Complement-mediated Cell Lysis Research Articles

The anesthetic management of mitral valve replacement in the patient with paroxysmal nocturnal hemoglobinuria - A case report -

Paroxysmal nocturnal hemoglobinuria (PNH) is a disorder of stem cells resulting in complement-mediated red cell lysis. Potential complications of cardiac surgery in patients with PNH include increased risk of infection due to neutropenia, hemolytic crisis as a result of complement activation during cardiopulmonary bypass, and bleeding if thrombocytopenic. We report a case of anesthetic management for mitral valve replacement surgery in a patient with PNH. Preoperative management included the administration of granulocyte-stimulating factor, red cell transfusion, steroid, and prophylactic antibiotics and prevention of thrombosis. Intraoperative and early postoperative management included filtering leukocyte from homologous blood, avoidance of plasma transfusion and replacement of platelet to control bleeding. Although the postoperative course was not smooth because of delayed wound healing, the patient had recovered well.

Analysis of the serine protease function of porcine factor I produced by liver cells for xenotransplantation

The use of a bioartificial liver with pig liver cells in the treatment of fulminant hepatic failure has already begun as a clinical trial in several countries. Therefore, studies on plasma complement regulatory proteins of the pig are necessary, because the liver produces them. Complement factor I is a serine protease that cleaves C3b and C4b. The DNA sequences of factor I have been reported in many species, with the noted exception of pigs. In this study, porcine factor I was cloned and an open reading frame of 1794 base pairs were identified. The predicted amino acid sequence maintained a relatively high homology compared to those of other mammals, especially in the serine protease (SP) region. The cell membrane-bound forms of the porcine and the human SP domain of factor I were constructed. Amelioration of complement-mediated cell lysis by these molecules was then tested, using several kinds of sera and Chinese hamster ovary (CHO) cell transfectants. Both molecules had a suppressing effect on pig, human and dog complements, indicating little species-specificity. Further studies of other plasma complement regulatory proteins produced from pig liver cells will need to be considered as the primary feature of the pig bioartificial liver.

The C-terminus of complement factor H is essential for host cell protection

Complement is a powerful self-amplifying system of innate immune defense with the capacity to eliminate microbes directly. Factor H is a central regulator in plasma which protects host tissue from complement mediated damage. Here we characterize the relevance of surface attached factor H, and study the regulatory activity of factor H on endothelial cells. Although these cells expressed membrane bound regulators, cell bound factor H contributed substantially to complement regulatory activity at the cell surface. Blockade of the C-terminus of factor H with monoclonal antibodies inhibited cell binding of this soluble regulator and resulted in enhanced complement activation on the cells. In the absence of factor H, increased deposition and slower inactivation of C3b resulted in higher amount of membrane attack complexes on the cell surface. When the membrane regulators CD55 and CD59 were removed by enzymatic treatment, complement mediated cell lysis was enhanced in the absence of factor H. Importantly, inhibition of the C-terminus did not compromise the regulatory function of factor H in fluid phase. Altogether these data point to a highly relevant, yet so far underestimated role of factor H for complement control at cellular surfaces, and reveal a decisive role of the factor H C-terminus in host cell recognition and protection.

CD52 over-expression affects rituximab-associated complement-mediated cytotoxicity but not antibody-dependent cellular cytotoxicity: Preclinical evidence that targeting CD52 with alemtuzumab may reverse acquired resistance to rituximab in non-Hodgkin lymphoma

In an attempt to define mechanisms by which B-cell non-Hodgkin lymphoma (NHL) may escape rituximab immunotherapy, we developed several rituximab-resistant cell lines (RRCL) generated from the rituximab-sensitive cell lines (RSCL) Raji and RL. Rituximab resistance was associated with CD20 downregulation and upregulation of CD52 and the complement inhibitory proteins (CIPs) CD55 and CD59. No significant alemtuzumab-associated complement-mediated cell lysis (CMC) or antibody-dependent cellular cytotoxicity (ADCC) was demonstrated in RSCL. In contrast, in vitro exposure of RRCL to alemtuzumab resulted in a significant degree of CMC and ADCC. Of note, in vitro blocking of CD52 with anti-CD52 F(ab’)2 fractions in RRCL improved rituximab-associated CMC as compared to unblocked RRCL. Our current data provides a basis for further evaluation of alemtuzumab-based clinical trials for patients with rituximab-resistant NHL.

