Complement in metabolic disease: metaflammation and a two-edged sword.

We are currently experiencing an enduring global epidemic of obesity and diabetes. It is now understood that chronic low-grade tissue inflammation plays an important role in metabolic disease, brought upon by increased uptake of a so-called Western diet, and a more sedentary lifestyle. Many evolutionarily conserved links exist between metabolism and the immune system, and an imbalance in this system induced by chronic over-nutrition has been termed 'metaflammation'. The complement system is an important and evolutionarily ancient part of innate immunity, but recent work has revealed that complement not only is involved in the recognition of pathogens and induction of inflammation, but also plays important roles in cellular and tissue homeostasis. Complement can therefore contribute both positively and negatively to metabolic control, depending on the nature and anatomical site of its activity. This review will therefore focus on the interactions of complement with mechanisms and tissues relevant for metabolic control, obesity and diabetes.

The role of complement inhibitors beyond controlling inflammation.

The complement system is an arm of innate immunity that aids in the removal of pathogens and dying cells. Due to its harmful, pro-inflammatory potential, complement is controlled by several soluble and membrane-bound inhibitors. This family of complement regulators has been recently extended by the discovery of several new members, and it is becoming apparent that these proteins harbour additional functions. In this review, the current state of knowledge of the physiological functions of four complement regulators will be described: cartilage oligomeric matrix protein (COMP), CUB and sushi multiple domains 1 (CSMD1), sushi domain-containing protein 4 (SUSD4) and CD59. Complement activation is involved in both the development of and defence against cancer. COMP expression is pro-oncogenic, whereas CSMD1 and SUSD4 act as tumour suppressors. These effects may be related in part to the complex influence of complement on cancer but also depend on unrelated functions such as the protection of cells from endoplasmic reticulum stress conveyed by intracellular COMP. CD59 is the main inhibitor of the membrane attack complex, and its deficiency leads to complement attack on erythrocytes and severe haemolytic anaemia, which is now amenable to treatment with an inhibitor of C5 cleavage. Unexpectedly, the intracellular pool of CD59 is crucial for insulin secretion from pancreatic ß-cells. This finding is one of several relating to the intracellular functions of complement proteins, which until recently were only considered to be present in the extracellular space. Understanding the alternative functions of complement inhibitors may unravel unexpected links between complement and other physiological systems, but is also important for better design of therapeutic complement inhibition.

Cartilage oligomeric matrix protein contributes to the development and metastasis of breast cancer.

Cartilage oligomeric matrix protein (COMP) is a soluble pentameric protein expressed in cartilage and involved in collagen organization. Tissue microarrays derived from two cohorts of patients with breast cancer (n=122 and n=498) were immunostained, revealing varying expression of COMP, both in the tumor cells and surrounding stroma. High levels of COMP in tumor cells correlated, independently of other variables, with poor survival and decreased recurrence-free survival. Breast cancer cells, MDA-MB-231, stably expressing COMP were injected into the mammary fat pad of SCID (CB-17/Icr-Prkdcscid/Rj) mice. Tumors expressing COMP were significantly larger and were more prone to metastasize as compared with control, mock-transfected, tumors. In vitro experiments confirmed that COMP-expressing cells had a more invasive phenotype, which could in part be attributed to an upregulation of matrix metalloprotease-9. Furthermore, microarray analyses of gene expression in tumors formed in vivo showed that COMP expression induced higher expression of genes protecting against endoplasmic reticulum stress. This observation was confirmed in vitro as COMP-expressing cells showed better survival as well as a higher rate of protein synthesis when treated with brefeldin A, compared with control cells. Further, COMP-expressing cells appeared to undergo a metabolic switch, that is, a Warburg effect. Thus, in vitro measurement of cell respiration indicated decreased mitochondrial metabolism. In conclusion, COMP is a novel biomarker in breast cancer, which contributes to the severity of the disease by metabolic switching and increasing invasiveness and tumor cell viability, leading to reduced survival in animal models and human patients.

