Complement Inhibitory Proteins Research Articles

Yes-associated Protein Mediates Acute Lung Injury in Adult but not Juvenile Mice

Objective: Adults with The Acute Respiratory Distress Syndrome (ARDS) have worse outcomes than children with Pediatric ARDS. This finding can be reproduced in animal models, suggesting a difference in Acute Lung Injury (ALI) mechanisms. Understanding these mechanistic differences could inform age-specific treatment. Hypothesis: Since Yes-associated protein (YAP) is important for lung development and reactive to mechanical stretch, we hypothesized that YAP is important for ALI mechanisms. Methods: To induce ALI, we intranasally instilled mice with Pseudomonas aeruginosa strain K (2.5 x 105 colony forming units (c.f.u.)) in juvenile (3-week-old) and adult (10-week-old) C57BL/6J mice. 24 h later, we collected bronchoalveolar lavage (BAL) fluid and measured protein, cell count, and c.f.u. To knockdown YAP expression, we injected siRNA 24 h before P. aeruginosa instillation. We removed the lungs and enriched for CD45+ and CD41+ fraction, and CD31+ fraction using Dynabeads sheep anti-rat IgG (Invitrogen). In separate experiments, lungs were separated into nuclear and cytosolic fractions for immunoblot analysis. For immunoprecipitation, the cytosolic fraction was added to beads coated with anti-YAP antibody (rabbit monoclonal). We washed the beads and eluted bound proteins, which were identified by label-free mass spectrometry. Data and results: While both juvenile and adult mice had significantly increased BAL cell counts and protein 24 h after P. aeruginosa instillation, adult mice had significantly greater cell counts and protein than juveniles. BAL c.f.u. were similar in both groups, suggesting equal instillation of P. aeruginosa. P. aeruginosa instillation increased whole lung YAP expression in both cytosolic and nuclear fractions in adult mice by >3-fold, but YAP expression was not increased in P. aeruginosa-treated juvenile mice. YAP protein increases occurred in CD31+ fraction (endothelial cells), but no YAP was quantified in CD45+/CD41+ fraction (leukocytes and platelets). Vascular knockdown of YAP expression by ~75% in adult mice blocked P. aeruginosa-induced BAL cell count increases. Proteomics analysis revealed 62 proteins that had significantly altered YAP binding upon P. aeruginosa treatment (47 with increased YAP binding). The complement factors H and I had a log2-fold-change for YAP binding of 4.56 and 2.26, respectively, upon P. aeruginosa treatment. Conclusions: Adult mice exhibited YAP-dependent Acute Lung Injury; juveniles did not. YAP-dependent mechanisms of injury may be secondary to YAP binding to the complement inhibitory proteins H and I. More work is needed to establish the cell-specificity of YAP-complement factor binding and to determine the location of complement signaling. HL148403, Parker B Francis, and Columbia University Department of Pediatrics Children’s Health Innovation Nucleation Fund to RFH; Ernest E Just Summer Scholarship to AMD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Daratumumab induces cell-mediated cytotoxicity of primary effusion lymphoma and is active against refractory disease

ABSTRACT Primary effusion lymphoma (PEL), an aggressive non-Hodgkin lymphoma caused by Kaposi sarcoma-associated herpesvirus (KSHV), lacks standard therapy and has a median survival of 10–22 months with combination chemotherapy. PEL is a tumor of plasmablast-like B cells generally expressing CD38, the target of daratumumab (Dara). Initially, we assessed PEL cells from eight patients and established that each expressed high levels of CD38 by flow cytometry. PEL cell lines were also evaluated and most had high CD38 expression. We then assessed Dara’s effects on complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of PEL cell lines as well as its clinical benefits on two patients with PEL. Despite high CD38 expression, Dara did not induce CDC of PEL cell lines, due in part to high levels of the complement-inhibitory proteins, CD55 and CD59. However, Dara induced significant and dose-dependent increases in ADCC, particularly in those lines with high CD38 levels. Two FDA-approved drugs, all trans-retinoic acid (ATRA) and pomalidomide (Pom), significantly increased surface CD38 levels in low-CD38 expressing PEL cell lines, resulting in increased Dara-induced ADCC. Two patients with refractory PEL were treated with Dara alone or in combination with Pom. One patient with leptomeningeal PEL had a complete response to Dara and Pom combination treatment. Others had improvement in performance status and resolution of malignant ascites with Dara alone. Together, these data support the use of Dara monotherapy or in combination with ATRA or Pom as a potential therapeutic option for PEL.

