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Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are characterized by small-vessel necrotizing inflammation, and prior to the advent of immunosuppressive therapy frequently had a fatal outcome. Treatment has transformed AAV into a relapsing/remitting disease with increased drug-related toxicities and organ damage. The use of glucocorticoids, cyclophosphamide and immunosuppressives (including azathioprine, mycophenolate and methotrexate) was optimized through a sequence of clinical trials establishing a standard of care against which subsequent targeted therapies could be developed. Improved understanding of pathophysiology has supported the development of B-cell depletion and complement inhibition in granulomatosis with polyangiitis and microscopic polyangiitis, and interleukin 5 inhibition for eosinophilic granulomatosis with polyangiitis, leading to the approval of newer agents for these conditions. There has been an increased attention on minimizing the adverse effects of treatment and on understanding the epidemiology of comorbidities in AAV. This review will focus on recent evidence from clinical trials, especially with respect to glucocorticoids, avacopan, plasma exchange, rituximab and mepolizumab, and their interpretation in the 2022 management recommendations by the European League of Associations of Rheumatology. [ABSTRACT FROM AUTHOR]

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis worldwide, with a potentially serious prognosis. At present, management of IgAN is primarily based on therapeutic lifestyle changes, and excellent blood pressure control and maximized supportive treatment with the combination of inhibition of the renin–angiotensin–aldosterone system with either inhibitors of angiotensin-converting enzyme or angiotensin II receptor blockers and inhibitors of sodium-glucose cotransporter-2, and possibly in the future also with endothelin antagonists. Supportive care currently represents the cornerstone of treatment of IgAN. Targeted-release formulation of budesonide should replace systemic corticosteroids in patients with higher proteinuria and active histological lesions. New treatment options are aimed at immunopathogenesis of IgAN including depletion or modulation of Galactose-deficient-Immunoglobulin A1–producing B cells, plasma cells, and the alternate and/or lectin pathway of complement. The exact place of monoclonal antibodies and complement inhibitors will need to be determined. This article reviews potential supportive therapies currently available for patients with IgAN. [ABSTRACT FROM AUTHOR]

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. Recent years have witnessed significant improvements in the understanding of the pathogenesis of IgAN and particularly, the pathogenic role of complement activation. The alternative complement pathway is the major complement cascade activator in IgAN, and glomerular C3 deposition has been shown to correlate with disease progression. In addition, several studies have provided insight into the pathogenic role of factor H–related proteins -1 and -5 in IgAN, as independent players in complement dysregulation. The lectin pathway has also been shown to be associated with the severity of IgAN. Glomerular deposition of C4d has been associated with increased histologic disease activity, faster decline in estimated glomerular filtration rate and higher risk of kidney failure. On the other hand, although overlooked in the Oxford classification, numerous studies have shown that the coexistence of thrombotic microangiopathy in IgAN is a significant indicator of a poorer prognosis. All the breakthroughs in the understanding of the contributing role of complement in IgAN have paved the way for the development of new complement-targeted therapies in this disease. Several ongoing trials are evaluating the efficacy of new agents against factor B (iptacopan, Ionis-FB-LRX), C3 (pegcetacoplan), factor D (vemircopan, pelecopan), C5 (ravulizumab, cemdisiran) and C5a receptor 1 (avacopan). In this study, we provide a comprehensive review of the role of complement in IgAN, including the emerging mechanisms of complement activation and the promising potential of complement inhibitors as a viable treatment option for IgAN. [ABSTRACT FROM AUTHOR]

The complement system plays a crucial role in innate immunity, providing essential defense against pathogens. However, uncontrolled or prolonged activation of the complement cascade can significantly contribute to kidney damage, especially in cases of glomerulonephritis. Immunoglobulin A nephropathy (IgAN), the most prevalent form of primary glomerulonephritis, has growing evidence supporting the involvement of complement alternative and lectin pathways. In fact, patients with IgAN experience complement activation within their kidney tissue, which may be involved in the development of glomerular damage and the progression of IgAN. Complement activation has emerged as a significant area of interest in IgAN, with numerous complement-targeting agents currently being explored within this field. Nevertheless, the exact mechanisms of complement activation and their role in IgAN progression require comprehensive elucidation. This review seeks to contextualize the proposed mechanisms of complement activation within the various stages ("hits") of IgAN pathogenesis, while also addressing the clinical implications and anticipated outcomes of complement inhibition in IgAN. [ABSTRACT FROM AUTHOR]

A refined understanding of the role of complement in the pathogenesis of glomerular and other kidney diseases has, over the past two decades, been matched by the development of novel, complement-targeting therapies. As we increasingly recognize the important role that complement activation across all three pathways—classical, lectin and alternative—plays in glomerular lesions both rare (e.g. C3 glomerulopathy) and common (e.g. immunoglobulin A nephropathy), we can identify avenues for precise, targeted approaches to modifying the natural history of these kidney diseases. In this review, we survey the evidence on using complement inhibition from the earliest, small-scale studies focusing on C5-targeting agents to more recent, large, multicenter, randomized trials utilizing complement blockade higher up in the complement pathway at the level of C3. We conclude by examining where the field of complement targeting therapy may be headed in light of these studies. [ABSTRACT FROM AUTHOR]

Background Severe coronavirus disease 2019 (COVID-19) has been increasingly recognized as a multisystem disease. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect literally any cell type that expresses its target receptor angiotensin-converting enzyme 2. However, COVID-19-associated organ dysfunction is not only mediated by direct viral effects but also by the interaction between the host's immune response, endotheliopathy, and microvascular coagulopathy. It has been proposed that the activation of the complement system plays a central role in the pathophysiology of severe COVID-19 and the associated endotheliopathy. Case summary A 76-year-old male patient with indeterminate cardiogenic shock in the setting of confirmed SARS-CoV-2 infection was admitted to our intensive care unit. Coronary angiography did not reveal a plausible explanation for his symptoms. The patient developed renal failure, neurological symptoms, severe thrombocytopenia, and a Coombs-negative haemolytic anaemia with schistocytes. All together the clinical picture was highly suggestive of a thrombotic microangiopathy (TMA) with microvascular cardiac involvement. Conventional therapeutic strategies including high-dose steroids and seven sessions of therapeutic plasma exchange were all unsuccessful. Interestingly, complement inhibition with Eculizumab as rescue approach led to a rapid clinical and laboratory improvement and the patients were discharged with normalized organ functions at Day 36. Conclusion The aetiology of cardiogenic shock observed in this patient cannot simply be explained by his focal and chronic coronary findings. Although viral myocarditis was not formally excluded, both the clinical features of TMA and the rapid resolution of all clinical signs and symptoms after pharmacological complement inhibition suggest a SARS-CoV-2-driven microangiopathic origin of heart failure. [ABSTRACT FROM AUTHOR]

This article discusses the advancements in immunonephrology, specifically in the diagnosis and treatment of immune-mediated kidney diseases. It highlights the need for biomarkers to assess disease activity and the potential of novel immunomodulators. The article also emphasizes the importance of specific biological drugs in understanding the pathophysiology of human kidney diseases. The supplement of the journal Nephrology Dialysis Transplantation includes various studies on glomerulonephritis, complement inhibitors, immunomodulatory drugs, infection prevention, and the role of chronic kidney disease therapy. The authors hope that these advancements will improve the management of immune-mediated kidney diseases. [Extracted from the article]

