Complement dependent cytotoxicity activity of therapeutic antibody fragments is acquired by immunogenic glycan coupling

Oligosaccharides are implicated in the development of the immune response notably in complement activation. Anti-tumoural immunotherapy by monoclonal antibodies (mAbs) offers some advantages to chemotherapy including cell targeting but some of them are inefficient to generate cytotoxicity dependent complement (CDC) known to be important in the antibody’s efficacy. The aim of this study is to give a CDC activity of mAb by linkage of a complement activating oligosaccharide to this antibody via a hetero-bifunctional linker allowing control of the conjugation reaction. We worked on non Hodgkin Burkitt’s lymphoma as cancer source, Fab fragments of rituximab devoid of complement activity as mAb and the trisaccharide Gal alpha(1→3)Gal beta(1→4)GlcNAc as immunogenic glycan. The bioconjugate Fab-Gal was characterized by biochemical methods and we demonstrated that the α-Gal epitope was recognized by seric immunoglobulins. After checking the recognition capacity of the Fab-Gal conjugate for the CD20 epitope, in vitro assays were performed to evaluate the activation of the complement cascade by the Fab-Gal conjugate. The effect of this bioconjugate was confirmed by the evaluation of the proliferation response of Burkitt’s cell line. The relative facility realization of this strategy represents new approaches to increase activities of mAbs.

Biología comparada del sistema de complemento en peces / Comparative biology of the complement system in fish / Biologia comparativa do sistema complemento em peixes

Dentro de la respuesta inmune humoral se encuentran componentes que mantienen la homeostasis de los organismos a través del control de agentes patógenos por medio de la opsonización, quimiotaxis de células fagocíticas facilitando el proceso de eliminación de lo extraño o sin su acompañamiento, en el caso de la formación de poros en la membrana celular. A un grupo de este conjunto de componentes de origen molecular proteico se denominósistema del complemento, el cual posee tres vías de activación (Clásica, Alternativa y Lectinas), funciona comoanafilatoxinas, reguladores y receptores. La presente revisión tiene como objetivo discutir acerca de los diferentes componentes del sistema del complemento en la escala animal enfocándose principalmente en peces teleósteos y mamíferos, como organismos modelos en busca de elucidar sus diferencias, homologías y respuestas.

Within the humoral immune response can be found components that maintain an organism’s homeostasis viacontrol of pathogenic agents using opsonization, chemotaxis of phagocytic cells which facilitates the processof elimination of foreign bodies, or in its absence, the formation of pores in the cellular membrane. One of these groups of components, of protein origin, is referred to as the complement system, which has 3 means of activation (Classic, Alternative, and Lectins) and functions as anaphylactic toxins, regulators and receptors. The aim of this review is to discuss the different components of the complement system in the animal kingdom, focusing principally on teleost fish and mammals, as model organisms in the search to elucidate their differences, homologies, and answers.

Dentro da resposta imune humoral encontram-se componentes que mantém a homeostase do organismo através docontrole de patógenos, por opsonização, quimiotaxia de células fagocíticas que facilita o processo de eliminaçãode corpos estranhos, ou na sua ausência, a formação de poros na membrana celular. Este conjunto de componentes moleculares de origem protéica são chamados de sistema complemento, que tem três vias de ativação (clássica, alternativa e lectinas), funciona como anafilatoxinas, reguladores e receptores. Esta revisão tem como objetivo discutir os vários componentes do sistema complemento na escala animal focando principalmente em peixes teleósteos e mamíferos como organismos modelos na busca de elucidar suas diferenças, homologias e respostas.

Complement-dependent lymphocytotoxicity crossmatch in deceased donor renal transplant: A single institutional experience.

Complement-dependent lymphocytotoxicity crossmatches (n=217) between 47 deceased donors and 150 potential renal recipients were retrospectively studied. A negative cross match was reported in 48 (22.1%), doubtful positive in 126 (58.1%), weakly positive in 32 (14.7%) and positive in 11 (5.1%). No autoantibodies were detected. Renal transplantation was performed in 35.5% of the potential recipients. There was no incidence of hyperacute rejection. The graft survival rate was 88% at 15 months of follow up. The study concludes that a negative pretransplant lympocytotoxicity crossmatch using the basic National Institute of Health technique eliminates hyperacute rejection, but carries drawbacks, which require modification and supplementation with more sensitive and specific assays.

Complex genetic mechanisms in glaucoma: An overview.

