COVID-19 cytokine storm: The anger of inflammation.

Patients with COVID-19 who require ICU admission might have the cytokine storm. It is a state of out-of-control release of a variety of inflammatory cytokines. The molecular mechanism of the cytokine storm has not been explored extensively yet. The attachment of SARS-CoV-2 spike glycoprotein with angiotensin-converting enzyme 2 (ACE2), as its cellular receptor, triggers complex molecular events that leads to hyperinflammation. Four molecular axes that may be involved in SARS-CoV-2 driven inflammatory cytokine overproduction are addressed in this work. The virus-mediated down-regulation of ACE2 causes a burst of inflammatory cytokine release through dysregulation of the renin-angiotensin-aldosterone system (ACE/angiotensin II/AT1R axis), attenuation of Mas receptor (ACE2/MasR axis), increased activation of [des-Arg9]-bradykinin (ACE2/bradykinin B1R/DABK axis), and activation of the complement system including C5a and C5b-9 components. The molecular clarification of these axes will elucidate an array of therapeutic strategies to confront the cytokine storm in order to prevent and treat COVID-19 associated acute respiratory distress syndrome.

Aliskiren inhibits renin-mediated complement activation.

Certain kidney diseases are associated with complement activation although a renal triggering factor has not been identified. Here we demonstrated that renin, a kidney-specific enzyme, cleaves C3 into C3b and C3a, in a manner identical to the C3 convertase. Cleavage was specifically blocked by the renin inhibitor aliskiren. Renin-mediated C3 cleavage and its inhibition by aliskiren also occurred in serum. Generation of C3 cleavage products was demonstrated by immunoblotting, detecting the cleavage product C3b, by N-terminal sequencing of the cleavage product, and by ELISA for C3a release. Functional assays showed mast cell chemotaxis towards the cleavage product C3a and release of factor Ba when the cleavage product C3b was combined with factor B and factor D. The renin-mediated C3 cleavage product bound to factor B. In the presence of aliskiren this did not occur, and less C3 deposited on renin-producing cells. The effect of aliskiren was studied in three patients with dense deposit disease and this demonstrated decreased systemic and renal complement activation (increased C3, decreased C3a and C5a, decreased renal C3 and C5b-9 deposition and/or decreased glomerular basement membrane thickness) over a follow-up period of four to seven years. Thus, renin can trigger complement activation, an effect inhibited by aliskiren. Since renin concentrations are higher in renal tissue than systemically, this may explain the renal propensity of complement-mediated disease in the presence of complement mutations or auto-antibodies.

NF-κB signaling pathway-enhanced complement activation mediates renal injury in trichloroethylene-sensitized mice.

Both NF-κB pathway and complement activation appear to be involved in kidney damage induced by trichloroethylene (TCE). However, any relationship between these two systems has not yet been established. The present study aimed to clarify the role of NF-κB in complement activation and renal injury in TCE-sensitized BALB/c mice. Mice were sensitized by an initial subcutaneous injection and repeated focal applications of TCE to dorsal skin at specified timepoints. NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) was injected (intraperitoneal) before the final two focal TCE challenges. In the experiments, mice had their blood and kidneys collected. Kidney function was evaluated via blood urea nitrogen (BUN) and creatinine (Cr) content; renal histology was examined using transmission electron microscopy (TEM). Kidney levels of phospho-p65 were assessed by Western blot and kidney mRNA levels of interleukin (IL)-1ß, IL-6, IL-17, tumor necrosis factor (TNF)-α, and p65 by real-time quantitative PCR. Presence of C3 and C5b-9 membrane attack complexes in the kidneys was evaluated via immunohistochemistry. The results showed there was significant swelling, vacuolar degeneration in mitochondria, shrinkage of microvilli, disappearance of brush borders, segmental foot process fusion, and glomerular basement membrane thickening (or disrobing) in kidneys from TCE-sensitized mice. In conjunction with these changes, serum BUN and Cr levels were increased and IL-1ß, IL-6, IL-17, and TNFα mRNA levels were elevated. Levels of p65 and phospho-p65 protein were also up-regulated, and there was significant C3 and C5b-9 deposition. PDTC pretreatment attenuated TCE-induced up-regulation of p65 and its phosphorylation, complement deposition, cytokine release, and renal damage. These results provide the first evidence that NF-κB pathway has an important role in TCE-induced renal damage mediated by enhanced complement activation in situ.