Factor H uptake regulates intracellular C3 activation during apoptosis and decreases the inflammatory potential of nucleosomes.

Factor H (FH) binds apoptotic cells to limit the inflammatory potential of complement. Here we report that FH is actively internalized by apoptotic cells to enhance cathepsin L-mediated cleavage of endogenously expressed C3, which results in increased surface opsonization with iC3b. In addition, internalized FH forms complexes with nucleosomes, facilitates their phagocytosis by monocytes and induces an anti-inflammatory biased cytokine profile. A similar cytokine response was noted for apoptotic cells coated with FH, confirming that FH diminishes the immunogenic and inflammatory potential of autoantigens. These findings were supported by in vivo observations from CFH(-/-) MRL-lpr mice, which exhibited higher levels of circulating nucleosomes and necrotic cells than their CFH(+/+) littermates. This unconventional function of FH broadens the established view of apoptotic cell clearance and appears particularly important considering the strong associations with genetic FH alterations and diseases such as systemic lupus erythematosus and age-related macular degeneration.

A novel method for direct measurement of complement convertases activity in human serum.

Complement convertases are enzymatic complexes that play a central role in sustaining and amplification of the complement cascade. Impairment of complement function leads directly or indirectly to pathological conditions, including higher infection rate, kidney diseases, autoimmune- or neurodegenerative diseases and ischaemia-reperfusion injury. An assay for direct measurement of activity of the convertases in patient sera is not available. Existing assays testing convertase function are based on purified complement components and, thus, convertase formation occurs under non-physiological conditions. We designed a new assay, in which C5 blocking compounds enabled separation of the complement cascade into two phases: the first ending at the stage of C5 convertases and the second ending with membrane attack complex formation. The use of rabbit erythrocytes or antibody-sensitized sheep erythrocytes as the platforms for convertase formation enabled easy readout based on measurement of haemolysis. Thus, properties of patient sera could be studied directly regarding convertase activity and membrane attack complex formation. Another advantage of this assay was the possibility to screen for host factors such as C3 nephritic factor and other anti-complement autoantibodies, or gain-of-function mutations, which prolong the half-life of complement convertases. Herein, we present proof of concept, detailed description and validation of this novel assay.

Interactions of histidine-rich glycoprotein with immunoglobulins and proteins of the complement system.

This study describes how the serum protein histidine-rich glycoprotein (HRG) affects the complement system. We show that HRG binds strongly to several complement proteins: C1q, factor H and C4b-binding protein and that it is found complexed with these proteins in human sera and synovial fluids of rheumatoid arthritis patients. HRG also binds C8 and to a lesser extent mannose-binding lectin, C4 and C3. However, HRG alone neither activates nor inhibits complement. Both HRG and C1q bind to necrotic cells and increase their phagocytosis. We found that C1q competes weakly with HRG for binding to necrotic cells whilst HRG does not compete with C1q. Furthermore, HRG enhances complement activation on necrotic cells measured as deposition of C3b. We show that HRG inhibits the formation of immune complexes of ovalbumin/anti-ovalbumin, whilst the reverse holds for C1q. Immune complexes formed in the presence of HRG show enhanced complement activation, whilst those formed in the presence of C1q show diminished complement activation. Taken together, HRG may assist in the maintenance of normal immune function by mediating the clearance of necrotic material, inhibiting the formation of insoluble immune complexes and enhancing their ability to activate complement, resulting in faster clearance.

Complement inhibitor C4b-binding protein in primary Sjögren's syndrome and its association with other disease markers.