Abstract P044: A genetically engineered mouse Sertoli cell line survives chronic humoral immune factors when grafted into diabetic mice

Abstract Patients with uncontrolled diabetes have an increased risk of developing cancer, therefore controlling their blood glucose and insulin levels is imperative. An effective treatment to accomplish this is transplantation of insulin-producing pancreatic islets. However, these patients must take chronic immunosuppressants to prevent graft rejection, which also increases cancer risk. An alternative to traditional transplantation is to transduce immunoregulatory cells such as Sertoli cells (SC), which survive as grafts without immune suppressants, to express insulin. We have previously transduced a mouse SC tumor line (MSC-1) to express human insulin (MSC1-HI), demonstrated that they survive (at 70-75%) when grafted into diabetic mice after 50 days, and confirmed their sustained expression of human insulin. Since complement is an important part of immune rejection, we hypothesize that one mechanism SC use to survive is inhibition of complement by expression of complement inhibitory proteins (CIP). We confirmed that primary mouse SC (pSC), MSC-1 cells, and MSC1-HI cells survive human complement activation in vitro while controls are killed, indicating that SC must be inhibiting complement, and microarray analyses of pSC and MSC-1 cells identified gene expression of 14 different CIP. We immunostained MSC-1 and MSC1-HI grafts to study deposition of C9, the lytic pore component of the complement membrane attack complex. In MSC1-HI grafts, C9 increased from about 10 positive cells/mm2 graft to roughly 40 positive cells/mm2 graft, and a dramatic increase in C9 deposition was observed on MSC-1 grafts—from about 4 positive cells/mm2 to well over 350 positive cells/mm2. Furthermore, we found that MSC1-HI grafts were significantly larger at day 50 than MSC-1 grafts. The MSC1-HI grafts also increased in size by almost 300% between days 20 and 50 post-transplantation as compared to MSC-1 grafts, which decreased in size by 64%. Thus, it appears that MSC-1 grafts are still rejecting while MSC1-HI grafts are growing, indicating some level of tumorigenesis. Indeed, insulin has been shown to be a growth factor for SC, hence we propose that increased expression of insulin in the MSC-1 cancer cell line is encouraging tumorigenesis in diabetic mice. Taken together, these data indicate that MSC1-HI cells undergo tumorigenesis and can survive and proliferate in spite of complement activation, which may be playing a role in MSC-1 graft reduction. Since complement can be a powerful anti-tumor and transplant rejection mechanism, understanding how these SC tumor cells evade complement killing has significance in both treating cancer and increasing grafted organ survival. As diabetes patients are more susceptible to tumors, further studying the relationship between insulin and tumorigenesis will increase knowledge to allow for more effective management of tumors. Citation Format: Rachel L. Washburn, Taylor Hibler, Jannette M. Dufour. A genetically engineered mouse Sertoli cell line survives chronic humoral immune factors when grafted into diabetic mice. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P044.

Vemircopan (ALXN2050) Monotherapy in Paroxysmal Nocturnal Hemoglobinuria: Interim Data from a Phase 2 Open-Label Proof-of-Concept Study

