The effect of anti­HLA class I antibodies on the immunological properties of human glomerular endothelial cells and their modification by mTOR inhibition or GCN2 kinase activation.

In antibody­mediated rejection (ABMR), the graft endothelium is at the forefront of the kidney transplant against the assault from the recipient's humoral immune system, and is a target of the latter. The present study investigated the effect of antibodies against human leukocyte antigen (HLA) class I (anti­HLAI) on the immunological properties of human glomerular endothelial cells. Additionally, the effect of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) inhibitor (everolimus), or the general control nonderepressible 2 kinase (GCN2K) activator (halofuginone) on anti­HLAI antibody­mediated alterations was assessed. Cell integrity was examined, an lactate dehydrogenase (LDH) release assay was performed and cleaved caspase­3 levels were determined. Furthermore, cell proliferation was analyzed by performing a bromodeoxyuridine assay and the cellular proteins involved in signal transduction or immune effector mechanisms were assessed via western blotting. IL­8, monocyte chemoattractive protein­1 (MCP­1), von Willebrand factor (vWF) and transforming growth factor­beta 1 (TGF­ß1) were assayed via ELISA. The results revealed that anti­HLAI triggered integrin signaling, activated mTOR and GCN2K, preserved cell integrity and promoted cell proliferation. Additionally, by increasing intercellular adhesion molecule 1 (ICAM­1), HLA­DR, IL­8 and MCP­1 levels, anti­HLAI enhanced the ability of immune cells to interact with endothelial cells thus facilitating graft rejection. Contrarily, by upregulating CD46 and CD59, anti­HLAI rendered the endothelium less vulnerable to complement­mediated injury. Finally, by enhancing vWF and TGF­ß1, anti­HLAI may render the endothelium prothrombotic and facilitate fibrosis and graft failure, respectively. According to our results, mTORC1 inhibition and GCN2K activation may prove useful pharmaceutical targets, as they prevent cell proliferation and downregulate ICAM­1, IL­8, MCP­1 and TGF­ß1. mTORC1 inhibition also decreases vWF.

The imitation game: a viral strategy to subvert the complement system.

Viruses are obligate parasites of cellular hosts and therefore are constantly confronted with the host immune system. Evasion of innate immunity mechanisms by viruses is paramount for the establishment of their infection. The complement system can directly neutralize viruses and also augments adaptive immune responses against them. This system, therefore, is central to host innate immune surveillance, and viruses have evolved a multitude of ways to escape its assault. A major strategy employed by viruses is the molecular mimicry of human complement regulators, namely regulators of complement activation (RCA) proteins and CD59. Herein, we outline up-to-date information on the structure, function and role of viral homologs of the human complement regulators in viral pathogenesis.

Immuno-Resolving Ability of Resolvins, Protectins, and Maresins Derived from Omega-3 Fatty Acids in Metabolic Syndrome.

Omega-3 fatty acid consumption has been suggested to be beneficial for the prevention of type 2 diabetes mellitus (T2DM). Its effects have been attributed to anti-inflammatory activity, with the inhibition of arachidonic acid metabolism playing a central role. However, a more recent view is that omega-3 fatty acids play an active role as the precursors of potent, specialized pro-resolving mediators (SPMs), such as resolvins, protectins, and maresins. Docosahexaenoic acid (DHA)- and eicosapentaenoic-acid-derived SPMs are identified in the adipose tissue but the levels of certain SPMs (e.g., protectin D1) are markedly reduced with obesity, suggesting adipose SPM deficiency, potentially resulting in unresolved inflammation. Supplementation of the biosynthetic intermediates of SPM (e.g., 17-hydroxy-DHA) or omega-3 fatty acids increases the level of adipose SPMs, reduces adipose inflammation (decrease in macrophage accumulation and change to less inflammatory macrophages), and enhances insulin sensitivity. The findings from studies using rodent obesity models must be translated to humans. It will be important to further elucidate the underlying mechanisms by which obesity reduces the levels of and the sensitivity to SPM in adipose tissues. This will enable the development of nutrition therapy to enhance the effects of omega-3 fatty acids in the prevention and/or treatment of T2DM.