Iron oxide nanoparticles induce cytokine secretion in a complement-dependent manner in a human whole blood model.

Iron oxide nanoparticles (IONPs) are promising nanomaterials for biomedical applications. However, their inflammatory potential has not been fully established. Here, we used a lepirudin anti-coagulated human whole blood model to evaluate the potential of 10 nm IONPs to activate the complement system and induce cytokine production. Reactive oxygen species and cell death were also assessed. The IONPs activated complement, as measured by C3a, C5a and sC5b-9, and induced the production of pro-inflammatory cytokines in a particle-dose dependent manner, with the strongest response at 10 µg/mL IONPs. Complement inhibitors at C3 (compstatin analog Cp40) and C5 (eculizumab) levels completely inhibited complement activation and secretion of inflammatory mediators induced by the IONPs. Additionally, blockade of complement receptors C3aR and C5aR1 significantly reduced the levels of various cytokines, indicating that the particle-induced secretion of inflammatory mediators is mainly C5a and C3a mediated. The IONPs did not induce cell death or reactive oxygen species, which further suggests that complement activation alone was responsible for most of the particle-induced cytokines. These data suggest that the lepirudin anti-coagulated human whole blood model is a valuable ex vivo system to study the inflammatory potential of IONPs. We conclude that IONPs induce complement-mediated cytokine secretion in human whole blood.

Eculizumab-C5 complexes express a C5a neoepitope in vivo: Consequences for interpretation of patient complement analyses.

The complement system has obtained renewed clinical focus due to increasing number of patients treated with eculizumab, a monoclonal antibody inhibiting cleavage of C5 into C5a and C5b. The FDA approved indications are paroxysmal nocturnal haemoglobinuria and atypical haemolytic uremic syndrome, but many other diseases are candidates for complement inhibition. It has been postulated that eculizumab does not inhibit C5a formation in vivo, in contrast to what would be expected since it blocks C5 cleavage. We recently revealed that this finding was due to a false positive reaction in a C5a assay. In the present study, we identified expression of a neoepitope which was exposed on C5 after binding to eculizumab in vivo. By size exclusion chromatography of patient serum obtained before and after infusion of eculizumab, we document that the neoepitope was exposed in the fractions containing the eculizumab-C5 complexes, being positive in this actual C5a assay and negative in others. Furthermore, we confirmed that it was the eculizumab-C5 complexes that were detected in the C5a assay by adding an anti-IgG4 antibody as detection antibody. Competitive inhibition by anti-C5 antibodies localized the epitope to the C5a moiety of C5. Finally, acidification of C5, known to alter C5 conformation, induced a neoepitope reacting identical to the one we explored, in the C5a assays. These data are important for interpretation of complement analyses in patients treated with eculizumab.

Comparative and correlative assessments of cytokine, complement and antibody patterns in paediatric type 1 diabetes.