A subgroup of patients suffering from primary Sjögren's syndrome (pSS) display unexplained low levels of complement components C3 and/or C4 which is associated with increased risk of non-Hodgkin's lymphoma. C4b-binding protein (C4BP) is a major fluid-phase complement inhibitor which can influence C4 and C3 levels. Therefore we analysed C4BP levels in the sera of patients with pSS to better understand the disturbances in complement in pSS. Associations with other disease markers were also investigated to define a possible role of C4BP as marker of high-risk disease course. Plasma levels of C4BP were analysed in pSS patients (n=86) and in controls (n=68) by ELISA. C4BP levels from 49 patients were correlated to disease activity markers and autoantibody profiles. We found that total C4BP plasma levels were significantly higher in pSS patients compared with controls. C4BP levels correlated to the acute phase response, to levels of C4 and C3 as well as to the CD4+/CD8+ T-cell ratio. C4BP levels were inversely related to IgG levels, extent of autoantibody production and global disease activity. C3dg levels, a marker of complement activation, displayed a negative correlation to C4 levels but interestingly not to C4BP levels. In conclusion, C4BP levels are increased in patients suffering from pSS proportional to their acute phase response. However, in the most active cases, with the most widespread autoantibody production, C4BP levels were decreased in parallel with levels of C3 and C4 and CD4+ T cells, suggesting that disturbed complement regulation may contribute to pathogenicity in pSS.

Complement activation and inhibition: a delicate balance.

Complement is part of the innate immune defence and not only recognizes microbes but also unwanted host molecules to enhance phagocytosis and clearance. This process of opsonisation must be tightly regulated to prevent immunopathology. Endogenous ligands such as dying cells, extracellular matrix proteins, pentraxins, amyloid deposits, prions and DNA, all bind the complement activator C1q, but also interact with complement inhibitors C4b-binding protein and factor H. This contrasts to the interaction between C1q and immune complexes, in which case no inhibitors bind, resulting in full complement activation. Disturbances to the complement regulation on endogenous ligands can lead to diseases such as age-related macular degeneration, neurological and rheumatic disorders. A thorough understanding of these processes might be crucial to developing new therapeutic strategies.

C4b-binding protein (C4BP) inhibits development of experimental arthritis in mice.

OBJECTIVES: To assess the human complement inhibitor C4b-binding protein (C4BP) for treatment of arthritis. METHODS: We have used two mouse models of rheumatoid arthritis (RA) to assess the therapeutic effect of C4BP on different phases of arthritis, the collagen antibody-induced arthritis (CAIA), an acute antibody-induced disease and the collagen-induced arthritis (CIA), which carries the full complexity of arthritis. RESULTS: Purified human C4BP injected intraperitoneally alleviated CAIA significantly in a manner similar to cobra venom factor that depletes complement due to massive activation. Furthermore, C4BP was injected before and after the disease development into CIA mice. In the former case, the disease onset was delayed and in the latter, the severity of the disease was reduced in animals treated with C4BP. However, C4BP did not affect the anti-CII antibody synthesis. C4BP present in mouse sera decreased activity of the classical but not the alternative pathway of the complement system when these were assessed in a fluid phase. However, C4BP was efficiently inhibiting the alternative pathway when present on the activating surface. Taken together, the disease ameliorating effect of C4BP appears to be related to inhibition of both pathways of complement. CONCLUSIONS: Although human C4BP was cleared relatively fast from the circulation and was only moderately affecting complement activity, its effect on the disease severity was substantial, suggesting that minor alterations in complement activity can have significant therapeutic value in RA.

Technical and biochemical factors affecting cerebrospinal fluid 5-MTHF, biopterin and neopterin concentrations.