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder characterized by a deficit of complement inhibitory proteins on the blood cell surface, causing intravascular hemolysis (IVH) and risk of thrombosis owing to terminal complement activation. Treatment of patients with PNH with complement C5 inhibitors has greatly improved clinical outcomes and survival; however, some patients taking C5 inhibitors develop clinically significant extravascular hemolysis (EVH) that may result in symptomatic anemia and require blood transfusions. Thus, when treating PNH, complete and sustained terminal complement inhibition is needed. Pharmacologic inhibition of factor D, the rate-limiting enzyme of the complement alternative pathway (AP), even as a monotherapy, may control IVH while preventing EVH, eventually improving anemia and the need for transfusions. The oral factor D inhibitor ALXN2040 (danicopan) showed efficacy as a monotherapy and an add-on treatment to the C5 inhibitor eculizumab, but there was suboptimal control of IVH in some patients when used as a monotherapy. A latter oral factor D inhibitor, ALXN2050 (vemircopan), has the same mechanism of action as danicopan but demonstrates increased potency and binding affinity for factor D. It also achieves immediate, complete, and sustained AP inhibition with twice-daily administration (BID). Vemircopan is being investigated in a phase 2 proof-of-concept study as a monotherapy to treat patients with PNH. Methods: This is an ongoing, phase 2, open-label study (NCT04170023) to assess the efficacy, safety, and PK/PD of vemircopan monotherapy in patients with PNH. The study comprises a 60-day screening period, a 12-week treatment period, and a 96-week long-term extension. Three patient groups are included: treatment-naïve, eculizumab switch, and danicopan monotherapy rollover. This interim analysis focuses on efficacy and safety data from the treatment-naïve group to establish proof of concept in PNH. Key inclusion criteria for the treatment-naïve group include lactate dehydrogenase (LDH) ≥1.5 × upper limit of normal (ULN), absolute reticulocyte count ≥100x109/L, and anemia (hemoglobin [Hgb] <10.5 g/dL). Patients receive vemircopan 120 mg BID, which can be escalated to 180 mg BID per investigator discretion based on guidelines in the protocol (Hgb has not increased by ≥1 g/dL by week 4 relative to baseline; blood transfusion needed; or LDH >1.5 × ULN by day 14 in 2 consecutive assessments). Primary endpoint is change from baseline to week 12 in Hgb. Key secondary endpoints include change from baseline to week 12 in LDH and Functional Assessment of Chronic Illness Therapy - Fatigue (FACIT-Fatigue) score. Safety is reported as treatment-emergent adverse events (TEAEs). Results: As of a data cutoff date of April 30, 2022, 11 treatment-naïve patients were enrolled (mean [SD] age, 44.4 [17.8] y; 63.6% male; 72.7% Asian, 54.5% received transfusions during screening period). Nine patients completed 12 weeks in the study and were included in this analysis. From baseline to week 12, mean (SD) Hgb increased by 3.9 (1.11) g/dL from 7.9 (1.29) g/dL to 11.8 (1.25) g/dL (Fig. 1A), LDH rapidly decreased by 81% from 7 × ULN to 1.4 × ULN (Fig. 1B), absolute reticulocyte count decreased from 212.4 (86.47) x 103/uL to 120.0 (51.44) x 103/uL, and a 13-point improvement in FACIT-Fatigue score was observed. No patients needed transfusions except 1 case on day 2 owing to low Hgb (5.1 g/dL). As of the data cutoff date, 31 TEAEs were reported (n=9 patients); most (25/31; 80.6%) were considered unrelated to study drug. No serious TEAEs, grade ≥3 TEAEs, discontinuations, or deaths were reported. The most common TEAE was headache (n=4; 36.4%). There were no thrombotic events, seizures, or meningococcal infections. Thus far, 7 of 11 patients received dose escalation from 120 mg BID to 180 mg BID, which was associated with increased Hgb levels. Conclusions: This interim analysis of treatment-naïve patients with PNH suggests that monotherapy with vemircopan controlled IVH (reduction in LDH to almost normal levels) and prevented EVH (clinically meaningful increases in Hgb values, reticulocyte count reductions). No new safety signals were identified during the 12-week evaluation period. This interim efficacy and safety analysis provides proof of concept in PNH and suggests that phase 3 trials of vemircopan in patients with PNH are warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Extracellular Vesicles Isolated from Plasma of Multiple Myeloma Patients Treated with Daratumumab Express CD38, PD-L1, and the Complement Inhibitory Proteins CD55 and CD59.

Daratumumab (DARA) has improved the outcome of treatment of multiple myeloma (MM). DARA acts via complement-dependent and -independent mechanisms. Resistance to DARA may result from upregulation of the complement inhibitory proteins CD55 and CD59, downregulation of the DARA target CD38 on myeloma cells or altered expression of the checkpoint inhibitor ligand programmed death ligand-1 (PD-L1) or other mechanisms. In this study, EVs were isolated from peripheral blood (PB) and bone marrow (BM) from multiple myeloma (MM) patients treated with DARA and PB of healthy controls. EV size and number and the expression of CD38, CD55, CD59 and PD-L1 as well as the EV markers CD9, CD63, CD81, CD147 were determined by flow cytometry. Results reveal that all patient EV samples express CD38, PD-L1, CD55 and CD59. The level of CD55 and CD59 are elevated on MM PB EVs compared with healthy controls, and the level of PD-L1 on MM PB EVs is higher in patients responding to treatment with DARA. CD147, a marker of various aspects of malignant behaviour of cancer cells and a potential target for therapy, was significantly elevated on MM EVs compared with healthy controls. Furthermore, mass spectrometry data suggests that MM PB EVs bind DARA. This study reveals a MM PB and BM EV protein signature that may have diagnostic and prognostic value.