A Novel Class of Vascular Endothelial Growth Factor-responsive Genes That Require Forkhead Activity for Expression

Recently, we have shown that transient phosphorylation and inhibition of the pro-apoptotic transcription factor, forkhead, by vascular endothelial growth factor (VEGF) is essential for endothelial cell (EC) survival and proliferation. The goal of the present study was to determine whether forkhead (FKHR) also plays a positive role in agonist-mediated gene induction. Human coronary artery ECs were transduced with adenovirus overexpressing constitutively active phosphorylation-resistant triple mutant FKHR or transfected with small interference RNA (siRNA) against FKHR. The cells were then treated in the absence or presence of VEGF and assayed for gene expression using quantitative real-time PCR and Northern blots analyses. The data revealed a novel set of VEGF-responsive genes that require FKHR activity for optimal expression in ECs, including bone morphogenic protein 2, cbp/p300-interacting transactivator 2, decay accelerating factor (DAF), vascular cell adhesion molecule-1 (VCAM-1), manganese superoxide dismutase, endothelial-specific molecule-1, RING1 and YY1-binding protein, and matrix metalloproteinase-10. Consistent with a positive role for FKHR in mediating VEGF induction of DAF and VCAM-1 mRNA, siRNA against FKHR attenuated the effect of VEGF on complement-mediated EC lysis and monocyte adhesion, respectively. VEGF induction of the forkhead-dependent genes was down-regulated by the NF-kappaB inhibitor, constitutively active Ad-IkappaB, and in some cases by the nuclear factor of activated T-cells (NF-AT) inhibitor, cyclosporin. Together, these findings suggest that the VEGF-forkhead signaling axis plays an important functional role in ECs beyond the regulation of cell survival/apoptosis and cell cycle.

Anti-ischemia/reperfusion of C1 inhibitor in myocardial cell injury via regulation of local myocardial C3 activity

C3 is common to all pathways of complement activation augmenting ischemia/reperfusion (I/R)-induced myocardial injury and cardiac dysfunction. Complement inhibition with the complement regulatory protein, C1 inhibitor (C1INH), obviously exerts cardioprotective effects. Here, we examine whether C1INH regulates C3 activity in the ischemic myocardial tissue. C1INH markedly suppressed C3 mRNA expression and protein synthesis in both a model of I/R-induced rat acute myocardial infarction (AMI) and the cultured rat H9c2 heart myocytes. At least, this regulation was at the transcriptional level in response to oxygen tension. In vitro, C3 deposition on, and binding to, the surface of rat myocardial cells were significantly blocked by C1INH treatment. C1INH could inhibit classical complement-mediated cell lysis via suppressing the biological activity of C3. Therefore, C1INH, in addition to inhibition of the systemic complement activation, prevents myocardial cell injury via a direct inhibitory role in the local myocardial C3 activity.

Porcine reproductive and respiratory syndrome virus-infected alveolar macrophages contain no detectable levels of viral proteins in their plasma membrane and are protected against antibody-dependent, complement-mediated cell lysis

Porcine reproductive and respiratory syndrome virus (PRRSV) can evade the host immune system, which results in prolonged virus replication for several weeks to several months. To date, the mechanisms of PRRSV immune evasion have not been investigated in detail. One possible immune-evasion strategy is to avoid incorporation of viral proteins into the plasma membrane of infected cells, as this prevents recognition by virus-specific antibodies and consequent cell lysis either by the classical complement pathway or by antibody-dependent, cell-mediated cytotoxicity. In this study, viral proteins were not observed in the plasma membrane of in vitro-infected macrophages by using confocal microscopy or flow cytometry. Subsequently, the sensitivity of PRRSV-infected macrophages towards antibody-dependent, complement-mediated cell lysis (ADCML) was determined by using an ADCML assay. A non-significant percentage of PRRSV-infected cells were killed in the assay, showing that in vitro PRRSV-infected macrophages are protected against ADCML. PRRSV proteins were not detected in the plasma membrane of in vivo-infected alveolar macrophages and ADCML was also not observed. Together, these data indicate that viral proteins are not incorporated into the plasma membrane of PRRSV-infected macrophages, which makes infected cells invisible to PRRSV-specific antibodies. This absence of viral proteins on the cell surface could explain the protection against ADCML observed for in vitro and in vivo PRRSV-infected macrophages, and may play a role in virus persistence.

