Downregulation of miRNA-26a by HIV-1 Enhances CD59 Expression and Packaging, Impacting Virus Susceptibility to Antibody-Dependent Complement-Mediated Lysis.

MicroRNAs (miRNAs) play important roles in the control of HIV-1 infection. Here, we performed RNA-seq profiling of miRNAs and mRNAs expressed in CD4+ T lymphocytes upon HIV-1 infection. Our results reveal significant alterations in miRNA and mRNA expression profiles in infected relative to uninfected cells. One of the miRNAs markedly downregulated in infected cells is miRNA-26a. Among the putative targets of miRNA-26a are CD59 receptor transcripts, which are significantly upregulated in infected CD4+ T cells. The addition of miRNA-26a mimics to CD4+ T cells reduces CD59 at both the mRNA and surface protein levels, validating CD59 as a miRNA-26a target. Consistent with the reported inhibitory role of CD59 in complement-mediated lysis (CML), knocking out CD59 in CD4+ T cells renders both HIV-1-infected cells and progeny virions more prone to antibody-dependent CML (ADCML). The addition of miRNA-26a mimics to infected cells leads to enhanced sensitivity of progeny virions to ADCML, a condition linked to a reduction in CD59 packaging into released virions. Lastly, HIV-1-mediated downregulation of miRNA-26a expression is shown to be dependent on integrated HIV-1 expression but does not involve viral accessory proteins. Overall, these results highlight a novel mechanism by which HIV-1 limits ADCML by upregulating CD59 expression via miRNA-26a downmodulation.

CD59 Protects Primary Human Cerebrovascular Smooth Muscle Cells from Cytolytic Membrane Attack Complex.

Cerebral amyloid angiopathy is characterized by a weakening of the small- and medium-sized cerebral arteries, as their smooth muscle cells are progressively replaced with acellular amyloid ß, increasing vessel fragility and vulnerability to microhemorrhage. In this context, an aberrant overactivation of the complement system would further aggravate this process. The surface protein CD59 protects most cells from complement-induced cytotoxicity, but expression levels can fluctuate due to disease and varying cell types. The degree to which CD59 protects human cerebral vascular smooth muscle (HCSM) cells from complement-induced cytotoxicity has not yet been determined. To address this shortcoming, we selectively blocked the activity of HCSM-expressed CD59 with an antibody, and challenged the cells with complement, then measured cellular viability. Unblocked HCSM cells proved resistant to all tested concentrations of complement, and this resistance decreased progressively with increasing concentrations of anti-CD59 antibody. Complete CD59 blockage, however, did not result in a total loss of cellular viability, suggesting that additional factors may have some protective functions. Taken together, this implies that CD59 plays a predominant role in HCSM cellular protection against complement-induced cytotoxicity. The overexpression of CD59 could be an effective means of protecting these cells from excessive complement system activity, with consequent reductions in the incidence of microhemorrhage. The precise extent to which cellular repair mechanisms and other complement repair proteins contribute to this resistance has yet to be fully elucidated.

CD59 Protects Primary Human Cerebrovascular Smooth Muscle Cells from Cytolytic Membrane Attack Complex.

Cerebral amyloid angiopathy is characterized by a weakening of the small and medium sized cerebral arteries, as their smooth muscle cells are progressively replaced with acellular amyloid ß, increasing vessel fragility and vulnerability to microhemorrhage. In this context, an aberrant overactivation of the complement system would further aggravate this process. The surface protein CD59 protects most cells from complement-induced cytotoxicity, but expression levels can fluctuate due to disease and vary between cell types. The degree to which CD59 protects human cerebral vascular smooth muscle (HCSM) cells from complement-induced cytotoxicity has not yet been determined. To address this shortcoming, we selectively blocked the activity of HCSM-expressed CD59 with an antibody and challenged the cells with complement, then measured cellular viability. Unblocked HCSM cells proved resistant to all tested concentrations of complement, and this resistance decreased progressively with increasing concentrations of anti-CD59 antibody. Complete CD59 blockage, however, did not result in total loss of cellular viability, suggesting that additional factors may have some protective functions. Taken together, this implies that CD59 plays a predominant role in HCSM cellular protection against complement-induced cytotoxicity. Over-expression of CD59 could be an effective means of protecting these cells from excessive complement system activity, with consequent reduction in the incidence of microhemorrhage. The precise extent to which cellular repair mechanisms and other complement repair proteins contribute to this resistance has yet to be fully elucidated.