Antibodies to myelin oligodendrocyte glycoprotein (MOG-Abs) define a distinct disease entity. Here we aimed to understand essential structural features of MOG required for recognition by autoantibodies from patients. We produced the N-terminal part of MOG in a conformationally correct form; this domain was insufficient to identify patients with MOG-Abs by ELISA even after site-directed binding. This was neither due to a lack of lipid embedding nor to a missing putative epitope at the C-terminus, which we confirmed to be an intracellular domain. When MOG was displayed on transfected cells, patients with MOG-Abs recognized full-length MOG much better than its N-terminal part with the first hydrophobic domain (P < 0.0001). Even antibodies affinity-purified with the extracellular part of MOG recognized full-length MOG better than the extracellular part of MOG after transfection. The second hydrophobic domain of MOG enhanced the recognition of the extracellular part of MOG by antibodies from patients as seen with truncated variants of MOG. We confirmed the pivotal role of the second hydrophobic domain by fusing the intracellular part of MOG from the evolutionary distant opossum to the human extracellular part; the chimeric construct restored the antibody binding completely. Further, we found that in contrast to 8-18C5, MOG-Abs from patients bound preferentially as F(ab')2 rather than Fab. It was previously found that bivalent binding of human IgG1, the prominent isotype of MOG-Abs, requires that its target antigen is displayed at a distance of 13-16 nm. We found that, upon transfection, molecules of MOG did not interact so closely to induce a Förster resonance energy transfer signal, indicating that they are more than 6 nm apart. We propose that the intracellular part of MOG holds the monomers apart at a suitable distance for bivalent binding; this could explain why a cell-based assay is needed to identify MOG-Abs. Our finding that MOG-Abs from most patients require bivalent binding has implications for understanding the pathogenesis of MOG-Ab associated disorders. Since bivalently bound antibodies have been reported to only poorly bind C1q, we speculate that the pathogenicity of MOG-Abs is mostly mediated by other mechanisms than complement activation. Therefore, therapeutic inhibition of complement activation should be less efficient in MOG-Ab associated disorders than in patients with antibodies to aquaporin-4 . [ABSTRACT FROM AUTHOR]

Summary: Carbamylation is a post‐translational modification that can be detected on a range of proteins, including immunoglobulin (Ig)G, in several clinical conditions. Carbamylated IgG (ca‐IgG) was reported to lose its capacity to trigger complement activation, but the mechanism remains unclear. Because C1q binds with high affinity to hexameric IgG, we analyzed whether carbamylation of IgG affects binding of C1q, hexamerization and complement‐dependent cytotoxicity (CDC). Synovial tissues of rheumatoid arthritis (RA) patients were analyzed for the presence of ca‐IgG in vivo. Synovial tissues from RA patients were analyzed for the presence of ca‐IgG using mass spectrometry (MS). Monomeric or hexameric antibodies were carbamylated in vitro and quality in solution was controlled. The capacity of ca‐IgG to activate complement was analyzed in enzyme‐linked immunosorbent (ELISAs) and cellular CDC assays. Using MS, we identified ca‐IgG to be present in the joints of RA patients. Using in vitro carbamylated antibodies, we observed that ca‐IgG lost its capacity to activate complement in both solid‐phase and CDC assays. Mixing ca‐IgG with non‐modified IgG did not result in effective inhibition of complement activation by ca‐IgG. Carbamylation of both monomeric IgG and preformed hexameric IgG greatly impaired the capacity to trigger complement activation. Furthermore, upon carbamylation, the preformed hexameric IgG dissociated into monomeric IgG in solution, indicating that carbamylation influences both hexamerization and C1q binding. In conclusion, ca‐IgG can be detected in vivo and has a strongly reduced capacity to activate complement which is, in part, mediated through a reduced ability to form hexamers. [ABSTRACT FROM AUTHOR]

Reepithelialization is crucial for effective wound repair in burn wounds. Reactive oxygen species (ROS) have shown to be important in this. Recent studies suggest that NOX proteins produce ROS in keratinocytes. In the present study, we have studied NOX proteins in burn wounds, including the effect of C1-esterase inhibitor (C1inh) hereon, which is the endogenous inhibitor of complement activity whereof we have shown previously that it also increased the rate of reepithelialization in burn wounds. Skin tissue derived from healthy control Wistar rats (n = 6) were compared with burn-injured rats, with (n = 7) or without C1inh treatment (n = 7). After 14 days, rats were terminated. From the burn-injured rats, the entire wound and nonburned skin from the hind leg, that is, internal control was excised. From the control rats, dorsal skin was excised. In these skin samples, NOX2 and NOX4 were analyzed immunohistochemically. In nonburned rats, NOX2 was found in keratinocytes in both the basal layer and suprabasal layer of the epidermis; and the number of NOX2-positive keratinocytes was 367/mm2 (254-378). In burned rats, the number of NOX2-positive keratinocytes was significantly increased in the newly forming epidermis in the burned area to 1019/mm2 (649-1172), especially in the suprabasal layer, but significantly decreased in remote nonburned skin to 22/mm2 (6-89). C1inh treatment counteracted these changes in epidermal NOX2 expression in burned rats, both in the burned area as in remote nonburned skin. No NOX4 expression was found in the epidermis in none of the groups. NOX2 expression was increased in keratinocytes in newly forming epidermis after burn injury. C1inh, a drug that increases the rate of reepithelialization, counteracted this effect. These results suggest a role for NOX2 in the reepithelialization of burn wounds. [ABSTRACT FROM AUTHOR]

Background The C5 complement inhibitor eculizumab is a first-line treatment in atypical haemolytic uraemic syndrome (aHUS). Therapy with eculizumab is associated with a highly increased risk for meningococcal infection. Therefore, vaccination is highly recommended before beginning treatment. Efficacy of quadrivalent meningococcal vaccines (MenACWY) in patients treated with the C5 complement inhibitor eculizumab in aHUS has not yet been determined. Methods Patients with aHUS received one dose of a MenACWY conjugate vaccine before eculizumab treatment commenced. Bactericidal titres against meningococcal serogroups A, C, W and Y were determined using baby rabbit complement in 25 patients. Results Full immune response to meningococcal vaccination was detected in five patients (20%), while seven patients (28%) showed no immune response in any of the tested serogroups. The remaining 13 patients showed incomplete immune response with proof of protective antibody titres for one to three serogroups without perceptible preference for any serogroup. Bactericidal titres after re-vaccination were available for 17 patients. Nine patients with incomplete immune response after first vaccinations showed protective antibody titres for all serogroups after re-vaccination. Kidney function had improved in >50% of patients at the time of re-vaccination compared with the time of first vaccination and immunosuppressive therapy was only applied to re-vaccinated patients following kidney transplantation. Conclusions Immunogenicity of first quadrivalent meninongococcal vaccination is insufficient in patients with aHUS. Booster response is promising, but incomplete. Therefore, establishing antibiotic prophylaxes seems pivotal. [ABSTRACT FROM AUTHOR]

In this issue of Clinical Kidney Journal , Plasse et al. report on the use of high-dose aliskiren as an adjunct therapy in a patient treated with eculizumab for haemolytic uraemic syndrome (HUS). This follows the recent description of the complement factor 3 (C3) activating activity of the enzyme renin and the successful therapeutic use of the direct renin inhibitor aliskiren in three cases of C3 glomerulopathy/dense deposit disease. We discuss the potential clinical and pathophysiological implications of these reports on nephropathies linked to complement, from HUS to C3 glomerulopathy to immunoglobulin A nephropathy as well as the concept of dual complement inhibition for kidney disease. [ABSTRACT FROM AUTHOR]