Complement insufficiency limits efficacy in a xenograft model of hyperacute rejection for cancer therapy.

Hyperacute rejection (HAR) is a rapid immunological response to an organ xenotransplant caused by recognition of endothelial galactose(alpha1,3)galactose (alphaGal) epitopes and complement-mediated cell lysis by host anti-alphaGal antibody ('natural antibody'). The alphaGal epitope is synthesised by a galactosyl transferase ((alpha1,3)GT) which humans lack. Because human cells transduced with (alpha1,3)GT are sensitised to natural antibody/complement-mediated lysis in human serum, delivery of (alpha1,3)GT to tumour vasculature in patients is a potential therapeutic strategy, by mimicking the pathophysiology of organ rejection. We therefore sought to develop an animal model of HAR for cancer therapy. Nude/(alpha1,3)GT knock-out mice allowed the growth of human tumour xenografts and the use of ecotropic retrovirus producer cells to generate expression of alphaGal on tumour vasculature. Lysis of alphaGal-positive murine endothelial cells with rabbit complement in conjunction with murine anti-alphaGal antibody in vitro was used to define the conditions necessary for HAR. However, tumour growth retardation and destruction of alphaGal-positive tumour endothelium were minimal after their administration, despite sera retaining post hoc cytolytic activity with fresh complement. The major limitation of this experimental system, of relevance to other therapeutic approaches, results from the use of a xenograft, in which additional xenoreactivities lead to complement insufficiency. Development of a tractable preclinical model in which to evaluate HAR for cancer therapy requires a syngeneic experimental system.

Apoptosis and complement-mediated lysis of myeloma cells by polyclonal rabbit antithymocyte globulin

AbstractCurrent monoclonal antibody therapies for multiple myeloma have had limited success, perhaps due to narrow target specificity. We have previously described the ability of polyclonal rabbit antithymocyte globulin (rATG) to induce caspase- and cathepsin-mediated apoptosis in human B and plasma cells. We now extend this observation to myeloma cells. Complement independent cell death was measured after addition of rATG (1-1000 μg/mL) to cultures of myeloma cell lines or primary CD138+ isolates from patient bone marrow aspirates. rATG induced significant levels of apoptosis in myeloma cells as assayed by caspase induction, annexin V binding, subdiploid DNA fragmentation, plasma-membrane permeability, and loss of mitochondrial-membrane potential. Addition of complement greatly augmented myeloma-cell death. Binding of rATG to individual myeloma cell-surface proteins, primarily CD38, CD52, CD126, and CD138, was demonstrated by competitive inhibition experiments with targeted monoclonal antibodies. Three pathways of cell death were identified involving caspase activation, cathepsin D, and the genistein sensitive tyrosine kinase pathway. F(ab′)2 fragments of rATG had reduced proapoptotic activity, which was restored by coincubation with Fc fragments, and anti-CD32 or anti-CD64 antibodies. We conclude that rATG is an effective agent for in vitro induction of apoptosis in multiple myeloma, and that exploratory clinical trials may be warranted.

Phenotypic expression of factor H mutations in patients with atypical hemolytic uremic syndrome

