Neisseria meningitidis Serogroup Z Meningitis in a Child With Complement C8 Deficiency and Potential Cross Protection of the MenB-4C Vaccine.

Complement deficient patients are susceptible to rare meningococcal serogroups. A 6-year-old girl presented with serogroup Z meningitis. This led to identification of a C8 deficiency. The MenB-4C vaccine induced cross-reactive antibodies to serogroup Z and increased in vitro opsonophagocytic killing and may thus protect complement deficient patients.

Occurrence of an incomplete C8 molecule in homozygous C8 deficiency in man.

Sera from unrelated individuals with recurrent Neisserial infections lacked C8 hemolytic activity, but contained a protein that is antigenically related to C8. Immunochemical analysis revealed complete identity of the C8-related protein of all three sera and a marked antigenic deficiency compared with normal C8. The C8-related protein was isolated from serum by adsorption to immobilized anti-C8 IgG, elution with 3 M guanidine, and subsequent gel filtration. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the abnormal protein resembled the alpha-gamma subunit of normal C8 with respect to mobility and its ability to be cleaved upon reduction into the alpha and gamma chains. The beta chain present in normal C8 was absent. Sedimentation equilibrium analysis indicated a molecular weight of 86,000 for the abnormal C8 protein, which is identical to that of the alpha-gamma subunit of normal C8. Amino acid analysis revealed no significant difference between the abnormal C8 and normal alpha-gamma. Unlike normal C8, the abnormal protein did not bind to EAC1-7 or to SC5b-7; however, upon addition to the deficient serum of beta chain isolated from normal C8, hemolytic activity was restored and formation of SC5b-9 occurred. We concluded that the dysfunctional C8 protein in the three individuals' serum is identical to the alpha-gamma subunit of normal C8 and that this form of C8 deficiency is distinct from the C8 deficiencies previously reported in which the entire three-chain protein is lacking.

Host defense against Neisseria meningitidis requires a complement-dependent bactericidal activity.

Some individuals, with severe or recurrent infection with Neisseria species, have been identified as lacking a component in the terminal attack sequence of complement (complement components 5 to 9). The relevance of the terminal attack sequence to various phases of host defense was tested with the use of the C-11 strain of meningococci and human serum genetically deficient in complement component 8 (C8-D). The C8-D serum was comparable to normal serum in supporting ingestion and intracellular killing by leukocytes but was not bactericidal in the fluid phase unless reconstituted with C8. Thus, serum complement-dependent bactericidal activity may be especially critical for the host's defense against invasive Neisseria species.

Functional Complement Analysis Can Predict Genetic Testing Results and Long-Term Outcome in Patients With Complement Deficiencies.

Background: Prevalence of complement deficiencies (CDs) is markedly higher in Slovenian primary immunodeficiency (PID) registry in comparison to other national and international PID registries. Objective: The purposes of our study were to confirm CD and define complete and partial CD in registered patients in Slovenia, to evaluate frequency of clinical manifestations, and to assess the risk for characteristic infections separately for subjects with complete and partial CD. Methods: CD was confirmed with genetic analyses in patients with C2 deficiency, C8 deficiency, and hereditary angioedema or with repeated functional complement studies and measurement of complement components in other CD. Results of genetic studies (homozygous subjects vs. heterozygous carriers) and complement functional studies were analyzed to define complete (complement below the level of heterozygous carriers) and partial CD (complement above the level of homozygous patients). Presence of characteristic infections was assessed separately for complete and partial CD. Results: Genetic analyses confirmed markedly higher prevalence of CD in Slovenian PID registry (26% of all PID) than in other national and international PID registries (0.5-6% of all PID). Complement functional studies and complement component concentrations reliably distinguished between homozygous and heterozygous CD carriers. Subjects with partial CD had higher risk for characteristic infections than previously reported. Conclusion: Results of our study imply under-recognition of CD worldwide. Complement functional studies and complement component concentrations reliably predicted risk for characteristic infections in patients with complete or partial CD. Vaccination against encapsulated bacteria should be advocated also for subjects with partial CD and not limited to complete CD.