Background Complement is thought to play a role in immunoglobulin A nephropathy (IgAN), though the activating mechanisms are unknown. This study focused on the gene expression of CD46 and CD55, two key molecules for regulating C3 convertase activity of lectin and alternative complement pathways at a cellular level. Methods The transcriptional expression in peripheral white blood cells (WBCs) of CD46 and CD55 was investigated in 157 patients enrolled by the Validation of the Oxford Classification of IgAN group, looking for correlations with clinical and pathology features and estimated glomerular filtration rate (eGFR) modifications from renal biopsy to sampling. Patients had a previous median follow-up of 6.4 (interquartile range 2.8–10.7) years and were divided into progressors and non-progressors according to the median value of their velocity of loss of renal function per year (−0.41 mL/min/1.73 m2/year). Results CD46 and CD55 messenger RNA (mRNA) expression in WBCs was not correlated with eGFR values or proteinuria at sampling. CD46 mRNA was significantly correlated with eGFR decline rate as a continuous outcome variable (P = 0.014). A significant difference was found in CD46 gene expression between progressors and non-progressors (P = 0.013). CD46 and CD55 mRNA levels were significantly correlated (P < 0.01), although no difference between progressors and non-progressors was found for CD55 mRNA values. The prediction of progression was increased when CD46 and CD55 mRNA expressions were added to clinical data at renal biopsy (eGFR, proteinuria and mean arterial blood pressure) and Oxford MEST-C (mesangial hypercellularity, endocapillary hypercellularity, segmental glomerulosclerosis, tubular atrophy/interstitial fibrosis, presence of any crescents) score. Conclusions Patients with progressive IgAN showed lower expression of mRNA encoding for the complement inhibitory protein CD46, which may implicate a defective regulation of C3 convertase with uncontrolled complement activation. [ABSTRACT FROM AUTHOR]

Clinical efficacy of complement C5a inhibition by IFX-1 in hidradenitis suppurativa: an open-label single-arm trial in patients not eligible for adalimumab Dear Editor, Circulating concentrations of the split complement factor C5a are significantly increased in hidradenitis suppurativa (HS).[1] C5a exerts strong chemotaxis and activation of neutrophils at the inflammatory areas so that blockade may be a promising therapeutic target. Six AEs were recorded during the treatment period: two HS exacerbations, one furuncle, one respiratory tract infection, one soft tissue infection and one increase of aspartate aminotransferase. [Extracted from the article]

Background: Emergence of antigenically drifted influenza A(H3N2) viruses resulted in reduced vaccine effectiveness in all age groups during the 2014-2015 influenza season. In children, inactivated influenza vaccine (IIV) elicited neutralizing antibodies (Abs) against drifted strains at significantly lower levels than against the vaccine strain. Little is known about the cross-reactivity of cell-mediated immunity against drifted strains in children.Methods: Children aged 3-17 years (n = 48) received IIV during the 2014-2015 influenza season. Peripheral blood mononuclear cells, collected before (on day 0) and after (on days 7 and 21) vaccination were evaluated for induction of cross-reactive plasmablasts, memory B cells, and cytokine-secreting CD4(+) and CD8(+) T cells against the vaccine and drifted A(H3N2) viruses by an enzyme-linked immunospot assay and flow cytometry.Results: IIV increased frequencies of plasmablasts and memory B cells. The overall induction of the T-cell response was not significant. Both B-cell and T-cell responses showed significant cross-reactivity against A(H3N2) viruses. Age and preexisting immunity affected virus-specific plasmablast responses and fold-change of T-cell responses, respectively. The proportion of T-helper type 1-prone (ie, interferon γ- or tumor necrosis factor α-secreting) CD4(+) T cell responses also increased with age.Conclusions: In children aged 3-17 years, B- and T-cell responses following IIV receipt showed significant cross-reactivity against A(H3N2) viruses during a vaccine mismatch season. [ABSTRACT FROM AUTHOR]

Background. In contrast to atypical haemolytic uraemic syndrome (aHUS), only single case reports and limited data have been published on systemic activation of the complement system and mutations in complement genes in paediatric enterohaemorrhagic Escherichia coli-induced HUS (EHEC-HUS). Methods. Complement activation (CH50, APH50, C3d, sC5b- 9) was analysed at four timepoints (Week 1, Week 2, Month 3 and Month 6 after primary diagnosis of HUS) in 25 children with EHEC-HUS. Seven patients received the complement C5 inhibitor eculizumab. Targeted next generation sequencing for a total of 89 genes involved in complement regulation and coagulation and haemostasis was performed in all patients. Results. Activity of classical (CH50) and alternative (APH50) complement pathways was normal or even elevated throughout the observation time, except for patients under eculizumab treatment. In contrast, the mean concentration of the soluble terminal complement complex (sC5b-9) was significantly elevated at the first timepoint (mean 498 ng/mL), dropping to normal values after 2 weeks. Initially elevated (42 mU/L) median C3d concentration reached normal levels fromWeek 2. Levels of sC5b-9 >320 ng/mL at the time of HUS diagnosis were associated with arterial hypertension, oedema and lower platelet counts, but not with the duration of dialysis. Genetic analysis revealed various changes that may have had a modifying impact on the clinical course. Conclusions. Complement activation at the acute phase of EHEC-HUS, indicated by increased levels of sC5b-9, predicts a poor outcome. Complement alterations appear to be more frequent in patients with EHEC-HUS than previously thought and are suspected to have a role in the severity of the disease. [ABSTRACT FROM AUTHOR]

Complement activation is of major importance in numerous pathological conditions. Therefore, targeted complement inhibition is a promising therapeutic strategy. C1-esterase inhibitor (C1-INH) controls activation of the classical pathway (CP) and the lectin pathway (LP). However, conflicting data exist on inhibition of the alternative pathway (AP) by C1-INH. The inhibitory capacity of C1-INH for the CP is potentiated by heparin and other glycosaminoglycans, but no data exist for the LP and AP. The current study investigates the effects of C1-INH in the presence or absence of different clinically used heparinoids on the CP, LP and AP. Furthermore, the combined effects of heparinoids and C1-INH on coagulation were investigated. C1-INH, heparinoids or combinations were analysed in a dose-dependent fashion in the presence of pooled serum. Functional complement activities were measured simultaneously using the Wielisa®-kit. The activated partial thrombin time was determined using an automated coagulation analyser. The results showed that all three complement pathways were inhibited significantly by C1-INH or heparinoids. Next to their individual effects on complement activation, heparinoids also enhanced the inhibitory capacity of C1-INH significantly on the CP and LP. For the AP, significant potentiation of C1-INH by heparinoids was found; however, this was restricted to certain concentration ranges. At low concentrations the effect on blood coagulation by combining heparinoids with C1-INH was minimal. In conclusion, our study shows significant potentiating effects of heparinoids on the inhibition of all complement pathways by C1-INH. Therefore, their combined use is a promising and a potentially cost-effective treatment option for complement-mediated diseases. [ABSTRACT FROM AUTHOR]

Active complement mediators play a key role in graft- versus-host diseases, but little attention has been given to the angiogenic balance and complement modulation during allograft acceptance. The complement cascade releases the powerful proinflammatory mediators C3a and C5a anaphylatoxins, C3b, C5b opsonins and terminal membrane attack complex into tissues, which are deleterious if unchecked. Blocking complement mediators has been considered to be a promising approach in the modern drug discovery plan, and a significant number of therapeutic alternatives have been developed to dampen complement activation and protect host cells. Numerous immune cells, especially macrophages, develop both anaphylatoxin and opsonin receptors on their cell surface and their binding affects the macrophage phenotype and their angiogenic properties. This review discusses the mechanism that complement contributes to angiogenic injury, and the development of future therapeutic targets by antagonizing activated complement mediators to preserve microvasculature in rejecting the transplanted organ. [ABSTRACT FROM AUTHOR]