Intravitreal Injection of Amyloid ß1-42 Activates the Complement System and Induces Retinal Inflammatory Responses and Malfunction in Mouse.

To investigate whether intravitreal injection of amyloid ß1-42 (Aß1-42) activates the complement system and induces retinal inflammatory responses and malfunction, Aß1-42 was applied intravitreally in mice. The expressions of key components of complement system were determined by real-time PCR. Retinal function was assessed by electroretinography. We found interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in Aß1-42 treated mice retinas increased from day 1 to day 7. Compared with control group, mRNA expression of C1qa and C3 in the Aß1-42 treated retinas increased at days 1 and 7. The level of CFB, CFD, or CFH increased at day 4 and day 7. Regulator of membrane attack complex (MAC), CD59a, increased from day 1 to day 7. The expression of the main complement components in Aß1-42 treated eyes increased at days 4 and 7. Therefore, our results suggested that exogenous Aß1-42 activated CP and AP of the complement system in mice retinas, induced retinal inflammatory responses, and caused retinal malfunction.

Pancreatic cancer-educated macrophages protect cancer cells from complement-dependent cytotoxicity by up-regulation of CD59.

Tumor-associated macrophages (TAMs) are versatile immune cells that promote a variety of malignant behaviors of pancreatic cancer. CD59 is a GPI-anchored membrane protein that prevents complement activation by inhibiting the formation of the membrane attack complex, which may protect cancer cells from complement-dependent cytotoxicity (CDC). The interactions between CD59, TAMs and pancreatic cancer remain largely unknown. A tissue microarray of pancreatic cancer patients was used to evaluate the interrelationship of CD59 and TAMs and their survival impacts were analyzed. In a coculture system, THP-1 cells were used as a model to study the function of TAMs and the roles of pancreatic cancer-educated macrophages in regulating the expression of CD59 in pancreatic cancer cells were demonstrated by real-time PCR, western blot and immunofluorescence staining. The effects of macrophages on regulating CDC in pancreatic cancer cells were demonstrated by an in vitro study. To explore the potential mechanisms, RNA sequencing of pancreatic cancer cells with or without co-culture of THP-1 macrophages was performed, and the results showed that the IL-6R/STAT3 signaling pathway might participate in the regulation, which was further demonstrated by target-siRNA transfection, antibody neutralization and STAT3 inhibitors. Our data revealed that the infiltration of TAMs and the expression of CD59 of pancreatic cancer were paralleled, and higher infiltration of TAMs and higher expression of CD59 predicted worse survival of pancreatic cancer patients. Pancreatic cancer-educated macrophages could protect cancer cells from CDC by up-regulating CD59 via the IL-6R/STAT3 signaling pathway. These findings uncovered the novel mechanisms between TAMs and CD59, and contribute to providing a new promising target for the immunotherapy of pancreatic cancer.

Immunophenotyping of Paroxysmal Nocturnal Hemoglobinuria (PNH).

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare but often debilitating disease which may lead to death in up to 35% of patients within 5 years if unrecognized and untreated. Detection of PNH and assessment of PNH clone size in RBC and WBC lineages by flow cytometric analysis has increased in importance due to the availability of novel therapies. These therapies typically block the hemolysis of red blood cells and thus significantly lower the morbidities and mortality associated with this disease. This chapter describes validated, state-of-the-art, high-sensitivity flow cytometric methodologies based on latest published testing guidelines for PNH.

Altered Expression of Complement Regulatory Proteins CD35, CD46, CD55, and CD59 on Leukocyte Subsets in Individuals Suffering From Coronary Artery Disease.

Studies conducted in animal models have suggested that membrane complement regulatory proteins play an important role in the pathophysiology of coronary artery disease (CAD). In this study, a total of 100 individuals, with stable CAD and 100 healthy controls, both groups predominantly male, were recruited. We evaluated the plasma levels of complement regulatory proteins (Cregs) CD35, CD46, CD55, and CD59 and their surface expression on granulocytes, lymphocytes, and monocytes by flow cytometry. The mRNA expression of these Cregs in total leukocytes was determined by quantitative PCR. The soluble forms of Cregs, C3c, Mannose binding protein-associated serine protease 2 (MASP-2), Platelet activating factor-acetyl hydrolase (PAF-AH), and inflammatory cytokines were quantified by ELISA. High plasma levels of C3c, indicative of complement activation, in addition to significantly low levels of Cregs, were observed in CAD patients. A significantly lower expression of CD46 and CD55 on the surface of lymphocytes, monocytes, and granulocytes and higher surface expression of CD35 and CD59 on granulocytes (p < 0.0001) was seen in CAD patients as compared to healthy donors. The high expression of CD59 on granulocytes positively correlated with the severity of disease and may serve as a potential marker of disease progression in CAD.