BACKGROUND: The diagnosis of pediatric neurologic disorders with a deficiency in the biosynthesis of either the neurotransmitters serotonin and dopamine, or the co-factor tetrahydrobiopterin or a cerebral 5-methyltetrahydrofolate (5-MTHF) deficiency, strongly relies on a robust analysis of neurotransmitter metabolites, pterins and 5-MTHF in the cerebrospinal fluid (CSF). The aim of this study was to investigate which technical and biochemical factors affect the CSF concentration of 5-MTHF, neopterin and biopterin in a pediatric population. METHODS: We studied effects of the ventriculo-spinal gradient, total protein concentration, pretreatment with ascorbic acid (in case of 5-MTHF analysis), pretreatment of CSF with trichloro acetic acid (TCA)/dithiotreitol (DTE) and oxidation with either iodine or manganese oxide (in case of pterin analysis), storage time and age of the patients. We included CSF samples from children until the age of 18 years and analysed 5-MTHF, neopterin, biopterin, homovanillic acid (HVA), 5-hydroxy-indoleacetic acid (5-HIAA) and total protein. RESULTS: The major findings of our study are: (1) CSF 5-MTHF, neopterin and biopterin concentrations are not affected by the ventriculo-spinal gradient; (2) pretreatment of CSF with ascorbic acid has negligible effects on 5-MTHF concentrations; (3) pretreatment of CSF with TCA/DTE and oxidation with iodine results in the most accurate determination of neopterin and biopterin; (4) when adjusted for age and total protein, CSF 5-MTHF correlated with 5-HIAA, but not with HVA; (5) the reference value of 5-MTHF in CSF in childhood is age-dependent (r=-0.634; p0.001); (6) we did not observe an age-dependency for neopterin and biopterin in CSF. CONCLUSION: 5-MTHF, neopterin and biopterin can be analysed in any volume of CSF that is collected. For correct analysis of pterins, CSF will have to be pretreated to stabilize the concentrations and stored properly, whereas such pretreatment is not necessary for 5-MTHF.

Role of complement and complement regulators in the removal of apoptotic cells.

Apoptosis, followed by rapid phagocytic clearance, is the primary mechanism by which organisms dispose of unwanted cells. The intracellular and extracellular composition of an apoptotic cell changes to decrease immunogenicity and enhance its uptake. By changing their extracellular composition, apoptotic cells acquire the capacity to bind complement initiation molecules such as C1q and MBL. Binding of these molecules can lead to complement activation. Membrane bound complement inhibitors are down-regulated during apoptosis, which would leave the cell less protected against complement activation; however, recent data show that fluid-phase complement inhibitors may compensate for this loss of regulation. Importantly, binding of complement is a process that mainly takes place during the late stages of apoptosis. Most cells will be cleared before that stage under steady state conditions, but during overwhelming apoptosis or impaired phagocytosis, apoptotic cells may remain in tissues for a longer time and acquire complement proteins. Based on the data from deficiencies of early complement components and the development of systemic lupus erythematosus with accumulation of dead cells, it is clear that, under certain conditions, apoptotic cells persist, becoming necrotic and overloading the scavenging capacities of the complement system. Although the complement system is also involved in inducing apoptosis in target cells, this review will focus on the role of complement in the clearance of apoptotic cells.

Immune evasion by acquisition of complement inhibitors: the mould Aspergillus binds both factor H and C4b binding protein.

Pathogenic fungi represent a major threat particularly to immunocompromised hosts, leading to severe, and often lethal, systemic opportunistic infections. Although the impaired immune status of the host is clearly the most important factor leading to disease, virulence factors of the fungus also play a role. Factor H (FH) and its splice product FHL-1 represent the major fluid phase inhibitors of the alternative pathway of complement, whereas C4b-binding protein (C4bp) is the main fluid phase inhibitor of the classical and lectin pathways. Both proteins can bind to the surface of various human pathogens conveying resistance to complement destruction and thus contribute to their pathogenic potential. We have recently shown that Candida albicans evades complement by binding both Factor H and C4bp. Here we show that moulds such as Aspergillus spp. bind Factor H, the splicing variant FHL-1 and also C4bp. Immunofluorescence and flow cytometry studies show that the binding of Factor H and C4bp to Aspergillus spp. appears to be even stronger than to Candida spp. and that different, albeit possibly nearby, binding moieties mediate this surface attachment.

Relapsing fever spirochetes Borrelia recurrentis and B. duttonii acquire complement regulators C4b-binding protein and factor H.