Mouse Sertoli Cells Inhibit Humoral-Based Immunity

Transplantation is used to treat many different diseases; however, without the use of immunosuppressants, which can be toxic to the patient, grafted tissue is rejected by the immune system. Humoral immune responses, particularly antibodies and complement, are significant components in rejection. Remarkably, Sertoli cells (SCs), immunoregulatory testicular cells, survive long-term after transplantation without immunosuppression. The objective of this study was to assess SC regulation of these humoral-based immune factors. Mouse SCs survived in vitro human complement (model of robust complement-mediated rejection) and survived in vivo as allografts with little-to-no antibody or complement fragment deposition. Microarray data and ELISA analyses identified at least 14 complement inhibitory proteins expressed by mouse SCs, which inhibit complement at multiple points. Interestingly, a mouse SC line (MSC-1), which was rejected by day 20 post transplantation, also survived in vitro human complement, showed limited deposition of antibodies and complement, and expressed complement inhibitors. Together this suggests that SC inhibition of complement-mediated killing is an important component of SC immune regulation. However, other mechanisms of SC immune modulation are also likely involved in SC graft survival. Identifying the mechanisms that SCs use to achieve extended survival as allografts could be utilized to improve graft survival.

Relationship between the expression of complement inhibitory proteins and therapeutic efficacy of antibodies in breast cancer.

Complement regulatory proteins (mCRPs) CD55, CD46 and CD59 have been proposed as key elements in therapeutic resistance against cancer. mCRP-expressing tumor cells, in addition to hindering trastuzumab, pertuzumab and sacituzumab-govitecan therapeutic activity in breast cancer, can regulate biological processes that promote tumor progression. This review describes the structure of mCRPs and analyzes their expression using transcriptomic databases from breast cancer patients, in addition to collecting information on mCRPs interactions and signaling in tumor cells. Given that mCRPs are relevant targets, several strategies that have been explored for their inhibition and regulation in order to increase therapeutic efficacy and prevent cancer resistance and progression are described.

Seeking the Sertoli cell complement inhibitory signature to prolong transplant survival

Abstract Clinical transplantation can treat various illnesses, although without the use of toxic immunosuppressants transplanted tissue is rejected by the immune system. Complement, an enzymatic cascade that leads to cytolysis by insertion of the membrane attack complex (MAC), is a significant component in transplant rejection. Interestingly, Sertoli cells (SC) survive long-term after transplantation across immune barriers without immunosuppression. This study used allotransplant (between same species) and xenotransplant (between different species) models to investigate the role of complement inhibition in SC extended survival. Since SC were resistant to in vitro complement killing, mouse or pig SC were transplanted into mice or rats, respectively. Grafts were collected and analyzed for MAC. MAC was not detected on SC transplants, indicating that SC inhibit complement before MAC deposition, possibly through expression of complement inhibitory proteins (CIP). Bioinformatic analyses of mouse or pig SC identified expression of 13 CIP. After expression of two key CIP, CD46 and CD55, were knocked down, SC were killed by complement in vitro. Taken together, these data indicate that the SC complement inhibitory signature is critical for SC graft survival. Understanding SC complement inhibition is important as it could lead to improved transplant survival with a decreased requirement of harsh immunosuppressants, making this procedure more attainable for patients. Supported by grants from the CH foundation, the South Plains Foundation, and the Mary Lou Clements-Mann Scholarship

Extracellular fibrinogen-binding protein (Efb), a key immune evasion protein of Staphylococcus aureus and a potential therapeutic target

Due to the continued growth of antibiotic-resistant Staphylococcus aureus strains, it is necessary to explore alternative targets for future therapeutic applications. For this reason, is important to understand the staphylococcal immune evasion mechanisms with a special focus on extracellular fibrinogen-binding protein (Efb) and Efb related proteins. Therefore, it was conducted a literature review to compile relevant information on this protein. It was found that Efb has three binding sites with biological relevance that could be used as therapeutic targets with specificity for fibrinogen, platelets, and complements. First, the fibrinogen-binding motifs also found in coagulase block neutrophil αMβ2 adherence to fibrinogen and attract fibrinogen to the bacterial surface, forming capsule-like structures that block phagocytosis. Second, Efb is a potent anti-thrombotic agent, probably related to its P-selectin binding capacity. Efb P-selectin binding blocks the interaction of P-selectin with the PSGL-1 receptor, thereby impairs the mechanisms of platelet-mediated leukocyte recruitment to the site of vascular injury. Third, the Efb complement binding domain, also found in other staphylococcal complement inhibitory proteins like Ecb, Sbi, and SCIN, is responsible for the evasion of the complement-mediated immune response. Efb reduces the formation of C3 convertase and the interaction with neutrophils, affect B-cells activation, and maturation. Efb binding sites have a clear implication on the virulence of Staphylococcus aureus in mastitis, wound infection, staphylococcal pneumonia, and infections related to implanted devices, and contributes to staphylococcal persistence in host tissues and abscess formation in the kidneys. Given the biological relevance of Efb binding sites in staphylococcal infections, they are promising vaccine targets. Additionally, due to the inhibitory effect of Efb on platelets and complements, Efb can be a potential therapeutic agent to treat diseases associated with thrombosis and abnormal complement activity.