One of the most widespread and effective environmental factors is the infection with enteroviruses (EVs) which accelerate ß cell destruction in type 1 diabetes (T1D). This study represented a comparison between diabetic EV+ and EV- children as well as correlation analysis between autoantibodies, T1D markers, cytokines, complement activation products and anti-coxsackievirus (CV) immunoglobulin (Ig)G. EV RNA was detected in Egyptian children with T1D (26·2%) and healthy controls (0%). Detection of anti-CV IgG in T1D-EV+ resulted in 64% positivity. Within T1D-EV+ , previously diagnosed (PD) showed 74 versus 56% in newly diagnosed (ND) children. Comparisons between populations showed increased levels of haemoglobin A1c (HbA1c), C-reactive protein (CRP), nitric oxide (NO), glutamic acid decarboxylase and insulin and islet cell autoantibodies [glutamic acid decarboxylase autoantibodies (GADA), insulin autoantibodies (IAA) and islet cell cytoplasmic autoantibodies (ICA), respectively], interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL -10, IL -12, IL -17, C3d and sC5-9 in T1D-EV+ versus T1D-EV- . Conversely, both IL-20 and transforming growth factor (TGF-ß) decreased in T1D-EV+ versus EV- , while IL-4, -6 and -13 did not show any changes. Correlation analysis showed dependency of accelerated autoimmunity and ß cell destruction on increased IFN-γ, IL-12 and IL-17 versus decreased IL-4, -6 and -13. In conclusion, IFN-γ, IL-12 and IL-17 played an essential role in exacerbating EV+ -T1D, while C3d, sC5b -9, IL-10 and -20 displayed distinct patterns.

Brain histopathology in patients with systemic lupus erythematosus: identification of lesions associated with clinical neuropsychiatric lupus syndromes and the role of complement.

OBJECTIVES: Neuropsychiatric (NP) involvement is a poorly understood manifestation of SLE. We studied post-mortem histopathology in relation to clinical NPSLE syndromes and complement deposition in brains of NPSLE and SLE patients and controls. Furthermore, we investigated the correlation between cerebral post-mortem histopathology and ex vivo 7 T MRI findings in SLE and NPSLE. METHODS: A nationwide search for autopsy material yielded brain tissue from 16 NPSLE and 18 SLE patients. Brains obtained from 24 patients who died of acute cardiac events served as controls. Apart from a histopathological evaluation, paraffin-embedded cortical tissue was stained for components of the classical, lectin and terminal complement pathways. RESULTS: Diffuse vasculopathy, microinfarction, macroinfarction, vasculitis and microthrombi occurred significantly more often in NPSLE than SLE patients and were absent in controls. Focal vasculopathy was found in both SLE patients and controls. Complement deposition was strongly associated with both SLE and NPSLE, but not with controls (P < 0.001). Microthrombi were found uniquely in NPSLE and were associated with C4d and C5b-9 deposits (P < 0.05). A 7 T MRI was unable to detect most small vessel injury that was visible histopathologically. CONCLUSION: Our study demonstrates that histopathological lesions in NPSLE represent a continuum, ranging from non-specific lesions such as focal vasculopathy, to more specific lesions including C4d- and C5b-9-associated microthrombi and diffuse vasculopathy related to clinical syndromes defining NPSLE. Complement deposition may be a key factor in the interaction between circulating autoantibodies and thromboischaemic lesions observed in NPSLE. Therefore, complement inhibition may have novel therapeutic potential in NPSLE.

Plasma Kallikrein-Kinin system mediates immune-mediated renal injury in trichloroethylene-sensitized mice.

Trichloroethylene (TCE) is a major environmental pollutant. An immunological response is a newly-recognized mechanism for TCE-induced kidney damage. However, the role of the plasma kallikrein-kinin system (KKS) in immune-mediated kidney injury has never been examined. This study aimed to explore the role of the key components of the KKS, i.e. plasma kallikrein (PK), bradykinin (BK) and its receptors B1R and B2R, in TCE-induced kidney injury. A mouse model of skin sensitization was used to explore the mechanism of injury with or without a PK inhibitor PKSI. Kidney function was evaluated by measuring blood urea nitrogen (BUN) and creatinine (Cr) in conjunction with histopathologic characterization. Plasma BK was determined by ELISA; Renal C5b-9 membrane attack complex was evaluated by immunohistochemistry. Expression of BK and PK in the kidney was detected by immunofluorescence. mRNA and protein levels of B1R and B2R were assessed by real-time qPCR and Western blot. As expected, numerous inflammatory cell infiltration and tubular epithelial cell vacuolar degeneration were observed in TCE-sensitized mice. Moreover, serum BUN and Cr and plasma BK were increased. In addition, deposition of BK, PK and C5b-9 were observed and B1R and B2R mRNA and proteins levels were up-regulated. Pre-treatment with PKSI, a highly selective inhibitor of PK, alleviated TCE-induced renal damage. In addition, PKSI attenuated TCE-induced up-regulation of BK, PK and its receptors and C5b-9. These results provided the first evidence that activation of the KKS contributed to immune-mediated renal injury induced by TCE and also helped to identify the KKS as a potential therapeutic target for mitigating chemical sensitization-induced renal damage.

