Inhibition of the different complement pathways has varying impacts on the serum bactericidal activity and opsonophagocytosis against Haemophilus influenzae type b

Defense against Haemophilus influenzae type b (Hib) is dependent on antibodies and complement, which mediate both serum bactericidal activity (SBA) and opsonophagocytosis. Here we evaluated the influence of capsule-specific antibodies and complement inhibitors targeting the central component C3, the alternative pathway (AP; fB, fD), the lectin pathway (LP; MASP-2) and the terminal pathway (C5) on both effector functions. Findings may be relevant for the treatment of certain diseases caused by dysregulation of the complement system, where inhibitors of complement factors C3 or C5 are used. Inhibitors against other complement components are being evaluated as potential alternative treatment options that may carry a reduced risk of infection by encapsulated bacteria. Serum and reconstituted blood of healthy adults were tested for bactericidal activity before and after vaccination with the Hib capsule-conjugate vaccine ActHIB. Most sera had bactericidal activity prior to vaccination, but vaccination significantly enhanced SBA titers. Independently of the vaccination status, both C3 and C5 inhibition abrogated SBA, whereas inhibition of the LP had no effect. AP inhibition had a major inhibitory effect on SBA of pre- vaccination serum, but vaccination mitigated this inhibition for all disease isolates tested. Despite this, SBA-mediated killing of some Hib isolates remained retarded. Even for the most serum-resistant isolate, SBA was the dominating defense mechanism in reconstituted whole blood, as addition of blood cells to the serum did not enhance bacterial killing. Limited Fc receptor-mediated opsonophagocytosis was unmasked when bacterial killing by the membrane attack complex was blocked. In the presence of C3 or C5 inhibitors, addition of post-vaccination, but not of pre-vaccination serum to the blood cells triggered opsonophagocytosis, leading to suppression of bacterial multiplication. Taken together, our data indicate that for host defense against Hib, killing by SBA is more efficient than by blood cell opsonophagocytosis. However, additional defense mechanisms, such as bacterial clearance by spleen and liver, may play an important role in preventing Hib-mediated sepsis, in particular for Hib isolates with increased serum-resistance. Results indicate potentially improved safety profile of AP inhibitors over C3 and C5 inhibitors as alternative therapeutic agents in patients with increased susceptibility to Hib infection

Alternative complement pathway inhibition does not abrogate meningococcal killing by serum of vaccinated individuals

Dysregulation of complement activation causes a number of diseases, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. These conditions can be treated with monoclonal antibodies (mAbs) that bind to the complement component C5 and prevent formation of the membrane attack complex (MAC). While MAC is involved in uncontrolled lysis of erythrocytes in these patients, it is also required for serum bactericidal activity (SBA), i.e. clearance of encapsulated bacteria. Therefore, terminal complement blockage in these patients increases the risk of invasive disease by Neisseria meningitidis more than 1000-fold compared to the general population, despite obligatory vaccination. It is assumed that alternative instead of terminal pathway inhibition reduces the risk of meningococcal disease in vaccinated individuals. To address this, we investigated the SBA with alternative pathway inhibitors. Serum was collected from adults before and after vaccination with a meningococcal serogroup A, C, W, Y capsule conjugate vaccine and tested for meningococcal killing in the presence of factor B and D, C3, C5 and MASP-2 inhibitors. B meningococci were not included in this study since the immune response against protein-based vaccines is more complex. Unsurprisingly, inhibition of C5 abrogated killing of meningococci by all sera. In contrast, both factor B and D inhibitors affected meningococcal killing in sera from individuals with low, but not with high bactericidal anti-capsular titers. While the anti-MASP-2 mAb did not impair SBA, inhibition of C3 impeded meningococcal killing in most, but not in all sera. These data provide evidence that vaccination can provide protection against invasive meningococcal disease in patients treated with alternative pathway inhibitors

In vaccinated individuals serum bactericidal activity against B meningococci is abrogated by C5 inhibition but not by inhibition of the alternative complement pathway

INTRODUCTION: Several diseases caused by the dysregulation of complement activation can be treated with inhibitors of the complement components C5 and/or C3. However, complement is required for serum bactericidal activity (SBA) against encapsulated Gram-negative bacteria. Therefore, C3 and C5 inhibition increases the risk of invasive disease, in particular by Neisseria meningitidis. As inhibitors against complement components other than C3 and C5 may carry a reduced risk of infection, we compared the effect of inhibitors targeting the terminal pathway (C5), the central complement component C3, the alternative pathway (FB and FD), and the lectin pathway (MASP-2) on SBA against serogroup B meningococci. METHODS: Serum from adults was collected before and after vaccination with the meningococcal serogroup B vaccine 4CMenB and tested for meningococcal killing. Since the B capsular polysaccharide is structurally similar to certain human polysaccharides, 4CMenB was designed to elicit antibodies against meningococcal outer membrane proteins. RESULTS: While only a few pre-vaccination sera showed SBA against the tested B meningococcal isolates, 4CMenB vaccination induced potent complement-activating IgG titers against isolates expressing a matching allele of the bacterial cell surface-exposed factor H-binding protein (fHbp). SBA triggered by these cell surface protein-specific antibodies was blocked by C5 and reduced by C3 inhibition, whereas alternative (factor B and D) and lectin (MASP-2) pathway inhibitors had no effect on the SBA of post-4CMenB vaccination sera. DISCUSSION: Compared to the SBA triggered by A,C,W,Y capsule polysaccharide conjugate vaccination, SBA against B meningococci expressing a matching fHbp allele was remarkably resilient against the alternative pathway inhibition