Voluntary wheel running prevents formation of membrane attack complexes and myelin degradation after peripheral nerve injury.

Regular aerobic activity is associated with a reduced risk of chronic pain in humans and rodents. Our previous studies in rodents have shown that prior voluntary wheel running can normalize redox signaling at the site of peripheral nerve injury, attenuating subsequent neuropathic pain. However, the full extent of neuroprotection offered by voluntary wheel running after peripheral nerve injury is unknown. Here, we show that six weeks of voluntary wheel running prior to chronic constriction injury (CCI) reduced the terminal complement membrane attack complex (MAC) at the sciatic nerve injury site. This was associated with increased expression of the MAC inhibitor CD59. The levels of upstream complement components (C3) and their inhibitors (CD55, CR1 and CFH) were altered by CCI, but not increased by voluntary wheel running. Since MAC can degrade myelin, which in turn contributes to neuropathic pain, we evaluated myelin integrity at the sciatic nerve injury site. We found that the loss of myelinated fibers and decreased myelin protein which occurs in sedentary rats following CCI was not observed in rats with prior running. Substitution of prior voluntary wheel running with exogenous CD59 also attenuated mechanical allodynia and reduced MAC deposition at the nerve injury site, pointing to CD59 as a critical effector of the neuroprotective and antinociceptive actions of prior voluntary wheel running. This study links attenuation of neuropathic pain by prior voluntary wheel running with inhibition of MAC and preservation of myelin integrity at the sciatic nerve injury site.

Estudo do SNP C1QA-A27GG e das proteínas reguladoras do complemento CD55 e CD59 como biomarcadores de resposta clínica do rituximabe no linfoma de grandes células B / [Study of SNP C1QA-A27GG and complement regulatory proteins CD55 and CD59 as biomarkers of clinical response to rituximab in lymphoma of large B cells]

A combinação do anti-CD20 rituximabe (R) e quimioterapia (CHOP-R) melhorou o desfecho dos pacientes com linfoma difuso de grandes células B (LDGCB), entretanto nem todos os pacientes respondem satisfatoriamente. Embora o mecanismo antitumoral do rituximabe in vivo não tenha sido claramente definido, acredita-se que uma das principais vias seja a citotoxicidade dependente de complemento (CDC). Um polimorfismo de nucleotídeo simples (SNP) no gene do componente do complemento Cq1 (C1QA-A276G) e a superexpressão das proteínas reguladoras do complemento (PRC) foram associados à resposta clínica de pacientes com linfomas B, entretanto estes biomarcadores não foram explorados em uma grande série de pacientes com LDGCB. Linhagens celulares derivadas de LNH-B foram empregadas para provar que a expressão de PRC contribui à inibição da CDC-R. Observamos também, que a resistência à CDC-R pode ser revertida por bloqueio funcional de CD55 e CD59 utilizando anticorpos neutralizantes. Uma coorte retrospectiva de 202 pacientes com LDGCB que receberam R-CHOP como primeira linha de tratamento, foi avaliada quanto: a expressão dos marcadores CD55 e CD59 por imunohistoquímica (IHQ) em material tumoral fixado e incluído em parafina, a distribuição dos genótipos C1QA-A276G determinados por PCR em tempo real e as características clínicas, resposta terapêutica e sobrevida. A taxa de sobrevida global (SG) e de sobrevida livre de progressão (SLP) da nossa coorte foi 69,8% e 66,3%, respectivamente...