Complement Activation in Patients with Focal Segmental Glomerulosclerosis.

BACKGROUND: Recent pre-clinical studies have shown that complement activation contributes to glomerular and tubular injury in experimental FSGS. Although complement proteins are detected in the glomeruli of some patients with FSGS, it is not known whether this is due to complement activation or whether the proteins are simply trapped in sclerotic glomeruli. We measured complement activation fragments in the plasma and urine of patients with primary FSGS to determine whether complement activation is part of the disease process. STUDY DESIGN: Plasma and urine samples from patients with biopsy-proven FSGS who participated in the FSGS Clinical Trial were analyzed. SETTING AND PARTICIPANTS: We identified 19 patients for whom samples were available from weeks 0, 26, 52 and 78. The results for these FSGS patients were compared to results in samples from 10 healthy controls, 10 patients with chronic kidney disease (CKD), 20 patients with vasculitis, and 23 patients with lupus nephritis. OUTCOMES: Longitudinal control of proteinuria and estimated glomerular filtration rate (eGFR). MEASUREMENTS: Levels of the complement fragments Ba, Bb, C4a, and sC5b-9 in plasma and urine. RESULTS: Plasma and urine Ba, C4a, sC5b-9 were significantly higher in FSGS patients at the time of diagnosis than in the control groups. Plasma Ba levels inversely correlated with the eGFR at the time of diagnosis and at the end of the study. Plasma and urine Ba levels at the end of the study positively correlated with the level of proteinuria, the primary outcome of the study. LIMITATIONS: Limited number of patients with samples from all time-points. CONCLUSIONS: The complement system is activated in patients with primary FSGS, and elevated levels of plasma Ba correlate with more severe disease. Measurement of complement fragments may identify a subset of patients in whom the complement system is activated. Further investigations are needed to confirm our findings and to determine the prognostic significance of complement activation in patients with FSGS.

Antibodies reactive to cleaved sites in complement proteins enable highly specific measurement of soluble markers of complement activation.

An emerging number of diseases and therapeutic approaches with defined involvement of the complement system justify a need for specific markers reflecting activation of particular effector arms of the complement cascade. Measurement of such soluble markers in circulation is a challenge since the specificity of antibodies must be limited to activated complement fragments but not predominant and ubiquitous parental molecules. Existing assays for the measurement of soluble, activated complement proteins are based on the detection of conformational neoepitopes. We tested an alternative approach based on detection of short linear neoepitopes exposed at the cleavage sites after activation of the actual complement component. Obtained antibodies reactive to C4d and C5b fragments enabled us to set up highly specific sandwich ELISAs, which ensured trustful measurements without false positive readouts characteristic for some of the widely used assays.

Exposure to the complement C5b-9 complex sensitizes 661W photoreceptor cells to both apoptosis and necroptosis.

The loss of photoreceptors is the defining characteristic of many retinal degenerative diseases, but the mechanisms that regulate photoreceptor cell death are not fully understood. Here we have used the 661W cone photoreceptor cell line to ask whether exposure to the terminal complement complex C5b-9 induces cell death and/or modulates the sensitivity of these cells to other cellular stressors. 661W cone photoreceptors were exposed to complete normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and Sirtuin 2 inhibition using 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furyl]-N-5-quinolinylacrylamide (AGK2). The sensitivity of 661W cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9. 661W cells underwent apoptotic cell death following exposure to C5b-9, as judged by poly(ADP-ribose) polymerase 1 cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but 661W cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9 significantly increased 661W sensitivity to staurosporine-induced apoptosis and necroptosis. These studies show that low levels of C5b-9 on 661W cells can induce apoptosis, and that C5b-9 specifically sensitizes 661W cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in photoreceptor loss, with implications for the molecular aetiology of retinal disease.