We investigated the phenotypic expression of factor H mutations in two patients with atypical hemolytic uremic syndrome (HUS). Factor H in serum was assayed by rocket immunoelectrophoresis, immunoblotting, and double immunodiffusion and in tissue by immunohistochemistry. Functional activity was analyzed by hemolysis of sheep erythrocytes and binding to endothelial cells. A homozygous mutation in complement control protein (CCP) domain 10 of factor H was identified in an adult man who first developed membranoproliferative glomerulonephritis and later HUS. C3 levels were very low. The patient had undetectable factor H levels in serum and a weak factor H 150 kDa band. Double immunodiffusion showed partial antigenic identity with factor H in normal serum owing to the presence of factor H-like protein 1. Strong specific labeling for factor H was detected in glomerular endothelium, mesangium and in glomerular and tubular epithelium as well as in bone marrow cells. A heterozygous mutation in CCP 20 of factor H was found in a girl with HUS. C3 levels were moderately decreased at onset. Factor H levels were normal and a normal 150 kDa band was present. Double immunodiffusion showed antigenic identity with normal factor H. Factor H labeling was minimal in the renal cortex. Factor H dysfunction was demonstrated by increased sheep erythrocyte hemolysis and decreased binding to endothelial cells. In summary, two different factor H mutations associated with HUS were examined: in one, factor H accumulated in cells, and in the other, membrane binding was reduced.

Safety of Etanercept in Psoriasis

Conventional systemic treatments for patients with psoriasis are associated with multiple adverse effects that require continuous monitoring. The introduction of new biological agents such as etanercept, a fully human fusion protein, has permitted individualisation of patients' treatment according to disease stage. The drug is a competitive inhibitor of tumour necrosis factor-alpha (TNFalpha) that prevents interaction between this cytokine and its cell surface receptors. Etanercept also modulates the activity of other inflammatory cytokines and does not induce complement-mediated cell lysis in vitro. The main source of information regarding etanercept safety comes from studies in patients with rheumatoid arthritis. The most common adverse effect during drug administration is mild injection site reactions. There is no increase in the overall incidence of infections compared with placebo, although there have been several reports of infections caused by intracellular organisms (Mycobacterium tuberculosis, Listeria monocytogenes, and Mycobacterium avium intracellulare). Therefore, combination of this drug with corticosteroids must be carefully monitored and should be avoided in patients with established sepsis. There are no data showing that treatment with etanercept results in an increase in the occurrence of malignant neoplasms. However, caution is recommended in use of etanercept in patients with a current or past history of demyelinating disease. Etanercept must be used with extreme caution in patients with heart failure because of several reports indicating a worsening or de novo occurrence of congestive heart failure while receiving the drug. Monitoring of autoantibodies is not currently considered necessary as they do not predict response, toxicity or autoimmune events. The presence of non-neutralising antibodies to the TNF receptor fragment or other protein components of etanercept has not been related to a decrease in drug response or adverse reactions. Etanercept does not generally modify the course of inflammatory bowel disease. When combined with other systemic therapies for psoriasis, current data do not show an increase in adverse events. In patients with hepatitis C viral infection, etanercept does not increase transaminase levels or viral load and in some instances has allowed the concomitant use of interferon which had previously been discontinued because of a worsening of psoriasis. Etanercept is rated as a US FDA category B drug in pregnancy. However, its use is not recommended in pregnant women unless the benefit-risk ratio greatly favours its use. Etanercept is not recommended for use in lactating women. Etanercept represents a relevant treatment for psoriasis, efficacious over many weeks and safe but special care should be taken to avoid the potential risks.

Rituximab Activity Is Potentiated by GM-CSF in Patients with Relapsed, Follicular Lymphoma: Results of a Phase II Study.