Combined inhibition of complement and CD14 is known to attenuate bacterial-induced inflammation, but the dependency of the bacterial load on this effect is unknown. Thus, we investigated whether the effect of such combined inhibition on Escherichia coli- and Staphylococcus aureus-induced inflammation was preserved during increasing bacterial concentrations. Human whole blood was preincubated with anti-CD14, eculizumab (C5-inhibitor) or compstatin (C3-inhibitor), or combinations thereof. Then heat-inactivated bacteria were added at final concentrations of 5 × 104−1 × 108/ml ( E. coli) or 5 × 107−4 × 108/ml ( S. aureus). Inflammatory markers were measured using enzyme-linked immunosorbent assay (ELISA), multiplex technology and flow cytometry. Combined inhibition of complement and CD14 significantly ( P < 0.05) reduced E. coli-induced interleukin (IL)-6 by 40-92% at all bacterial concentrations. IL-1β, IL-8 and macrophage inflammatory protein (MIP)-1α were significantly ( P < 0.05) inhibited by 53-100%, and the effect was lost only at the highest bacterial concentration. Tumour necrosis factor (TNF) and MIP-1β were significantly ( P < 0.05) reduced by 80-97% at the lowest bacterial concentration. Monocyte and granulocyte CD11b were significantly ( P < 0.05) reduced by 63-91% at all bacterial doses. Lactoferrin was significantly ( P < 0.05) attenuated to the level of background activity at the lowest bacterial concentration. Similar effects were observed for S. aureus, but the attenuation was, in general, less pronounced. Compared to E. coli, much higher concentrations of S. aureus were required to induce the same cytokine responses. This study demonstrates generally preserved effects of combined complement and CD14 inhibition on Gram-negative and Gram-positive bacterial-induced inflammation during escalating bacterial load. The implications of these findings for future therapy of sepsis are discussed. [ABSTRACT FROM AUTHOR]

Background. Patients infected with influenza A(H7N9) virus present with acute lung injury (ALI) that is due to severe pneumonia and systemic inflammation. It is often fatal because there are few effective treatment options. Complement activation has been implicated in the pathogenesis of virus-induced lung injury; therefore, we investigated the effect of targeted complement inhibition on ALI induced by H7N9 virus infection. Methods. A novel neutralizing specific antihuman C5a antibody (IFX-1) was used. This antibody blocked the ability of C5a to induce granulocytes to express CD11b while not affecting the ability of C5b to form the membrane attack complex. African green monkeys were inoculated with H7N9 virus and treated intravenously with IFX-1. Results. The virus infection led to intense ALI and systemic inflammatory response syndrome (SIRS) in association with excessive complement activation. Anti-C5a treatment in H7N9-infected monkeys substantially attenuated ALI: It markedly reduced the lung histopathological injury and decreased the lung infiltration of macrophages and neutrophils. Moreover, the treatment decreased the intensity of SIRS; the body temperature changes were minimal and the plasma levels of inflammatory mediators were markedly reduced. The treatments also significantly decreased the virus titers in the infected lungs. Conclusions. Antihuman C5a antibody treatment remarkably reduced the ALI and systemic inflammation induced by H7N9 virus infection. Complement inhibition may be a promising adjunctive therapy for severe viral pneumonia. [ABSTRACT FROM AUTHOR]

Whether C5 blocking may improve the outcomes of patients developing chemotherapy-induced thrombotic microangiopathy (TMA) remains elusive. Lung fibrosis is a well-known complication of bleomycin, whereas TMAs are very rare (<20 cases described). Here, we report an exceptional case of a male patient that developed acute respiratory distress syndrome and TMA following administration of bleomycin, cisplatin and etoposide. Refractoriness to plasma exchanges prompted us to use eculizumab as salvage therapy. Eculizumab led to complete remission of the TMA before Day 2. However, the patient progressed towards refractory respiratory failure, suggesting that pathophysiological mechanisms of bleomycin-induced lung fibrosis and TMA differ. [ABSTRACT FROM AUTHOR]

Background An outbreak of haemolytic uraemic syndrome (HUS) due to Shiga toxin-secreting Escherichia coli (STEC) O104:H4 from contaminated fenugreek sprouts occurred in June 2011 near Bordeaux, France. In the context of this outbreak, all patients were treated with the monoclonal anti-C5 antibody, eculizumab. Methods The diagnosis of HUS was made based on haemolytic anaemia, low platelet count and acute kidney injury. Data were obtained from initial gastrointestinal symptoms to the end of follow-up 10 weeks after the start of eculizumab. Results Among 24 cases of STEC gastroenteritis, HUS developed in nine patients (eight adults and one child), 6 (median; range 3–12) days after digestive symptoms begun. The median (range) highest or lowest biological values were platelet count 26 (range 14–93) G/L; haemoglobin 6.6 (range 5–10.7) g/dL; LDH 1520 (range 510–2568) IU/L; creatinine 152 (range 48–797) µmol/L. All patients had extra-renal complications (liver 9, pancreas 5, brain 3 and heart 3). Two patients were dialysed, and one was ventilated. After failure of plasma exchange to increase platelets in the first three patients, eculizumab was administered in all nine patients, 0–4 days after HUS diagnosis (median 1 day). One patient with very severe neurological HUS received immunoadsorption. Outcome was favourable in all patients, with rapid normalization of haemoglobin, platelets, LDH levels, renal function and neurological improvement. There were no deaths and no serious adverse events related to eculizumab. Conclusions Early treatment of O104:H4 STEC-HUS by eculizumab was associated with a rapid and efficient recovery. Controlled prospective evaluation of eculizumab in STEC-HUS is warranted. [ABSTRACT FROM PUBLISHER]

Background Shiga toxin-induced haemolytic uraemic syndrome (STEC-HUS) is an acute multisystem disorder characterized by renal failure, neurological dysfunction, haemolysis and intravascular thrombosis. Circulating microparticles originating from a number of cell types including thrombocytes and leucocytes are elevated in paediatric patients. In vitro data also suggest modification of leucocyte death by Shiga toxin. Here, we investigated microparticle generation and leucocyte cell death in vivo in adult STEC-HUS patients during acute disease and recovery. Methods Multi-colour flow cytometry and immunofluorescence were used to assess microparticle concentration and provenience thrombocyte microparticle seeding to leucocytes and leucocyte cell death in adult STEC-HUS patients treated at a tertiary care centre during the STEC-HUS outbreak in Germany in 2011. Results Plasma microparticle concentrations of both platelet and leucocyte origin were elevated during acute STEC-HUS. Platelet microparticles (MP) were detected on a high proportion of monocytes and granulocytes. Among therapeutic interventions, plasma exchange reduced platelet marker expression on leucocytes, inhibition of complement had only moderate impact on the number of circulating MP and did not alter platelet microparticle binding to leucocytes. Numbers of apoptotic and necrotic monocytes and granulocytes were significantly increased in patients with STEC-HUS compared to healthy controls. Complement inhibition significantly increased the number of circulating apoptotic cells. Monocyte apoptosis on admission was significantly higher in patients subsequently assigned to plasma exchange or admitted to the intensive care unit. Conclusions In STEC-HUS, elevated numbers of circulating MP and dead leucocytes were detected. Monocyte and granulocyte deaths are novel markers of acute STEC-HUS that may actively contribute to tissue destruction by liberation of pro-inflammatory enzymes and cytokines. [ABSTRACT FROM PUBLISHER]

The article discusses research which examines the chromosomal instability and growth inhibition of cultured marsupial (opossum) cells following DNA polymerase α inhibitor treatment. The study showed that dehydroaltenusin inhibits euthorian and metatherian DNA replication. Further, it revealed that dehyroaltenusin clearly suppressed the growth of metatherian cultured cells.