Identification of heme oxygenase 1 (HO-1) as a novel negative regulator of mobilization of hematopoietic stem/progenitor cells.

Activation of complement cascade (ComC) play and important role in mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB). While there are vast experimental data on the mechanisms and factors that induce or promote mobilization of HSPCs, there is relatively less data on negative regulators of this process. We demonstrate for the first time that heme oxygenase-1 (HO-1) that has a well-documented anti-inflammatory potential plays an important and heretofore unrecognized role in retention of HSPCs in BM niches by i) modulating negatively activation of mobilization promoting ComC, ii) maintaining stromal derived factor-1 (SDF-1) level in the BM microenvironment and iii) attenuating chemotactic responsiveness of HSPCs to SDF-1 and sphingosine-1 phosphate (S1P) gradients in PB. Furthermore, our data showing a positive mobilizing effect by a non-toxic small-molecule inhibitor of HO-1 (SnPP) suggest that blockade of HO-1 would be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate better the molecular mechanisms responsible for the potential effect of HO-1 in homing of HSPCs after transplantation.

Exploitation of the complement system by oncogenic Kaposi's sarcoma-associated herpesvirus for cell survival and persistent infection.

During evolution, herpesviruses have developed numerous, and often very ingenious, strategies to counteract efficient host immunity. Specifically, Kaposi's sarcoma-associated herpesvirus (KSHV) eludes host immunity by undergoing a dormant stage, called latency wherein it expresses a minimal number of viral proteins to evade host immune activation. Here, we show that during latency, KSHV hijacks the complement pathway to promote cell survival. We detected strong deposition of complement membrane attack complex C5b-9 and the complement component C3 activated product C3b on Kaposi's sarcoma spindle tumor cells, and on human endothelial cells latently infected by KSHV, TIME-KSHV and TIVE-LTC, but not on their respective uninfected control cells, TIME and TIVE. We further showed that complement activation in latently KSHV-infected cells was mediated by the alternative complement pathway through down-regulation of cell surface complement regulatory proteins CD55 and CD59. Interestingly, complement activation caused minimal cell death but promoted the survival of latently KSHV-infected cells grown in medium depleted of growth factors. We found that complement activation increased STAT3 tyrosine phosphorylation (Y705) of KSHV-infected cells, which was required for the enhanced cell survival. Furthermore, overexpression of either CD55 or CD59 in latently KSHV-infected cells was sufficient to inhibit complement activation, prevent STAT3 Y705 phosphorylation and abolish the enhanced survival of cells cultured in growth factor-depleted condition. Together, these results demonstrate a novel mechanism by which an oncogenic virus subverts and exploits the host innate immune system to promote viral persistent infection.

Humoral immune mechanism of liver injury in giant cell hepatitis with autoimmune hemolytic anemia.