Combination treatment with lipoteichoic acids isolated from Lactobacillus plantarum and Staphylococcus aureus alleviates atopic dermatitis via upregulation of CD55 and CD59.

The innate immune complement system helps clear invading pathogens by forming membrane attack complexes (MACs) on their surface. Abnormal activation of the complement system may aggravate atopic dermatitis (AD) symptoms in AD patients. Here, we investigated the anti-AD effects of LTAs isolated from Lactobacillus plantarum (pLTA) and Staphylococcus aureus (aLTA) by examination of complement regulatory proteins (CRPs). Combination treatment with pLTA and aLTA increased CD55 and CD59 production in HaCaT cells. The regulation of CD55 and CD59 was mediated by p38 mitogen-activated protein kinase (p38) signaling pathways in pLTA- and aLTA-treated cells. Complement-dependent cytotoxicity (CDC) and bactericidal assays revealed that combination treatment with pLTA and aLTA down-regulated the complement system. In experiments using an irritant contact dermatitis (ICD)-induced mouse model, the levels of MAC and C3 convertase (C3C) were lower in serum collected from pLTA- and aLTA-injected mice than in serum from mice who were untreated or received pLTA or aLTA alone. Combination treatment also inhibited IgE and CCL2 levels in ICD mice. On the other hand, IFN-γ level was significantly increased, indicating that combination treatment switches the Th2 response to a Th1 response. Our results suggest that combination treatment with LTAs could be a good therapeutic approach to alleviate AD by reducing formation of MACs and inducing a Th1 response.

Complement deficiencies and dysregulation: Pathophysiological consequences, modern analysis, and clinical management.

Complement defects are associated with an enhanced risk of a broad spectrum of infectious as well as systemic or local inflammatory and thrombotic disorders. Inherited complement deficiencies have been described for virtually all complement components but can be mimicked by autoantibodies, interfering with the activity of specific complement components, convertases or regulators. While being rare, diseases related to complement deficiencies are often severe with a frequent but not exclusive manifestation during childhood. Whereas defects of early components of the classical pathway significantly increase the risk of autoimmune disorders, lack of components of the terminal pathway as well as of properdin are associated with an enhanced susceptibility to meningococcal infections. The impaired synthesis or function of C1 inhibitor results in the development of hereditary angioedema (HAE). Furthermore, complement dysregulation causes renal disorders such as atypical hemolytic uremic syndrome (aHUS) or C3 glomerulopathy (C3G) but also age-related macular degeneration (AMD). While paroxysmal nocturnal hemoglobinuria (PNH) results from the combined deficiency of the regulatory complement proteins CD55 and CD59, which is caused by somatic mutation of a common membrane anchor, isolated CD55 or CD59 deficiency is associated with the CHAPLE syndrome and polyneuropathy, respectively. Here, we provide an overview on clinical disorders related to complement deficiencies or dysregulation and describe diagnostic strategies required for their comprehensive molecular characterization - a prerequisite for informed decisions on the therapeutic management of these disorders.

Staphylococcal phosphatidylinositol-specific phospholipase C potentiates lung injury via complement sensitisation.