The article discusses research which examines the effectiveness of ϵ-viniferin in red wine for inhibiting vascular smooth muscle cell (VSMC) migration and proliferation. The study showed that ϵ-viniferin is more effective in inhibiting VSMC proliferation compared to its monomer resveratrol. It revealed that nitric oxide compounds increased the expression of the antioxidant enzyme.

Primary cultures of human hepatocytes were used to investigate whether the dietary isothiocyanates, sulforaphane (SFN), and phenethyl isothiocyanate (PEITC) can reduce DNA adduct formation of the hepatocarcinogen aflatoxin B1 (AFB). Following 48 h of pretreatment, 10 and 50μM SFN greatly decreased AFB-DNA adduct levels, whereas 25μM PEITC decreased AFB-DNA adducts in some but not all hepatocyte preparations. Microarray and quantitative reverse transcriptase (RT)-PCR analyses of gene expression in SFN and PEITC-treated hepatocytes demonstrated that SFN greatly decreased cytochrome P450 (CYP) 3A4 mRNA but did not induce the expression of either glutathione S-transferase (GST) M1 or GSTT1. The protective effects of SFN required pretreatment; cotreatment of hepatocytes with SFN and AFB in the absence of pretreatment had no effect on AFB-DNA adduct formation. When AFB-DNA adduct formation was evaluated by GST genotype, the presence of one or two functional alleles of GSTM1 was associated with a 75% reduction in AFB-DNA adducts, compared with GSTM1 null. In conclusion, these results demonstrate that the inhibition of AFB-DNA adduct formation by SFN is dependent on changes in gene expression rather than direct inhibition of catalytic activity. Transcriptional repression of genes involved in AFB bioactivation (CYP3A4 and CYP1A2), but not transcriptional activation of GSTs, may be responsible for the protective effects of SFN. Although GSTM1 expression was not induced by SFN, the presence of a functional GSTM1 allele can afford substantial protection against AFB-DNA damage in human liver. The downregulation of CYP3A4 by SFN may have important implications for drug interactions. [ABSTRACT FROM PUBLISHER]

Induced pluripotent stem cell technology has attracted enormous interest for potential application in regenerative medicine. Here, we report that a specific glycogen synthase kinase 3 (GSK-3) inhibitor, CHIR99021, can induce the reprogramming of mouse embryonic fibroblasts transduced by only two factors, Oct4 and Klf4. When combined with Parnate (also named tranylcypromine), an inhibitor of lysine-specific demethylase 1, CHIR99021 can cause the reprogramming of human primary keratinocyte transduced with the two factors, Oct4 and Klf4. To our knowledge, this is the first time that human iPS cells have been generated from somatic cells without exogenous Sox2 expression. Our studies suggest that the GSK-3 inhibitor might have a general application to replace transcription factors in both mouse and human reprogramming. [ABSTRACT FROM AUTHOR]

Background. Emerging research suggests that genetically distinct strains of Mycobacterium tuberculosis may modulate the immune system differently. This may be of importance in high-burden settings where ⩾1 genetic group of M. tuberculosis confers significant morbidity. Methods. A dynamic mathematical model was constructed to evaluate how different degrees of cross-immunity among M. tuberculosis groups could affect epidemics of drug-resistant tuberculosis (TB). Results. Simulated populations with immunogenically distinct TB strain groups experienced a heightened risk of drug-resistant TB, compared with populations without such strain diversity, even when the same rates of case detection and treatment success were achieved. The highest risks of infection were observed in populations in which HIV was prevalent. Drug-resistant strains with very low transmission fitness could still propagate in environments with reduced cross-immunity among different strain groups, even after common targets for case detection and treatment success are reached. Conclusions. It is possible that the propagation of drug-resistant strains could depend not only on the rate of development of resistance and the fitness of the drug-resistant strains but, also, on the diversity of the strains in the region. The risk of infection with drug-resistant strains could be amplified in locations where there is reduced cross-immunity between originating strain groups. This amplification may be most profound during the first few decades of TB treatment expansion. [ABSTRACT FROM AUTHOR]

A faster and simpler method to monitor the photoinactivation process of Escherichia coli involving the use of recombinant bioluminescent bacteria is described here. Escherichia coli cells were transformed with luxCDABE genes from the marine bioluminescent bacterium Vibrio fischeri and the recombinant bioluminescent indicator strain was used to assess, in real time, the effect of three cationic meso-substituted porphyrin derivatives on their metabolic activity, under artificial (40 W m−2) and solar irradiation (≈620 W m−2). The photoinactivation of bioluminescent E. coli is effective (>4 log bioluminescence decrease) with the three porphyrins used, the tricationic porphyrin Tri-Py+-Me-PF being the most efficient compound. The photoinactivation process is efficient both with solar and artificial light, for the three porphyrins tested. The results show that bioluminescence analysis is an efficient and sensitive approach being, in addition, more affordable, faster, cheaper and much less laborious than conventional methods. This approach can be used as a screening method for bacterial photoinactivation studies in vitro and also for the monitoring of the efficiency of novel photosensitizer molecules. As far as we know, this is the first study involving the use of bioluminescent bacteria to monitor the antibacterial activity of porphyrins under environmental conditions. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Contact of blood with artificial surfaces and air as well as ischemia/reperfusion injury to the heart and lungs mediate systemic and local inflammation during cardiopulmonary bypass (CPB). Activation of complement and coagulation cascades leads to and accompanies endothelial cell damage. Therefore, endothelial-targeted cytoprotection with the complement inhibitor and endothelial protectant dextran sulfate (DXS, MW 5000) may attenuate CBP-associated myocardial and pulmonary injury. Methods: Eighteen pigs (DXS, n =10; phosphate buffered saline [PBS], n =8) underwent standard cardiopulmonary bypass. After aortic cross-clamping, cardiac arrest was initiated with modified Buckberg blood cardioplegia (BCP), repeated after 30 and 60min with BCP containing either DXS (300mg/10ml, equivalent to 5mg/kg) or 10ml of PBS. Following 30min reperfusion, pigs were weaned from CPB. During 2h of observation, cardiac function was monitored by echocardiography and invasive pressure measurements. Inflammatory and coagulation markers were assessed regularly. Animals were then sacrificed and heart and lungs analyzed. Results: DXS significantly reduced CK-MB levels (43.4±14.8ng/ml PBS, 35.9±11.1ng/ml DXS, p =0.042) and significantly diminished cytokine release: TNFalpha (1507.6±269.2pg/ml PBS, 222.1±125.6pg/ml DXS, p =0.0071), IL1beta (1081.8±203.0pg/ml PBS, 110.7±79.4pg/ml DXS, p =0.0071), IL-6 (173.0±91.5pg/ml PBS, 40.8±19.4pg/ml DXS, p =0.002) and IL-8 (304.6±81.3pg/ml PBS, 25.4±14.2pg/ml DXS, p =0.0071). Tissue endothelin-1 levels were significantly reduced (6.29±1.90pg/100mg PBS, 3.55±1.15pg/100mg DXS p =0.030) as well as thrombin–anti-thrombin formation (20.7±1.0μg/ml PBS, 12.8±4.1μg/ml DXS, p =0.043). Also DXS reduced cardiac and pulmonary complement deposition, neutrophil infiltration, hemorrhage and pulmonary edema (measured as lung water content, 81±3% vs 78±3%, p =0.047), indicative of attenuated myocardial and pulmonary CPB-injury. Diastolic left ventricular function (measured as dp/dt min), pulmonary artery pressure (21±3mmHg PBS, 19±3mmHg DXS, p =0.002) and right ventricular pressure (21±1mmHg PBS, 19±3mmHg DXS p =0.021) were significantly improved with the use of DXS. Conclusions: Addition of DXS to the BCP solution ameliorates post-CPB injury and to a certain extent improves cardiopulmonary function. Endothelial protection in addition to myocyte protection may improve post-CPB outcome and recovery. [Copyright &y& Elsevier]