Effects of Human, Caprine, and Bovine Milk Fat Globules on Microbiota Adhesion and Gut Microecology.

Milk fat is an essential nutrient for infant development. The effects and mechanisms of human, caprine, and bovine milk fat globules (MFGs) on the gut microbiota were investigated in this study. Human MFGs enhance the efficacy of probiotics by inhibiting pathogen function. Akkermansia and Bifidobacterium were identified as the dominant microbiota by human MFGs. Mucin and complement inhibitory proteins in human MFGs were found to inhibit different pathogens. Caprine MFGs directly promoted the colonization of probiotics and the emergence of the biomarker Allobaculum. Mucin 1 in caprine MFGs was primarily responsible for inducing probiotic adhesion. Bovine MFGs increased the abundance of Oscillospira, which reduces the risk of obesity. Due to the enrichment of cell-cell junction proteins and the lack of mucin, the regulation of gut microecology by bovine MFGs was not readily apparent. In short, this study paves the way for the development of functional infant formula.

Determinants of response to daratumumab in Epstein-Barr virus-positive natural killer and T-cell lymphoma

BackgroundThe potential therapeutic efficacy of daratumumab in natural killer T-cell lymphoma (NKTL) was highlighted when its off-label usage produced sustained remission in a patient with highly refractory disease. This is...

Daratumumab Efficiently Targets NK/T Cell Lymphoma with High CD38 Expression

CD38 expression can be detected on a majority of NK/T cell lymphoma (NKTCL). Based on the clinical data of 94 patients with ENKTL, high CD38 surface expression was an adverse prognostic factor for progression free survival, and these patients had significantly inferior outcomes (Wang, 2015). This corroborates with our own studies showing that relapsed and refractory NKTCL patients almost always exhibit high CD38 expression, thereby proposing that it may be worthwhile to target CD38 as a novel therapeutic option.Daratumumab is a human anti-CD38 monoclonal antibody which has shown significant anti-cancer activity in multiple myeloma. In this study, we investigated the potential cytotoxic effects and mechanisms of actions of Daratumumab in a panel of 10 NKTCL cell lines. Firstly, we identified that cell lines can be stratified into CD38Hi, CD38Mid and CD38Lo as quantified by mRNA expression and CD38 protein surface expression which was qualitatively analysed by FACS and IHC. Daratumumab is able to induce antibody dependent cellular cytotoxicity (ADCC) in a CD38-dependent manner and in a dose-dependent fashion. We also observe a greater induction of antibody dependent cellular phagocytosis (ADCP) activation in correlation with CD38 expression. Furthermore, complement-dependent cytotoxicity (CDC) was significantly triggered by Daratumumab in CD38Hi and CD38Mid cells. However, we found evidence of an additional level of regulation provided by the expression of complement inhibitory proteins(CIPs) CD46, CD55 and CD59 on NKTCL cell lines which can limit and inhibit the induction of CDC regardless of CD38 surface expression levels.All-trans retinoic acid (ATRA) is a vitamin A derivative that binds to retinoic acid receptor alpha leading to the upregulation of CD38 expression. Treatment with ATRA increases CD38 mRNA and surface protein expression in all CD38Hi-Lo NKTCL cell lines. Interestingly CD38Lo cell lines showed the greatest fold increase of CD38 mRNA and surface protein expression. Notably, addition of ATRA to SNK-10, a CD38LoCD55Hi cell line, prior to Daratumumab treatment was able to improve the induction of ADCC but not CDC. This suggests a critical role for CIPS in mediating the induction of CDC in NKTCL.Subsequent investigation for the efficacy of Daratumumab in vivo is currently being performed.We also discovered a synergistic cytotoxic effect in NKTCL CD38Hi cell lines mediated by L-Asparaginase (a component of the current chemotherapeutic regimen in NKTCL) treatment in combination with Daratumumab. Altogether,these results propose a rationale therapeutic option for single or combined Daratumumab treatment in the clinic especially for relapsed and refractory patients. DisclosuresJin:Janssen China R&D: Employment. Yu:Janssen China R&D: Employment. Chen:Janssen China R&D: Employment. Yang:Janssen China R&D: Employment. Chng:Janssen China R&D: Research Funding.

