Intracellular complement activation-An alarm raising mechanism?

It has become increasingly apparent that the complement system, being an ancient defense mechanism, is not operative only in the extracellular milieu but also intracellularly. In addition to the known synthetic machinery in the liver and by macrophages, many other cell types, including lymphocytes, adipocytes and epithelial cells produce selected complement components. Activation of e.g. C3 and C5 inside cells may have multiple effects ranging from direct antimicrobial defense to cell differentiation and possible influence on metabolism. Intracellular activation of C3 and C5 in T cells is involved in the maintenance of immunological tolerance and promotes differentiation of T helper cells into Th1-type cells that activate cell-mediated immune responses. Adipocytes are unique in producing many complement sensor proteins (like C1q) and Factor D (adipsin), the key enzyme in promoting alternative pathway amplification. The effects of complement activation products are mediated by intracellular and cell membrane receptors, like C3aR, C5aR1, C5aR2 and the complement regulator MCP/CD46, often jointly with other receptors like the T cell receptor, Toll-like receptors and those of the inflammasomes. These recent observations link complement activation to cellular metabolic processes, intracellular defense reactions and to diverse adaptive immune responses. The complement components may thus be viewed as intracellular alarm molecules involved in the cellular danger response.

Dysfunction of complement receptors CR3 (CD11b/18) and CR4 (CD11c/18) in pre-eclampsia: a genetic and functional study.

OBJECTIVE: To study genetic variants and their function within genes coding for complement receptors in pre-eclampsia. DESIGN: A case-control study. SETTING: Pre-eclampsia is a common vascular disease of pregnancy. The clearance of placenta-derived material is one of the functions of the complement system in pregnancy. POPULATION: We genotyped 500 women with pre-eclamptic pregnancies and 190 pregnant women without pre-eclampsia, as controls, from the FINNPEC cohort, and 122 women with pre-eclamptic pregnancies and 1905 controls from the national FINRISK cohort. METHODS: The functional consequences of genotypes discovered by targeted exomic sequencing were explored by analysing the binding of the main ligand iC3b to mutated CR3 or CR4, which were transiently expressed on the surface of COS-1 cells. MAIN OUTCOME MEASURES: Allele frequencies were compared between pre-eclamptic pregnancies and controls in genetic studies. The functional consequences of selected variants were measured by binding assays. RESULTS: The most significantly pre-eclampsia-linked CR3 variant M441K (P = 4.27E-4, OR = 1.401, 95% CI = 1.167-1.682) displayed a trend of increased adhesion to iC3b (P = 0.051). The CR4 variant A251T was found to enhance the adhesion of CR4 to iC3b, whereas W48R resulted in a decrease of the binding of CR4 to iC3b. CONCLUSIONS: Results suggest that changes in complement-facilitated phagocytosis are associated with pre-eclampsia. Further studies are needed to ascertain whether aberrant CR3 and CR4 activity leads to altered pro- and anti-inflammatory cytokine responses in individuals carrying the associated variants, and the role of these receptors in pre-eclampsia pathogenesis. TWEETABLE ABSTRACT: Genetic variants of complement receptors CR3 and CR4 have functional consequences that are associated with pre-eclampsia.

Tumour-cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma.

BACKGROUND: The incidence of epidermal keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is increasing worldwide. OBJECTIVES: To study the role of the complement classical pathway components C1q, C1r and C1s in the progression of cSCC. METHODS: The mRNA levels of C1Q subunits and C1R and C1S in cSCC cell lines, normal human epidermal keratinocytes, cSCC tumours in vivo and normal skin were analysed with quantitative real-time polymerase chain reaction. The production of C1r and C1s was determined with Western blotting. The expression of C1r and C1s in tissue samples in vivo was analysed with immunohistochemistry and further investigated in human cSCC xenografts by knocking down C1r and C1s. RESULTS: Significantly elevated C1R and C1S mRNA levels and production of C1r and C1s were detected in cSCC cells, compared with normal human epidermal keratinocytes. The mRNA levels of C1R and C1S were markedly elevated in cSCC tumours in vivo compared with normal skin. Abundant expression of C1r and C1s by tumour cells was detected in invasive sporadic cSCCs and recessive dystrophic epidermolysis bullosa-associated cSCCs, whereas the expression of C1r and C1s was lower in cSCC in situ, actinic keratosis and normal skin. Knockdown of C1r and C1s expression in cSCC cells inhibited activation of extracellular signal-related kinase 1/2 and Akt, promoted apoptosis of cSCC cells and significantly suppressed growth and vascularization of human cSCC xenograft tumours in vivo. CONCLUSIONS: These results provide evidence for the role of tumour-cell-derived C1r and C1s in the progression of cSCC and identify them as biomarkers and putative therapeutic targets in cSCC. What's already known about this topic? The incidences of actinic keratosis, cutaneous squamous cell carcinoma (cSCC) in situ and invasive cSCC are increasing globally. Few specific biomarkers for progression of cSCC have been identified, and no biological markers are in clinical use to predict the aggressiveness of actinic keratosis, cSCC in situ and invasive cSCC. What does this study add? Our results provide novel evidence for the role of complement classical pathway components C1r and C1s in the progression of cSCC. What is the translational message? Our results identify complement classical pathway components C1r and C1s as biomarkers and putative therapeutic targets in cSCC.