Relapsing fever is a rapidly progressive and severe septic disease caused by certain Borrelia spirochetes. The disease is divided into two forms, i.e., epidemic relapsing fever, caused by Borrelia recurrentis and transmitted by lice, and the endemic form, caused by several Borrelia species, such as B. duttonii, and transmitted by soft-bodied ticks. The spirochetes enter the bloodstream by the vector bite and live persistently in plasma even after the development of specific antibodies. This leads to fever relapses and high mortality and clearly indicates that the Borrelia organisms utilize effective immune evasion strategies. In this study, we show that the epidemic relapsing fever pathogen B. recurrentis and an endemic relapsing fever pathogen, B. duttonii, are serum resistant, i.e., resistant to complement in vitro. They acquire the host alternative complement pathway regulator factor H on their surfaces in a similar way to that of the less serum-resistant Lyme disease pathogen, B. burgdorferi sensu stricto. More importantly, the relapsing fever spirochetes specifically bind host C4b-binding protein, a major regulator of the antibody-mediated classical complement pathway. Both complement regulators retained their functional activities when bound to the surfaces of the spirochetes. In conclusion, this is the first report of complement evasion by Borrelia recurrentis and B. duttonii and the first report showing capture of C4b-binding protein by spirochetes.

Streptococcal M protein: structural studies of the hypervariable region, free and bound to human C4BP.

Streptococcus pyogenes is a Gram-positive bacterium that causes several diseases, including acute tonsillitis and toxic shock syndrome. The surface-localized M protein, which is the most extensively studied virulence factor of S. pyogenes, has an approximately 50-residue N-terminal hypervariable region (HVR) that plays a key role in the escape of the host immunity. Despite the extensive sequence variability in this region, many HVRs specifically bind human C4b-binding protein (C4BP), a plasma protein that inhibits complement activation. Although the more conserved parts of M protein are known to have dimeric coiled-coil structure, it is unclear whether the HVR also is a coiled coil. Here, we use nuclear magnetic resonance (NMR) to study the conformational properties of HVRs from M4 and M22 proteins in isolation and in complex with the M protein binding portion of C4BP. We conclude that the HVRs of M4 and M22 are folded as coiled coils and that the folded nucleus of the M4 HVR has a length of approximately 27 residues. Moreover, we demonstrate that the C4BP binding surface of M4-N is found within a region of four heptad repeats. Using molecular modeling, we propose a model for the structure of the M4 HVR that is consistent with our experimental information from NMR spectroscopy.

Dissecting the regions of virion-associated Kaposi's sarcoma-associated herpesvirus complement control protein required for complement regulation and cell binding.

Complement, which bridges innate and adaptive immune responses as well as humoral and cell-mediated immunity, is antiviral. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a lytic cycle protein called KSHV complement control protein (KCP) that inhibits activation of the complement cascade. It does so by regulating C3 convertases, accelerating their decay, and acting as a cofactor for factor I degradation of C4b and C3b, two components of the C3 and C5 convertases. These complement regulatory activities require the short consensus repeat (SCR) motifs, of which KCP has four (SCRs 1 to 4). We found that in addition to KCP being expressed on the surfaces of experimentally infected endothelial cells, it is associated with the envelope of purified KSHV virions, potentially protecting them from complement-mediated immunity. Furthermore, recombinant KCP binds heparin, an analogue of the known KSHV cell attachment receptor heparan sulfate, facilitating infection. Treating virus with an anti-KCP monoclonal antibody (MAb), BSF8, inhibited KSHV infection of cells by 35%. Epitope mapping of MAb BSF8 revealed that it binds within SCR domains 1 and 2, also the region of the protein involved in heparin binding. This MAb strongly inhibited classical C3 convertase decay acceleration by KCP and cofactor activity for C4b cleavage but not C3b cleavage. Our data suggest similar topological requirements for cell binding by KSHV, heparin binding, and regulation of C4b-containing C3 convertases but not for factor I-mediated cleavage of C3b. Importantly, they suggest KCP confers at least two functions on the virion: cell binding with concomitant infection and immune evasion.