In this study, we show that berberine chloride (BBR) has antimicrobial activities against all 43 tested strains of Staphylococcus aureus, an important human and animal pathogen. However, the response mechanisms of S. aureus to BBR are still poorly understood. Affymetrix GeneChips were used to determine the global transcription of S. aureus triggered by treatment with subinhibitory concentrations of BBR. 468 genes were up-regulated and 262 genes were down-regulated upon exposure to BBR. There was elevated transcription of various transporter genes, including genes involved in multidrug resistance, members of the multidrug and toxin extrusion family, the ferrous iron transporter, the amino acid transporter, the Na+/H+ antiporter, and the potassium cation transporter. Measurements of active transport were used to demonstrate a phenotypic correlation between efflux transporter overexpression and inhibition of BBR uptake. Furthermore, BBR induced the expression of urease genes, sortase enzyme, and iron-regulated surface determinant genes, but repressed transcription of a gene encoding arylamine N-acetyltransferase activity (N315-SA2490). To our knowledge, this is the first analysis of a genome-wide transcription profile of S. aureus cells in response to BBR treatment. These results will pave the way to exploring the mechanisms of BBR against S. aureus. [ABSTRACT FROM AUTHOR]

The metabolic pathway involving dihydroxyacetone is poorly characterized although novel enzymes associated with this metabolite have recently been demonstrated. The role of GldA in dihydroxyacetone and methylglyoxal metabolism was investigated by purifying the enzyme and characterizing its catalytic ability using nuclear magnetic resonance (NMR) spectroscopy. At neutral pH, the enzyme exhibits much higher affinities towards dihydroxyacetone, methylglyoxal, and glycolaldehyde than glycerol with Km values of 0.30, 0.50, 0.85, and 56 mM, respectively. This is consistent with NMR data with crude extracts, showing that the conversion from dihydroxyacetone to glycerol by GldA is far more efficient than the reverse reaction. Dihydroxyacetone was found to be lethal at higher concentration with an LC50 value of 28 mM compared with 0.4 mM of methylglyoxal, while lactaldehyde was found to exhibit significant growth inhibition in Escherichia coli cells. The toxicity of dihydroxyacetone appears to be due to its intracellular conversion to an aldehyde compound, presumably methylglyoxal, since the glyoxalase mutant becomes sensitive to dihydroxyacetone. Based on information that gldA is preceded in an operon by the ptsA homolog and talC gene encoding fructose 6-phosphate aldolase, this study proposes that the primary role of gldA is to remove toxic dihydroxyacetone by converting it into glycerol. [ABSTRACT FROM AUTHOR]

A novel compound designated zafrin [4β-methyl-5, 6, 7, 8 tetrahydro-1 (4β-H)-phenanthrenone] was isolated from a crude extract of a marine bacterium identified as Pseudomonas stutzeri. Zafrin showed strong antibacterial activity against both Gram-positive and Gram-negative bacteria. The compound was purified and its structure was elucidated by spectroscopic methods including 1H-nuclear magnetic resonance (NMR), 13C-NMR, 1D-NMR and 2D-NMR spectroscopy. It could be demonstrated that a purified solution of zafrin was active against several human pathogens, including Staphylococcus aureus, and Salmonella typhi. By contrast, zafrin did not inhibit the growth of eukaryotic organisms Candida albicans and Schizosaccharomyces pombe. The minimal inhibitory concentration for Gram-positive bacteria ranged from 50 to 75 μg mL−1 and varied between 75 and 125 μg mL−1 for Gram-negative bacteria. Zafrin lysed Bacillus subtilis cells grown in an osmotically protected medium, suggesting that it does not act upon the cell wall. Further investigation using B. subtilis indicated that the compound is bactericidal and is likely to target the cell membrane. [ABSTRACT FROM AUTHOR]

The isoprenoid farnesol was previously shown to induce morphological features characteristic of apoptosis in the filamentous fungus Aspergillus nidulans. This study demonstrates that under similar liquid media growth conditions, farnesol also triggers apoptosis in the plant pathogenic fungus Fusarium graminearum. However, unlike A. nidulans, F. graminearum spores treated with farnesol exhibited altered germination patterns and most (>60%) lysed upon prolonged exposure. Given the economic importance of F. graminearum as a pathogen of small grains, this study proposes that farnesol may have potential value as an antifungal compound. [ABSTRACT FROM AUTHOR]

The complement inhibitor Factor H has three distinct binding sites for C3b and for heparin, but in solution uses specifically the most C-terminal domain, i.e. short consensus repeats (SCR) 20 for ligand interaction. Two novel monoclonal antibodies (mABs C14 and C18) that bind to the most C-terminal domain SCR 20 completely blocked interaction of Factor H with the ligands C3b, C3d, heparin and binding to endothelial cells. In contrast, several mAbs that bind to the N-terminus and to the middle regions of the molecule showed no or minor inhibitory effects when assayed by enzyme-linked immunosorbent assay (ELISA) and ligand interaction assays. This paradox between a single functional binding site identified for native Factor H versus multiple interaction sites reported for deletion constructs is explained by a compact conformation of the fluid phase protein with one accessible binding site. On zymosan particles mAbs C14 and C18 blocked alternative pathway activation completely. Thus demonstrating that native Factor H makes the first and initial contact with the C terminus, which is followed by N terminally mediated complement regulation. These results are explained by a conformational hypothetical model: the native Factor H protein has a compact structure and only one binding site accessible. Upon the first contact the protein unfolds and exposes the additional binding sites. This model does explain how Factor H mediates recognition functions during complement control and the clustering of disease associated mutations in patients with haemolytic uraemic syndrome that have been reported in the C-terminal recognition domain of Factor H. [ABSTRACT FROM AUTHOR]

Aims Pexelizumab, a monoclonal antibody inhibiting C5, reduced 90 day mortality and shock in the COMplement inhibition in Myocardial infarction treated with Angioplasty (COMMA) trial without apparent reductions in infarct size. Inflammation is a critical component of ST-elevation myocardial infarction (STEMI); this substudy examines prognostic values of selected markers and treatment effects. [ABSTRACT FROM PUBLISHER]

Lactoferrin is an important biological molecule with many functions such as modulation of the inflammatory response, iron metabolism and antimicrobial defense. One effect of lactoferrin is the inhibition of the classical complement pathway. This study reports that antimicrobial peptides derived from the N-terminal region from both human and bovine lactoferrin, lactoferricin H and lactoferricin B, respectively, inhibit the classical complement pathway. No inhibitory effect of these peptides was observed on the alternative complement pathway in an AP50 assay. However, lactoferricin B reduced the inhibitory properties of serum against Escherichia coli in a concentration dependent manner. These results suggest that the N-terminal region of lactoferrin is the important part in the inhibition of complement activation and that these peptides possess other important properties than their antimicrobial effect. [Copyright &y& Elsevier]