Profiling Complement System Components in Primary CNS Vasculitis.

Complement activation has been implicated in the pathogenesis of many vasculitic syndromes such as anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides. Using an array-based multiplex system, we simultaneously quantified serum and CSF levels of activated and regulatory complement system proteins in patients with primary CNS vasculitis (PACNS; n = 20) compared to patients with non-inflammatory conditions (n = 16). Compared to non-inflammatory controls, levels of C3a, C5a, and SC5b-9, indicative for general activation of the complement system, of C4a, specific for the activation of the classical pathway, Ba and Bb, reflective for alternative complement activation as well as concentrations of complement-inhibitory proteins factor H and factor I were unchanged in patients with PACNS. Our study does not support the hypothesis that complement activation is systemically increased in patients with PACNS.

Xenogeneic Sertoli cells express complement inhibitory proteins necessary for their survival of hyperacute rejection

Abstract Transplantation is used to treat organ failure associated with various diseases, however there is a severe shortage of organs acceptable for transplant. Utilization of pig organs offers an endless supply of transplantable tissue, but xenografts are rapidly destroyed by hyperacute rejection (HAR). The primary humoral mediator of HAR is the activation of complement (C″) by preformed-antibody binding to xenoantigens, causing a proteolytic cascade that results in cell lysis by membrane attack complex (MAC) insertion. Sertoli cells (SCs) are immunoregulatory testicular cells that survive as xenografts for an extended period without the use of immunosuppressive drugs. The goal of this study was to examine the mechanisms for SC survival of C″. Neonatal porcine SCs (NPSCs) transplanted into rats survived as xenografts while porcine islets (NPIs) did not. Immunohistochemistry of the surviving grafts show deposited C3, but not MAC, suggesting that NPSCs inhibit C″ after activation. An in vitro human serum (HS)-C″ cytotoxicity assay confirmed that NPSCs survive activated human C″. Analysis of NPSC expression of complement inhibitory proteins (CIP) revealed higher levels of membrane cofactor protein (MCP) and decay accelerating factor (DAF) as compared to NPIs, which are killed by C″. Knocking down expression of MCP and DAF using shRNA confirmed their importance in NPSC survival of C″. Less than 10% of these cells survived exposure to the HS-C″ cytotoxicity assay, indicating MCP and DAF are important in NPSC xenograft survival in vitro.

Gene expression in Staphylococcus aureus skin infection

Gene expression in Staphylococcus aureus changes during infection to survive its host. Therefore, to find new strategies to combat staphylococcal infections, it is important to understand the mechanisms that this pathogen uses to adapt to its host and how the host responds to the presence of staphylococcal cells. We have reviewed two studies of gene expression in Staphylococcus aureus during skin infections, one study using a rabbit skin infection model and the other study using a diabetic skin infection model in mice. We compared the two gene expression profiles to find similarities and differences. Many genes did not show any differences in gene expression in S. aureus during the skin infection compared to the control groups. However,19 genes were upregulated in both systems include chaperones (e.g., groES, groEL, grpE, dnaK9), sodM, hrcA, sbi, and the gene encoding a cadmium-exporting ATPase protein. Also, four genes were downregulated in both systems including a gene that encodes a hydrolase and three genes for hypothetical proteins. Also, there was a group of genes expressed in different ways in the two systems. The gene expression of sarU, transcriptional regulators of the LysR family, Cro family, crp family, TetR family, tenA, and many hypothetical proteins were upregulated in the rabbit system but downregulated in the mouse system. The genes rps, rpl, rpm, and several others involved, for example, in translation and transcription were downregulated in the rabbit system but upregulated in the mouse system. Many genes that showed significant changes in overall gene expression in the rabbit model were unaffected in the mouse model. For example, in the rabbit skin infection model increased important gene regulators like agr and sarV, while some stress-response genes (e.g., sigB and lexA) were downregulated. The gene expression of several staphylococcal genes encoding virulence factors such as fibronectin-binding proteins, hemolysins, coagulases, complement inhibitory proteins, Emp, and many exotoxins were upregulated while clumping factor A was downregulated. Besides, some genes showed expression changes in the mouse model, but not in the rabbit model. For example, sarA, rot, ecb, ctsR, spx, many ribosomal proteins, and hypothetical proteins increased, while cap5k, lysE, rusA, and many hypothetical proteins decreased in the mouse model but they were unaffected in the rabbit model. On the other hand, the host responded to the S. aureus infection by inducing the expression of genes encoding host inflammatory cytokines, receptors, genes associated with neutrophil adhesion and migration, inflammation, and immune cell trafficking. In conclusion, the level of gene expression changed both in the pathogen and the host during the skin infection. The information of gene expression can make significant contributions to understand which genes are involved in the infection process, which can be targeted for antimicrobial chemotherapy.