The hyphal and yeast forms of Candida albicans bind the complement regulator C4b-binding protein.

Candida albicans, an important pathogenic yeast, activates all three pathways of the complement system. To understand how this yeast evades the effects of the activated system, we have analyzed the binding of the classical pathway inhibitor C4b-binding protein (C4BP) by C. albicans. Purified native as well as recombinant C4BP bound dose dependently to the yeast and hyphal forms, as shown by multiple methods, such as confocal microscopy, flow cytometry, a novel enzyme-linked immunosorbent assay, absorption from human serum, and direct binding assays with purified proteins. A prominent binding site was identified at the tip of the germ tube, a structure that is considered important for tissue penetration and pathogenesis. The binding site in C4BP was localized to the two N-terminal complement control protein domains by using recombinant deletion constructs and site-specific monoclonal antibodies. As the alternative pathway inhibitors factor H and FHL-1 also bind to C. albicans, the binding of all three plasma proteins was compared. Simultaneous binding of the classical regulator C4BP and the alternative pathway regulator factor H was demonstrated by confocal microscopy. In addition, FHL-1 competed for binding with C4BP, suggesting that these two related complement regulators bind to the same structures on the yeast surface. The surface-attached C4BP maintains its complement regulatory activities and inactivates C4b. The surface-attached human C4BP serves multiple functions relevant for immune evasion and likely pathogenicity. It inhibits complement activation at the yeast surface and, in addition, mediates adhesion of C. albicans to host endothelial cells.

Strategies developed by bacteria and virus for protection from the human complement system.

The complement system is an important part of innate immunity providing immediate protection against pathogens without a need for previous exposure. Its importance is clearly shown by the fact that patients lacking complement components suffer from fulminant and recurring infections. Complement is an explosive cascade, and in order to control it there are inhibitors present on every human cell and also circulating in blood. However, many infectious agents have developed strategies to prevent clearance and destruction by complement. Some pathogens simply hijack the host's complement inhibitors, while others are able to produce their own homologues of human inhibitors. Knowledge of these mechanisms on a molecular level may aid development of vaccines and novel therapeutic strategies that would be more specific than the use of antibiotics that, apart from causing resistance problems, also affect the normal flora, the outcome of which could be devastating. In this study the structural requirements and functional consequences of interactions between the major soluble inhibitor of complement C4b-binding protein and Neisseria gonorrhoeae, Bordetella pertussis, Streptococcus pyogenes, Escherichia coli K1, Moraxella catarrhalis and Candida albicans are described. Furthermore, a novel inhibitor produced by Kaposi's sarcoma-associated herpesvirus is identified and characterized in detail: KCP. It is shown that KCP inhibits classical C3-convertase and presents activated complement factors C4b and C3b for destruction by a serine proteinase, factor I. Using molecular modelling and site-directed mutagenesis, it was possible to localize sites on the surface of KCP required for complement inhibition and it is concluded that KCP uses molecular mechanisms identical to human inhibitors.

Structural and functional studies of complement inhibitor C4b-binding protein.