Glaucomas comprise a group of hereditary optic neuropathies characterized by progressive and irreversible visual field loss and damage to the optic nerve head. It is a complex disease with multiple molecular mechanisms underlying its pathogenesis. Genetic heterogeneity is the hallmark of all glaucomas and multiple chromosomal loci have been linked to the disease, but only a few genes have been characterized, viz. myocilin (MYOC), optineurin (OPTN), WDR36 and neurotrophin-4 (NTF4) in primary open angle glaucoma (POAG) and CYP1B1 and LTBP2 in congenital and developmental glaucomas. Case-control-based association studies on candidate genes involved in different stages of glaucoma pathophysiology have indicated a very limited involvement. The complex mechanisms leading to glaucoma pathogenesis indicate that it could be attributed to multiple genes with varying magnitudes of effect. In this review, we provide an appraisal of the various efforts in unraveling the molecular mystery in glaucoma and also some future directions based on the available scientific knowledge and technological developments.

Sistema imunitário: Parte I. Fundamentos da imunidade inata com ênfase nos mecanismos moleculares e celulares da resposta inflamatória / Immune system: Part I. Fundamentals of innate immunity with emphasis on molecular and cellular mechanisms of inflammatory response

O sistema imunológico é constituído por uma intrincada rede de órgãos, células e moléculas, e tem por finalidade manter a homeostase do organismo, combatendo as agressões em geral. A imunidade inata atua em conjunto com a imunidade adaptativa e caracteriza-se pela rápida resposta à agressão, independentemente de estímulo prévio, sendo a primeira linha de defesa do organismo. Seus mecanismos compreendem barreiras físicas, químicas e biológicas, componentes celulares e moléculas solúveis. A primeira defesa do organismo frente a um dano tecidual envolve diversas etapas intimamente integradas e constituídas pelos diferentes componentes desse sistema. A presente revisão tem como objetivo resgatar os fundamentos dessa resposta, que apresenta elevada complexidade e é constituída por diversos componentes articulados que convergem para a elaboração da resposta imune adaptativa. Destacamos algumas etapas: reconhecimento molecular dos agentes agressores; ativação de vias bioquímicas intracelulares que resultam em modificações vasculares e teciduais; produção de uma miríade de mediadores com efeitos locais e sistêmicos no âmbito da ativação e proliferação celulares, síntese de novos produtos envolvidos na quimioatração e migração de células especializadas na destruição e remoção do agente agressor, e finalmente a recuperação tecidual com o restabelecimento funcional do tecido ou órgão.

The immune system consists of an intricate network of organs, cells, and molecules responsible for maintaining the body's homeostasis and responding to aggression in general. Innate immunity operates in conjunction with adaptive immunity and is characterized by rapid response to aggression, regardless of previous stimulus, being the organism first line of defense. Its mechanisms include physical, chemical and biological barriers, cellular components, as well as soluble molecules. The organism first line of defense against tissue damage involves several steps closely integrated and constituted by different components of this system. The aim of this review is to restore the foundations of this response, which has high complexity and consists of several components that converge to articulate the development of adaptive immune response. We selected some of the following steps to review: perception and molecular recognition of aggressive agents; activation of intracellular pathways, which result in vascular and tissue changes; production of a myriad of mediators with local and systemic effects on cell activation and proliferation, synthesis of new products involved in the chemoattraction and migration of cells specialized in destruction and removal of offending agent; and finally, tissue recovery with restoration of functional tissue or organ.

Mechanism of Humoral and Cellular Immune Modulation Provided by Porcine Sertoli Cells

The understanding of main mechanisms that determine the ability of immune privilege related to Sertoli cells (SCs) will provide clues for promoting a local tolerogenic environment. In this study, we evaluated the property of humoral and cellular immune response modulation provided by porcine SCs. Porcine SCs were resistant to human antibody and complement-mediated formation of the membrane attack complex (38.41+/-2.77% vs. 55.02+/-5.44%, p=0.027) and cell lysis (42.95+/-1.75% vs. 87.99 +/-2.25%, p<0.001) compared to immortalized aortic endothelial cells, suggesting that porcine SCs are able to escape cellular lysis associated with complement activation by producing one or more immunoprotective factors that may be capable of inhibiting membrane attack complex formation. On the other hand, porcine SCs and their culture supernatant suppressed the up-regulation of CD40 expression (p<0.05) on DCs in the presence of LPS stimulation. These novel findings, as we know, suggest that immune modulatory effects of porcine SCs in the presence of other antigen can be obtained from the first step of antigen presentation. These might open optimistic perspectives for the use of porcine SCs in tolerance induction eliminating the need for chronic immunosuppressive drugs.