BACKGROUND AND AIMS: Giant cell hepatitis with autoimmune hemolytic anemia (GCH-AHA) is presumed to be an autoimmune disease, but the mechanism of liver injury is unknown. We proposed that in CGH-AHA, the humoral limb of autoimmunity is the dominant force driving progressive liver injury. METHODS: We studied 6 cases of GCH-AHA and 6 cases of autoimmune hepatitis (AIH) with early childhood onset (3 type 1 and 3 type 2). Liver biopsies were graded for portal and periportal inflammation and for giant cells. Immunohistochemistry characterized cellular inflammation and complement involvement in injury by showing C5b-9 complex in hepatocytes. RESULTS: Clinical and biochemical features at presentation were generally similar; however, the absence of autoantibodies and the presence of Coombs positivity did distinguish GCH-AHA from early-onset AIH. Liver biopsy pathology in CGH-AHA showed giant cells and little inflammation, whereas AIH showed the opposite. C5b-9 staining showed high-grade complement-mediated pan-lobular hepatocyte injury in all of the cases with GCH-AHA, whereas little C5b-9 was seen in hepatocytes in cases with AIH. Inflammation in GCH-AHA comprised mainly lobular macrophages and neutrophils, whereas portal and periportal T-cell and B-cell inflammation characterized cases with AIH. Most cases with AIH responded to therapy with prednisone and azathioprine, whereas most cases with GCH-AHA responded only to rituximab. CONCLUSIONS: Widespread complement-mediated hepatocyte injury and typical C3a and C5a complement-driven liver inflammation along with Coombs-positive hemolytic anemia in GCH-AHA provide convincing evidence that systemic B-cell autoimmunity plays a central pathologic mechanism in the disease. Our findings support B-cell-directed immunotherapy as a first-line treatment of GCH-AHA.

Quantification of complement C5b-9 binding to cells by flow cytometry.

Interaction of the complement system, directly or indirectly (e.g., via antibodies), with cells activates the early and late complement components and culminates in the deposition of a membrane-spanning C5b-9 complex on the cell surface. At a high copy number, this C5b-9 will activate cell death, whereas at a low copy number, it will transmit various signals into cells. Quantification of C5b-9 deposition is useful for assessments of the capacity of cells and antibodies to activate complement. By using an antibody that identifies a novel antigen of the C5b-9 complex, the amount of C5b-9 complexes on cells can be quantified by flow cytometry. The detailed protocol is described in this chapter.

The effects of Eculizumab on the pathology of malignant atrophic papulosis.

BACKGROUND: Degos disease is a frequently fatal and incurable occlusive vasculopathy most commonly affecting the skin, gastrointestinal tract and brain. Vascular C5b-9 deposition and a type I interferon (IFN) rich microenvironment are held to be pathogenetically important in the evolution of the vascular changes. We recently discovered the use of eculizumab as a salvage drug in the treatment of near fatal Malignant atrophic papulosis (MAP). The effects of eculizumab on the pathology of MAP are explored. METHODS: Archival skin and gastrointestinal biopsy material was procured over a 2.5-year period before and after eculizumab therapy in our index case. Routine light microscopy and immunohistochemical assessment for C3d, C4d, C5b-9, MxA and caspase 3 were examined. Direct immunofluorescent studies were also conducted on select biopsy material. RESULTS: The patient had received eculizumab as a emergent life saving measure and following rapid improvement he continued with biweekly infusions for 4 years. Although improved he continues to have signs and symptoms of persistent abdominal disease. Pre-Eculizumab biopsies showed an active thrombotic microangiopathy associated with a high type I interferon signature and extensive vascular deposits of C5b-9 in skin and gastrointestinal biopsies. Endothelial cell apoptosis as revealed by Caspase 3 expression was noted. Inflammation comprising lymphocytes and macrophages along with mesenchymal mucin was observed as well. Post-eculizumab biopsies did not show active luminal thrombosis but only chronic sequelae of prior episodes of vascular injury. There was no discernible caspase 3 expression. After 12 months of therapy, C5b-9 was no longer detectable in tissue. The high type I IFN signature and inflammation along with mucin deposition was not altered by the drug. In addition, there was little effect of the drug on the occlusive fibrointimal arteriopathy which appears to be one characterized by extensive myofibroblastic expansion of the intima potentially as revealed by staining for smooth muscle actin without immunoreactivity for desmin and myogenin. CONCLUSIONS: Complement activation and enhanced endothelial cell apoptosis play an important role in the thrombotic complications of MAP. However, the larger vessel proliferative intimal changes appear to be independent of complement activation and may be on the basis of other upstream mechanisms. Monitoring C5b-9 deposition in tissue is likely not of great value in assessing treatment response to eculizumab given the persistence of C5b-9 in tissue for several months despite clinically effective C5 blocking therapy. A more integrated approach addressing upstream and downstream pathways in addition to those attributable to complement activation are critical for the successful treatment of MAP. Eculizumab may be used as salvage therapy in critically ill patients with thrombotic microangiopathy.