C4b-binding protein (C4BP) is a potent inhibitor of the classical pathway of the complement system. This large plasma glycoprotein consists of seven identical alpha-chains and a unique beta-chain held together by disulphide bridges. Both types of subunits are composed almost exclusively of complement control protein domains (CCPs). Using homology-based computer modelling and mutagenesis of recombinant proteins we have localized binding sites for several ligands of C4BP: complement factor C4b, heparin and vitamin K-dependent anticoagulant protein S (PS). We found that C4b requires CCP1-3 of the alpha-chain for binding. The interaction is ionic in nature and mediated by a cluster of positively charged amino acids present on the interface between CCP1 and CCP2 of the alpha-chain. Loss of C4b-binding resulted in a loss of all inhibitory functions of C4BP within the classical pathway of complement. Binding of heparin required CCPs 1-3 of the alpha-chain, with CCP2 being the most important, as well as the cluster of positively charged amino acids involved in binding of C4b. The interaction between C4BP and PS is of very high affinity and conveyed by a cluster of surface exposed hydrophobic amino acids localized on CCP1 of the beta-chain. Furthermore, C4BP is captured on the surface of several pathogens, which may contribute to their serum resistance and pathogenicity. We have localized interaction of C4BP with Neisseria gonorrhoeae, Bordetella pertussis, Streptococcus pyogenes and Escherichia coli to various regions of the alpha-chain.

A pilot case control follow-up study on hearing in children treated with tobramycin in the newborn period.

OBJECTIVE: To assess the occurrence of hearing loss in children due to neonatal exposure to long courses of tobramycin and/or high tobramycin serum concentrations. METHODS: This was a pilot case-control study in 3-4-year old children. Data on tobramycin administration were abstracted from the patient files of an earlier study. Patients exposed in the neonatal period to either long courses (>7 days) or high serum concentrations of tobramycin constituted the study group. The control group consisted of patients without tobramycin exposure. Patients were matched for other risk factor according to criteria of the joint committee on infant hearing. All patients underwent the following investigations: otoscopy and pneumatic otoscopy, followed by impedance audiometry, to exclude middle ear effusion. Click-evoked oto-acoustic emissions (ce-OAE) as well as distortion product oto-acoustic emissions (dp-OAE), tested at f2 frequencies ranging from 1 to 10 kHz, were measured to assess hearing. All patients with abnormal ce-OAE results underwent brainstem electric response audiometry (BERA) as well. Since aminoglycoside ototoxicity is usually bilateral, results were compared per patient and not per ear. RESULTS: A total of 29 patients were tested. Eleven patients were excluded due to middle ear effusion. Data for 18 patients were analyzed. In the tobramycin treated group (n=9) both ce-OAE and dp-OAE (at all tested frequencies) were not detectable in six ears of three patients. All other patients had normal ce-OAE's as well as normal dp-OAE's in this frequency range. Difference between the tobramycin treated and control group for OAE as well as dp-OAE showed a trend (P=0.08). In all three patients with undetectable emissions BERA confirmed a cochlear loss of 60-70 dB at 3 kHz in both ears. These three patients had the longest total exposure to tobramycin: 20-24 days and 84-92 mg/kg, respectively. No relation to either peak or trough serum concentrations could be detected. CONCLUSION: There was no statistical relation between hearing loss and tobramycin exposure, probably due to sample size. Our results do indicate a need for a case-control follow-up study of hearing in neonates exposed to long courses of aminoglycosides.

Bordetella pertussis binds to human C4b-binding protein (C4BP) at a site similar to that used by the natural ligand C4b.

Human complement regulators are important targets for pathogenic microorganisms. In one such interaction, Bordetella pertussis binds human C4b-binding protein (C4BP), a high-molecular-weight plasma protein that acts as inhibitor of the classical pathway of complement activation. At least two different B. pertussis surface components, one of which is the virulence factor filamentous hemagglutinin (FHA), contribute to the binding. We used a set of C4BP mutants and monoclonal antibodies to characterize the region in C4BP that binds B. pertussis and analyzed the salt sensitivity of the interaction. These studies indicated that positively charged residues at the interface between complement control protein modules 1-2 in the C4BP alpha-chain are important for binding, and that the site in C4BP that binds B. pertussis is very similar, but not identical, to the C4b-binding site. Bacteria-bound C4BP retained its complement regulatory function and B. pertussis selectively bound C4BP in human plasma, indicating that binding occurs also in vivo. Together, these findings indicate that B. pertussis exploits a site in C4BP, resembling that used by the natural ligand C4b.