Rituximab, a monoclonal antibody that targets the CD20 antigen, has shown single-agent activity in patients with previously untreated and relapsed follicular lymphoma (FL). Mechanisms of rituximab-mediated antineoplastic activity include complement-mediated cell (CDC) lysis, apoptosis, and the induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Granulocyte macrophage colony stimulating factor (GM-CSF) is a biologic agent capable of enhancing proliferation of cell populations and cell surface receptors necessary for rituximab activity. It was hypothesized that the combination of GM-CSF and rituximab would substantially improve the treatment efficacy of rituximab in relapsed, FL patients. The current study assessed the clinical response, biologic response, and safety of combination rituximab and GM-CSF in patients with relapsed FL. Response and toxicity were defined using previously determined standard criteria (Cheson B, et al. J Clin Oncol. 1999; 17:1244–1253) and World Health Organization standard criteria. A total of 46 patients, with a median age of 60 years (range, 37–80 years), received rituximab and GM-CSF between January 2000 and October 2000. This analysis focuses on 34 patients with relapsed or progressive FL. GM-CSF was administered at 5 mg/kg/day on Days 1–8 and rituximab was administered at 375 mg/m2/week on Day 5 of each 21-day cycle for a total of 4 cycles. Throughout the treatment and follow-up periods, all patients underwent clinical examinations, bone marrow biopsies, standard biologic assays (eg, lactate dehydrogenase, β2-macroglobulin, creatinine, and liver enzymes), complete blood cell counts, immunophenotyping, and polymerase chain reaction analysis for bcl-2 expression. Response was evaluated at Days 30, 60, 90, and every 4 months until progression. Of the 34 enrolled patients, 1 patient was excluded due to transformation and 4 patients withdrew from the study due to toxicity. The complete response (CR) rate was 45% and the overall response rate was 70% (15 CR + CR unconfirmed and 8 partial responses [PR]) for the 33 intent-to-treat patients with relapsed FL. CR was reached in 63% (7 of 11) patients who received ≥3 lines of previous therapy (range, 3–7 lines). The median progression-free survival was 16.7 months. A significant increase (P <0.01) in cell populations involved in ADCC (white blood cells, neutrophils, and macrophages) was demonstrated after rituximab/GM-CSF therapy. In an effort to determine if a subset of patients may be more responsive to the rituximab/GM-CSF combination, expression of dendritic cell subpopulations 1 and 2 (DC-1 and DC-2) were measured before and after treatment. Patients who achieved a CR had significantly lower DC-2 expression than patients achieving a PR (P = 0.012). In conclusion, the relatively high CR rate, combined with the longer than expected duration of response in this treatment refractory population, suggests that the combination of rituximab plus GM-CSF is a safe and effective regimen in the treatment of FL patients and warrants further investigation.

Cloning and Expression of a Functionally Active Truncated N‐Glycosylated KSHV ORF4/KCP/Kaposica in the Methylotrophic Yeast Pichia pastoris

Kaposi's sarcoma herpesvirus (KSHV) is a typical DNA virus that is associated with a number of proliferative diseases including Kaposi's sarcoma. The KSHV open reading frame (ORF) 4 encodes a complement regulatory protein (Kaposi complement control protein, KCP) that binds complement components and inhibits the complement-mediated lysis of cells infected by the virus, thus providing a strategy for evasion of the host complement system. Based on primary sequence analysis and comparison with other functionally and structurally similar proteins, oligonucleotide primers were designed to amplify by polymerase chain reaction (PCR) three regions of the predicted ORF 4 from human herpes virus-8 (HHV-8) DNA isolated from a primary effusion lymphoma cell line. The PCR products were inserted by ligation into the expression vector pPIC9 to generate three recombinant plasmids for heterologous expression in the yeast, Pichia pastoris, to produce separately the four N-terminal sushi domains (KCP-S, small), KCP protein lacking the putative transmembrane-binding domain (KCP-M, medium), and the full-length protein (KCP-F, full). Expression of the viral proteins was confirmed by SDS-PAGE, immunologic detection, and Western blot analyses using a rabbit polyclonal antibody directed against a selected peptide region that is common to all three recombinant KCPs. KCP-S directly from expression media could inhibit complement-mediated lysis of sensitized sheep erythrocytes by approximately 60% in a hemolysis assay. This result confirms previous reports that recombinant KCP is twice as efficient in inhibiting the classic pathway-mediated lysis of erythrocytes than is the vaccinia virus complement control protein, which also contains four sushi domains.

Effect of glycosylphosphatidylinositol specific phospholipase D gene expression levels on complement mediated killing of leukemic cells in patients with chronic myeloid leukemia