Complement receptor 1-related gene/protein y (Crry) is a potent murine complement regulator that inhibits C3 convertases. Transgenic mice that overexpress soluble Crry (sCrry), directed systemically by the metallothionein-I promoter, have been used as an animal model for chronic blockade of complement activation. Recently we have found that alternative pathway (AP) activity in Crry transgenic mice was not inhibited as much as expected. To elucidate the mechanism of this effect, we evaluated the AP activities and levels of sCrry and AP complement components in transgenic and non-transgenic mice. In transgenic mice, expression of sCrry was induced by feeding zinc sulphate solution to 70·1 ± 42·7 µg/ml mean serum level. Its corresponding level of purified sCrry inhibited 49% of AP activity of normal mice serum; however, the actual AP activities in transgenic mice were not decreased when compared to non-transgenic mice (130·2 ± 9·0% versus 113·0 ± 35·4%). Expressed sCrry was functional, as immunoprecipitation and removal of sCrry from transgenic sera with rabbit anti-Crry polyclonal antibody resulted in enhanced AP activity, consistent with initial levels of sCrry. We then compared the changes to C3, factor B, factor H and factor D serum levels in transgenic and non-transgenic mice after induction of sCrry expression. Of these only C3 was increased after zinc feeding in transgenic mice compared to non-transgenic mice (142·8 ± 14·1% versus 121·4 ± 15·1%, P = 0·023). These results suggest that the inhibitory effect of chronic exposure to sCrry is compensated by concomitant alteration in C3 levels. This result also suggests the presence of a complement regulatory protein controls the level of serum C3, which has potential importance in the design and interpretation of studies involving chronic use of complement inhibitors. [ABSTRACT FROM AUTHOR]

Previous studies have shown that DAF (or CD55), a cell surface inhibitor of autologous C3 activation, is present in tears and that > 90% of the C3 convertase regulatory activity in tear fluid resides in this protein (Lass JH et al., Invest Ophth Vis Sci 1990; 31:1136–48). This study investigated whether (i) the membrane cofactor protein (MCP or CD46), an additional factor that regulates C3 activation, and (ii) the membrane inhibitor of reactive lysis (MIRL or CD59), a cell surface regulator that acts to prevent formation of the membrane attack complex, are also present in tears, and if so, are functional. Two-site immunoradiometric assays showed that MCP is present in tears at low levels (42 + 8 ng/ml, n = 8) while CD59 is present at levels (222 + 78 ng/ml, n = 14) comparable to those of DAF (325 + 289 ng/ml, n = 12). The concentrations of CD59 (i) were increased two-fold or more in closed eye tears, and (ii) were decreased in reflex tears. Western blotting showed that CD59 protein in tears migrates with an apparent mol. wt similar to membrane CD59 protein. Phenyl–Sepharose adsorption and Triton X-114 partitioning of tear CD59 as well as of tear DAF however, showed that both proteins are devoid of GPI anchors. Assays using cobra venom factor-activated human serum and guinea pig erythrocytes showed that CD59 is functionally active in inhibiting autologous C5b-9-mediated lysis and, under constitutive conditions, accounts for > 85% of the C9 inhibitory activity in tear fluid. [ABSTRACT FROM AUTHOR]

Normal human serum contains an inhibitor of complement which is distinguished by its small size of 500 daltons. the low molecular weight inhibitor (LMWI). When LMWI was present during incubation of zymosan or cobra venom factor with serum, formation of complement reactive complexes was blocked as measured by failure of these mixtures to lyse susceptible erythrocytes from patients with paroxysmal nocturnal haemoglobinuria (PNH). Addition of LMWI to pre-formed complexes had no effect on their subsequent haemolytic activity. When dialysis was used to remove LMWI from reaction mixtures, it was shown that LMWI had not irreversibly altered any of the complement components. Purified components were used to demonstrate that LMWI prevented factor D activation of cobra venom factor-factor B complexes. LMWI also strongly inhibited binding of 125I-factor B to human erythrocytes bearing C3b and had little or nor effect on binding of 125I-factor H to the C3b bearing cells. Factor B binding to C3b was equally inhibited on normal and PNH erythrocytes. Thus, a dialysable fraction from normal human serum prevents activation of human complement by blocking formation, but not the activity of the C3/C5 convertase. These low molecular weight inhibitors result in inhibition of factor B binding to C3b and inhibition of factor D activation of C3bB complexes. [ABSTRACT FROM AUTHOR]

Humoral and cell-mediated immune responses to Salmonella typhi were studied in 10 chronic typhoid carriers, and in healthy controls. Carriers showed impaired cellular reactivity to S. typhi antigens in the leucocyte migration inhibition test (LMI), Carriers did not show a generalized depression of cell-mediated immunity in that delayed hypersensitivity skin test responses to recall antigens, peripheral blood T cell numbers, and lymphocyte transformation responses to mitogens were normal. Lymphocyte transformation in the presence of S. typhi antigen occurred to a greater extent than normal in four of six subjects tested and suggested the possibility of dissociated defects of cellular immunity. Carriers showed normal humoral immunity, as judged by antibodies to the flagellar and somatic antigens of S. typhi and S. paratyphi and to Vi antigens of S, typhi. The results suggest that the carrier state may be the consequence of a specific defect in cell-mediated immune responses to S, typhi. [ABSTRACT FROM AUTHOR]

Normal human serum and urine were found to contain a low molecular weight complement inhibitor (LMWI). LMWl was separated from serum by dialysis in membrane tubing or through an Amicon PM10, and then concentrated on an Amicon UM05 membrane. On Bio-Gel P-2 filtration. LMWI was eluted just after the column calibration marker, stachyose hydrate (666.6 daltons), and was estimated to be 500 daltons. Both pathways of complement activation were susceptible to modulation by LMWI. Addition of LMWI reduced the haemolysis of sheep erythrocytes sensitized with antibody, rabbit erythrocytes and guinea-pig erythrocytes bearing human C3 and C4. Formation of EAC&142sline; from EACI4and guinea-pig C2 was blocked, indicating a failure to generate the classical pathway C3 convertase; however, the lysis of preformed EAC&142sline; was not suppressed. Conversion of factor B and C3 did not occur when LMWI was present during zymosan activation of serum. This indicates that the inhibitor either prevented, or acted at a step prior to, the cleavage of factor B by factor D. LMWI did not prevent formation of erythrocyte-C567 intermediates nor their subsequent lysis by C8 and C9. Thus, serum contains a 500-dalton Inhibitor which modulates the activities of both complement pathways at an early step in each of the activation sequences. LMWI may serve as a regulator of the inflammatory process by suppressing C3 convertase formation and generation of complement-derived, biologically reactive molecules. [ABSTRACT FROM AUTHOR]

Minor elevation of the concentration of the C3b inactivator (KAF) in whole serum (by 15-25%) markedly inhibits the capacity of the serum to support complement activation by inulin, aggregated IgG and by low concentrations at CVF. However, there was no effect when large concentrations of CVF were used nor was the spontaneous ageing of C3 slowed by increased KAF concentrations. These findings show that the activity of the C3b feedback cycle can be reduced by raising the KAF concentration above physiological levels. This finding may provide a mechanism for damping down complement activation locally in vivo. It seems that the spontaneous ageing of C3 in vitro does not involve a KAF- inhibitable step and therefore cannot be involved to explain the `C3 tickover' in vitro. [ABSTRACT FROM AUTHOR]

Synthetic conjugates of β-lactoglobulin and reducing sugar prepared by Maillard reaction acquire the capacity of inactivating haemolytic complement in human serum. The C-inactivating potential is related to the mean number of (lysine)-ε-amino-1-deoxy-2-ketose residues incorporated in the model compound. These findings are compared to results obtained by skin-testing similar artificial allergens in atopic subjects. [ABSTRACT FROM AUTHOR]

A series of experiments is reported to identify and characterize the inactivation process in operation when drinking water, heavily contaminated with a Kenyan isolate of Escherichia coli , is stored in transparent plastic bottles that are then exposed to sunlight. The roles of optical and thermal inactivation mechanisms are studied in detail by simulating conditions of optical irradiance, water turbidity and temperature, which were recorded during a series of solar disinfection measurements carried out in the Kenyan Rift Valley. Optical inactivation effects are observed even in highly turbid water (200 ntu) and at low irradiances of only 10 mW cm-2 . Thermal inactivation is found to be important only at water temperatures above 45 °C, at which point strong synergy between optical and thermal inactivation processes is observed. The results confirm that, where strong sunshine is available, solar disinfection of drinking water is an effective, low cost method for improving water quality and may be of particular use to refugee camps in disaster areas. Strategies for improving bacterial inactivation are discussed. [ABSTRACT FROM AUTHOR]