Genetic polymorphism in C8 beta-chains. Evidence for two unlinked genetic loci for the eighth component of human complement (C8).

Genetic polymorphism in the beta-subunit of the eighth component of human complement, C8, was defined by isoelectric focusing of serum in polyacrylamide gel in the presence of urea and development of specific patterns of hemolysis in an overlay gel containing antibody-sensitized erythrocytes and C8 beta-chain-deficient serum. Bands of hemolysis induced by serum from unrelated Caucasians suggested autosomal codominant inheritance of three structural alleles at a single locus, C82: C82 degrees A (acidic), C82 degrees B (basic), and C82 degrees A1 (very acidic) with frequencies of 0.952, 0.044, and 0.004, as well as the probable null allele C82 degrees Q0. The distribution of phenotypes agreed with the Hardy-Weinberg equilibrium. The previously described genetic polymorphism in human C8 defined with the use of "complete" C8 (C8 alpha-gamma-chain)-deficient serum was distinct from and independent of the inherited structural variation at C82. Therefore, the locus for C8 alpha-gamma-chains has been redesignated C81, and has the alleles C81 degrees A, C81 degrees A1, and C81 Q0. Linkage studies failed to show close linkage between the two loci for C8, C81, and C82, and between C82 and the major histocompatibility complex or C6.

Two types of dysfunctional eighth component of complement (C8) molecules in C8 deficiency in man. Reconstitution of normal C8 from the mixture of two abnormal C8 molecules.

Restoration of hemolytic activity was examined in sera from seven unrelated eighth component of complement (C8)-deficient subjects. The sera fell into two groups, depending on whether hemolytic activity was restored by the addition of the beta-chain (group 1) or the alpha-gamma-subunit (group 2) purified from normal human C8. Antigenic analysis of these sera by double-immunodiffusion using anti-human C8 confirmed previous findings of a dysfunctional C8 in the four sera of group 1 and established the presence of a different dysfunctional C8 in one of the sera of group 2 when tested at a high concentration. Further characterization of the dysfunctional C8 molecules in the two sera by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that group 1 sera were missing the beta-subunit and group 2 sera were missing the alpha-gamma-subunit of the C8 molecule. Sera from either of these two groups alone did not produce hemolysis in hemolytic plates containing sheep erythrocytes coated with antibody and complement components up to C7 (EAC1-7) and C9. When sera from the two groups were added to adjacent wells in the hemolytic plates, a zone of hemolysis developed between the wells. The contribution of C8 alpha-gamma from the sera of group 1 and of C8 beta from those of group 2 to the lysis of EAC1-7 in the presence of C9 was confirmed by the inhibitory effect of specific antibodies against the two C8 subunits. In experiments in which hemolytic activity was reconstituted by mixing sera from group 1 with sera from group 2, the serum source of C8 beta (group 2) was the limiting reagent. The dysfunctional C8 molecule in this serum was able to bind to EAC1-7. Chromatographic analysis demonstrated that the generation of hemolytic activity in the mixture of the two sera resulted from the reconstitution of the C8 molecule rather than the sequential action of the two C8 subunits.

Absence of the eighth component of complement in association with systemic lupus erythematosus-like disease.

A 56-yr-old black woman with absence of the eighth component of complement and a disease compatible with systemic lupus erythematosus is described. Her disease is characterized by the presence of photosensitive "malar" rash, alopecia, polyarthritis, and nephrotic syndrome. Hemolytic and immunochemical assays of the serum complement components were normal, except for C8 which was undetectable. Hemolytic activity could be restored to normal by the addition of functionally pure C8. In vitro tests to investigate the functional integrity of the classical and alternative pathways indicated that the functions mediated by activation of C3 and C5 were intact whereas heatlabile bactericidal activity was totally absent. Addition of C8 restored this function to normal levels. One of two brothers of the proband was shown to have serum C8 levels approaching 50% of normal indicating the hereditary nature of the defect. HLA typing studies showed that the normal brother had identical haplotypes to the proband while the proposed heterozygous brother only shared one haplotype with the patient, suggesting that the gene controlling the C8 defect was not closely linked to the major histocompatibility complex. If the association of a connective tissue disease and absence of a terminal component of complement is not coincidental, these results suggest that C8 deficiency may be associated with a subtle defect in the defense mechanisms to viral infection leading to viral persistance and perhaps to diseases such as systemic lupus erythematosus where chronic viral infections have been implicated.