Background To explore the disparity in glycosylphosphatidylinositol phospholipase D (GPI-PLD) expression levels between mononuclear cells of chronic myeloid leukemia (CML) and healthy controls, and clarify the certain relation of GPI-PLD expression levels to complement mediated killing of leukemic cells. Methods Competitive RT-PCR was used to detect quantitatively the GPI-PLD mRNA in mononuclear cells. GPI-anchored CD55 and CD59 were analyzed by flow cytometry and Western blotting. Complement-mediated lysis was assessed by staining method of trypan blue dye. Results The GPI-PLD activities and their mRNA copies in CML patients were significantly lower than those in healthy adults. At the tenth day after treatment with bone marrow transplantation (BMT), the GPI-PLD activities and copies of GPI-PLD mRNA almost recovered to the expression levels of healthy subjects. The expression of both CD55 and CD59 in CML patients were significantly higher than those in healthy subjects. After treatment with insulin (10 − 7 mol/l) plus glucose (16.7 mmol/l) for 48 h, the cellular GPI-PLD activity and mRNA levels in K562 cells derived from the leukemic cells of a CML patient all increased about 3-fold. Simultaneously, the GPI-anchored CD55 and CD59 on cell surfaces were released into the culturing medium, and the killing rate of complement-mediated K562 cell lysis increased almost 3 times. Conclusion The decreased GPI-PLD expression may reduce the release of GPI-anchored CD55 and CD59 in leukemia cells and finally decrease complement mediated killing of these cells in chronic phase of CML.

Attribution of the Various Inhibitory Actions of the Streptococcal Inhibitor of Complement (SIC) to Regions within the Molecule

Some strains of Streptococcus pyogenes secrete a virulence factor called the streptococcal inhibitor of complement (SIC) function. SIC is a polyfunctional protein that interacts with a number of host proteins and peptides, especially with those that are involved in host defense systems. In addition to inhibiting the complement-mediated lysis of cells, SIC inhibits lysozyme, secretory leukocyte proteinase inhibitor, and beta-defensins. SIC also binds to proteins associated with the cytoskeleton and thereby may cause cytoskeletal derangement. The SIC molecule has three distinct structural domains constituting the N-proximal short repeat region (SRR), the central long repeat region (LRR), and the C-proximal proline-rich region (PRR). To map various functions to the structural domains, we have analyzed recombinant subclones expressing various parts of SIC and elastase-generated discrete fragments of SIC for binding to various ligands and for determining their biological properties. The results demonstrate the following. (a) SRR alone was sufficient to confer inhibition of complement function. (b) Anti-defensin and anti-lysozyme activities were mapped to the SRR plus LRR. (c) The LRR plus PRR harbored ezrin binding activity.

Alemtuzumab induces enhanced apoptosis in vitro in B-cells from patients with chronic lymphocytic leukemia by antibody-dependent cellular cytotoxicity

Alemtuzumab, a monoclonal anti-CD52 antibody has been shown to be highly effective in B-cell chronic lymphocytic leukemia, even in fludarabine-refractory disease. The mechanism of action is currently unknown, but may rely on complement-mediated cell lysis and antibody-dependent cellular cytotoxicity. The aim of this study was to assess the proapoptotic activity of alemtuzumab in chronic lymphocytic leukemia and to describe pathways potentially underlying this effect. Peripheral blood mononuclear cells from 21 chronic lymphocytic leukemia patients were treated in vitro in the absence of complement with fludarabine alone, alemtuzumab alone, or with the additional presence of a cross-linking anti-Fc-antibody. Apoptosis was quantified after 24 h by flow cytometry analysis. Expression of several pro- and anti-apoptotic proteins was determined at different time points. Apoptosis of peripheral blood mononuclear cells treated with alemtuzumab alone was significantly enhanced compared to untreated cells suggesting a minor potentially cytotoxic mechanism by direct signaling independent from antibody-dependent cellular cytotoxicity. The presence of a cross-linking anti-Fc-antibody induced the formation of cell clusters and enhanced apoptosis significantly suggesting a potential role of antibody-dependent cellular cytotoxicity in alemtuzumab induced apoptosis. Alemtuzumab activated a CD52-dependent signaling pathway which induced a significant increase in caspase 3 and 8 expression. Alemtuzumab significantly enhances apoptosis in chronic lymphocytic leukemia cells in vitro, especially in combination with a cross-linking anti-Fc-antibody, this effect being mediated by a caspase-dependent pathway.