Viral complement regulators: the expert mimicking swindlers.

The complement system is a principal bastion of innate immunity designed to combat a myriad of existing as well as newly emerging pathogens. Since viruses are obligatory intracellular parasites, they are continuously exposed to host complement assault and, therefore, have imbibed various strategies to subvert it. One of them is molecular mimicry of the host complement regulators. Large DNA viruses such as pox and herpesviruses encode proteins that are structurally and functionally similar to human regulators of complement activation (RCA), a family of proteins that regulate complement. In this review, we have presented the structural and functional aspects of virally encoded RCA homologs (vRCA), in particular two highly studied vRCAs, vaccinia virus complement control protein (VCP) and Kaposi's sarcoma-associated herpesvirus complement regulator (kaposica). Importance of these evasion molecules in viral pathogenesis and their role beyond complement regulation are also discussed.

The use of MTT [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2H- tetrazolium bromide]-reduction as an indicator of the effects of strain-specific, polyclonal rabbit antisera on Candida albicans and C. krusei.

There is only scanty data on the effects of specific antibody, with or without complement, on Candida albicans or Candida krusei in cell-free systems in vitro, although previously published work has shown that specific antibody mediates anti- Candida immunity in vivo by inhibition of adherence to host cells or surfaces and by the promotion of phagocytosis and intra-phagocytic killing. The MTT (3-[4, 5-dimethyl-2-thiazolyl] -2, 5-diphenyl -2H- tetrazolium bromide)-reduction method as a test of the viability of fungi was used to investigate the effect of complement, normal serum and immune serum on these two species of Candida that are of increasing importance as opportunistic pathogens. We report that normal rabbit serum or strain-specific, polyclonal anti- Candida rabbit antibody, with or without guinea pig complement, did not cause the reduction of total cell-mass or of the viability of either C. albicans or C. krusei, in vitro as determined by the MTT-reduction test. Complement alone without specific antibody, also, had no such effect on these two Candida species.

In vitro and in situ activation of the complement system by the fungus Lacazia loboi

Since there are no studies evaluating the participation of the complement system (CS) in Jorge Lobo's disease and its activity on the fungus Lacazia loboi, we carried out the present investigation. Fungal cells with a viability index of 48 percent were obtained from the footpads of BALB/c mice and incubated with a pool of inactivated serum from patients with the mycosis or with sterile saline for 30 min at 37 °C. Next, the tubes were incubated for 2 h with a pool of noninactivated AB+ serum, inactivated serum, serum diluted in EGTA-MgCl2, and serum diluted in EDTA. The viability of L. loboi was evaluated and the fungal suspension was cytocentrifuged. The slides were submitted to immunofluorescence staining using human anti-C3 antibody. The results revealed that 98 percent of the fungi activated the CS by the alternative pathway and no significant difference in L. loboi viability was observed after CS activation. In parallel, frozen histological sections from 11 patients were analyzed regarding the presence of C3 and IgG by immunofluorescence staining. C3 and IgG deposits were observed in the fungal wall of 100 percent and 91 percent of the lesions evaluated, respectively. The results suggest that the CS and immunoglobulins may contribute to the defense mechanisms of the host against L. loboi.

Considerando que não existe nenhum estudo avaliando a participação do sistema complemento (SC) na doença de Jorge Lobo e sua atividade sobre o fungo Lacazia loboi, realizamos o presente trabalho. Os fungos foram obtidos dos coxins plantares de camundongos BALB/c com índice de viabilidade de 48 por cento e, em seguida, foram incubados com pool de soro inativado de pacientes ou com solução salina estéril (SSE) por 30 min, a 37 °C. Os tubos foram incubados, por 2 h, com pool de soro AB+ sem inativar, inativado, diluído em EGTA-MgCl2 e EDTA. A viabilidade do L. loboi foi avaliada e a suspensão fúngica foi citocentrifugada. As lâminas foram submetidas à técnica de imunofluorescência empregando o anticorpo anti-C3 humano. Os resultados revelaram que 98 por cento dos fungos ativaram o SC pela via alternativa e que não houve diferença significante na viabilidade do L. loboi após ativação do SC. Em paralelo, cortes histológicos congelados de 11 pacientes foram avaliados quanto à presença de C3 e IgG, pela técnica de imunofluorescência. Foram encontrados depósitos de C3 e de IgG na parede dos fungos em 100 por cento e 91 por cento das lesões avaliadas, respectivamente. Os resultados sugerem que o SC e as imunoglobulinas poderiam contribuir nos mecanismos de defesa do hospedeiro contra o L. loboi.

