1 could be an effective vaccine for ovarian cancer patients.

Antibodies in cerebral cavernous malformations react with cytoskeleton autoantigens in the lesional milieu.

Previous studies have reported robust inflammatory cell infiltration, synthesis of IgG, B-cell clonal expansion, deposition of immune complexes and complement within cerebral cavernous malformation (CCM) lesions. B-cell depletion has also been shown to reduce the maturation of CCM in murine models. We hypothesize that antigen(s) within the lesional milieu perpetuate the pathogenetic immune responses in CCMs. This study aims to identify those putative antigen(s) using monoclonal antibodies (mAbs) derived from plasma cells found in surgically removed human CCM lesions. We produced human mAbs from laser capture micro-dissected plasma cells from four CCM patients, and also germline-reverted versions. CCM mAbs were assayed using immunofluorescence on central nervous system (CNS) tissues and immunocytochemistry on human primary cell lines. Antigen characterization was performed using a combination of confocal microscopy, immunoprecipitation and mass spectrometry. Affinity was determined by enzyme-linked immunosorbent assay, and specificity by multi-color confocal microscopy and quantitative co-localization. CCM mAbs bound CNS tissue, especially endothelial cells and astrocytes. Non-muscle myosin heavy chain IIA (NMMHCIIA), vimentin and tubulin are three cytoskeleton proteins that were commonly targeted. Selection of cytoskeleton proteins by plasma cells was supported by a high frequency of immunoglobulin variable region somatic hypermutations, high affinity and selectivity of mAbs in their affinity matured forms, and profoundly reduced affinity and selectivity in the germline reverted forms. Antibodies produced by plasma cells in CCM lesions commonly target cytoplasmic and cytoskeletal autoantigens including NMMHCIIA, vimentin and tubulin that are abundant in endothelial cells and astrocytes. Binding to, and selection on autoantigen(s) in the lesional milieu likely perpetuates the pathogenetic immune response in CCMs. Blocking this in situ autoimmune response may yield a novel treatment for CCM.

Impact and production of Non-HLA-specific antibodies in solid organ transplantation.

Organ transplantation is an effective way to treat end-stage organ disease. Extending the graft survival is one of the major goals in the modern era of organ transplantation. However, long-term graft survival has not significantly improved in recent years despite the improvement of patient management and advancement of immunosuppression regimen. Antibody-mediated rejection is a major obstacle for long-term graft survival. Donor human leucocyte antigen (HLA)-specific antibodies were initially identified as a major cause for antibody-mediated rejection. Recently, with the development of solid-phase-based assay reagents, the contribution of non-HLA antibodies in organ transplantation starts to be appreciated. Here, we review the role of most studied non-HLA antibodies, including angiotensin II type 1 receptor (AT1 R), K-α-tubulin and vimentin antibodies, in the solid organ transplant, and discuss the possible mechanism by which these antibodies are stimulated.

Biphasic Feline Mammary Carcinomas Including Carcinoma and Malignant Myoepithelioma.

Feline mammary tumors are usually malignant and aggressive carcinomas. Most cases are simple monophasic carcinomas (1 epithelial population), and additional phenotyping is usually not needed. In this study, we describe 10 malignant mammary tumors from 9 female cats that had unusual histomorphology: they appeared biphasic, with 2 distinct cell populations. Initially, they were morphologically diagnosed as either carcinosarcoma (1/10) or malignant pleomorphic tumor (9/10) of the mammary gland, as the latter did not match any previously described histological subtype. Immunohistochemistry (IHC) was performed for pancytokeratin, cytokeratins 8 and 18, cytokeratin 14, cytokeratins 5 and 6, vimentin, p63, calponin, alpha-smooth muscle actin, Ki-67, ERBB2, estrogen receptor alpha, and progesterone receptor. In 7 of 10 cases, the biphasic nature was confirmed and, on the basis of the IHC results, they were classified as carcinoma and malignant myoepithelioma (4/10), ductal carcinoma (1/10), and carcinosarcoma (2/10). The other 3 of 10 cases were monophasic based on IHC. In the cases of carcinoma and malignant myoepithelioma, the malignant myoepithelial cells were 100% positive for vimentin (4/4) and variably positive for p63, calponin, and cytokeratins (4/4). These findings show that, although rare, biphasic mammary carcinomas do occur in cats. In dogs and humans, tumors composed of malignant epithelial and myoepithelial cells have a less aggressive behavior than certain simple carcinomas, and therefore, their identification might also be clinically significant in the cat.

Antibodies against carbamylated vimentin exist in systemic lupus erythematosus and correlate with disease activity.

OBJECTIVES: Antibodies against carbamylated protein (anti-CarP) were found to be a promising marker to evaluate joint damage and disease activity in patients with rheumatoid arthritis (RA). However, whether anti-CarP antibodies were present in systemic lupus erythematosus (SLE) remained ambiguous. We have therefore undertaken this study to assess the levels of serum anti-CarP antibodies and to evaluate their clinical value in SLE. METHODS: Serum levels of antibodies against carbamylated-vimentin (anti-Carp) were measured by enzyme immunosorbent assay in 100 patients with SLE, 76 with RA, 17 with primary Sjögren syndrome (pSS), and 68 healthy controls. Data analyses between anti-Carp antibodies and other laboratory measures were performed using SPSS 24 software for Windows. RESULTS: The levels of serum anti-CarP antibodies in patients with SLE were significantly higher than those in healthy controls. In addition, anti-CarP antibodies were present in SLE patients lacking the disease-specific antibodies, including anti-Smith-negative patients (24.4%, 21/86), anti-dsDNA-negative patients (29.3%, 12/41), anti-nucleosome-negative patients (21.4%, 9/42), and antiribosomal P protein antibody-negative patients (23.7%, 18/76). There were significant differences between the anti-CarP-positive and anti-CarP-negative SLE patients in clinical and laboratory features, such as age, erythrocyte sedimentation rate (ESR), C-reactive protein, rheumatoid factor, third-generation cyclic citrullinated peptide (CCP3), anticardiolipin, D-dipolymer, complement 3, immunoglobulin G (IgG), red blood cell count (RBC) and hemoglobin. After adjusting for age and disease duration, the high levels of anti-CarP antibodies were still correlated with low RBC, hemoglobin and high ESR, IgG and CCP3. Active SLE patients demonstrated higher anti-CarP IgG than inactive patients. Moreover, the levels of anti-CarP were significantly higher in SLE patients with arthralgia and/or arthritis than in those without joint involvement. CONCLUSIONS: Anti-CarP antibodies were present in SLE patients and associated with the disease severity. These might provide a potential supplement to other specific autoantibodies for diagnosis of SLE and serve as a promising marker for measuring joint damage in the disease.