The introduction of the anti-CD20 rituximab to the chemotherapy (CHOP) has significantly improved the treatment outcome of diffuse large B-Cell lymphoma (DLBCL) patients, however, not all patients respond satisfactorily. Even though the antitumor mechanism of rituximab in vivo is not clearly understood, it is thought that complement-dependent cytotoxicity (CDC-R) is one of the major pathways. A single-nucleotide polymorphism (SNP) in the component of complement C1q (C1QA-A276G) and overexpression of complement regulatory proteins (CRP) were associate to the clinical response of B-lymphomas patients, nevertheless, these markers have not been explored in a large series of patients with DLBCL. Cell lines from NHL-B were employed to prove that the expression of CRP contributes to inhibition of CDC-R. We also observed that resistance to CDC-R could be reversed by functional blockade of CD55 and CD59 using neutralizing antibodies. A retrospective cohort of 202 patients with DLBCL receiving R-CHOP as first-line treatment was evaluated for: the expression of CD55 and CD59 markers by immunohistochemistry (IHC) in fixed paraffin embedded tumor material, the distribution of genotypes C1QA-A276G determined by real time PCR and the clinical characteristics, response to therapy and survival. The rate of overall survival (OS) and progression-free survival (PFS) of our cohort was 69.8% and 66.3%, respectively. The application of IPI and IPI-R coefficients, successfully distinguish groups of patients with PFS (P <0.001) and OS (P <0.001)...

Frequency of paroxysmal nocturnal hemoglobinuria in patients attended in Belém, Pará, Brazil

BACKGROUND: Paroxysmal nocturnal hemoglobinuria is a hematological disease with complex physiopathology. It is genetically characterized by a somatic mutation in the PIG-A gene (phosphatidylinositol glycan anchor biosynthesis, class A), in which the best known antigens are DAF (decay accelerating factor or CD55) and MIRL (membrane inhibitor of reactive lysis or CD59). OBJECTIVE: To determine the frequency of paroxysmal nocturnal hemoglobinuria in patients attended at the HEMOPA foundation from November 2008 to July 2009. METHOD: Thirty patients, with ages ranging from two to 79 years old and suspected of having paroxysmal nocturnal hemoglobinuria were examined. All patients were immunophenotyped by flow cytometry for the CD5, CD59, CD16 and CD45 antigens. RESULTS: Paroxysmal nocturnal hemoglobinuria was identified in nine of the thirty patients investigated. Another 3 cases had inconclusive results with CD59-negative labeling only for neutrophils. The highest frequency of paroxysmal nocturnal hemoglobinuria patients (7/9) and inconclusive cases (2/3) were between 19 years old and 48 years old, with a median of 28 years. CONCLUSION: These results show the importance of flow cytometry to identify cases in which patients are deficient in only one antigen (CD59).

The role of CD55/CD59 complement regulatory proteins on peripheral blood cells of systemic lupus erythematosus patients / O papel das proteínas reguladoras do complemento CD55/CD59 em células de sangue periférico de pacientes com lúpus eritematoso sistêmico

CD55 e CD59 são proteínas de membrana ancoradas por glicosilfosfatidilinositol que apresentam propriedades reguladoras da ativação da cascata do complemento. Essa regulação ocorre através da inibição da C3 convertase pelo CD55 e prevenção da etapa final de polimerização do complexo de ataque à membrana pelo CD59. Deficiência na expressão dessas proteínas pode estar associada a uma maior ativação do sistema complemento, inclusive do complexo de ataque à membrana, levando à morte celular. Pacientes com lúpus eritematoso sistêmico, com anemia hemolítica e linfopenia, parecem apresentar uma deficiência adquirida de CD55 e CD59. Contudo, os mecanismos que modulam essa diminuída expressão continuam desconhecidos e o seu impacto nas manifestações do lúpus eritematoso sistêmico precisa ser mais bem estudado.

CD55 and CD59 are glycosylphosphatidylinositol-anchored proteins with regulatory properties on the activating cascades of the complement system. This regulation occurs through inhibition of the C3-convertase formation by CD55, and prevention of the terminal polymerization of the membrane attack complex by CD59. Deficiency in the expression of these proteins can be associated with increased susceptibility to complement-mediated cell death. Systemic lupus erythematosus patients with hemolytic anemia and lymphopenia seem to have an acquired deficiency of CD55 and CD59 proteins. However, the mechanisms involved in this deficiency and its impact on the clinical manifestation of SLE needs to be further investigated.