Distribution of exogenous complement factor H in mice in vivo.

Factor H is an important regulator of complement activation in plasma and on cell surfaces in both humans and mice. If FH function is compromised, inappropriate complement activation on self-surfaces can have disastrous effects as seen in the kidney diseases atypical haemolytic uremic syndrome (aHUS) and C3 glomerulopathy. As FH constructs have been proposed to be used in treatment for these diseases, we studied the distribution of exogenous FH fragments in mice. Full-length mFH, mFH1-5 and mFH18-20 fragments were radiolabelled, and their distribution was examined in WT, FH-/- and FH-/- C3-/- mice in vivo. Whole body scintigraphy revealed accumulation of radioactivity in the abdominal part of the mice, but also to the thyroid gland and urinary bladder. At organ level in WT mice, some full-length FH accumulated in internal organs, but most of it remained in the circulation. Both of the mFH fragments accumulated in the kidneys and were excreted in urine. For mFH1-5, urinary secretion is the likely cause for the accumulation. Concentration of mFH18-20 to kidneys was slower, and at tissue level, mFH18-20 was localized at the proximal tubuli in WT and FH-/- C3-/- mice. No C3-independent binding to glomeruli was detected. In conclusion, these results show that glomerular glycosaminoglycans and sialic acids alone do not collect FH in kidneys. Deposition of C3 fragments is also needed, which implies that in aHUS, the problem is in simultaneous recognition of C3 fragments and glycosaminoglycans or sialic acids by FH, not just the inability of FH to recognize glomerular endothelium as such.

C4B gene influences intestinal microbiota through complement activation in patients with paediatric-onset inflammatory bowel disease.

Complement C4 genes are linked to paediatric inflammatory bowel disease (PIBD), but the mechanisms have remained unclear. We examined the influence of C4B gene number on intestinal microbiota and in-vitro serum complement activation by intestinal microbes in PIBD patients. Complement C4A and C4B gene numbers were determined by genomic reverse transcription-polymerase chain reaction (RT-PCR) from 64 patients with PIBD (Crohn's disease or ulcerative colitis). The severity of the disease course was determined from faecal calprotectin levels. Intestinal microbiota was assessed using the HITChip microarray. Complement reactivity in patients was analysed by incubating their sera with Yersinia pseudotuberculosis and Akkermansia muciniphila and determining the levels of C3a and soluble terminal complement complex (SC5b-9) using enzyme immunoassays. The microbiota diversity was wider in patients with no C4B genes than in those with one or two C4B genes, irrespective of intestinal inflammation. C4B and total C4 gene numbers correlated positively with soluble terminal complement complex (TCC, SC5b-9) levels when patient serum samples were stimulated with bacteria. Our results suggest that the C4B gene number associates positively with inflammation in patients with PIBD. Multiple copies of the C4B gene may thus aggravate the IBD-associated dysbiosis through escalated complement reactivity towards the microbiota.

Identification of Glyceraldehyde-3-Phosphate and Alcohol Dehydrogenases as Autoantigens in Doberman Hepatitis.