Circulating complement activation in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis.

Studies in animal models suggest that complement activation is crucial in the pathogenesis of anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). Here we investigate the circulating complement activation profile of 66 patients with active stage AAV compared to that of 54 patients with AAV in remission. Plasma levels of C3a, C5a, soluble C5b-9, and Bb, all determined by enzyme-linked immunosorbent assay, were significantly higher in active stage than in remission of AAV, while plasma levels of properdin were significantly lower in the former than the latter disease stage. There was no significant difference in the plasma levels of C4d between active stage and remission. The plasma level of Bb in patients with active AAV significantly correlated with the proportion of total and cellular crescents in the renal biopsy, the erythrocyte sedimentation rate, and the Birmingham Vasculitis Activity Scores. Thus, systemic activation of complement by the alternative pathway takes place in human AAV. Circulating Bb might be a useful biomarker in assessing disease activity of AAV.

Effects of PI3-k/Akt short hairpin RNA on proliferation, fibronectin production and synthesis of thrombospondin-1 and transforming growth factor-beta1 in glomerular mesangial cells induced by sublytic C5b-9 complexes.

OBJECTIVES: To explore proliferation of glomerular mesangial cells (GMC) and secretion of extracellular matrix (fibronectin induced by sublytic C5b-9 complexes), and then ascertain the role of phosphatidylinositol 3-kinase (PI3-k)/Akt signal pathway in these processes, by using small hairpin RNAs. MATERIAL AND METHODS: The expression of cyclin D(2), (3)H-thymidine into DNA and production of fibronectin including thrombospondin-1 and transforming growth factor-beta(1) in the GMCs stimulated by sublytic C5b-9 or transfected with expression vectors of PI3-k and Akt short hairpin RNA or LY294002 (PI3-k inhibitor) were measured by Real-time quantitative polymerase chain reaction (PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and (3)H-thymidine incorporation ((3)H-TdR), respectively. RESULTS: The expression of cyclin D(2), (3)H-thymidine into DNA and fibronectin in the GMCs stimulated by sublytic C5b-9 could all be increased, and the elevations of these parameters mentioned above were also markedly reduced in the GMCs transfected with vectors of PI3-k and Akt short hairpin RNA or LY294002, respectively. CONCLUSIONS: These data indicate that sublytic C5b-9 can promote proliferation of GMCs and secretion of fibronectin as well as synthesis of thrombospondin-1 and transforming growth factor-beta(1). The PI3-k/Akt signal pathway in these reactions, mediated by sublytic C5b-9 complexes, may play at least a partial role.

Complement activation in experimental and human temporal lobe epilepsy.

We investigated the involvement of the complement cascade during epileptogenesis in a rat model of temporal lobe epilepsy (TLE), and in the chronic epileptic phase in both experimental as well as human TLE. Previous rat gene expression analysis using microarrays indicated prominent activation of the classical complement pathway which peaked at 1 week after SE in CA3 and entorhinal cortex. Increased expression of C1q, C3 and C4 was confirmed in CA3 tissue using quantitative PCR at 1 day, 1 week and 3-4 months after status epilepticus (SE). Upregulation of C1q and C3d protein expression was confirmed mainly to be present in microglia and in a few hippocampal neurons. In human TLE with hippocampal sclerosis, astroglial, microglial and neuronal (5/8 cases) expression of C1q, C3c and C3d was observed particularly within regions where neuronal cell loss occurs. The membrane attack protein complex (C5b-C9) was predominantly detected in activated microglial cells. The persistence of complement activation could contribute to a sustained inflammatory response and could destabilize neuronal networks involved.