Human lung cancer expresses cell membrane complement inhibitory proteins (CIP). We investigated whether human lung cancer cell lines also express cell-membrane CIP molecules and whether the biology of CIP molecules in these cell lines differs from that of CIP in normal human respiratory epithelium in culture. The cell lines ChaGo K-1 and NCI-H596 were compared with normal human nasal epithelium in primary cultures in respect to the level of cell membrane CIP expression of membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55) and CD59, in respect to the level of cell resistance to complement-mediated lysis, and in respect to the contribution of cell membrane CIP to cell resistance against complement-mediated lysis. We found, using flow cytometry, that both human lung cancer cell lines expressed MCP, DAF and CD59, as did normal nasal epithelial cells. However, normal cells showed a large subpopulation of low DAF-expressing cells (60% of all cells) and a smaller subpopulation of high DAF-expressing cells (40%), while the lung cancer cell lines showed only one cell population, of high DAF expression. In addition, both lung cancer cell lines expressed higher MCP levels, and NCI-H596 cells showed higher levels of CD59. Cell resistance to complement-mediated lysis of both lung cancer cell lines was much higher than that of normal cells. Fifty percent normal human serum, under the same concentrations of complement activators, induced lysis of less than a mean of 10% of lung cancer cells, while lysing up to a mean of 50% of nasal epithelial cells. Lung cancer cell resistance to complement was due to its ability to prevent significant activation of complement upon its cell membrane, as manifested by a failure of complement activators to increase cell membrane deposition of C3-related fragments. The exact mechanism for this resistance remains obscure. Unexpectedly, neutralizing antibodies, anti-MCP and anti-DAF were entirely ineffective and anti-CD59... [ABSTRACT FROM AUTHOR]

It has been previously reported that the expression of the complement receptor, CR1, on erythrocytes is reduced in patients with RA and that the reduced expression of CR1 is related to disease activity. In this study we investigate the role of other regulatory proteins, i.e. decay-accelerating factor (DAF) and CD59 (membrane inhibitor of reactive lysis), in the pathogenesis of RA by checking the expression of DAF and CD59 on erythrocytes of RA patients to establish whether reduced expression of DAF and CD59 on erythrocytes could be related to increased ability of erythrocytes to activate complement in RA. Flow cytometry was used to measure the expression of DAF and CD59 on erythrocytes from RA patients as well as the deposition of C3 fragments occurring in vivo or after in vitro complement activation. Significantly reduced expression of DAF and CD59 was observed on erythrocytes of RA patients. A significant inverse relationship was observed between DAF expression and in vitro complement activation, whereas no significant relationship between CD59 and complement activation was observed. Finally, we demonstrated an inverse relationship between CH50 activity and DAF expression. Thus, determination of DAF on erythrocytes can emerge as an additional tool in the assessment of extent of complement activation in RA. [ABSTRACT FROM AUTHOR]

Immunotherapy using MoAbs is inefficient due to limited activation of human effectors by mouse antibodies and multiple protective mechanisms available to host cells against autologous complement. We have used chemically engineered antibody constructs and human complement in vitro to specifically target and kill neoplastic B lymphoid cells (Raji). Fab'γFcγ2 chimaeric antibody (specific for human CD37) was used to activate the classical pathway of human complement on Raji cells, whilst CD59 was neutralized using one of two different bispecific F(ab'γ)2 antibody constructs which contained both cell-targeting (anti-CD19 or anti-CD38) and CD59-neutralizing moieties. When either bispecific construct was used to neutralize CD59, 15-25% of cells were lysed. If CD55 was also neutralized using specific antibody, Raji cells were efficiently killed (70% lysis). When added to a mixture of target (Raji) and bystander (K562) cells, one bispecific antibody (anti-CD38 × anti-CD59) could be specifically delivered to Raji, avoiding significant uptake on CD59-expressing bystander cells (K562). The second bispecific antibody (anti-CD19 × anti-CD59) bound equally well to either cell type. Cell-specific targeting was dependent upon combination of a low-affinity anti-CD59 Fab'γ with a high-affinity antitumour cell Fab'γ, When Raji and K562 cells were mixed and incubated with a combination of the engineered constructs and ami-CD55 antibodies, Raji cell lysis (30-40%) was observed in the absence of K562 killing. We propose that combinations of these constructs may be of use for treatments such as ex vivo purging of autologous bone marrow or in vivo targeting of tumour cells. [ABSTRACT FROM AUTHOR]

Hyperacute rejection triggered by activation of the recipient's complement system represents the major barrier to successful xenotransplantation. Transfer of human membrane-associated complement regulators to donor organs has been suggested as one strategy to interfere with complement-mediated hyperacute xenograft rejection. Pigs are discussed as potential organ donors. We therefore investigated a putative protective function of the membrane-bound complement inhibitor CD59 in a pig-to-human in vitro model of hyperacute xenograft rejection. Aortic porcine endothelial cells were transfected with human CD59 cDNA. Expression of human CD59 was demonstrated by cytofluorimetric and RNA analysis. Removal of CD59 from the cell surface by phosphatidylinositol-specific phospholipase C (PI-PLC) demonstrated its production as a glycosyl phosphatidylinositol (GPI)-anchored protein. Functional activity of the transfected CD59 was tested by a lactate dehydrogenase (LDH) release assay for complement-mediated lysis. Porcine endothelial cells expressing human CD59 were significantly protected from lysis by human serum complement compared with CD59- cells. The protective effect was abolished by preincubating the cells with anti-CD59 antibodies or PI-PLC. We calculated by Scatchard analysis that the established CD59+ cell line expressed a CD59 level comparable to that of human endothelial cells. Our results recommend the production of pigs transgenic for CD59. [ABSTRACT FROM AUTHOR]

Extracts of the herb Ephedra sinica have long been used in traditional Chinese medicine for the treatment of, among other conditions, acute nephritis. In preliminary studies it was shown that extracts of ephedra caused inhibition of complement in vitro. We thus set out to isolate the active component(s) of this herb, to examine the complement-inhibiting capacity in sera from different species, and to characterize [he mechanism(s) by which it inhibits complement. Aqueous extraction of the herb followed by fractionation using thin layer chromatography (TLC) demonstrated that complement-inhibiting activity resided within a single band, hereafter termed the complement-inhibiting component (CIC), which represents an as yet uncharacterized polyanionic carbohydrate molecule, TLC-purified CIC inhibited the classical complement pathway in all species tested (human, pig, guinea pig, rat and rabbit). Using erythrocyte intermediates and sera pecifically depleted of individual components it was apparent that CIC inhibited C2. This- finding was confirmed using purified human C2, CIC causing a dose-dependent loss of C2 haemolytic activity. At much higher doses, CIC also showed some inhibiting effect in the terminal pathway, and this was shown to be due to inhibition of C9. In the alternative pathway CIC also showed inhibitory activity, although its site of action in this pathway remains unresolved. In Chinese medicine the herb is taken orally, though no studies of complement levels in patients taking the herb have been reported. Preliminary data indicate that oral administration in rats causes a partial inhibition of serum complement activity. Given the current enthusiasm for complement inhibition as a therapy for inflammatory diseases, this non-toxic, naturally occurring agent might be of therapeutic value. [ABSTRACT FROM AUTHOR]