An autoimmune background is suspected for Doberman hepatitis (DH). It is based on the finding of mononuclear cell infiltrates in the liver, strong female bias, association to the homozygous risk factor dog leucocyte antigen (DLA) allele DRB1*00601 and aberrant major histocompatibility complex (MHC) class II expression on hepatocytes that correlates with the degree of inflammation in the liver. The aim of this study was to search for autoantibodies against liver-related antigens associated with DH. Twenty-five Dobermans with subclinical DH (SDH), 13 that clinically manifest DH (CDH) and 17 healthy controls were studied. Immunoblotting analysis detected specific antibodies in the DH sera. By mass spectrometry the targets were identified as liver-related enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and alcohol dehydrogenase (ADH). Using ELISA, anti-GAPDH IgG was detected in 36% (9/25) of SDH dogs and 69.2% (9/13) of the CDH dogs compared to healthy controls (0/17) (P < 0.0005). Anti-ADH IgG was detected in 72% (18/25) of SDH dogs and 76.9% (10/13) of CDH dogs and only in one (1/17) control (P < 0.0005). The finding of novel autoantigens, GAPDH and ADH strengthen the hypothesis that DH is an autoimmune disease of the liver. These findings suggest that DH could be diagnosed by screening for autoantibodies against the defined antigens.

Microbes bind complement inhibitor factor H via a common site.

To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19-20 (FH19-20). We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii). We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens via utilization of a "superevasion site."

Autoimmunity, Not a Developmental Defect, is the Cause for Subfertility of Autoimmune Regulator (Aire) Deficient Mice.

Autoimmune regulator's (AIRE) best characterized role is in the generation immunological tolerance, but it is also involved in many other processes such as spermatogenesis. Loss-of-function mutations in AIRE cause a disease called autoimmune polyendocrinopathy, candidiasis and ectodermal dystrophy (APECED; also called autoimmune polyendocrinopathy syndrome type 1, APS-1) that is dominated by various autoimmune manifestations, mainly endocrinopathies. Both patients with APECED and Aire(-/-) mice suffer from varying levels of infertility, but it is not clear if it is a result of an autoimmune tissue damage or more of a developmental defect. In this study, we wanted to resolve whether or not the reduced fertility of Aire(-/-) mice is dependent on the adaptive immune system and therefore a manifestation of autoimmunity in these mice. We generated lymphopenic mice without Aire expression that were devoid of the autoimmune manifestations previously reported in immunocompetent Aire(-/-) mice. These Aire(-/-) Rag1(-/-) mice regained full fertility. This confirms that the development of infertility in Aire(-/-) mice requires a functional adaptive immune system. We also show that only the male Aire(-/-) mice are subfertile, whereas Aire(-/-) females produce litters normally. Moreover, the male subfertility can be adoptively transferred with lymphocytes from Aire(-/-) donor mice to previously fertile lymphopenic Aire(-/-) recipients. Our data show that subfertility in Aire(-/-) mice is dependent on a functional adaptive immune system thus confirming its autoimmune aetiology.

A novel mutation W388X underlying properdin deficiency in a Finnish family.

Properdin deficiency is a rare immunological disorder inherited as an X-chromosomal recessive trait. Properdin deficiency poses a significant risk for severe meningococcal infections. About 20 mutations have been reported to underlie properdin deficiency. Here we report a large Finnish family with a novel mutation in the properdin gene (CFP). Based on the total absence of properdin activity in a 14-year-old male patient with an infection resembling meningococcal bacteraemia, the coding region and splice sites of the gene were sequenced. The mutation is located in exon 9 and changes guanine to adenine at nucleotide 1164 (c.1164G>A) that causes tryptophan to change to a premature stop codon (W388X). The mother of the patient was shown to be a carrier of the mutation. In total, the mutation was identified in six females and three young males in the family. The mutation must be inherited from the grandfather who had died of an unknown infectious disease. This is the first mutation of the properdin gene identified in Finland.

High pentraxin-3 plasma levels associate with thrombocytopenia in acute Puumala hantavirus-induced nephropathia epidemica.