Microglial trogocytosis and the complement system regulate axonal pruning in vivo.

Partial phagocytosis-called trogocytosis-of axons by microglia has been documented in ex vivo preparations but has not been directly observed in vivo. The mechanisms that modulate microglial trogocytosis of axons and its function in neural circuit development remain poorly understood. Here, we directly observe axon trogocytosis by microglia in vivo in the developing Xenopus laevis retinotectal circuit. We show that microglia regulate pruning of retinal ganglion cell axons and are important for proper behavioral response to dark and bright looming stimuli. Using bioinformatics, we identify amphibian regulator of complement activation 3, a homolog of human CD46, as a neuronally expressed synapse-associated complement inhibitory molecule that inhibits trogocytosis and axonal pruning. Using a membrane-bound complement C3 fusion protein, we demonstrate that enhancing complement activity enhances axonal pruning. Our results support the model that microglia remodel axons via trogocytosis and that neurons can control this process through expression of complement inhibitory proteins.

CD38 deletion of human primary NK cells eliminates daratumumab-induced fratricide and boosts their effector activity

AbstractMultiple myeloma (MM) is a plasma cell neoplasm that commonly expresses CD38. Daratumumab (DARA), a human monoclonal antibody targeting CD38, has significantly improved the outcome of patients with relapsed or refractory MM, but the response is transient in most cases. Putative mechanisms of suboptimal efficacy of DARA include downregulation of CD38 expression and overexpression of complement inhibitory proteins on MM target cells as well as DARA-induced depletion of CD38high natural killer (NK) cells resulting in crippled antibody-dependent cellular cytotoxicity (ADCC). Here, we tested whether maintaining NK cell function during DARA therapy could maximize DARA-mediated ADCC against MM cells and deepen the response. We used the CRISPR/Cas9 system to delete CD38 (CD38KO) in ex vivo expanded peripheral blood NK cells. These CD38KO NK cells were completely resistant to DARA-induced fratricide, showed superior persistence in immune-deficient mice pretreated with DARA, and enhanced ADCC activity against CD38-expressing MM cell lines and primary MM cells. In addition, transcriptomic and cellular metabolic analysis demonstrated that CD38KO NK cells have unique metabolic reprogramming with higher mitochondrial respiratory capacity. Finally, we evaluated the impact of exposure to all-trans retinoic acid (ATRA) on wild-type NK and CD38KO NK cell function and highlighted potential benefits and drawbacks of combining ATRA with DARA in patients with MM. Taken together, these findings provide proof of concept that adoptive immunotherapy using ex vivo expanded CD38KO NK cells has the potential to boost DARA activity in MM.