Induction of Long-Term Protective Antiviral Endogenous Immune Response by Short Neutralizing Monoclonal Antibody Treatment

Long-term immune control of viral replication still remains a major challenge in retroviral diseases. Several monoclonal antibodies (MAbs) have already shown antiviral activities in vivo, including in the clinic but their effects on the immune system of treated individuals are essentially unknown. Using the lethal neurodegeneration induced in mice upon infection of neonates by the FrCas(E) retrovirus as a model, we report here that transient treatment by a neutralizing MAb shortly after infection can, after an immediate antiviral effect, favor the development of a strong protective host immune response containing viral propagation long after the MAb has disappeared. In vitro virus neutralization- and complement-mediated cell lysis assays, as well as in vivo viral challenges and serum transfer experiments, indicate a clear and essential contribution of the humoral response to antiviral protection. Our observation may have important therapeutic consequences as it suggests that short antibody-based therapies early after infection should be considered, at least in the case of maternally infected infants, as adjunctive treatment strategies against human immunodeficiency virus, not only for a direct effect on the viral load but also for favoring the emergence of an endogenous antiviral immune response.

Increased sensitivity of early apoptotic cells to complement-mediated lysis.

Opsonization of apoptotic cells with complement proteins contributes to their clearance by phagocytes. Little is known about the lytic effects of complement on apoptotic cells. Sensitivity of cells treated with anti-Fas antibody (Jurkat cells), staurosporine or etoposide (Raji cells) to lysis by complement was examined. As shown here, early apoptotic cells are more sensitive to lysis by antibody and complement than control cells. More complement C3 and C9 bound to apoptotic than to control cells, even though antibody binding was similar. Enhanced killing and C3/C9 deposition were blocked by benzyloxy-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor. Complement-mediated lysis of early apoptotic cells was also prevented by inhibitors of caspases 6, 8, 9 or 10. In contrast, caspase inhibitors had no effect on the lysis of non-apoptotic Jurkat and Raji cells. Early apoptotic Jurkat cells were also more sensitive to lysis by the pore formers streptolysin O and melittin. Sensitivity of Jurkat Bcl-2 transfectants to lysis by complement was analyzed. Enhanced Bcl-2 expression was associated with reduced C3 deposition and lower sensitivity to complement-mediated lysis. These results demonstrate that at an early stage in apoptosis, following caspase activation, cells become sensitive to necrotic-type death by complement and other pore formers. Furthermore, they suggest that Bcl-2 is actively protecting Jurkat cells from complement-mediated lysis.

Complement component C5 is not involved in scrapie pathogenesis

During transmissible spongiform encephalopathy (TSE) infections the accumulation of abnormal prion protein within the brain is often accompanied by severe neurodegeneration. Studies have implicated complement, including the membrane attack complex (MAC, C5b–C9), in inducing pathology in some neurodegenerative diseases. Recent studies show the MAC is localized on neurons in the brains of TSE patients implicating complement-mediated cell lysis in TSE neuropathology. To determine the role of the MAC in TSEs, we compared scrapie pathogenesis in C5-deficient and C5-sufficient mice. C5-deficient mice developed clinical scrapie with incubation periods similar to C5-sufficient mice. Furthermore, the severity of the neuropathology was not significantly different between C5-deficient and C5-sufficient mice. These data show that C5, and the MAC, are not involved in TSE pathogenesis.

Modulation of a “CD59‐like” Protein in Naegleria fowleri Amebae by Bacteria1

Found in soil and freshwater habitats, Naegleria fowleri are free-living amebae that cause a fatal disease in humans called Primary Amebic Meningoencephalitis. In the natural environment, amebae feed on bacteria. In the infected host, the amebae lyse and ingest nerve tissue. Recently, we have established that N. fowleri expresses a "CD59-like" surface protein, but the function of this protein in the ameba has not been elucidated. In mammalian cells, CD59 is a complement-regulatory protein that inhibits complement-mediated lysis of cells expressing this protein. In the present study, expression of the "CD59-like" protein in response to bacteria and bacterial toxins was investigated by Western immunoblot analysis. Co-culture of N. fowleri with log phase Escherichia coli or Pseudomonas aeruginosa resulted in differential expression of the "CD59-like" protein. Co-cultures of amebae and bacteria were examined by electron microscopy. The results of our study implicate a possible protective role of the "CD59-like" protein in response to bacterial predators and bacterial toxins, because amebae remained intact after co-culture with bacteria.