Our aim was to investigate whether plasma levels of the long pentraxin-3 (PTX3) associate with the severity of Puumala hantavirus-induced nephropathia epidemica (NE). Sixty-one prospectively identified consecutively hospitalized NE patients were examined. Plasma PTX3, interleukin (IL)-6, terminal complement complex SC5b-9, complement component C3, C-reactive protein (CRP), creatinine, sodium, kynurenine, and tryptophan levels, as well as the blood cell count, were determined for up to five consecutive days after hospitalization. Receiver operating characteristic (ROC) analysis revealed that the maximum PTX3 level >101.6 ng/ml (high PTX3) showed a sensitivity of 71% and a specificity of 89% for detecting platelet level <50 × 10(9)/l, with an area under the curve (AUC) value of 0.78 (95% confidence interval [CI] 0.63-0.94). High PTX3 level was also associated with several other variables reflecting the severity of the disease: patients with high PTX3 level had higher maximum blood leukocyte (16.1 vs. 9.7 × 10(9)/l, p < 0.001), plasma IL-6 (16.9 vs. 9.0 pg/ml, p = 0.007), and creatinine (282 vs. 124 µmol/l, p = 0.007) levels than patients with low maximum PTX3 level. They also had longer hospital stays (8 vs. 5 days, p = 0.015) compared to patients with low PTX3 level. High plasma PTX3 levels are associated with thrombocytopenia and the overall severity of NE.

Lipopolysaccharides and outer membrane proteins as main structures involved in complement evasion strategies of non-typhoidal Salmonella strains.

Non-typhoidal Salmonella (NTS) infections pose a serious public health problem. In addition to the typical course of salmonellosis, an infection with Salmonella bacteria can often lead to parenteral infections and sepsis, which are particularly dangerous for children, the elderly and immunocompromised. Bacterial resistance to serum is a key virulence factor for the development of systemic infections. Salmonella, as an enterobacterial pathogen, has developed several mechanisms to escape and block the antibacterial effects of the complement system. In this review, we discuss the relevance of outer membrane polysaccharides to the complement evasion mechanisms of NTS strains. These include the influence of the overall length and density of the lipopolysaccharide molecules, modifications of the O-antigen lipopolysaccharide composition and the role of capsular polysaccharides in opsonization and protection of the outer membrane from the lytic action of complement. Additionally, we discuss specific outer membrane protein complement evasion mechanisms, such as recruitment of complement regulatory proteins, blocking assembly of late complement components to form the membrane attack complex and the proteolytic cleavage of complement proteins.

Association of Doberman hepatitis to canine major histocompatibility complex II.

Doberman hepatitis (DH) is a chronic and progressive inflammatory liver disease that mainly affects female dogs. The high incidence of chronic hepatitis in Dobermans is suggestive of a genetic predisposition. DH is characterized by mononuclear cell infiltration and copper accumulation in the liver and major histocompatibility complex (MHC) class II antigen expression in the hepatocytes. In dogs, the MHC is referred to as the dog leukocyte antigen (DLA) system. In this study, the potential role of DLA genes in DH was investigated by sequence-based typing in the exon 2 of DLA-DRB1, -DQA1 and -DQB1. The case group comprised 37 Dobermans with subclinical or clinical DH. The control group consisted of 37 healthy Dobermans, with normal liver enzyme values and without immunosuppressive medication. The control dogs were over 10 years old to include dogs with the lowest genetic risk of DH. Our results indicate that Dobermans with homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 [odds ratio (OR) = 14.9, confidence limit (CL) = 3.1-71.7, P < 0.00005], especially with homozygosity for DLA-DRB1*00601 (P < 0.0005), are susceptible to DH. The DQ heterodimer DLA-DQA1*00901/DQB1*00101 and the allele DLA-DRB1*01501 appear to confer protection against DH (P < 0.001). Allele and haplotype frequencies were compared using chi-squared statistics. The disease shows a complex pattern of inheritance, but the observed DLA class II association with DH suggests a role for the immune system in the development of the disease.

Evaluation of DLA promoters in Doberman hepatitis.

Doberman hepatitis (DH) is associated with homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 indicating a role for the immune system in the development of the disease. The dog leucocyte antigen (DLA) class II expression is controlled at the transcriptional level with proximal promoters. Differential expression of DLA class II molecules of antigen-presenting cells is reported to affect susceptibility to or protection from different immune-mediated diseases. The aim of this study was to evaluate, whether the variation in promoter areas of homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 Dobermans could explain why some dogs become afflicted with DH and others do not. Our findings suggest that promoter variants are not associated as risk modifiers in homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 Dobermans, but additional factors are needed. Nevertheless, our study indicates that the whole DLA block is associated to the disease.

Defective central tolerance in Aire-deficient mice is not sufficient to induce symptomatic autoimmunity during lymphopenia-induced T cell proliferation.