Two distinct abnormalities in patients with C8 alpha-gamma deficiency. Low level of C8 beta chain and presence of dysfunctional C8 alpha-gamma subunit.

The sera from three C8 alpha-gamma deficient patients previously reported to have a selective C8 alpha-gamma defect were analyzed by SDS-PAGE and Western blot using two polyclonal antisera to C8 alpha-gamma and a monoclonal antibody to C8 alpha. All three sera exhibited C8 alpha-gamma bands that dissociated into alpha and gamma chains under reducing conditions. Quantitation of the alpha-gamma subunit in these sera by a sensitive ELISA revealed an amount approximately 1% of that found in normal human serum. A similar assay performed with a specific antiserum to C8 beta showed unexpectedly low levels of C8 beta in these sera, which were confirmed by hemolytic titration of C8 beta. The remarkable differences between C8 alpha-gamma and C8 beta in the C8 alpha-gamma deficient sera was that in spite of their comparable immunochemical levels, C8 beta still exhibited functional activity whereas C8 alpha-gamma was totally inactive. That the residual C8 alpha-gamma was inactive was also proved by its inability to show lytic bands in an overlay system after SDS-PAGE and subsequent removal of SDS. The implications of these findings for a novel concept of C8 deficiency are discussed.

Human deficiency of the eighth component of complement. The requirement of C8 for serum Neisseria gonorrhoeae bactericidal activity.

The serum of a 23-yr-old woman with prolonged disseminated gonococcal infection syndrome failed to normally promote hemolysis of sensitized sheep red blood cells (RBC). The patient's serum was deficient in the eight component of complement (C8) as determined by functional assays, immunoelectrophoresis, and quantitative immunoprecipitation. Functional titers of each of her other complement components were normal. No serum inhibitors of C8 were detected. The patient's serum supported activation of both the classical and alternate complement pathways. Her fresh serum lacked any bactericidal activity against Neisseria gonorrhoeae, but addition of purified C8 or complement donor serum restored bactericidal activity as well as RBC hemolytic activity. Her serum gave normal opsonization of yeast particles and staphylococci and had normal capacity to coat sensitized RBC with C8 and C4 and to generate chemotactic activity. No defects were observed in the patient's blood coagulation mechanisms. Complement-mediated bacterial lysis may be important in human defense against bacteremic Neisseria infections.

[Complement protein hereditary deficits during purulent meningitis: study of 61 adult Tunisian patients]. / Déficits héréditaires en protéines du complément au cours des méningites purulentes: étude de 61 patients adultes tunisiens et revue de la littérature.

Sixty one Tunisian adult patients with bacterial meningitis were screened for complement deficiency. Functional activity of the classical and the alternative pathways of complement (CH50 and AP50 respectively) were measured according to standard haemolytic procedures. Serum concentrations of C3 and C4 were determined by nephelometry. Late complement component (C5-C9) and properdin concentrations were assessed by double-ligand EISA. Complement deficiency was found in eight patients (13%): Seven had late complement component deficiency (three C7 deficiency, two C5 deficiency, one C6 deficiency and one C8 deficiency) and one had partial properdin deficiency. Patients with late complement component deficiency had a mean age of 24 years (range 17-32 years). All deficient patients had meningococcal meningitis. Recurrent meningitis was reported in half of the patients. Our findings demonstrated a high prevalence of complement deficiency in Tunisia suggesting that screening for hereditary complement deficiency should be performed in case of bacterial meningitides and meningococcal disease patients.

Invasive meningococcal disease in three siblings with hereditary deficiency of the 8(th) component of complement: evidence for the importance of an early diagnosis.