Dysfunction of innate immunity and associated pathology in neonates.

The neutrophils and complement system are the critical elements of innate immunity mainly due to participation in the first line of defense against microorganisms by means of phagocytosis, lysis of bacteria and activation of naive B-lymphocytes. In this report we provide an overview of the up to date information regarding the neutrophil and complement system's functional ability in newborn infants in association with the maternal conditions that exist during the intrauterine stage, gestational age and post-neonatal pathology. The neonates' capacity to control the neutrophil and complement protein activation process has also been discussed because of the evidence that uncontrolled activation of these immune elements provides a significant contribution to the tissue damage and subsequent pathology. The authors are confident that despite the many unanswered questions this review updates their knowledge and points the need for further research to clarify the role of the age-associated dysfunction of neutrophils and complement system in the infection and inflammation related pathology of newborn infants.

Role of Complement Regulatory Proteins in the Survival of Murine Allo-transplanted Sertoli Cells

Sertoli cells (SC) are known to contain immunoprotective properties, which allow them to survive as allografts without the use of immunosuppressive drugs. Experiments were designed to determine which factors are related to prolonged survival of allogeneic SC. Balb/c derived Sertoli (TM4) and colon cancer (CT-26) cell lines were implanted beneath the kidney capsule of non-immunosuppressed C57BL/6 mice and compared their survival as allografts. Compared to TM4 graft, which survived more than 7 days after transplantation, CT-26 showed massive infiltration of polymorphonuclear cells, necrosis and enlargement of draining lymph nodes. Cultured cell lines showed no differences in their expression patterns of FasL, TGF beta1, clusterin and two complement regulatory proteins (CRP, i.e., membrane cofactor protein, MCP; decay accelerating factor, DAF), but protectin (CD59), another member of CRP was expressed only on TM4. These results suggest that CD59 and unknown factors may contribute to the prolonged survival of SC in non-immunoprivileged sites.

Complement-mediated tail degradation of Neodiplostomum seoulense cercariae

The furcocercus cercariae of Neodiplostomum seoulense (Digenea: Neodiplostomidae) penetrate the skins of tadpoles and shed their tails. The speculated mechanism of this tail loss was physical efforts required to produce a vigorous zigzag motion during skin penetration; no other mechanism has been proposed. We examined the relationship between the host serum and cercarial tail loss. Cercariae of N. seoulense were collected from experimentally infected Segmentina hemisphaerula, and lots of 300 cercariae were cultured in medium 199 contained several types of sera. Cercarial tail degradation was induced in all media, but all the cercariae cultured except those cultured in media containing fetal bovine serum (FBS) died within 48 hr. After 72 hr cultivation in media containing FBS, cercarial tail degradation was induced in 67.0%; in continuous cultivation 13.3% of larvae survived for 7 days. Tail degradation did not occur in the absence of serum and when serum was heat inactivated at 56 degrees C for 30 min. The addition of 20 mM ethylenediaminetetraacetic acid (EDTA) blocked cercarial tail degradation completely. Moreover, the addition of 20 mM MgCl2 restored tail degradation blocked by EDTA. These results suggest that the alternative complement pathway is related with the N. seoulense cercarial tail degradation induced by serum.

Viral mimicry of the complement system.

The complement system is a potent innate immune mechanism consisting of cascades of proteins which are designed to fight against and annul intrusion of all the foreign pathogens. Although viruses are smaller in size and have relatively simple structure, they are not immune to complement attack. Thus, activation of the complement system can lead to neutralization of cell-free viruses, phagocytosis of C3b-coated viral particles, lysis of virus-infected cells, and generation of inflammatory and specific immune responses. However, to combat host responses and succeed as pathogens, viruses not only have developed/adopted mechanisms to control complement, but also have turned these interactions to their own advantage. Important examples include poxviruses, herpesviruses, retroviruses, paramyxoviruses and picornaviruses. In this review, we provide information on the various complement evasion strategies that viruses have developed to thwart the complement attack of the host. A special emphasis is given on the interactions between the viral proteins that are involved in molecular mimicry and the complement system.

Los microbios y el hombre, ¿enemigos o amigos? / Microorganisms and man: enemies or friends?