Transcriptional regulator autoimmune regulator (AIRE) controls thymic negative selection but it is also expressed in secondary lymphoid organs. The relative contribution of AIRE's central and peripheral function to the maintenance of tolerance is unclear. We transferred mature lymphocytes from Aire(-/-) or wild-type donors to Aire(+/+) lymphopenic recipients, which allowed us to gauge the autoreactivity inherent in the cells originating in an Aire(-/-) thymus. In the ensuing lymphopenia-induced proliferation (LIP), the recipients of cells from Aire(-/-) showed definite T cell hyperproliferation and developed autoantibodies at a higher frequency than the recipients of wild-type cells. However, neither of the recipient groups developed clinical symptoms, and pathological tissue infiltrates were also absent. The recipients of Aire(-/-) cells showed hyperproliferation and increased accumulation of regulatory T cells (Tregs), especially in tissues susceptible to inflammation triggered by LIP. These data are consistent with the view that T cells developing in the absence of Aire are autoreactive. However, overt autoimmunity was prevented, most likely by the suppressive function of Treg cells in the Aire-sufficient recipients. Our results support the importance of the peripheral AIRE expression in the maintenance of immunological tolerance.

Enhanced influenza A H1N1 T cell epitope recognition and cross-reactivity to protein-O-mannosyltransferase 1 in Pandemrix-associated narcolepsy type 1.

Narcolepsy type 1 (NT1) is a chronic neurological disorder having a strong association with HLA-DQB1*0602, thereby suggesting an immunological origin. Increased risk of NT1 has been reported among children or adolescents vaccinated with AS03 adjuvant-supplemented pandemic H1N1 influenza A vaccine, Pandemrix. Here we show that pediatric Pandemrix-associated NT1 patients have enhanced T-cell immunity against the viral epitopes, neuraminidase 175-189 (NA175-189) and nucleoprotein 214-228 (NP214-228), but also respond to a NA175-189-mimic, brain self-epitope, protein-O-mannosyltransferase 1 (POMT1675-689). A pathogenic role of influenza virus-specific T-cells and T-cell cross-reactivity in NT1 are supported by the up-regulation of IFN-γ, perforin 1 and granzyme B, and by the converging selection of T-cell receptor TRAV10/TRAJ17 and TRAV10/TRAJ24 clonotypes, in response to stimulation either with peptide NA175-189 or POMT1675-689. Moreover, anti-POMT1 serum autoantibodies are increased in Pandemrix-vaccinated children or adolescents. These results thus identify POMT1 as a potential autoantigen recognized by T- and B-cells in NT1.

Activation of the alternative pathway of complement by monoclonal lambda light chains in membranoproliferative glomerulonephritis.

Immunopathological evidence suggests that activation of the alternative pathway of complement (AP) is involved in membranoproliferative glomerulonephritis (MPGN) and in immunoglobulin A nephropathy. In this report we describe an AP dysfunction-associated factor that was isolated from the serum and urine of a patient with hypocomplementemic MPGN. Extensive glomerular deposits of C3, properdin, and of the terminal complement components were observed in the kidney of the patient. In her serum the AP hemolytic activity was virtually absent. When mixed with fresh normal serum, the patient's serum induced a 96% C3 conversion during a 30-min incubation at +37 degrees C. This activity was found to be due to a circulating factor that by immunochemical characterization proved to be a 46-kD monoclonal immunoglobulin lambda light (L) chain dimer (lambda L). Purified lambda L, but not control lambda or kappa L chains from patients with L chain disease, activated the AP in a dose- and ionic strength-dependent manner. Functionally, lambda L was differentiated from C3 nephritic factor (an autoantibody against the AP C3 convertase, C3bBb) by its inability to bind to and stabilize the C3bBb enzyme. Instead, lambda L was observed to interact directly with the AP control factor H. Thus, lambda L represents a novel type of immunoglobulin-related AP-activating factor with the capacity to initiate alternative complement pathway activation in the fluid phase.

Human protectin (CD59), an 18-20-kD homologous complement restriction factor, does not restrict perforin-mediated lysis.

Human protectin (CD59) is an 18-20-kD membrane glycoprotein that restricts lysis of human erythrocytes and leukocytes by homologous complement. By directly incorporating protectin into membranes of heterologous cells we observed that protectin did not prevent perforin-mediated killing, whereas complement killing was effectively restricted. Further, no significant enhancement of cell-mediated killing or target killing by purified perforin was observed with anti-protectin antibodies. Thus, in contrast with complement lysis, restriction of lysis by protectin does not apply to cell-mediated killing.