Identification of QTL genes for BMD variation using both linkage and gene-based association approaches

Low bone mineral density (BMD) is a risk factor for osteoporotic fracture with a high heritability. Previous large scale linkage study in Northern Chinese has identified four significant quantitative trait loci (QTL) for BMD variation on chromosome 2q24, 5q21, 7p21 and 13q21. We performed a replication study of these four QTL in 1,459 Southern Chinese from 306 pedigrees. Successful replication was observed on chromosome 5q21 for femoral neck BMD with a LOD score of 1.38 (nominal p value = 0.006). We have previously identified this locus in a genome scan meta-analysis of BMD variation in a white population. Subsequent QTL-wide gene-based association analysis in 800 subjects with extreme BMD identified CAST and ERAP1 as novel BMD candidate genes (empirical p value of 0.032 and 0.014, respectively). The associations were independently replicated in a Northern European population (empirical p value of 0.01 and 0.004 for CAST and ERAP1, respectively). These findings provide further evidence that 5q21 is a BMD QTL, and CAST and ERAP1 may be associated with femoral neck BMD variation

Poly(DL-lactide)-b-poly(N,N-dimethylamino-2-ethyl methacrylate): Synthesis, characterization, micellization behavior in aqueous solutions, and encapsulation of the hydrophobic drug dipyridamole

We synthesized a series of well-defined poly(DL-lactide)-b-poly(N,N- dimethylamino-2-ethyl methacrylate) (PDLLAb-PDMAEMA) amphiphilic diblock copolymers by employing a three-step procedure: (a) ring-opening polymerization (ROP) of DL-lactide using n-decanol and stannous octoate, Sn(Oct)2, as the initiating system, (b) reaction of the PDLLA hydroxyl end groups with bromoisobutyryl bromide, and (c) atom transfer radical polymerization, ATRP, of DMAEMA with the newly created bromoisobutyryl initiating site. The aggregation behavior of the prepared block copolymers was investigated by dynamic light scattering and ζ potential measurements at 25 °C in aqueous solutions of different pH values. The hydrophobic drug dipyridamole was efficiently incorporated into the copolymer aggregates in aqueous solutions of pH 7.40. High partition coefficient values were determined by fluorescence spectroscopy. © 2010 American Chemical Society

Micellization behavior of complex comblike block copolymer architectures

Complex comblike block copolymers of various architectures containing polystyrene, PS, and polyisoprene, PI, or polybutadiene, PB, components were synthesized by anionic polymerization high-vacuum techniques and the macromonomer strategy. Their micellization behavior was studied in n-decane, a selective solvent for the PI or PB blocks, and in N,N′-dimethylacetamide, DMA, selective for the PS blocks. Utilizing static and dynamic light scattering techniques, parameters such as the aggregation number and the hydrodynamic radius were determined. On the basis of these results, structural parameters of the micelles, i.e., core and corona radii as well as core-corona interfacial area, were calculated. The thermal stability of the micelles was also examined in both selective solvents. The macromolecular architecture was found to have a considerable effect on the micellization behavior of the block copolymers. © 2007 American Chemical Society

Alternative Complement Pathway Inhibition Does Not Abrogate Meningococcal Killing by Serum of Vaccinated Individuals

Dysregulation of complement activation causes a number of diseases, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. These conditions can be treated with monoclonal antibodies (mAbs) that bind to the complement component C5 and prevent formation of the membrane attack complex (MAC). While MAC is involved in uncontrolled lysis of erythrocytes in these patients, it is also required for serum bactericidal activity (SBA), i.e. clearance of encapsulated bacteria. Therefore, terminal complement blockage in these patients increases the risk of invasive disease by Neisseria meningitidis more than 1000-fold compared to the general population, despite obligatory vaccination. It is assumed that alternative instead of terminal pathway inhibition reduces the risk of meningococcal disease in vaccinated individuals. To address this, we investigated the SBA with alternative pathway inhibitors. Serum was collected from adults before and after vaccination with a meningococcal serogroup A, C, W, Y capsule conjugate vaccine and tested for meningococcal killing in the presence of factor B and D, C3, C5 and MASP-2 inhibitors. B meningococci were not included in this study since the immune response against protein-based vaccines is more complex. Unsurprisingly, inhibition of C5 abrogated killing of meningococci by all sera. In contrast, both factor B and D inhibitors affected meningococcal killing in sera from individuals with low, but not with high bactericidal anti-capsular titers. While the anti-MASP-2 mAb did not impair SBA, inhibition of C3 impeded meningococcal killing in most, but not in all sera. These data provide evidence that vaccination can provide protection against invasive meningococcal disease in patients treated with alternative pathway inhibitors