Staphylococcus aureus is frequently isolated from patients with community-acquired pneumonia and acute respiratory distress syndrome (ARDS). ARDS is associated with staphylococcal phosphatidylinositol-specific phospholipase C (PI-PLC); however, the role of PI-PLC in the pathogenesis and progression of ARDS remains unknown. Here, we showed that recombinant staphylococcal PI-PLC possesses enzyme activity that causes shedding of glycosylphosphatidylinositol-anchored CD55 and CD59 from human umbilical vein endothelial cell surfaces and triggers cell lysis via complement activity. Intranasal infection with PI-PLC-positive S. aureus resulted in greater neutrophil infiltration and increased pulmonary oedema compared with a plc-isogenic mutant. Although indistinguishable proinflammatory genes were induced, the wild-type strain activated higher levels of C5a in lung tissue accompanied by elevated albumin instillation and increased lactate dehydrogenase release in bronchoalveolar lavage fluid compared with the plc- mutant. Following treatment with cobra venom factor to deplete complement, the wild-type strain with PI-PLC showed a reduced ability to trigger pulmonary permeability and tissue damage. PI-PLC-positive S. aureus induced the formation of membrane attack complex, mainly on type II pneumocytes, and reduced the level of CD55/CD59, indicating the importance of complement regulation in pulmonary injury. In conclusion, S. aureus PI-PLC sensitised tissue to complement activation leading to more severe tissue damage, increased pulmonary oedema, and ARDS progression.

Characterization of a CD59 in orange-spotted grouper (Epinephelus coioides).

CD59, a multifunctional glycoprotein, not only plays a regulatory role in complement cascades, but also participates in modulation of teleostean immunity. In this study, full length sequence of EcCD59 was obtained, comprising a 5'UTR of 163 bp, an ORF of 354 bp and a 3'UTR of 559 bp. EcCD59 gene encoded a polypeptide of 117 amino acids. Tissue-specific analysis revealed that the highest expression of EcCD59 mRNA was observed in muscle. Vibrio alginolyticus challenge can significantly increase EcCD59 mRNA expression in liver, kidney and spleen. EcCD59 distribution was detected by a combined approach using GFP-overexpression, immunofluorescence and ELISA assay, indicating that EcCD59 may be predominantly aggregated in cellular membrane. Both EcCD59 and EcCD59delGPI can directly bind to V. alginolyticus and decrease the in vitro growth of V. alginolyticus. Additionally, vibrio injection experiment indicated that the binding of EcCD59 or EcCD59delGPI to V. alginolyticus can restrict its growth rate in vivo. In this study, we found that EcCD59 may be involved in immune defense against vibrio infection in a complement-independent manner.

Analysis of mutation in the rat Pig-a assay: I) studies with bone marrow erythroid cells.

We have established a flow cytometry-based Pig-a assay for rat bone marrow erythroid cells (BMEs). The BME Pig-a assay uses a DNA-specific stain and two antibodies: one against the transmembrane transferrin receptor (CD71 marker) and the other against the GPI-anchored complement inhibitory protein (CD59 marker). In F344 male rats treated acutely with a total of 120 mg/kg of N-ethyl-N-nitrosourea (ENU) the frequency of CD59-deficient phenotypically mutant BMEs increased approximately 24-fold compared to the rats concurrently treated with the vehicle. Such an increase of mutant BMEs coincides with increases of CD59-deficient reticulocytes measured in rats treated with similar doses of ENU. Sequence analysis of the endogenous X-linked Pig-a gene of CD59-deficient BMEs revealed that they are Pig-a mutants. The spectrum of ENU-induced Pig-a mutations in these BMEs was consistent with the in vivo mutagenic signature of ENU: 73% of mutations occurred at A:T basepairs, with the mutated T on the nontranscribed strand of the gene. T→A transversion was the most frequent mutation followed by T→C transition; no deletion or insertion mutations were present in the spectrum. Since BMEs are precursors of peripheral red blood cells, our findings suggest that CD59-deficient erythrocytes measured in the flow cytometric erythrocyte Pig-a assay develop from BMEs containing mutations in the Pig-a gene. Thus, the erythrocyte Pig-a assay detects mutation in the Pig-a gene. Environ. Mol. Mutagen. 59:722-732, 2018. © 2018 Wiley Periodicals, Inc.

Cell-Derived Viral Genes Evolve under Stronger Purifying Selection in Rhadinoviruses.