Arterial oxygenation can fall critically during intramedullary nailing of pathological femoral fractures.

BACKGROUND: The intramedullary nailing of a femoral shaft metastatic fracture or an impending fracture has been complicated by hemodynamic accidents in up to 13% of patients. In previous studies, otherwise healthy patients pulled well through the nailing despite high pulmonary shunting and vascular tone and right ventricular strain. We hypothesized that unfavorable hemodynamic and oxygenation trends milder than catastrophic ones can be found intraoperatively in most patients with a pathological fracture and cancer-affected lungs. METHODS: Eleven patients with a femoral metastatic fracture or an impending fracture were studied in general anesthesia. Radial artery and fast-response pulmonary artery catheters were inserted. Pre-, intra-, and postoperatively, 26 variables were measured and/or calculated up to 20 hours. Reamed nailing with a gamma nail was performed. RESULTS: At awake baseline, the mean pulmonary arterial pressure was 20 mm Hg ± 7 mm Hg and the shunt flow was 19% ± 6%. As response to the nailing, shunting increased from 14% ± 7% (mechanically ventilated) to 23% ± 10% (p < 0.05), and mean pulmonary arterial pressure increased to 29 mm Hg ± 6 mm Hg (p < 0.001). Oxygenation deteriorated to a level typical of acute lung injuries (Pao2/FIO2 242 mm Hg ± 73 mm Hg; p < 0.05). Intraoperatively, the oxygen delivery was poor, and acidosis developed (base excess, -2.9, p < 0.05). CONCLUSION: The baseline condition of patients with a pathological femoral fracture was comparable with that of healthy patients subjected to femoral fracture surgery. After reaming, arterial oxygenation deteriorated, being clinically poor for the rest of the study. We suggest increased inspiratory oxygen concentration intra- and postoperatively and maintenance of oxygen delivery by transfusions as needed, especially because hypoxia stimulates the growth of cancer.

Successful liver-kidney transplantation in two children with aHUS caused by a mutation in complement factor H.

A 12-month-old boy and his 16-year-old aunt became acutely ill 6 months apart and were diagnosed to have atypical hemolytic uremic syndrome (aHUS). Genetic analysis revealed heterozygous R1215Q mutation in complement factor H (CFH) in both patients. The same mutation was found in five healthy adult relatives indicating incomplete penetrance of the disease. The patients developed terminal renal failure and experienced reversible neurological symptoms in spite of plasma exchange (PE) therapy. In both cases, liver-kidney transplantation was successfully performed 6 months after the onset of the disease. To minimize complement activation and prevent thrombotic microangiopathy or overt thrombotic events due to the malfunctioning CFH, extensive PE with fresh frozen plasma was performed pre- and perioperatively and anticoagulation was started a few hours after the operation. No circulatory complications appeared and all four grafts started to function immediately. Also, no recurrence or other major clinical setbacks have appeared during the postoperative follow-up (15 and 9 months) and the grafts show excellent function. While more experience is needed, it seems that liver-kidney transplantation combined with pre- and perioperative PE is a rational option in the management of patients with aHUS caused by CFH mutation.

Responses to rituximab vary among follicular lymphoma B cells of different maturation stages.

The chimeric anti-CD20 monoclonal antibody rituximab has been used for the treatment of non-Hodgkin's lymphomas with varying responses. Rituximab has been demonstrated to act by direct complement-dependent cytotoxity (CDC) and by inducing apoptosis, complement-, and antibody-dependent cellular cytotoxity. In the present study, we determined whether rituximab's effector mechanisms differed between two human follicular lymphoma cell lines that originate from different maturation stages of B cell germinal centre (GC) development. The tested HF-1 and HF-4b lymphoma cells represent GC centrocytes and centroblasts, respectively. Both cell lines responded to rituximab treatment by undergoing apoptosis yet the HF-1 cells were more sensitive. A major difference was seen in the proliferation response as only the proliferation of HF-1 cells was inhibited by rituximab. In the presence of normal human serum (NHS) rituximab almost completely inhibited DNA synthesis and induced necrosis of both cell lines because of CDC. Our results show that the CD20-positive HF-1 and HF-4b cells respond differentially to rituximab-induced apoptosis and inhibition of proliferation but similarly to complement-mediated killing. The increased sensitivity of the HF-1 cell line to apoptosis and inhibition of proliferation may reflect a tendency of centrocytic cells for negative selection and a role for CD20 in this process.