BACKGROUND: Deficiency of the eighth component of complement (C8) is a very rare primary immunodeficiency, associated with invasive, recurrent infections mainly caused by Neisseria species. We report functional and immunochemical C8 deficiency diagnosed in three Albanian siblings who presented with severe meningococcal infections at the age of 15 years, 4 years and 17 months, respectively. The youngest suffered serious complications (necrosis of fingers and toes requiring amputation). METHODS: Functional activity of the classical, alternative and mannose-binding lectin complement pathways was measured in serum from the 3 siblings and their parents (37-year-old woman and 42-year-old man). Forty healthy subjects (20 males and 20 females aged 4-38 years) served as normal controls. Serum complement factors were measured by haemolytic assays and immunoblotting. Sequence DNA analysis of the C8B gene was performed. RESULTS: Analyses of the three complement pathways revealed no haemolytic activity and also absence of C8beta in serum samples from all three siblings. The genetic analysis showed that the three siblings were homozygous for the p.Arg428* mutation in the C8B gene on chromosome 1p32 (MIM 120960). The parents were heterozygous for the mutation and presented normal complement activities. A 2-year follow-up revealed no further infective episodes in the siblings after antibiotic prophylaxis and meningococcal vaccination. CONCLUSIONS: Complement deficiencies are rare and their occurrence is often underestimated. In presence of invasive meningococcal infection, we highlight the importance of complement screening in patients and their relatives in order to discover any genetic defects which would render necessary prophylaxis to prevent recurrent infections and severe complications.

Recurrent Neisseria meningitidis bacteremia. Association with deficiency of the eighth component of complement (C8) in a Sephardic Jewish family.

A 24-year-old man had repeated episodes of meningococcal meningitis. Selective deficiency of the eighth component of complement (C8) was demonstrated in the patient, his twin brother, and in one of five siblings. As the parents were first cousins of normal phenotype, this pattern is suggestive of an autosomal recessive heredity. The present report brings the total number of patients given the diagnosis of C8 deficiency to 14, and calls attention to the existence of this condition in Jews of Sephardic (Mediterranean) origin.

Beta chain deficiency in three patients with dysfunctional C8 molecules.

Structural and functional studies were performed on a dysfunctional C8 molecule present in the serum of two siblings and an unrelated individual. The C8 in these three sera exhibited a pattern of partial immunologic identity with C8 in normal serum but was devoid of functional activity. The C8 was immunoprecipitated from the three sera and from a control serum with an antihuman C8 antiserum and analyzed by SDS-PAGE using highly purified human C8 as a reference. A selective absence of a band of 62,000 mol. wt was observed in the immunoprecipitates from the sera containing dysfunctional C8. Experiments performed with the purified alpha-gamma and beta subunits showed that the hemolytic activity of the C8 deficient sera could be reconstituted by the addition of the beta chain but not the alpha-gamma dimer. Binding of the dysfunctional C8 to C567 was excluded by the following observations: (1) EAC1-7 treated with the C8 deficient sera and then washed could not be lysed after the addition of the beta subunit and C9; and (2) the abnormal molecules did not interfere with the consumption of normal C8 by the soluble complex SC5b-7.

Purification and characterization of the eighth and ninth components of carp complement.

Complement components corresponding to mammalian C8 and C9 were isolated from carp (Cyprinus carpio) serum. Carp C8 (M(r) 146,000) proved to be a gamma-globulin composed of three polypeptide chains (alpha-chain, M(r) 62,000; beta-chain, M(r) 62,000; gamma-chain, M(r) 22,000). The alpha-chain was disulfide-linked to the gamma-chain and the beta-chain was non-covalently associated with the alpha-gamma chain, in fair agreement with mammalian C8. However, the N-terminal amino acid sequences of the three subunits showed no homology with those of human C8. Carp C9 was an alpha-globulin composed of a single polypeptide (M(r) 91,000) and the N-terminus was blocked. Carp serum depleted of C8 did not hemolyse either carp antibody-sensitized sheep erythrocytes or non-sensitized rabbit erythrocytes, while C9-depleted carp serum did not hemolyse the former, but did hemolyse the latter target cells, as in the case of C9-depleted human serum.