Like many other large double-stranded DNA (dsDNA) viruses, herpesviruses are known to capture host genes to evade host defenses. Little is known about the detailed natural history of such genes, nor do we fully understand their evolutionary dynamics. A major obstacle is that they are often highly divergent, maintaining very low sequence similarity to host homologs. Here we use the herpesvirus genus Rhadinovirus as a model system to develop an analytical approach that combines complementary evolutionary and bioinformatic techniques, offering results that are both detailed and robust for a range of genes. Using a systematic phylogenetic strategy, we identify the original host lineage of viral genes with high confidence. We show that although host immunomodulatory genes evolve rapidly compared to other host genes, they undergo a clear increase in purifying selection once captured by a virus. To characterize this shift in detail, we developed a novel technique to identify changes in selection pressure that can be attributable to particular domains. These findings will inform us on how viruses develop strategies to evade the immune system, and our synthesis of techniques can be reapplied to other viruses or biological systems with similar analytical challenges.IMPORTANCE Viruses and hosts have been shown to capture genes from one another as part of the evolutionary arms race. Such genes offer a natural experiment on the effects of evolutionary pressure, since the same gene exists in vastly different selective environments. However, sequences of viral homologs often bear little similarity to the original sequence, complicating the reconstruction of their shared evolutionary history with host counterparts. In this study, we use a genus of herpesviruses as a model system to comprehensively investigate the evolution of host-derived viral genes, using a synthesis of genomics, phylogenetics, selection analysis, and nucleotide and amino acid modeling.

Proinflammatory CX3CR1+CD59+Tumor Necrosis Factor-Like Molecule 1A+Interleukin-23+ Monocytes Are Expanded in Patients With Ankylosing Spondylitis and Modulate Innate Lymphoid Cell 3 Immune Functions.

OBJECTIVE: Gut-derived innate lymphoid cell 3 (ILC3) has been shown to participate in the pathogenesis of ankylosing spondylitis (AS). CX3 CR1+ mononuclear phagocytes (MNPs) have been demonstrated to modulate ILC3 function in the gut. This study was undertaken to investigate the role of proinflammatory CX3 CR1+CD59+ MNPs in modulating ILC3 function in AS patients. METHODS: MNP subsets in the blood of AS patients and controls were analyzed by flow cytometry. The presence of CX3 CR1+CD59+ cells in tissue was confirmed by confocal microscopy. Expression of the proinflammatory chemokines CX3 CL1 and CCL2 and decoy receptor 6 (DcR-6) was analyzed. Peripheral CX3 CR1+CD59+ cells were cocultured with ILC3, and changes in their frequency were evaluated by flow cytometry. Transcriptome analysis of circulating CX3 CR1+ monocytes was also performed. RESULTS: DcR-6 deficiency and CCL2 overexpression were observed in inflamed tissues from AS patients. In the gut, the proinflammatory CX3 CR1+CD59+ MNP population was expanded, correlated with the presence of bacteria, and produced high levels of tumor necrosis factor-like molecule 1A (TL1A) and interleukin-23 (IL-23). MNPs positive for CD11b, CD11c, and major histocompatibility complex class II, predominantly expressing CX3 CR1, were also expanded in the small intestines of treatment-naive SKG relative to BALB/c mice. The frequency of gut-derived CX3 CR1+CD59+CCR9+TL1A+IL-23+ MNPs was significantly higher in the peripheral blood and synovial fluid of AS patients than controls. CCR9+CX3 CR1+CD59+ monocytes were also expanded in AS synovial and bone marrow samples. Transcriptome analysis of isolated CX3 CR1+CD59+ monocytes demonstrated a specific proinflammatory profile in AS. Isolated proinflammatory CX3 CR1+CD59+ MNPs from AS patients induced the expansion and activation of ILC3. CONCLUSION: Proinflammatory CX3 CR1+CD59+TL1A+IL-23+ MNPs are expanded in AS patients and display a specific proinflammatory transcriptome profile. Given the ability of these cells to support ILC3 expansion, they may promote a sustained proinflammatory status in AS.

Generation of GTKO Diannan Miniature Pig Expressing Human Complementary Regulator Proteins hCD55 and hCD59 via T2A Peptide-Based Bicistronic Vectors and SCNT.