Dysphagia with Blood Disorder? an Unusual Presentation for Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired hemolytic disorder of the multipotent myeloid stem cells, due to underexpression of surface complement inhibitory proteins CD55 and CD59, affecting 0.13/100,000 patients per year. Besides anemia and thrombosis, PNH can also cause smooth muscle dystonias owing to various mechanisms in the body. Studies have described smooth muscle dystonias presenting as recurrent abdominal pain, lethargy and erectile dysfunction, but intermittent dysphagia is rare with PNH. We describe a case of dysphagia with negative work up as initial presentation of PNH. 16-year-old female with no significant past medical history presented to the clinic with 1 week of ongoing fatigue and difficulty swallowing. She never had any similar complaints in the past nor in her family. The dysphagia was progressively worsening, initially for solid, then with liquids, accompanied by nausea, vomiting and globus sensation in the middle chest. No prior psychiatric history, binging-purging behavior, chest pain, shortness of breath, heat/cold intolerance, weight changes, bladder or bowel dysfunction. She also denied any recent travels, allergies, transfusions, sick contacts, or over-the-counter medication use. Initial labwork showed Hb 11.2 g/dL, MCV 80 fL, WBC 9 x103 K/uL, and platelet 219,000 K/uL. Metabolic panel, troponin, BNP, ANA, rheumatoid factor, lyme serology, vitamin B12, folate and iron studies were within normal limits. Mentzer index &amp;lt;13, serum protein electrophoresis ruled out Thalassemia. LDH was elevated and Haptoglobin decreased. Direct and indirect Coomb's antibody test were negative. Peripheral smear showed normocytic normochromic anemia with normal morphology of RBCs. Flow cytometry showed lack of CD55 and CD59. Esophagogastroduodenoscopy, done for dysphagia, to rule out luminal obstruction was unremarkable. CT chest with contrast ruled out external compression on esophagus. Videofluoroscopic swallow study revealed segmental, dynamic obstruction to both solids and liquids in the lower third of the esophagus. Dysphagia was attributed to PNH and patient was started on eculizumab. She responded to the medication in 3 weeks, during which outpatient symptomatic therapy was given for her anemia along with soft semisolid diet. In PNH, the RBCs may have varying degree of CD59 and glycosyl phosphatidylinositol (GPI) anchor protein deficiency, with unaffected cells described as type I, partially affected cells called type II, and complete lack of the proteins deemed type III red cells. PNH red cell type III and type II red cells are 15 to 25 times and 3 to 5 times more sensitive to complement-mediated lysis than the type I normal red cells. This variability in the severity of the deficiency, as well as in the proportion of the cell population affected, defines the extent of the clinical manifestations of the disease. Approximately 40% of patients have a combination of types I, II and III PNH cells With hemolysis, after exceeding haptoglobin's binding capacity, the free hemoglobin binds any available nitrous oxide (NO) in the blood. Hemolysis also reduces endogenous NO generation by conversion of l-arginine, which is a component in NO generation, to ornithine, thereby further reducing the systemic availability of NO. NO is endogenous smooth muscle relaxor via activation of cGMP. Decreases in NO levels are hypothesized to cause smooth muscle contraction, manifesting as abdominal pain, erectile dysfunction and rarely, dysphagia in the lower two-third of the esophagus which is predominantly smooth muscle. These symptoms occur more commonly in patients with large PNH clone sizes and, therefore, higher hemolytic rates. In this case study, our focus was to recognize PNH among differentials for new onset dysphagia due to complement-mediated intravascular hemolysis causing gastrointestinal dysmotility. In doing so, invasive investigations may be avoided. Advance studies may be needed to completely understand the causes and correlate between PNH, intravascular hemolysis and dysphagia. Disclosures No relevant conflicts of interest to declare.

TAK-573, an Anti-CD38/Attenuated Ifnα Fusion Protein, Has Clinical Activity and Modulates the Ifnα Receptor (IFNAR) Pathway in Patients with Relapsed/Refractory Multiple Myeloma

TAK-573, an Anti-CD38/Attenuated Ifnα Fusion Protein, Has Clinical Activity and Modulates the Ifnα Receptor (IFNAR) Pathway in Patients with Relapsed/Refractory Multiple Myeloma

The gut anti-complement activity of Aedes aegypti: Investigating new ways to control the major human arboviruses vector in the Americas

Aedes aegypti is the main urban vector of dengue virus, chikungunya virus and Zika virus due to its great dispersal capacity and virus susceptibility. A. aegypti feed on plant-derived sugars but females need a blood meal for egg maturation. Haematophagous arthropods need to overcome host haemostasis and local immune reactions in order to take a blood meal. In this context, molecules present in the saliva and/or intestinal contents of these arthropods must contain inhibitors of the complement system (CS). CS salivary and/or intestinal inhibitors are crucial to protect gut cells of haematophagous arthropods against complement attack. The present work aimed to investigate the anti-complement activity of A. aegypti intestinal contents on the alternative, classical and lectin pathways of the human complement system. Here we show that A. aegypti gut contents inhibited the human classical and the lectin pathways but not the alternative pathway. The A. aegypti gut content has a serine protease able to specifically cleave and inactivate human C4, which is a novel mechanism for human complement inactivation in haematophagous arthropods. The gut of female A. aegypti was capable of capturing human serum factor H (a negative complement modulator), unlike males. C3 molecules in recently blood-fed female A. aegypti remain in their original state, being inactivated to iC3b soon after a blood feed. A transmission-blocking vaccine using these complement inhibitory proteins as antigens has the potential to interfere with the insect's survival, reproductive fitness and block their infection by the arboviruses they transmit to humans.