A Small Molecule that Induces Intrinsic Pathway Apoptosis with Unparalleled Speed.

Apoptosis is generally believed to be a process that requires several hours, in contrast to non-programmed forms of cell death that can occur in minutes. Our findings challenge the time-consuming nature of apoptosis as we describe the discovery and characterization of a small molecule, named Raptinal, which initiates intrinsic pathway caspase-dependent apoptosis within minutes in multiple cell lines. Comparison to a mechanistically diverse panel of apoptotic stimuli reveals that Raptinal-induced apoptosis proceeds with unparalleled speed. The rapid phenotype enabled identification of the critical roles of mitochondrial voltage-dependent anion channel function, mitochondrial membrane potential/coupled respiration, and mitochondrial complex I, III, and IV function for apoptosis induction. Use of Raptinal in whole organisms demonstrates its utility for studying apoptosis in vivo for a variety of applications. Overall, rapid inducers of apoptosis are powerful tools that will be used in a variety of settings to generate further insight into the apoptotic machinery.

Meningococcal disease in patients with late complement component deficiency: studies in the U.S.S.R.

The purpose of this study was to examine the occurrence of late complement component deficiency (LCCD) states in the USSR. Thirty deficient individuals were detected: 27 with C8 beta and 3 with C7 deficiency. Among individuals with a first episode of meningococcal infection, about 1% had LCCD, whereas among patients with recurrent bacterial meningitis the prevalence of LCCD rose to approximately 50%. This corresponds to a prevalence for LCCD of approximately 12 per 100,000 in the general population. The individuals with LCCD identified in this study experienced about 77 episodes of meningococcal disease and acute bacterial meningitis. Mathematical analysis of the morbidity from meningococcal disease in individuals with LCCD demonstrated that the probability of disease did not change with the age of the patient and was not affected by prior episodes of infection. This finding suggest that in contrast to the situation in the general population, prior infection fails to protect the deficient individual from recurrent disease. In comparison to complement-sufficient persons, the course of disease in individuals with LCCD is less severe, as shown by a reduction in the number of episodes of endotoxic shock and mortality as well as their more rapid recovery. These findings suggest that exuberant complement activation and concomitant formation of membrane attack complexes during meningococcal infection in complement-sufficient patients plays an important role in the activation and injury of peripheral blood cells and endothelial cells during endotoxic shock.

Deafness, complement deficiencies and immunoglobulin status in patients with meningococcal diseases due to uncommon serogroups.

The prevalence of deafness and complement deficiencies in association with meningococcal disease caused by uncommon serogroups of meningococci was studied in 30 patients (Group A) and 30 controls with Serogroup B disease (Group B). In Group A 8 patients (26.6%) had hearing impairment in contrast to only 1 patient (3.3%) in Group B (P < 0.01). Complement deficiency was detected in 8 patients (26.6%) of Group A whereas none of the Group B patients showed a defect in the complement system (P < 0.01). Association between complement deficiencies and meningococcal disease was detected for Serogroups Y (n = 5; 16.6%) and W135 (n = 3; 10.0%). Localization of the defects revealed only complement deficiencies of the classical pathway (C8-beta or C7 defects). The levels of Ig and IgG subclasses were found to be within the normal range for all patients. Our results suggest that meningococcal diseases caused by uncommon serogroups are more often associated with deafness and late complement component defects.

Deficiency of the eighth component of complement associated with recurrent meningococcal meningitis--case report and literature review.

The authors report a case of deficiency of the eighth component of complement in a young adult with a history of three episodes of meningitis; one of them proved to be meningococcal. The literature was reviewed and meningitis due to Neisseria meningitidis strains causing disease in complement-deficient and complement-sufficient patients was demonstrated. Meningococcal disease may be the first manifestation of complement deficiency; screening for complement function must be considered for those with invasive meningococcal disease, with posterior evaluation of the components of the terminal pathway of complement.