Pig-to-human organ transplantation has drawn attention in recent years due to the potential use of pigs as an alternative source of human donor organs. While GGTA1 knockout (GTKO) can protect xenografts from hyperacute rejection, complement-dependent cytotoxicity might still contribute to this type of rejection. To prolong the xenograft survival, we utilized a T2A-mediated pCMV-hCD55-T2A-hCD59-Neo vector and transfected the plasmid into GTKO Diannan miniature pig fetal fibroblasts. After G418 selection combined with single-cell cloning culture, four colonies were obtained, and three of these were successfully transfected with the hCD55 and hCD59. One of the three colonies was selected as donor cells for somatic cell nuclear transfer (SCNT). Then, the reconstructed embryos were transferred into eight recipient gilts, resulting in four pregnancies. Three of the pregnant gilts delivered, yielding six piglets. Only one piglet carried hCD55 and hCD59 genetic modification. The expression levels of the GGTA1, hCD55, and hCD59 in the tissues and fibroblasts of the piglet were determined by q-PCR, fluorescence microscopy, immunohistochemical staining, and western blotting analyses. The results showed the absence of GGTA1 and the coexpression of the hCD55 and hCD59. However, the mRNA expression levels of hCD55 and hCD59 in the GTKO/hCD55/hCD59 pig fibroblasts were lower than that in human 293T cells, which may be caused by low copy number and/or CMV promoter methylation. Furthermore, we performed human complement-mediated cytolysis assays using human serum solutions from 0 to 60%. The result showed that the fibroblasts of this triple-gene modified piglet had greater survival rates than that of wild-type and GTKO controls. Taken together, these results indicate that T2A-mediated polycistronic vector system combined with SCNT can effectively generate multiplex genetically modified pigs, additional hCD55 and hCD59 expression on top of a GTKO genetic background markedly enhance the protective effect towards human serum-mediated cytolysis than those of GTKO alone. Thus, we suggest that GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pig will be a more eligible donor for xenotransplantation.

CD59: a promising target for tumor immunotherapy.

CD59 has been identified as a glycosylphosphatidylinositol-anchored membrane protein that acts as an inhibitor of the formation of the membrane attack complex to regulate complement activation. Recent studies have shown that CD59 is highly expressed in several cancer cell lines and tumor tissues. CD59 also regulates the function, infiltration and phenotypes of a variety of immune cells in the tumor microenvironment. Herein, we summarized recent advances related to the functions and mechanisms of CD59 in the tumor microenvironment. Therapeutic strategies that seek to modulate the functions of CD59 in the tumor microenvironment could be a promising direction for tumor immunotherapy.

Structural Analysis of CD59 of Chinese Tree Shrew: A New Reference Molecule for Human Immune System Specific CD59 Drug Discovery.

BACKGROUND: Chinese tree shrews (Tupaia belangeri chinensis) bear several characteristics that are considered to be very crucial for utilizing in animal experimental models in biomedical research. Subsequent to the identification of key aspects and signaling pathways in nervous and immune systems, it is revealed that tree shrews acquire common as well as unique characteristics, and hence offer a genetic basis for employing them as a prospective model for biomedical research. CD59 glycoprotein, commonly referred to as MAC-inhibitory protein (MAC-IP), membrane inhibitor of reactive lysis (MIRL), or protectin, is encoded by the CD59 gene in human beings. It is the member of the LY6/uPAR/alpha-neurotoxin protein family. OBJECTIVES: With this initial point, the objective of this study was to determine a comparative composite based structure of CD59 of Chinese tree shrew. The additional objective of this study was to examine the distribution of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, hydrophobicity molecular surface analysis and electrostatic potential analysis with the assistance of several bioinformatical analytical tools. METHODS: CD59 Amino acid sequence of Chinese tree shrew was collected from the online database system of National Centre for Biotechnology Information. SignalP 4.0 online server was employed for detection of signal peptide instance within the protein sequence of CD59. Molecular model structure of CD59 protein was generated by the Iterative Threading ASSEmbly Refinement (I-TASSER) suite. The confirmation for three-dimensional structural model was evaluated by structure validation tools. Location of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, and hydrophobicity molecular surface analysis was performed with the help of Chimera tool. Electrostatic potential analysis was carried out with the adaptive Poisson-Boltzmann solver package. Subsequently validated model was used for the functionally critical amino acids and active site prediction. The functionally critical amino acids and ligand- binding site (LBS) of the proteins (modeled) were determined using the COACH program. RESULT: Analysis of Ramachandran plot for Chinese tree shrew depicted that overall, 100% of the residues in homology model were observed in allowed and favored regions, sequentially leading to the validation of the standard of generated protein structural model. In case of CD59 of Chinese tree shrew, the total score of G-factor was found to be -0.66 that was generally larger than the acceptable value. This approach suggests the significance and acceptability of the modeled structure of CD59 of Chinese tree shrew. The molecular model data in cooperation to other relevant post model analysis data put forward molecular insight into protecting activity of CD59 protein molecule of Chinese tree shrew. CONCLUSION: In the present study, we have proposed the first molecular model structure of uncharted CD59 of Chinese tree shrew by significantly utilizing the comparative composite modeling approach. Therefore, the development of a structural model of the CD59 protein was carried out and analyzed further for deducing molecular enrichment technique. The collaborative effort of molecular model and other relevant data of post model analysis carry forward molecular understanding to protecting activity of CD59 functions towards better insight of features of this natural lead compound.