Todos los microorganismos (bacterias, parásitos, hongos y virus) han constituido desde siempre un panel de agresores para el hombre, siendo responsables directos o indirectos de múltiples patologías de origen infeccioso que se desarrollan enfrentando a los mecanismos de la inmunidad. El presente artículo se divide en dos partes: la primera constituye una resumida actualización de los eventos inmunes y la segunda una discusión de la relación microbios-hombre en términos evolutivos replanteando desde esa óptica el clásico antagonismo. En ese sentido, un dinámico equilibrio entre ambos, puede ser alternativamente alterado por uno de los responsables con una consecuente compensación a cargo de la contraparte. Así, tomando como referencia el permanente cambio en las estrategias de evasión microbiana, puede especularse que los mecanismos inespecíficos junto a las barreras naturales pueden haber sido la defensa ancestral del hombre. Más adelante la inflamación inicial puede haber sido mejorada mediante la generación de moléculas activadoras del complemento. Posteriormente, las moléculas de histocompatibilidad y los receptores antigénicos de los linfocitos B y de los linfocitos T pueden haber sido generadas para montar los eventos específicos que hoy se conocen. La participación de células fundamentales y accesorias en forma conjunta ha hecho necesaria la expresión de moléculas de adhesión y la generación y ampliación del espectro de citoquinas y sus receptores. La producción de anticuerpos puede haber inducido el mejoramiento efector de sistemas moleculares como el complemento y así juntos, eficientizar la primitiva fagocitosis. La evolución del linaje T puede haber sido más compleja, habida cuenta que debieron generarse subpoblaciones o sets citotóxicos por un lado y orquestadores y reguladores por otro. Por otra parte la diversidad de reconocimiento que se hizo necesaria a nivel de los receptores específicos se desarrolló con un gasto mínimo de información genética, pero posibilitó la aparición de fenómenos no deseados con participación de efectores inmunes, lo que impuso la aparición de críticos mecanismos reguladores donde participan sinérgicamente todos los sistemas biológicos...

Efecto in vitro del suero de la trucha arco iris, oncorhynchus mykiss, sobre metacercarias del género tylodelphys trematoda displostomatidae / The in vitro effect of rainbow trout, oncorhynchus mykiss, serum in tylodelphys sp trematoda displostomatidae metacercariae

Here, it has been described that normal fresh sera from rainbow trout (oncorhynchus mykiss) were able to Iyse Tylodlphys sp. metacercariae isolated form Galaxias maculatus (Jenyns, 1842) (Teleostei, Galaxiidae) brain. This effect was time dependent, and progressively increased since 1 h up to 18 h incubation. There were oscillations in the ability of different sera samples to lyse these parasites. Similar values were achieved with other salmonid sera, and also with normal human sera pool. The lytic activity was abolished by heat-inactivated sera, by zymosan treatment and by incubation in the presence of EDTA. However, the mean values obtained in the presence of EGTA-Mg++ did not significantly differ from those found with sera as a source of complement. The results of this study suggested the role of complement alternative pathway on Tylodephys sp. in vitro lysis, under our experimental conditions

O sistema complemento e a resposta imunológica ao HIV / The complement system and the immune response to HIV

Objetivo: O objetivo do presente artigo é descrever os principais mecanismos pelos quais o sistema complemento está envolvido na infecçäo pelo HIV. Métodos: Através de um levantamento bibliográfico sobre o tema, os autores discutem os estudos realizados para avaliar os distúrbios do sistema complemento em pacientes HIV positivos, tanto na faixa etária pediátrica como no adulto, em diversos estagios. Resultados: Verifica-se que o sistema complemento se encontra ativado pelas vias clássica e alternativa, sem correlaçäo com o estagio da doença. A ativaçäo da via clássica ocorre através da ligaçäo de C1q e gp41/gp120 do vírus, permitindo a infecçäo de células que näo expressam CD4, contribuindo para disseminaçäo da doença. Portanto, os dados de literatura sugerem que o sistema complemento näo é eficaz no controle da infecçäo pela HIV

Complement-fixing abilities and IgG subclasses of autoantibodies in epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita (EBA) is an autoimmune-mediated subepidermal bullous disease in which the target of the autoantibodies is type VII collagen, a major component of anchoring fibrils. The purpose of this study was to evaluate the complement-fixing abilities and IgG subclass distribution of autoantibodies in EBA, and to also attempt to investigate the relation between inflammation, complement fixation and IgG subclass distribution in EBA patients. Only 2 sera of 18 patients (11%) showed weak complement-fixing abilities. IgG1 and IgG4 were the most frequently and intensely stained IgG subclasses in EBA sera. We could not find any relationship between the clinico-pathologic types, complement-fixing abilities and IgG subclasses in EBA. These results suggested that complement activation may not be a key factor of bulla formation in EBA.