Eculizumab treatment: stochastic occurrence of C3 binding to individual PNH erythrocytes.

BACKGROUND: C5 blockade by eculizumab prevents complement-mediated intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH). However, C3-bound PNH red blood cells (RBCs), arising in almost all treated patients, may undergo extravascular hemolysis reducing clinical benefits. Despite the uniform deficiency of CD55 and of CD59, there are always two distinct populations of PNH RBCs, with (C3+) and without (C3-) C3 binding. METHODS: To investigate this paradox, the phenomenon has been modeled in vitro by incubating RBCs from eculizumab untreated PNH patients with compatible sera containing eculizumab, and by assessing the C3 binding after activation of complement alternative pathway. RESULTS: When RBCs from untreated patients were exposed in vitro to activated complement in the context of C5-blockade, there was the prompt appearance of a distinct C3+ PNH RBC population whose size increased with time and also with the rate of complement activation. Eventually, all PNH RBCs become C3+ to the same extent, without differences between old and young (reticulocytes) PNH RBCs. CONCLUSIONS: This study indicates that the distinct (C3+ and C3-) PNH RBC populations are not intrinsically different; rather, they result from a stochastic all-or-nothing phenomenon linked to the time-dependent cumulative probability of each individual PNH red cell to be exposed to levels of complement activation able to trigger C3 binding. These findings may envision novel approaches to reduce C3 opsonization and the subsequent extravascular hemolysis in PNH patients on eculizumab.

Carboxymethyl chitosan nanoparticles coupled with CD59-specific ligand peptide for targeted delivery of C-phycocyanin to HeLa cells.

The combination of nanotechnology and medicine will be the next generation of vehicles for targeted drug delivery. Carboxymethyl chitosan loaded with the anticancer drug C-phycocyanin and the CD59-specific ligand peptide for cancer cell targeting were used to create C-phycocyanin/carboxymethyl chitosan-CD59-specific ligand peptide nanoparticles using the ionic-gelation method. Optimal synthesis conditions, selected by response surface methodology, comprised the ratio carboxymethyl chitosan:C-phycocyanin = 3:1, and carboxymethyl chitosan and CaCl2 concentrations of 2.0 and 1.0 mg/mL, respectively. The resulting nanoparticles were spherical, with diameters of approximately 200 nm; the entrapment efficient was about 65%; and the drug loading was about 20%. The release of C-phycocyanin from C-phycocyanin/carboxymethyl chitosan nanoparticles was pH sensitive and had a sustainable effect in vitro. Guided by the CD59-specific ligand peptide, the nanoparticles efficiently targeted the surface of HeLa cells and had an obvious inhibitory effect on HeLa cell proliferation as determined by methyl thiazolyl tetrazolium assays. The nanoparticles were hemocompatible and induced apoptosis by upregulation of cleaved caspase-3 and cleaved polyADP-ribose polymerase proteins, and downregulation of Bcl-2 proteins. Our study provides a novel approach to the research and development of marine drugs, and support for targeted therapy using anticancer drugs.